CN101208087A

CN101208087A - Methods of using [3.2.0] heterocyclic compounds and analogs thereof

Info

Publication number: CN101208087A
Application number: CNA2005800477467A
Authority: CN
Inventors: 迈克尔·帕拉迪诺; 芭芭拉·克里斯蒂娜·波茨; 文卡塔·拉米·雷迪·马舍拉; 萨斯基亚·狄奥多拉·科尔内利亚·内特博姆
Original assignee: Nereus Pharmaceuticals Inc
Current assignee: Nereus Pharmaceuticals Inc
Priority date: 2004-12-03
Filing date: 2005-12-02
Publication date: 2008-06-25
Also published as: ZA200704461B

Abstract

Disclosed are methods of treating cancer, inflammatory conditions, and/or infectious disease in an animal comprising: administering to the animal, a therapeutically effective amount of a heterocyclic compound. The animal is a mammal, preferably a human or a rodent.

Description

The application process of [3.2.0] heterocyclic compound and analog thereof

Relevant patent information

The application requires the 60/633rd of December in 2004 submission on the 3rd according to 35 U.S.C. § 119 (e), the 60/643rd of submission on January 13rd, No. 379 1, the 60/658th of submission on March 4th, No. 922 1, the 60/676th, No. 553 U.S. Provisional Application No. that No. 884 and on April 29th, 2005 submit to; The title of above-mentioned each provisional application is " application process of [3.2.0] heterocyclic compound and analog thereof "; Introduce by the mode of reference at this full content each application.

Invention field

The present invention relates to some chemical compound and preparation method thereof and the application in chemistry and field of medicaments.Embodiment of the present invention disclosed herein relate to the method for using heterocyclic compound.In some embodiments, described chemical compound is as proteasome (proteasome) inhibitor.In other embodiments, described chemical compound is used for the treatment of inflammation, cancer and infectious disease.

Description of Related Art

In the U.S., cancer is to cause main causes of death.Although having prominent achievement aspect the new method of finding the treatment cancer, main treatment is selected to remain surgical operation, chemotherapy and X-ray therapy, and these three kinds of Therapeutic Method can use separately or use in conjunction.But surgical operation and X-ray therapy are usually only useful to the cancer of quite limited type, and they have limitation aspect the patient of having spread in the treatment cancer.Chemotherapy generally is used for the treatment of metastatic carcinoma or such as the patient of leukemic dispersivity cancer.Although chemotherapy has therapeutic value, because patient's cancerous cell becomes chemotherapeutant is had drug resistance, so it often can not cure diseases.Part reason may be that cancerous cell becomes chemotherapeutant is had drug resistance, and the common use in conjunction of this class therapeutic agent is treated the patient.

Similarly, the infectious disease that is for example caused by antibacterial, fungus and protozoacide also becomes day by day and is difficult to treatment and healing.Such as increasing antibacterial, fungus and protozoacide current antibiotic and chemotherapeutic agent are progressively produced drug resistance.This quasi-microorganism comprises bacillus (Bacillus), leishmania (Leishmania), plasmodium (Plasmadium) and trypanosomicide (Trypanosoma).

In addition, increasing disease and medical condition are divided into diseases associated with inflammation.This class disease comprises that the morbid state such as asthma arrives cardiovascular disease.Although new therapy and Medical Technology progress are arranged, this class disease still worldwide affects increasing people.

Therefore, there is demand, with treatment cancer, diseases associated with inflammation and infectious disease for new chemotherapy, antibacterial and anti-inflammatory agents.For this reason, different researcheres, institute and company are carrying out consistent efforts, to attempt to differentiate new, possible effectively chemotherapy and antimicrobial agent.

Marine natural products is the new potential anticancer agent and the abundant source of antimicrobial agent.The ocean is a huge compound system, and the microorganism in the diversified extreme condition environment that appears at pressure, salinity and temperature moves in wherein.Marine microorganism so evolutionary development have gone out on the metabolism and physiological unique property, these characteristics not only make them survive in extreme and changeable environment, and can make them produce special metabolite, these metabolites are (Okami, Y.1993 the JMar Biotechnol 1:59) that do not observe from the metabolite of Lu Sheng microorganism.The exemplary configuration type of this class metabolite comprises the chemical compound in the synthetic source of terpene, peptide, acetogenin and mixed biologic.Many antitumor, antibacterium, antifungal, antiinflammatory or immunosuppressant activity (Bull, A.T.et al.2000 Microbiol Mol Biol Rev 64:573 of all demonstrating in these molecules; Cragg, G.M.﹠amp; D.J.Newman2002 Trends Pharmacol Sci 23:404; Kerr, R.G.﹠amp; S.S.Kerr 1999 ExpOpin Ther Patents 9:1207; Moore, B.S 1999 Nat Prod Rep 16:653; Faulkner, D.J.2001 Nat Prod Rep 18:1; Mayer, A.M.﹠amp; V.K.Lehmann2001 Anticancer Res 21:2489), this has proved the practicality of utilizing this resource (marine natural products resource): can therefrom isolate invaluable disease therapeuticing medicine.In addition, help to solve chemical sproof problem the separating of new anticancer and antimicrobial agent different with the mechanism of action of the medicine of selling in the market, comprising to those drug resistance based on drug mechanism, this type of drug resistance has been engineered to the pathogen that is used for the bio-terrorism purposes.

Summary of the invention

Embodiment disclosed herein is usually directed to chemical compound, comprises heterocyclic compound and analog thereof.Some embodiments relate to the purposes of chemical compound as proteasome inhibitor.

In other embodiments, described chemical compound is used for the treatment of neoplastic disease, for example, suppresses the growth of tumor, cancer and other tumprigenicity tissue.Therapeutic Method disclosed herein can be used for treating any patient that tumor growth thing, cancer or optimum or virulent tumprigenicity growth-gen arranged under a cloud, and (this paper employed " tumor " or " kinds of tumors " comprise tumor, cancer, the tumprigenicity cell of diffusion and the growth of the tumprigenicity of limitation).The example of this class growth-gen includes but are not limited to: breast carcinoma; Osteosarcoma, angiosarcoma, fibrosarcoma and other sarcoma; Leukemia; The venous sinus tumor; Ovarian cancer, carcinoma of ureter, bladder cancer, the cancer of carcinoma of prostate and other urogenital system; Colon cancer, esophageal carcinoma and gastric cancer and other gastrointestinal cancer; Pulmonary carcinoma; Lymphoma; Myeloma; Cancer of pancreas; Hepatocarcinoma; Renal carcinoma; The endocrine cancer; Skin carcinoma; Melanoma; Hemangioma; And brain or central nervous system's (CNS) cancer.Generally speaking, the described tumor or the growth-gen of desire treatment can be any tumor or cancer, former or secondary.Some embodiment relates to the method for the treatment of neoplastic disease in the animal body.These methods can comprise, for example, and with the patient's administration of the chemical compound of effective dose to this treatment of needs.Other embodiment has related to described chemical compound and has been used for the treatment of the medicine of neoplastic disease or the purposes in the medicament in preparation.

Described chemical compound can with treatment administering drug combinations or the application such as chemotherapy, X-ray therapy and biotherapy.In some embodiments, described chemical compound can with chemotherapeutant co-administered or use.The chemotherapeutic example of this class comprises alkaloid, alkylating agent, antibiotic, antimetabolite, enzyme, hormone, platinum compounds, immunotherapy (antibody, T-cell, epi-position), BRMs or the like.Example comprises vincristine, vinblastine, vindesine, Paclitaxel (paclitaxel), Docetaxel, topoisomerase enzyme inhibitor etoposide (etoposide (VP-16), teniposide (VM-26)), camptothecine, chlormethine (cyclophosphamide), nitroso ureas, carmustine, lomustine, dacarbazine, melamine methylol, plug is for group and ametycin, dactinomycin (actinomycin D), anthracycline antibiotics (daunorubicin, daunomycin, daunorubicin), doxorubicin (amycin), idarubicin (idarubicin), anthraquinone class (anthracenedione) (mitoxantrone), bleomycin (Bleomycin Sulphate), plicamycin (mithramycin, antifol (methotrexate (Folex, Mexate)), purine antimetabolite (Ismipur (6-MP, purinethol) and 6-thioguanine (6-TG).At two kinds of main cancer therapy drugs of this kind apoplexy due to endogenous wind is Ismipur and 6-thioguanine, chlorine Deoxyadenosine and pentostatin, pentostatin (2 '-deoxycoformycin), the pyrimidine antagonist, fluorine pyrimidine (5-fluorouracil (Adrucil), floxuridine (FdUrd) (floxuridine)), cytosine arabinoside (Cytosar, ara-C), fludarabine, the L-asparaginase, hydroxyurea, glucocorticoids, estrogen antagonist, tamoxifen, the non-steroid antiandrogen, flutamide, aromatase inhibitor Anastrozole (Arimidex (Arirnidex)), cisplatin, 6-mercaptopurine and thioguanine, methotrexate, cyclophosphamide, cytosine arabinoside, the L-asparaginase, steroid: prednisone and dexamethasone.For example such as the proteasome inhibitor of bortezomib (Bortezomib) also can with chemical compound use in conjunction of the present invention.Biological example can comprise such as at the reagent of the TRAIL antibody of TRAIL, such as the integrin of α-V-β-3 (α V β 3) and/or relate to other cytokine/somatomedin of angiogenesis, VEGF, EGF, FGF and PDGF.In some respects, described chemical compound can be with antibody coupling or is together carried.Above-mentioned integrated processes can be used for treating various disease states, comprises cancer and neoplastic disease, inflammation and infected by microbes.

In other embodiments, described chemical compound is used for the treatment of the diseases associated with inflammation state.Some embodiment relates to the method for the treatment of diseases associated with inflammation state in the animal body, and these methods comprise, for example, and with the patient's administration of the chemical compound of effective dose to the described treatment of needs.Other embodiment has related to chemical compound and has been used for the treatment of the medicine of inflammation or the purposes in the medicament in preparation.

In certain embodiments, described chemical compound can be used for treating infectious disease.Infectious pathogen can be microorganism, for example, antibacterial, fungus, protozoacide and visible algae of microscopically or virus.And described infectious pathogen also can be B.anthracis (anthrax).In some embodiments, described infectious pathogen is a parasite.For example, described infectious pathogen can be plasmodium, leishmania and trypanosomicide.Some embodiment relates to the method for the treatment of infectious pathogen in the animal body.These methods comprise, for example, and with the patient's administration of the chemical compound of effective dose to the described treatment of needs.Other embodiment has related to chemical compound and has been used for the treatment of the medicine of infectious pathogen or the purposes in the medicament in preparation.

Some embodiments have related to chemical compound, the acceptable salt of its medicine and esters prodrug thereof the application in cancer, inflammation and infectious disease treatment of the structure with general formula I:

General formula I

Wherein dotted line is represented singly-bound or two key, and R wherein ₁Can be independently selected from single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl; acyl group; acyloxy; oxyl ketonic oxygen base; aryloxycarbonyl oxygen base; cycloalkyl; cycloalkenyl group; oxyl; the ring oxyl; aryl; heteroaryl; aryl oxyl carbonyl; oxyl carbonyl acyl group; amino; amino carbonyl; amino carbonyl oxygen base; nitro; azido; phenyl; the cycloalkyl acyl group; hydroxyl; alkylthio group; arylthio; oxygen base sulfonyl; carboxyl; cyano group; sulfo-; sulfoxide; sulfone; sulphonic acid ester; thiocyano; boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl.Wherein n equals 1 or 2, and if n equal 2, R then ₁Can be identical or different;

R wherein ₂Can be selected from single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group (comprise, for example, hexahydrobenzyl alcohol), oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl;

R wherein ₃Can be selected from single replacement of halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and the haloalkyl that comprises multi-haloalkyl; Each E wherein ₁, E ₂, E ₃And E ₄Can be and replace or unsubstituted hetero atom.

In some embodiments, preferred R ¹For replacing or unsubstituted C ₁-C ₅Alkyl.For example, preferable methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group and amyl group.In some embodiments, R ¹It or not replacement or unsubstituted.The C of straight chain ₆Alkyl.

Other embodiment relates to the method for the treatment of neoplastic disease in the animal body, and these methods can comprise, for example, with the general formula compound that is selected from general formula I-VI of treatment effective dose and the acceptable salt of medicine thereof and esters prodrug to patient's administration.

Other embodiments relate to the pharmaceutical composition that comprises the general formula compound that is selected from general formula I-V.Described pharmaceutical composition also can comprise antimicrobial.

Other embodiment also relates to the method for anticancer growth, and this method can comprise, for example, cancerous cell is contacted with the esters prodrug with general formula compound that is selected from general formula I-VI and the acceptable salt of medicine thereof.

Other embodiment relates to the active method of Profilin enzyme body, and this method comprises makes cell contact with the esters prodrug with general formula compound that is selected from general formula I-VI and the acceptable salt of medicine thereof.

Other embodiment relates to and suppresses the activatory method of nuclear Factor-Kappa B (NF-κ B), and this method comprises makes cell contact with the esters prodrug with general formula compound that is selected from general formula I-VI and the acceptable salt of medicine thereof.

Some embodiments relate to the method for the treatment of the diseases associated with inflammation state, comprise the patient's administration to the described treatment of needs of the general formula compound that is selected from general formula I-V of effective dose.

Other embodiment relates to the method for the treatment of the disease that is caused by microorganism, comprises the patient's administration to the described treatment of needs of the general formula compound that is selected from general formula I-V of effective dose.

Brief Description Of Drawings

Be incorporated herein and accompanying drawing exemplary illustration some preferred embodiment of the present invention only of a book part as an illustration.With the other parts of description, they all are used for explaining to those skilled in the art the optimal way of some chemical compound of preparation the present invention.In the accompanying drawings:

Fig. 1 shows the chemical constitution of Salinosporamide A.

Fig. 2 shows the general tropical distribution of Salinospora.The collection position of " X " expression Salinospora.

Fig. 3 shows the bacterium colony of Salinospora.

Fig. 4 shows the typical 16S rDNA sequence of Salinospora.Vertical bar is represented the signature nucleotide of Salinospora, and this signature nucleotide is distinguished in Salinospora and its nearest relevant species.

Fig. 5 shows Omuralide, a kind of catabolite of metabolite-lactacystin of microorganism.Fig. 5 also shows the chemical compound with formula II-16, is also referred to as Salinosporamide A.

Fig. 6 shows the cytotoxicity of the macrophage of fatefulue toxin mediation.The chemical compound of NPI-0052 expression II-16.

Fig. 7 has described chemical compound with structural formula II-20 ¹H NMR collection of illustrative plates.

Fig. 8 has described chemical compound with structural formula II-24C ¹H NMR collection of illustrative plates.

Fig. 9 has described chemical compound with structural formula II-19 ¹H NMR collection of illustrative plates.

Figure 10 has described chemical compound with structural formula II-2 ¹H NMR collection of illustrative plates.

Figure 11 has described the mass spectrum of the chemical compound with structural formula II-2.

Figure 12 has described chemical compound with structural formula II-3 ¹H NMR collection of illustrative plates.

Figure 13 has described the mass spectrum of the chemical compound with structural formula II-3.

Figure 14 has described chemical compound with structural formula II-4 ¹H NMR collection of illustrative plates.

Figure 15 has described the mass spectrum of the chemical compound with structural formula II-4.

Figure 16. chemical compound with structural formula II-5A has been described ¹H NMR collection of illustrative plates.

Figure 17 has described the mass spectrum of the chemical compound with structural formula II-5A.

Figure 18 has described chemical compound with structural formula II-5B ¹H NMR collection of illustrative plates.

Figure 19 has described the mass spectrum of the chemical compound with structural formula II-5B.

Figure 20 described have structural formula IV-3C chemical compound at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 21 described have structural formula IV-3C chemical compound at C ₆D ₆/ DMSO-d ₆In the 1HNMR collection of illustrative plates

Figure 22 has described chemical compound with structural formula II-13C ¹H NMR collection of illustrative plates.

Figure 23 has described chemical compound with structural formula II-8C ¹H NMR collection of illustrative plates.

Figure 24 has described chemical compound with structural formula II-25 ¹H NMR collection of illustrative plates.

Figure 25 has described chemical compound with structural formula II-21 ¹H NMR collection of illustrative plates.

Figure 26 has described chemical compound with structural formula II-22 ¹H NMR collection of illustrative plates.

Figure 27 shows the active inhibition of chymotrypsin-like of rabbit myopsin body.

Figure 28 shows the PGPH and the active inhibition of Caspase sample (Caspase-like) of rabbit myopsin body.

Figure 29 shows the active inhibition of the chymotrypsin-like of human red blood cell proteasome.

Figure 30 shows that II-16 handles the effect to the LLVY-AMC substrate cutting of chymase mediation.

Figure 31 shows nuclear Factor-Kappa B (NF-κ the B)/activity of luciferase and cytotoxicity characteristic spectrum of II-16.

Figure 32 is presented at the maintenance of the I κ B α of the minimizing of the I κ B α degraded that II-16 causes among HEK293 cell (A) and the HEK293 NF-κ B/ luciferase reporter gene clone (B) and phosphorylation.

After Figure 33 shows that II-16 is to HEK293 cell (A) and HEK293 NF-κ B/ luciferase reporter gene clone (B) processing, cyclin, the accumulation of p21 and p27.

Figure 34 is presented in the Jurkat cell, and II-16 is to the activation of Caspase-3 (Caspase-3).

Figure 35 is presented in the Jurkat cell, and II-16 is to the cutting of PARP.

Figure 36 is presented in the RAW264.7 cell, and II-16 is to the inductive Cytotoxic inhibition of LeTx.

Figure 37 shows that II-16 handles the effect to PARP in RPMI 8226 and the PC-3 cell and preceding Caspase-3 (Pro-Caspase-3) cutting.

Figure 38 shows that II-16 causes the dose dependent cutting of PARP and preceding Caspase-3 to the processing of RPMI 8226.

Figure 39 shows II-16, II-17, and II-18 is in external inhibition to proteasome.

Figure 40 shows the activity of the proteasome among the PWBL for preparing from the mice of II-16 treatment.

Figure 41 is presented in the PWBL analysis, the therapeutic effect of epoxomicin.

Figure 42 shows the comparison of internal analysis.

Figure 43 is presented in the mice that LPS handles, and Plasma TNF levels descends.

Figure 44 describes and is used for explanation at HEK293 NF-κ B/Luc cell, formula II-2, and II-3 and II-4 are to the analysis result of the effect of the luciferase activity of NF-κ B mediation.

Figure 45. description is used for explanation at HEK293 NF-κ B/Luc cell, and formula II-5A and II-5B are to the analysis result of the effect of the luciferase activity of NF-κ B mediation.

Figure 46 has described and has been used for formula II-2, and II-3 and II-4 are to the analysis result of the active effect of chymotrypsin-like of rabbit 20S proteasome.

Figure 47 has described formula II-5A and the II-5B active effect to the chymotrypsin-like of rabbit 20S proteasome.

Figure 48 has described formula II-2, the Cytotoxic effect of the anti-LeTx mediation of II-3 and II-4.

Figure 49 has described chemical compound with structural formula II-17 ¹H NMR collection of illustrative plates.

Figure 50 has described chemical compound with structural formula II-18 ¹H NMR collection of illustrative plates.

Figure 51 has described the chemical compound of formula II-26 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 52 has described the ORTEP figure that the computer of the chemical compound of formula II-26 produces.

Figure 53 has described the chemical compound of formula II-27 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 54 has described the chemical compound of formula II-28 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 55 has described the chemical compound of formula II-29 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 56 has described the chemical compound of formula II-30 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 57 has described the chemical compound of formula II-44 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 58 has described the chemical compound of formula I-7 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 59 has described the chemical compound of formula II-47 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 60 has described the chemical compound of formula II-38 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Figure 61 has described the chemical compound of formula II-50 at DMSO-d ₆In ¹H NMR collection of illustrative plates.

Detailed description of preferred embodiments

The present invention has quoted numerous lists of references.With the present invention quoted, comprise that each list of references full content of the United States Patent (USP) that this paper quotes introduces in this description by the mode of reference.

Embodiment of the present invention comprise, but be not limited to provide the preparation method of chemical compound, described chemical compound comprises chemical compound, for example, comprise chemical compound described herein and analog thereof, and provide and produce for example acceptable antimicrobial compositions of medicine, anti-cancer composition and anti-inflammatory method for compositions.Described method can comprise described compositions by quite high yield, and wherein said chemical compound and/or its derivant are one of active component in these compositionss.Other embodiment relates to provides the noval chemical compound that can't be obtained by present available method.In addition, embodiment relates to the method for the treatment of cancer, inflammation and infectious disease, especially treats the method for the infectious disease of those infected person.In some embodiments, one or more general formula compounds in the chemical compound group, or one or more chemical compound can specifically be got rid of outside any one or several different methods of treatment morbid state as herein described.As an illustrative example, formula II-16 chemical compound can be excluded in certain embodiments in the general therapeutic cancer, for example treats outside the method for cancer of particular type.Described method for example can comprise the step that the member with a class noval chemical compound of effective dose carries out administration.Embodiment preferred relates to the preparation and the using method of described chemical compound and this compounds disclosed by the invention, but these chemical compounds are not all to be necessary in all embodiments of the present invention, and these purposes can realize.

For the chemical compound that the present invention describes, each spatial carbon atom can be R or S configuration.Though illustrational in this application specific chemical compound is described with concrete configuration, also can expect at any given chiral centre place the opposite spatial chemistry or the chemical compound of its mixture being arranged.When in the derivant among the present invention chiral centre being arranged, be to be understood that described chemical compound comprises whole possible stereoisomers.

Compound of Formula I

Some embodiments provide the method for a compounds and these chemical compounds of preparation and acceptable salt of medicine and esters prodrug, and wherein said chemical compound is represented with general formula I:

General formula I

In certain embodiments, substituent R ₁, R ₂And R ₃Can comprise single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant independently: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl (comprise; for example, hexahydrobenzyl alcohol), cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group, sulfo-, sulfoxide, sulfone, sulphonic acid ester, thiocyano, boric acid and ester thereof and comprise the haloalkyl of multi-haloalkyl.In addition, in certain embodiments, each E ₁, E ₂, E ₃And E ₄Can be the hetero atom of hetero atom or replacement, for example, be selected from the hetero atom of nitrogen, sulfur and oxygen respectively.

In some embodiments, n can equal 1 or equal 2, and when n equaled 2, substituent group can be identical or different.In addition, in some embodiments, R ₃Be not hydrogen.

In some embodiments, preferred R ₁Be that replace or unsubstituted C ₁-C ₅Alkyl.For example, preferable methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group and amyl group.In some embodiments, R ₁Be not that replace or unsubstituted, unbranched C ₆Alkyl.

In some embodiments, work as R ₁One of substituent group is ethyl or chloroethyl and R ₃During for methyl, R ₂Be not hexamethylene-2-thiazolinyl methanol.

Preferably, R ₂Be formoxyl.For example, described chemical compound can have following structure I-1:

General formula I-1

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, the structure of general formula I-1 can have following spatial chemistry:

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, R ₂Can be methanol.For example, described chemical compound can have following structure I-2:

General formula I-2

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

As an example, the structure of general formula I-2 can have following spatial chemistry:

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

As the exemplary compounds of general formula I, can be chemical compound with following structure I-3:

General formula I-3

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

The chemical compound of general formula I-3 can have following spatial chemistry:

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Another exemplary compounds of general formula I can be the chemical compound with following structure I-4:

General formula I-4

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, the chemical compound of general formula I-4 can have following spatial chemistry:

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Another exemplary compounds of general formula I is the chemical compound with following structure I-5:

General formula I-5

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

For example, the chemical compound of general formula I-5 can have following spatial chemistry:

R ₈Can comprise, for example, hydrogen, fluorine, chlorine, bromine and iodine.

In some embodiments, the R of general formula I ₂It for example can be hexamethylene-2-thiazolinyl ylidenylmethyl (cyclohex-2-enylidenemethyl).For example, described chemical compound can have the structure of following general formula I-6:

General formula I-6

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, the chemical compound of general formula I-6 can have following spatial chemistry:

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

In going back an embodiment, the R of general formula I ₂It for example can be hexamethylene-2-thiazolinyl methyl.For example, chemical compound can have the structure of following general formula I-7:

General formula I-7

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Preferably, the chemical compound of general formula I-7 can have following spatial chemistry:

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

In other embodiments, R ₂It can be the cyclohexyl alkylamine.

In other embodiments, R ₂It also can be C-cyclohexyl-benzylidene amino.In other embodiments, R ₂It can be cyclohexane extraction formaldehyde O-oxime.

In addition, in some embodiments, R ₂It can be the cycloalkyl acyl group.

General formula I I chemical compound

Other embodiment provides the method for a compounds and these chemical compounds of preparation and acceptable salt of medicine and esters prodrug, and wherein said chemical compound is represented with general formula I I:

General formula I I

In certain embodiments, substituent R ₁, R ₃And R ₄Can comprise single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant independently: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl; acyl group; acyloxy; oxyl ketonic oxygen base; aryloxycarbonyl oxygen base; cycloalkyl; cycloalkenyl group; oxyl; the ring oxyl; aryl; heteroaryl; aryl oxyl carbonyl; oxyl carbonyl acyl group; amino; amino carbonyl; amino carbonyl oxygen base; nitro; azido; phenyl; the cycloalkyl acyl group; hydroxyl; alkylthio group; arylthio; oxygen base sulfonyl; carboxyl; cyano group; sulfo-; sulfoxide; sulfone; sulphonic acid ester; thiocyano; boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl.In addition, in certain embodiments, each E ₁, E ₂, E ₃And E ₄Can be replacement or unsubstituted hetero atom, for example, be selected from the hetero atom of nitrogen, sulfur and oxygen or the hetero atom of replacement.

In some embodiments, n can equal 1, and in other embodiments, it can equal 2.When n equaled 2, substituent group can be identical or different.In addition, in some embodiments, R ₃Be not hydrogen.M can equal 1 or 2, and when m equals 2, R ₄Can be identical or different.

E ₅For example can be OH, O, OR ₁₀, S, SR ₁₁, SO ₂R ₁₁, NH, NH ₂, NOH, NHOH, NR ₁₂And NHOR ₁₃, R wherein _10-13For example can comprise the replacement of halogen, following any group independently or not replace form: alkyl, aromatic radical, assorted aromatic radical or the like.R in addition ₁Can be CH ₂CH ₂X, wherein X for example can be H, F, Cl, Br and I.R ₃It can be methyl.In addition, R ₄Can comprise cyclohexyl.In addition, each E ₁, E ₃And E ₄Can be O and E ₂Can be NH.Preferably, R ₁Can be CH ₂CH ₂X, wherein X is selected from H, F, Cl, Br and I; R wherein ₄Can comprise cyclohexyl; R wherein ₃Can be methyl; And each E wherein ₁, E ₃And E ₄Can be O and E ₂Can be NH.

In some embodiments, work as R ₁One of substituent group is ethyl or chloroethyl, and R ₃When being methyl, R ₂Be not chlorine hexamethylene-2-thiazolinyl methanol.

In some embodiments, preferred R ₁Be that replace or unsubstituted C ₁-C ₅Alkyl.For example, preferable methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group and amyl group.In some embodiments, R ₁Be not that replace or C unsubstituted, straight chain ₆Alkyl.

For example, the exemplary compounds of general formula I I has following structure I I-1:

General formula I I-1

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Exemplary spatial chemistry can be as follows:

In preferred embodiments, general formula I I chemical compound has any in the following structure:

Following is respectively the exemplary spatial chemistry with chemical compound of structure I I-2, II-3 and II-4:

In other embodiments, R wherein ₄Can comprise 7-oxa--bicyclo-[4.1.0] heptan-2-base.The exemplary compounds of general formula I I is following structure I I-5:

General formula I I-5

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Following is the examples for compounds with general formula I I-5 structure:

General formula I I-5A and II-5B

In another embodiment, has a R at least ₄Can comprise and replacing or unsubstituted branched alkyl.For example, the chemical compound of general formula I I can be following structure I I-6:

General formula I I-6

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Following is the exemplary spatial chemistry with chemical compound of general formula I I-6 structure:

As other example, the chemical compound of general formula I I can be following structure I I-7:

General formula I I-7

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Following is the exemplary spatial chemistry with chemical compound of general formula I I-7 structure:

In other embodiments, has a R at least ₄Can be cycloalkyl and E ₅Can be oxygen.The exemplary compounds of general formula I I can be following structure I I-8:

General formula I I-8

R ₈For example can comprise hydrogen (II-8A), fluorine (II-8B), chlorine (II-8C), bromine (II-8D) and iodine (II-8E).

Following is the exemplary spatial chemistry with chemical compound of general formula I I-8 structure:

In some embodiments, E ₅Can be for generating the amine oxide of oxime.The exemplary compounds of general formula I I has following structure I I-9:

General formula I I-9

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine; R for example can be hydrogen, replacement or unsubstituted alkyl, aryl or heteroaryl or the like.

Following is the exemplary spatial chemistry with chemical compound of general formula I I-9 structure:

The other exemplary compounds of general formula I I has following structure I I-10:

General formula I I-10

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Following is the exemplary spatial chemistry with chemical compound of general formula I I-10 structure:

In some embodiments, E ₅Can be NH ₂The exemplary compounds of general formula I I has following structure I I-11:

General formula I I-11

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Following is the exemplary spatial chemistry with chemical compound of general formula I I-11 structure:

In some embodiments, has a R at least ₄Can comprise cycloalkyl and E ₅Can be NH ₂The exemplary compounds of general formula I I has following structure I I-12:

General formula I I-12

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Following is the exemplary spatial chemistry with chemical compound of general formula I I-12 structure:

The other exemplary compounds of general formula I I has following structure I I-13:

General formula I I-13

R ₈For example can comprise hydrogen (II-13A), fluorine (II-13B), chlorine (II-13C), bromine (II-13D) and iodine (II-13E).

Following is the exemplary spatial chemistry with chemical compound of general formula I I-13 structure:

The other exemplary compounds of general formula I I has following structure I I-14:

General formula I I-14

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Following is the exemplary spatial chemistry with chemical compound of general formula I I-14 structure:

In some embodiments, the chemical compound of general formula I I for example can comprise R ₄At least one is a cyclenes.In addition, in some embodiments, described chemical compound for example can comprise E ₅The position is a hydroxyl.Another exemplary compounds of general formula I I has following structure I I-15:

General formula I I-15

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Exemplary spatial chemistry can be as follows:

Following is respectively the exemplary spatial chemistry with chemical compound of structure I I-16, II-17, II-18 and II-19:

Formula II-16, II-17, II-18 and II-19 chemical compound can be by following fermentation of illustrating, synthetic or semi-synthetic and separation/purification method acquisitions.In addition, formula II-16, II-17, II-18 and II-19 chemical compound can be used as and can be described as " initiation material " and prepare other chemical compound as herein described.

In some embodiments, general formula I I chemical compound for example can comprise that methyl group is as R ₁Other exemplary compounds, formula II-20 has following structure and spatial chemistry:

In some embodiments, general formula I I chemical compound for example can comprise that ethoxy is as R ₁Other exemplary compounds, formula II-21 has following structure and spatial chemistry:

In some embodiments, the hydroxyl of formula II-21 can be esterified, thus R ₁For example can comprise ethyl propionate.Exemplary compounds, formula II-22 has following structure and spatial chemistry:

In some embodiments, general formula I I chemical compound for example can comprise that ethyl group is as R ₃Another exemplary compounds of general formula I I has following structure I I-23:

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.Exemplary spatial chemistry can be as follows:

In some embodiments, the chemical compound of general formula I I-23 can have following structure and the spatial chemistry by formula II-24C exemplary illustration, wherein R ₈Be chlorine:

In some embodiments, the chemical compound of general formula I I-15 can have the following spatial chemistry by the chemical compound exemplary illustration of formula II-25, wherein R ₈Be chlorine:

In some embodiments, general formula I I-15 chemical compound can have the following spatial chemistry by formula II-26 chemical compound exemplary illustration, wherein R ₈Be chlorine:

In some embodiments, general formula I I chemical compound can have following structure and the spatial chemistry by formula II-27 exemplary illustration, wherein R ₁Be ethyl:

In some embodiments, general formula I I chemical compound can have following structure and the spatial chemistry by formula II-28 exemplary illustration, wherein R ₁Be methyl:

In some embodiments, general formula I I chemical compound for example can comprise that the azido ethyl is as R ₁Another exemplary compounds, formula II-29 has following structure and spatial chemistry:

In some embodiments, general formula I I chemical compound for example can comprise that propyl group is as R ₁Another exemplary compounds, formula II-30 has following structure and spatial chemistry:

An exemplary compounds also, formula II-31 and II-32 have following structure and spatial chemistry:

Formula II-31 and II-32

Other exemplary compounds, formula II-33, II-34, II-35 and II-36 have following structure and spatial chemistry:

Formula II-33 to II-36

In some embodiments, general formula I I chemical compound for example can comprise that cyanoethyl is as R ₁For example, formula II-37 chemical compound has following structure and spatial chemistry:

In another embodiment, general formula I I chemical compound for example can comprise that ethyl thiocyanate is as R ₁For example, formula II-38 chemical compound has following structure and spatial chemistry:

In some embodiments, general formula I I chemical compound for example can comprise that thiol is as R ₁Another exemplary compounds, general formula I I-39 has following structure and spatial chemistry, wherein the alkyl or aryl of R=H, alkyl, aryl or replacement:

In another exemplary compounds, the sulfur in the general formula I I-39 chemical compound can be oxidized to sulfoxide (n=1) or sulfone (n=2), for example, and as shown in the general formula I I-40 chemical compound:

In some embodiments, the substituent R in the general formula I I chemical compound ₁Can comprise leaving group, for example, halogen, as shown in Compound I I-18 or the II-19, or another leaving group, as sulphonic acid ester.The methane sulfonate (methanesulfonates (mesylate)) that example is formula II-41:

In some embodiments, the substituent R in the general formula I I chemical compound ₁Can comprise electron acceptor.This electron acceptor for example can be a lewis acid, as boric acid or its ester.Exemplary compounds, general formula I I-42 has following structure and spatial chemistry, wherein for example, n=0,1,2,3,4,5 or 6, and for example, R=H or alkyl:

Another exemplary compounds of general formula I I-42 is a formula II-42A chemical compound, wherein n=2 and R=H and formula II-42B chemical compound, wherein n=1 and R=H:

Substituent R in some general formula I I chemical compounds ₁Comprise in the embodiment of electron acceptor that this electron acceptor for example can be the Michael receptor.Exemplary compounds, general formula I I-43 has following structure, n=0,1,2,3,4,5 or 6 wherein, and wherein Z is an electron withdraw group, and for example, CHO, COR, COOR, CONH ₂, CN, NO ₂, SOR, SO ₂R or the like:

Another exemplary compounds of general formula I I-43 is formula II-43A chemical compound, wherein n=1 and Z=CO ₂CH ₃:

In some embodiments, chemical compound can be the esters or the thio-acid esters prodrug of general formula I I chemical compound.For example, formula II-44 chemical compound (the thioester prodrug of formula II-16 chemical compound) has following structure and spatial chemistry:

In some embodiments, general formula I I chemical compound for example can comprise that alkenyl group is as R ₁, for example, vinyl (ethylenyl).Another exemplary compounds, formula II-46 has following structure and spatial chemistry:

In some embodiments, chemical compound can be the esters or the thio-acid esters prodrug of general formula I I chemical compound.For example, formula II47 chemical compound (the thioester prodrug of formula II-17 chemical compound) has following structure and spatial chemistry:

In some embodiments, chemical compound can be the esters or the thio-acid esters prodrug of general formula I I chemical compound.For example, formula II-48 chemical compound has following structure and spatial chemistry:

Other exemplary compounds, formula II-49 can have following structure and spatial chemistry:

In some embodiments, chemical compound can be the esters or the thio-acid esters prodrug of general formula I I chemical compound.For example, formula II-50 chemical compound (ester prodrugs of formula II-16 chemical compound) has following structure and stereochemical structure:

Compound of formula III

Other embodiment provides a compounds and has produced the method for this compounds and acceptable salt of medicine and esters prodrug, and wherein said chemical compound is represented with general formula III:

General formula III

In certain embodiments, substituent R ₁For example can comprise hydrogen, halogen, single replacement of following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl; acyl group; acyloxy; oxyl ketonic oxygen base; aryloxycarbonyl oxygen base; cycloalkyl; cycloalkenyl group; oxyl; the ring oxyl; aryl; heteroaryl; aryl oxyl carbonyl; oxyl carbonyl acyl group; amino; amino carbonyl; amino carbonyl oxygen base; nitro; azido; phenyl; hydroxyl; alkylthio group; arylthio; oxygen base sulfonyl; carboxyl; cyano group; sulfo-; sulfoxide; sulfone; sulphonic acid ester; thiocyano; boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl.For example, n can equal 1 or 2.

In certain embodiments, R ₄For example can be single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl.For example, m can equal 1 or 2.When m equaled 2, described substituent group can be identical or different.And, each E ₁, E ₂, E ₃, E ₄And E ₅For example can be to replace or unsubstituted hetero atom.For example, this hetero atom can be nitrogen, sulfur and oxygen.

In some embodiments, work as R ₁When one of substituent group is ethyl or chloroethyl, R ₂Be not hexamethylene-2-thiazolinyl methanol.

General formula I V chemical compound

Other embodiment provides a compounds and has produced the method for this compounds and acceptable salt of medicine and esters prodrug, and wherein said chemical compound is represented with general formula I V:

General formula I V

In certain embodiments, substituent R ₁, R ₃And R ₅Can comprise independently that hydrogen, halogen, the single of following residue replace polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group, sulfo-, sulfoxide, sulfone, sulphonic acid ester, thiocyano, boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl.And, each E ₁, E ₂, E ₃, E ₄And E ₅Can be the hetero atom of hetero atom or replacement, for example, nitrogen, sulfur and oxygen.In some embodiments, R ₃Be not hydrogen.N equals 1 or 2.When n equaled 2, described substituent group can be identical or different.M also can be 0,1,2,3,4,5,6,7,8,9,10 or 11.When m greater than 1 the time, described substituent group can be identical or different.

In some embodiments, described substituent R ₅For example can produce disubstituted cyclohexane extraction.The exemplary compounds of general formula I V is the following exemplary stereochemical structure VI-1 that contains and do not contain:

General formula I V-1

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.Substituent R ₆And R ₇Can comprise single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant independently: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl.R in addition ₆And R ₇Can be identical or different.

For example, the exemplary compounds of general formula I V has following structure I V-2:

General formula I V-2

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Exemplary spatial chemistry can be as follows:

For example, the exemplary compounds of general formula I V has following structure I V-3;

General formula I V-3

R ₈For example can comprise hydrogen (IV-3A), fluorine (IV-3B), chlorine (IV-3C), bromine (IV-3D) and iodine (IV-3E).

Exemplary configurations and spatial chemistry can be as follows:

Other exemplary configurations and spatial chemistry can be as follows:

Formula IV-3C

For example, the exemplary compounds of general formula I V has following structure I V-4:

General formula I V-4

R ₈For example can comprise hydrogen, fluorine, chlorine, bromine and iodine.

Exemplary spatial chemistry can be as follows:

General formula V chemical compound

Some embodiments provide a compounds and have produced the method for this compounds and acceptable salt of medicine and esters prodrug, and wherein said chemical compound is represented with general formula V:

General formula V

In certain embodiments, substituent R ₁And R ₅Can comprise hydrogen, halogen, single replacement of following residue, polysubstituted or unsubstituted variant independently: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl; acyl group; acyloxy; oxyl ketonic oxygen base; aryloxycarbonyl oxygen base; cycloalkyl; cycloalkenyl group; oxyl; the ring oxyl; aryl; heteroaryl; aryl oxyl carbonyl; oxyl carbonyl acyl group; amino; amino carbonyl; amino carbonyl oxygen base; nitro; azido; phenyl; hydroxyl; alkylthio group; arylthio; oxygen base sulfonyl; carboxyl; cyano group; sulfo-; sulfoxide; sulfone; sulphonic acid ester; thiocyano; boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl.In certain embodiments, each E ₁, E ₂, E ₃, E ₄And E ₅Can be the hetero atom of hetero atom or replacement, for example, nitrogen, sulfur and oxygen.N equals 1 or 2, and when n equaled 2, substituent group can be identical or different.Preferably, m can for, for example, 0,1,2,3,4,5,6,7,8,9,10 or 11.When m greater than 1 the time, R ₅Can be identical or different.

General formula VI chemical compound

Some embodiments provide a compounds and have produced the method for this compounds and acceptable salt of medicine and esters prodrug, and wherein said chemical compound is represented with general formula VI:

General formula VI

R wherein ₁Can be independently selected from single replacement of following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, phenyl, cycloalkyl acyl group, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, sulfo-, sulfoxide, sulfone borate and the haloalkyl that comprises multi-haloalkyl.N equals 1 or 2, and if n equal 2, R then ₁Can be identical or different;

R wherein ₂Can be selected from hydrogen, halogen, single replacement of following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl (comprise, for example, hexahydrobenzyl alcohol), cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group, sulfo-, sulfoxide, sulfone, sulphonic acid ester, thiocyano, boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl;

R wherein ₃Can be selected from single replacement of halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, the ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, the cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group, sulfo-, sulfoxide, sulfone, sulphonic acid ester, thiocyano, boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl; Each E wherein ₁, E ₂, E ₃And E ₄Can be for replacing or unsubstituted hetero atom.

In some embodiments, work as R ₁One of substituent group is ethyl or chloroethyl, and R ₃During for methyl, R ₂Be not hexamethylene-2-thiazolinyl methanol.

In some embodiments, preferred R ₁Be that replace or unsubstituted C ₁-C ₅Alkyl.For example, preferable methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group and amyl group.

R wherein ₁₄Can be selected from single replacement of halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl; acyl group; acyloxy; oxyl ketonic oxygen base; aryloxycarbonyl oxygen base; cycloalkyl; cycloalkenyl group; oxyl; the ring oxyl; aryl; heteroaryl; aryl oxyl carbonyl; oxyl carbonyl acyl group; amino; amino carbonyl; amino carbonyl oxygen base; nitro; azido; phenyl; the cycloalkyl acyl group; hydroxyl; alkylthio group; arylthio; oxygen base sulfonyl; carboxyl; cyano group; sulfo-; thioester; sulfoxide; sulfone; sulphonic acid ester; thiocyano and the haloalkyl that comprises multi-haloalkyl.

In some embodiments, preferably, R ₁₄Alkyl hydrosulfide and E for alkyl hydrosulfide or replacement ₃Be oxygen.

For example, in some embodiments, some chemical compounds of general formula VI can have the following structure that is called general formula VI-1:

R wherein ₁Can be independently selected from single replacement of following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, phenyl, cycloalkyl acyl group, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, sulfo-, sulfoxide, sulfone, borate and the haloalkyl that comprises multi-haloalkyl.N equals 1 or 2, and if n equal 2, R then ₁Can be identical or different;

R wherein ₃Can be selected from single replacement of halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, the ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, the cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group, sulfo-, sulfoxide, sulfone, sulphonic acid ester, thiocyano, boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl; Each E wherein ₁, E ₂, E ₃, E ₄And E ₅Can be for replacing or unsubstituted hetero atom.

R wherein ₅Can be independently selected from hydrogen, halogen, single replacement of following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl; acyl group; acyloxy; oxyl ketonic oxygen base; aryloxycarbonyl oxygen base; cycloalkyl; cycloalkenyl group; oxyl; the ring oxyl; aryl; heteroaryl; aryl oxyl carbonyl; oxyl carbonyl acyl group; amino; amino carbonyl; amino carbonyl oxygen base; nitro; azido; phenyl; the oxygen base; hydroxyl; alkylthio group; arylthio; oxygen base sulfonyl; carboxyl; cyano group; sulfo-; sulfoxide; sulfone; sulphonic acid ester; thiocyano; boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl; wherein m is 0; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10 or 11; if and m is greater than 1, then R ₅Can be identical or different; Wherein said substituent R ₅Can form ring; Each E wherein ₁, E ₂, E ₃, E ₄And E ₅Can be for replacing or unsubstituted hetero atom.

For example, described chemical compound has following structure VI-1A:

Formula VI-1A

Exemplary spatial chemistry can be as follows:

For example, the exemplary compounds of general formula VI has following structure and spatial chemistry VI-1B:

Formula VI-1B

Another example, the chemical compound of general formula VI have following structure and spatial chemistry VI-1C:

Formula VI-1C

Some embodiment also provides the acceptable salt of medicine and the esters prodrug of general formula I-VI chemical compound, and the method by method acquisition disclosed herein and the described chemical compound of purification is provided.

Term " esters prodrug ", especially when the esters prodrug that refers to by the synthetic compound of Formula I of method disclosed herein, this term refers to the chemical derivative of described chemical compound, and it transforms (for example in blood or hydrolysis in the tissue) in vivo fast and generates this chemical compound.Term " esters prodrug " refers to the derivant of described chemical compound disclosed herein, and it is to form by in several groups that add multiple formation ester that is hydrolyzed or thioester under physiological condition any one.The example of esters prodrug group comprises pivoyloxymethyl, acetate methyl, 2-phthalidylidene, 2,3-indanyl and methoxy and thioester and other this class group well known in the art, comprise (5-R-2-oxo-1,3-Dioxol-4-yl) methyl group.Can prepare other prodrug by the corresponding thioester for preparing described chemical compound, for example, by preparing, for example with phenylmercaptan., cysteine or derivatives thereof or propanethiol reaction with suitable thiol reactant.Other example of esters prodrug group for example can find in following document: " the Pro-drugs as Novel DeliverySystems " of T.Higuchi and V.Stella (" as the prodrug of the delivery system of novelty ") the 14th volume, A.C.S.SymposiumSeries, American Chemical Society (1975); " Bioreversible Carriers inDrug Design:Theory and Application " (" bioreversible carrier in the drug design: principle and application "), E.B.Roche edits, Pergamon Press:New York, 14-21 (1987) (example that is used as the esters of prodrug is provided for the chemical compound that comprises carboxylic group).The mode of above-mentioned each list of references by reference is incorporated herein its full content.

Term used herein " esters prodrug " also refers in vivo and transforms fast, for example, generates the chemical derivative of this chemical compound of described chemical compound by the hydrolysis in blood.

Term used herein " the acceptable salt of medicine ", and comprise the acceptable salt time-like of medicine general formula I-VI chemical compound and by method disclosed by the invention generation and synthetic general formula I-VI chemical compound when referring specifically to, this term refers to the acceptable salt of any medicine of this chemical compound, and preferably refers to the acid-adducting salt of this chemical compound.The example of the preferred acceptable salt of medicine is alkali metal salt (sodium or a potassium); Alkali salt (calcium or magnesium); Or from ammonia or from the acceptable organic amine of medicine deutero-ammonium salt, for example C ₁-C ₇Alkylamine, cyclohexylamine, triethanolamine, ethylenediamine or three (methylol) aminomethane.For passing through the synthetic basic amine chemical compound of the described method of this embodiment, the example of the preferred acceptable salt of medicine is acceptable mineral acid of medicine or organic acid acid-adducting salt, for example, halogen acids, sulphuric acid, phosphoric acid or aliphatic or aromatic carboxylic acid or sulfonic acid, for example acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, p-methyl benzenesulfonic acid or LOMAR PWA EINECS 246-676-2.

Preferred pharmaceutical composition disclosed herein comprises acceptable salt of medicine and the esters prodrug thereof by the general formula I-VI chemical compound of method acquisition disclosed herein and purification.Therefore, if the manufacturing of pharmaceutical preparation relates to fully the mixing of active component of drug excipient and salt form, so preferred the use be the drug excipient of non-alkalescence, just acidity or neutral excipient.

It should also be understood that phrase " chemical compound with comprise this compound compositions " or any similar phrase mean the chemical compound that comprises any appropriate format that is suitable for drug conveying, the present invention goes through this chemical compound in addition.For example, in certain embodiments, described chemical compound or comprise the acceptable salt of medicine that this compound compositions can comprise this chemical compound.

In one embodiment, described chemical compound can be used for treating microbial diseases, cancer and inflammation.Disease is interpreted as having contained infectious disease and autoimmune disease, noninfectious disease and chronic disease state widely.In preferred embodiments, disease causes by microorganism, for example, and antibacterial, fungus and protozoacide.Using method also can comprise chemical compound or comprise the step of this compound compositions to the individual administration of suffering from infectious disease or cancer.Described chemical compound or compositions can be according to the effective dose administrations of concrete infectious disease, cancer or diseases associated with inflammation state of treatment.

Described infectious disease can be, for example, and the disease that causes by bacillus such as anthrax bacillus and bacillus cereus (B.cereus).Infectious disease can be the disease that is caused by protozoacide, for example, and leishmania, plasmodium or trypanosomicide.Described chemical compound or compositions can with together administration of medicine acceptable carrier, diluent, excipient or the like.

Described cancer can be, for example, and multiple myeloma, colorectal carcinoma, carcinoma of prostate, breast carcinoma, nonsmall-cell lung cancer, ovarian cancer, melanoma or the like.

Described diseases associated with inflammation state can be, for example, and rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, apoplexy, myocardial infarction, reperfusion injury or the like.

Term used herein " halogen atom " is meant the stable atom of arbitrary radiation in the 7th hurdle of the periodic table of elements, i.e. fluorine, chlorine, bromine or iodine, preferred bromine and chlorine.

That term as used herein " alkyl " is meant is any straight chain or side chain, replace or unsubstituted saturated hydrocarbons, preferred C ₁-C ₂₄, and more preferably C ₁-C ₆Hydrocarbon, most preferable, ethyl, propyl group, isopropyl, butyl, isobutyl group, tert-butyl and amyl group.In the saturated hydrocarbons of described replacement, preferred C ₁-C ₂₄, and C most preferably ₁-C ₆The saturated hydrocarbons and the amino hydrocarbon that replaces of single halo, dihalo and perhalogeno.

Term " replacement " is its common connotation, as the connotation described in the numerous same time patents in the association area.Referring to, for example, the 6th, 509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443 and 6,350, No. 759 United States Patent (USP)s are incorporated herein by reference its full content at this.Specifically; the definition that replaces is with the 6th; 509; the definition that provides in No. 331 United States Patent (USP)s is equally wide in range; the 6th; 509; the term " alkyl of replacement " of No. 331 United States Patent (USP) definition; make it refer to alkyl; preferred 1 to 10 carbon atom of this alkyl; has 1 to 5 substituent group; preferred 1 to 3 substituent group, described substituent group is selected from oxyl; the oxyl that replaces; cycloalkyl; the cycloalkyl that replaces; cycloalkenyl group; the cycloalkenyl group that replaces; acyl group; acyl amino; acyloxy; amino; the amino that replaces; aminoacyl; aminoacyl oxygen base; oxygen base acylamino-; cyano group; halogen; hydroxyl; carboxyl; carboxyalkyl; ketone group; the thioketone base; mercaptan; the sulfo-oxyl; the sulfo-oxyl that replaces; aryl; aryloxy; heteroaryl; heteroaryl oxygen base; heterocyclic radical; the heterocyclyloxy base; hydroxylamino; oxyl amino; nitro;--the SO-alkyl;--the alkyl that SO-replaces;--the SO-aryl;--the SO-heteroaryl;--SO ₂-alkyl,--SO ₂The alkyl of-replacement,--SO ₂-aryl and--SO ₂-heteroaryl.Other above-mentioned listed patent also provides the standard definition of the term " replacement " that is fully understood by those of skill in the art.

Term " cycloalkyl " refers to any non-aromatic hydrocarbon ring, preferably has the ring of five to 12 atomic buildings.Term " acyl group " refers to the alkyl or aryl that derives from keto acid, preferred acetyl group.

That term used herein " thiazolinyl " is meant is any straight chain or side chain, replace or unsubstituted unsaturated hydrocarbons, comprises many unsaturated hydrocarbons, preferably contains C ₁-C ₆Straight chain, monounsaturated and diunsaturated unsubstituted hydrocarbon, and most preferably monounsaturated dihalogenated hydrocarbon.Term " cycloalkenyl group " refers to any non-aromatic hydrocarbon ring, preferably contains the ring of five to 12 atomic buildings.

Term " aryl ", " aryl of replacement ", " heteroaryl " and " heteroaryl of replacement " that the present invention uses refer to the aromatic hydrocarbon ring, preferably have five, six or seven atoms, the most preferably ring of six atomic buildings." heteroaryl " and " heteroaryl of replacement " refers to such aromatic hydrocarbon ring, in this aromatic hydrocarbon ring at least one as oxygen, the hetero atom of sulfur or nitrogen-atoms with at least one carbon atom this ring in.Term " heterocycle " or " heterocyclic " refer to and contain one or more heteroatomic any cyclic compounds.The aryl of described replacement, heterocycle and heteroaryl can be replaced by any substituent group, comprise aforesaid and as known in the art substituent group.

That term " oxyl " refers to is any straight chain or side chain, replace or unsubstituted, saturated or unsaturated ethers, preferred C ₁-C ₆Not substituent ether straight chain, saturated, preferred methoxyl group, and dimethyl, diethyl, methyl-isobutyl group and methyl-tertbutyl ether also are preferred.Term " ring oxyl " refers to any non-aromatic hydrocarbon ring, preferably has the ring of five to 12 atomic buildings.Term " oxyl carbonyl " refers to any straight chain, side chain, cyclic, saturated, undersaturated, the aliphatic or aromatic oxyl that links to each other with carbonyl group.Example comprise methoxycarbonyl group, ethoxy carbonyl group, propyl group oxygen base carbonyl group, isopropyl oxygen base carbonyl group, butoxy carbonyl group, the second month in a season-butoxy carbonyl group, uncle-butoxy carbonyl group, cyclopentyloxy carbonyl group, cyclohexyl oxygen base carbonyl group, benzyloxycarbonyl group, pi-allyl oxygen base carbonyl group, phenyl oxygen base carbonyl group, pyridine radicals oxygen base carbonyl group or the like.

The term " pure " that the present invention uses, " purification ", " purification basically " refer to the chemical compound described in the embodiment with " isolating " and do not contain other different with it chemical compound, if this chemical compound exists with its naturalness, then this chemical compound is associated with other different with it chemical compound of its naturalness and this.Chemical compound is described to " pure ", " purification ", " purification basically " and " isolating " in certain embodiments of the invention, then this chemical compound can comprise at least 0.5%, 1%, 5%, 10% or 20% weight portion of given sample, and most preferably comprises at least 50% or 75% weight portion.

Term " derivant ", " variant " or other similar term refer to the chemical compound into the analog of other chemical compound.

Can obtain and some chemical compound of purification general formula I-VI, perhaps can be by the chemical compound of the mentioned purification of this paper by these chemical compounds of semi-synthetic acquisition.Usually, not to this qualification, the chemical compound of general formula I I-15, preferably, formula II-16, II-17, II-18 and II-19 can synthesize acquisition or obtain by fermentation.Exemplary fermentation operation is provided hereinafter.In addition, the chemical compound of general formula I I-15, preferably, formula II-16, II-17, II-18 and II-19 can be used as initial compounds in case obtain multiple other chemical compound of describing of synthetic the present invention.It is exemplary unrestriced synthetic that this paper provides.

Formula II-16 formula II-17

Formula II-18 formula II-19

Current salt ferment (about 350-400mg/L) production II-16 chemical compound by high yield, and the change of condition produces new analog in fermented product extract.Fig. 1 shows the chemical constitution of II-16.Can produce other analog by directed biosynthesis.Directed biosynthesis refers to by add the modification that biosynthetic precursor analog obtains natural product in the fermented product of producing microbial.(Lam, et al., J Antibiot (Tokyo) 44:934 (1991), Lam, et al., J Antibiot (Tokyo) 54:1 (2001) introduces its full content in this mode by reference).

Be exposed in the analog of acetic acid, phenylalanine, valine, butanoic acid, shikimic acid and halogen producing culture, preferably do not comprise to cause the generation of new analog by chlorine.The new analog that produces can be detected in crude extract easily by HPLC and LC-MS.For example, contain the culture medium of sodium bromide, bromo-analog of variable concentrations in processing after, in the shake-flask culture thing, can successfully produce the formula II-18 of the titer of 14mg/L.

Second approach that produces new analog is to pass through biotransformation.Bioconversion reaction is by enzyme or contains the chemical reaction of the whole-cell catalytic of these enzymes.Referring to Zaks, A., Curr Opin ChemBiol 5:130 (2001).The natural product of microorganism is the ideal substrate of bioconversion reaction, because they are synthetic by series of enzymatic reactions in microbial cell.Referring to Riva, S., Curr Opin Chem Biol 5:106 (2001).

The structure of given described chemical compound for example comprises the structure of general formula I I-15, and possible biosynthetic origin is S-acetyl-coenzyme-A, ethyl malonyl coenzyme A (ethylmalonyl-CoA), phenylalanine and chlorine.The ethyl malonyl coenzyme A derives from butyryl coenzyme A, and butyryl-coenzyme A derives from valine or crotonyl-CoA.Referring to Liu, et al., Metab Eng 3:40 (2001).Phenylalanine derives from shikimic acid.

Chemical compound formula I-7, II-16, II-17, II-18, II-20, II-24C, II-26, II-27 and II-28 Generation

The generation of chemical compound formula I-7, II-16, II-17, II-18, II-20, II-24C, II-26, II-27 and II-28 can realize by following method: under condition of the present invention, be preferably under buried aerobic conditions, in the appropriate nutrition culture medium, cultivate the natural variant bacterial strain NPS21184 of bacterial strain CNB476 and bacterial strain CNB476, up in fermented product, detecting a large amount of chemical compounds; By from fermentation liquid, extracting active component with suitable solvent; Concentrate the solvent that contains required component; Material after will concentrating is then isolated described chemical compound in chromatographic isolation other metabolite from also be present in this culture medium.

Fig. 2 shows more worldwide collection positions of culture (CNB476), and this culture is also referred to as Salinospora.Fig. 3 shows the bacterium colony of Salinospora.Fig. 4 shows the typical 16S rDNA sequence of Salinospora.Strip is represented the isolated signature nucleotide from its nearest relevant species with Salinospora.

Culture (CNB476) is deposited in Rockville on June 20th, 2003, the American Type Culture Collection of MD (American Type Culture Collection) (ATCC) and be appointed as ATCC patent preserving number PTA-5275.Bacterial strain NPS21184 is the natural variant of bacterial strain CNB476, and it is as the single bacterium colony separator that is derived from bacterial strain CNB476.Bacterial strain NPS21184 has been deposited in ATCC on April 27th, 2005.All requirements of budapest treaty are satisfied in this ATCC preservation.Described culture also maintains and can be from 10480Wateridge Circle, obtains among the San Diego, the Nereus medicinal fungus storehouse of CA 92121 (Pharmaceutical Culture Collection).Except the specified microorganisms that the present invention describes, be to be understood that also and can cultivate mutant, as by using mutant chemistry or that the mutagenic agent that comprises X-ray or the like physics produces, and the organism that changes its gene structure by Protocols in Molecular Biology is to produce initial compounds formula II-16, II-17 and II-18.

The fermentation of bacterial strain CNB476 and bacterial strain NPS21184

Be of value to the generation that can finish chemical compound under the temperature of producing the satisfied growth of organism, for example 16 ℃ to 40 ℃, but preferably under 32 ℃, ferment at 22 ℃.Can hatch aqueous culture medium a period of time to finish the generation of chemical compound, this can be monitored by high pressure liquid chromatography (HPLC), and for example, the preferred time period is about 2 days to 10 days, on gyrate shaker, turn round, be preferably 150rpm to 250rpm with about 50rpm to 400rpm.Also can be by in such as the bioreactor of the fermentation tank system that is fit to described production strain growth, cultivating the generation that this production bacterial strain is finished described chemical compound.

Those of ordinary skill in the art can finish described microbial growth by using proper culture medium.Put it briefly, the source of carbon comprises glucose, fructose, mannose, maltose, galactose, mannitol and glycerol, other sugar and sugar alcohol, starch and other carbohydrate or such as the carbohydrate derivates of glucosan, crystal glucose and complicated nutrient oatmeal for example, corn flour, millet, corn etc.The exact amount of the carbon source that uses in described culture medium partly depends on other composition in this culture medium, still, for example can use the carbohydrate amount of 0.5% to 25% culture medium weight ratio satisfactorily.For example, can use these carbon sources or in same culture medium, can unite and use a plurality of such carbon sources separately.Some carbon source that preferred use hereinafter provides.

The source of nitrogen comprises aminoacid, for example glycine, arginine, threonine, methionine or the like, ammonium salt and complicated source, for example yeast extract, corn immersion, soluble distillate, Semen sojae atricolor powder, cotton seeds powder, fish flour, peptone or the like.For example, can use separately or unite the multiple source of using nitrogen with the amount of 0.5% to 25% culture medium weight ratio.

In the nutrition inorganic salt that can join in the culture medium, inorganic salt commonly used is to produce sodium, potassium, magnesium, calcium, phosphorus, sulfur, chlorine, carbonate and similar ionic inorganic salt.Also comprise trace meter for example cobalt, manganese, ferrum, molybdenum, zinc, cadmium or the like.

The biological activity of chemical compound and application

Some embodiments relate to cancer, inflammation and infectious disease, particularly influence people's treatment of diseases method.Described method can comprise, for example, and with the step of the member's administration in the class noval chemical compound of effective dose.Therefore, described chemical compound disclosed herein can be used for treating cancer, inflammation and infectious disease.

Described chemical compound has multiple biological activity.For example, this chemical compound has chemical sensitization activity, antimicrobial, anti-inflammatory, radiation sensitization and active anticancer.

Described chemical compound has proteasome and suppresses active.Proteasome suppresses the active ability that can produce this chemical compound as anticarcinogen, antiinflammatory and antimicrobial whole or in part.

Described proteasome is the multimeric protein enzyme, and it is by its chymotrypsin-like, trypsin-like and peptidyl glutamyl hydrolase polypeptide (PGPH; Be also referred to as Caspase sample activity) the intracellular protein of active degradation.Described 26S proteasome comprises the Proteolytic enzyme core and one or two 19S that are called the 20S proteasome and regulates subunit.This 20S proteasome produces the proteolytic activity at many substrates, and these substrates comprise impaired protein, transcription factor NF-KB and inhibitor I κ B, signaling molecule, tumor-inhibiting factor and cell cycle regulatory factors.Three kinds of different proteinase activities are arranged: 1) chymotrypsin-like in described proteasome; 2) peptidyl glutamyl hydrolase polypeptide (PGPH) activity trypsin-like and 3).

As an example, the chemical compound (EC of formula II-16 ₅₀2nM) than Omuralide (EC ₅₀52nM) chymotrypsin-like of the described rabbit myopsin of more effective inhibition body is active and also can suppress the chymotrypsin-like activity (EC of the deutero-proteasome of human red blood cell ₅₀About 250pM).Fig. 5 shows the catabolite Omuralide and the formula II-16 chemical compound of lactacystin.With respect to the catalytic activity that suppresses chymase, formula II-16 compound exhibits goes out the active significant preferred property of the chymotrypsin-like that suppresses described proteasome.The chemical compound of formula II-16 has shown that also the trypsin-like of low nM suppresses active (about 10nM), but is suppressing aspect the PGPH activity of described proteasome relatively poor effect (EC is arranged ₅₀About 350pM).

Research has in addition characterized the effect of the chemical compound of the present invention's description, comprises the research of formula II-16 to the effect of NF-κ B/I κ B signal pathway.With tumor necrosis factor-α (TNF-α) phosphorylation of I κ B α and the degraded of proteasome mediation, NF-kB activation are subsequently brought out in the processing of HEK293 cell (human embryo kidney (HEK)).In order to determine that proteasome suppresses, then stimulated in 1 hour with TNF-α with formula II-16 chemical compound pretreatment HEK293 cell.Promote the accumulating of I κ B α of phosphorylation with the processing of formula II-16 chemical compound, this shows the I κ B α degraded that has suppressed the proteasome mediation.

In addition, produced stable HEK293 clone (NF-κ B/Luc 293), it carries the luciferase reporter gene under the adjusting of 5xNF-κ B binding site.With TNF-α the stimulation of NF-κ B/Luc 293 has been increased luciferase activity, this is the result of NF-kB activation, has then reduced activity with the pretreatment of formula II-16 chemical compound.The degraded of accumulating and reduce total I κ B α of the I κ B α of phosphorylation in Western engram analysis display type II-16 compound promoted NF-κ B/Luc 293 cells.Formula II-16 chemical compound also demonstrates the increase cyclin, the level of p21 and p27.

Tumor cell is more responsive than normal cell to proteasome inhibitor.In addition, proteasome suppresses to have increased the sensitivity of cancerous cell to anticarcinogen.By detecting the cytotoxic activity that the cytotoxic activity of multiple cancerous cell line is detected the chemical compound that comprises formula II-16 of the present invention's description.For example, at National Cancer Institute 60 human tumor cell lines are carried out examination and detect formula II-16 chemical compound.Formula II-16 compound exhibits cytotoxic activity optionally, its average GI ₅₀Value (obtaining 50% growth inhibiting concentration) is less than 10nM.Maximum efficiency [both LC have been observed to SK-MEL-28 melanoma and MDA-MB-235 breast cancer cell ₅₀(concentration)＜10nM] with 50% cell lethality.

Comprise the melanoma (B16-F10) of people's colorectum (HT-29 and LoVo), prostate (PC3), mammary gland (MDA-MB-235), lung (NCI-H292), ovary (OVCAR3), acute T-chronic myeloid leukemia (Jurkat), Mus and one group of cell line 48h post-evaluation cytotoxic activity of normal person fibroblast (CCD-27sk) with Salinosporamide A processing.To HT-29, LoVo, PC3, MDA-MB-231, NCI-H292, OVCAR3, Jurkat and B16-F10 cell are responsive, EC ₅₀Value is respectively 47,69,78,67,97,69,10 and 33nM.On the contrary, to the EC of CCD-27sk cell ₅₀Value is 196nM.With Salinosporamide A at about EC ₅₀Concentration causes the cutting of Caspase-3 activation and PARP to the processing of Jurkat cell, confirms apoptosis-induced effect.

Estimate the anti-anthrax activity of described chemical compound with the inductive cytotoxicity analysis of external LeTx.As an example, this result shows that formula II-16 chemical compound is the inductive Cytotoxic effective inhibitor of LeTx of mouse macrophage sample RAW264.7 cell.Separately the processing of RAW264.7 cell is compared with LeTx, the processing of RAW264.7 cell is caused long (the average EC of 10 multiplications of the viability of the cell that LeTx handles with formula II-16 chemical compound ₅₀＜4nM).

The potential chemical sensitization effect of formula II-16 chemical compound

Research has in addition characterized the effect (see embodiment) of chemical compound described herein to NF-κ B/I κ B signal pathway.In stimulated cells not, transcription factor nuclear factor-kappa B (NF-κ B) is arranged in Cytoplasm with the disactivation composite form with Profilin I κ B (inhibitor of NF-κ B).Multiple stimulation can cause I κ B phosphorylation by the I kappa b kinase, causes ubiquitinization (ubiquitination) and Degradation by proteasome subsequently.Along with the degraded of I κ B, NF-κ B transposition is interior and regulator gene expression to nuclear, and this has influenced and has comprised the many cell processes that suppress apoptosis.Can activate NF-κ B in the CCL188 that comprises the LoVo cell such as the chemotherapeutant of CPT-11 (Irinotecan Irinotecan), cause these cells that apoptotic ability takes place and reduce.Painter，R.B.Cancer Res 38：4445(1978)。Velcade ^TMIt is the dipeptides ylboronic acid, chymotrypsin-like activity (the Lightcap of its Profilin enzyme body, et al., Clin Chem 46:673 (2000), Adams, et al., Cancer Res 59:2615 (1999), Adams, Curr Opin Oncol14:628 (2002)), and increase trypsin and PGPH activity.Nearest Velcade ^TM(PS-341; Millennium Pharmaceuticals, Inc.) licensed as proteasome inhibitor, it has demonstrated the direct toxicity of cancerous cell and has also improved in the external LoVo cell and suppress cytotoxic activity via the CPT-11 in the LoVo xenograft models of the I κ B degraded of proteasome.Blum，et al.，Ann Intern Med 80：249(1974)。In addition, find Velcade ^TMCan suppress the expression of short angiogenesis (proangiogenic) chemotactic factor in the squamous cell carcinoma/relevant proto-oncogene-α (Growth Related Oncogene-alpha, GRO-α) of cytokine growth and VEGF (VEGF).May realize by suppressing described NF-kB pathway.Dick，et al.，JBiol Chem 271：7273(1996)。These data show that proteasome suppresses not only can reduce the survival and the growth of tumor cell, also can reduce angiogenesis.

Anti-anthrax activity

The other potential application of proteasome inhibitor comes from recently the research about biophylaxis classification A material (biodefense Category A agent) B.anthracis (anthrax).The anthrax bacillus spore is inhaled into and lives with in the lung, and they are taken in by macrophage there.In this macrophage, spore germinates, duplicates, causes this cell to be killed.But before killing this cell, the macrophage migration of infection is in lymph node, and when dead there, they discharge their inclusions, and this organism is entered in the blood system, further duplicate the justacrine lethal toxin.Hanna，et al.，Proc Natl Acad Sci USA 90：10198(1993)。Anthrax toxin is to form the related indication reason of anthrax.Two protein that in the pathogeny of anthrax, play a crucial role be protective antigen (PA, 83kDa) and lethal factor (LF, 90kDa), they jointly are called lethal toxin (LeTx).LF has the enzyme function, but needs PA to finish its biological effect.One of PA and LF can not cause death separately, and still, when they made up, when in the intravenous injection precession object, PA and LF caused death.Kalns，et al.，Biochem Biophys ResCommun 297：506(2002)，Kalns，et al.，Biochem Biophys Res Commun 292：41(2002)。

Protective antigen, the receptors bind component of anthrax toxin is responsible for lethal factor is transported in the host cell.The heptamer (see figure 6) that the PA oligomerize circularizes.Each heptamer that combines with its receptor at cell surface has in conjunction with the ability that reaches most three LF molecules.By receptor-mediated endocytosis, the complex that forms between PA heptamer and the LF is taken in to cell.After endocytosis, LF is released in the endochylema, there its attack various kinds of cell target.Mogridge，et al.，Biochemistry 41：1079(2002)，Lacy，et al.，JBiolChem 277：3006(2002)，Bradley，et al.，Nature 414：225(2001)。

Lethal factor (LF) is the metalloproteases that zinc relies on, and it can divide and make the signal protein inactivation of mitogen activated protein kinase kinases family (MAPKK) in endochylema.Duesbery，et al.，Science 280：734(1998)，Bodart，et al.，Cell Cycle 1：10(2002)，Vitale，et al.，J Appl Microbiol 87：288(1999)，Vitale，et al.，Biochem J 352 Pt 3：739(2000)。In seven different known mapk kinases, six have demonstrated by LF and have cut.In cell, the transduction of mapk kinase approach participates in making these albumen become the multiple signal of cell death, propagation and the differentiation of highly significant target.But some stops some inhibitor of the inductive cell death of LeTx can not stop MAPKK to be cut by LF, and this activity that shows this inhibitor is not enough to inducing cell death.Kim，et al.，J Biol Chem 278：7413(2003)，Lin，et al.，CurrMicrobiol 33：224(1996)。

Studies show that described Profilin enzyme physical ability stops the inductive cell death of LeTx.Tang，etal.，Infect Immun 67：3055(1999)。The data show proteasome activity is essential for the RAW264.7 macrophage that kills of LeTx mediation, and proteasome inhibitor protection RAW264.7 avoids LeTx.Proteasome suppresses not block the MEK1 cutting, and this shows that the LeTx approach is not to be blocked in the upstream of MEK1 cutting in these research.In addition, do not increase with proteasome activity in the cell of LeTx processing.These data show as chemical compound described herein, and novel, effective protein proteins enzyme body inhibitor also can stop the inductive cell death of LeTx, as shown in Figure 6.

The receptor of PA has been differentiated and has been expressed by many cell types.Escuyer，et al：，IyafectInaraun 59：3381(1991)。Lethal toxin is cultivated in the strain in a few cell of macrophage activity, causes cell death in several hrs.Hanna，et al.，Proc Natl Acad Sci USA 90：10198(1993)，Kim，et al.，JBiol Chem 278：7413(2003)，Lin，et al.，CurrMicrobiol 33：224(1996)。LeTx can induce through the mouse macrophage sample RAW264.7 of extracorporeal treatment and the necrosis and the apoptosis of J774A.1 cell.

Described result shows that chemical compound described herein is the inductive Cytotoxic effective inhibitor of LeTx of mouse macrophage sample RAW264.7 cell.For example, compare, the processing of RAW264.7 cell is caused long (the average EC of 10 multiplications of the cell viability of LeTx processing with formula II-16 chemical compound with the LeTx individual processing ₅₀＜4nM), and therefore provide valuable therapy for anthrax infects.For example, formula II-16 promotes the survival of RAW264.7 macrophage in the presence of LeTx, and this shows that this chemical compound and derivant thereof provide valuable clinical treatment for anthrax infects.

Pharmaceutical composition

In one embodiment, described chemical compound disclosed herein is used for pharmaceutical composition.Can randomly produce this chemical compound by method disclosed by the invention.This chemical compound can for example be used for pharmaceutical composition, and this pharmaceutical composition comprises to be prepared to be used to store and the medicine acceptable carrier of administration subsequently.Embodiment also relates to above-mentioned disclosed product and the chemical compound that contains medicine effective quantity in medicine acceptable carrier or diluent.The acceptable carrier or the diluent that are used for the treatment of purposes are known in pharmaceutical field, and for example at Remington ' sPharmaceutical Sciences (Lei Mingdun pharmacopedics), 18th Ed., Mack Publishing Co., Easton, PA (1990)) is described in, its full content introduced in this mode by reference.Antiseptic, stabilizing agent, dyestuff even correctives all can be provided in this pharmaceutical composition.The ester that for example, can add sodium benzoate, ascorbic acid and P-hydroxybenzoic acid as antiseptic.In addition, can use antioxidant and suspending agent.

Described compositions (the particularly compositions of general formula I-VI) can be carried out prescription and with the tablet that acts on oral administration, capsule or elixir; The suppository that is used for rectally; The sterile solution, the suspensoid that are used for drug administration by injection; Be used for through the paster of skin administration and subcutaneous deposit or the like.Injection can be prepared as following conventionally form: as solution or suspension, be fit in liquid, to make the solid dosage forms of solution or suspension before the injection or as Emulsion.The excipient that is fit to for example is water, saline, glucose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride or the like.In addition, if desired, described injecting drug use pharmaceutical composition can comprise a spot of avirulence adminicle, for example wetting agent, pH buffer agent or the like.If desired, also can use absorption to strengthen preparation (for example, liposome).

Be used for the aqueous solution that non-pharmaceutical preparation through enteral administration comprises the described reactive compound that exists with water-soluble form.In addition, also the suspension of described reactive compound can be prepared as suitable oily injection suspension.Lipophilic solvent that is fit to or carrier comprise such as Semen Sesami wet goods fatty oil or other such as soybean oil, grape-fruit seed oil or Semen Armeniacae Amarum wet goods organic oil or such as synthetic fatty acid esters such as ethyl oleate or triglyceride, or liposome.The aqueous injection suspension can comprise the material that increases this suspension viscosity, for example sodium carboxymethyl cellulose, Sorbitol or glucosan.Randomly, described suspension can comprise suitable stabilizing agent or increase the reagent of the dissolubility of described chemical compound with permission preparation highly concentrated solution.

Can obtain to be used for oral pharmaceutical preparation by following method: described reactive compound is mixed with solid excipient, the gained mixture of at random milling, and this mixture is processed into granule, if desired, after adding the auxiliary agent that is fit to, obtain tablet or sugar-coat agent nuclear.The excipient that is fit to comprises lactose, sucrose, mannitol or Sorbitol particularly such as the filler of sugar; Cellulose preparation is as corn starch, wheaten starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone (PVP).Can add disintegrating agent if desired, as crospolyvinylpyrrolidone, agar or alginic acid or such as the alginate of sodium alginate.The sugar-coat agent is examined the bag quilt that is fit to.For this purpose, can use spissated sugar juice, this sugar juice can randomly contain arabic gum, Talcum, polyvinylpyrrolidone, carbopol gel (carbopol gel), Polyethylene Glycol and/or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture.In order to discern or represent the feature of the various combination of active compound doses, can in tablet or dragee coatings, add dyestuff or pigment.For this purpose, can use spissated sugar juice, this sugar juice can randomly contain arabic gum, Talcum, polyvinylpyrrolidone, carbopol gel (carbopol gel), Polyethylene Glycol and/or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture.In order to discern or represent the feature of the various combination of active compound doses, can in tablet or dragee coatings, add dyestuff or pigment.Can use method as known in the art to make these preparations and (for example see U.S. Patent number 5,733,888 (composition for injection); 5,726,181 (being insoluble in the chemical compound of water); 5,707,641 (treatment effective protein proteins matter or peptides); 5,667,809 (lipotropy reagent); 5,576,012 (polymerization agent of solubilising); 5,707,615 (anti-virus formulations); 5,683,676 (drug particles); 5,654,286 (topical formulations); 5,688,529 (oral suspensionses); 5,445,829 (slow releasing preparation); 5,653,987 (liquid preparations); 5,641,515 (controlled release preparations) and 5,601,845 (spherical preparation)); Be incorporated herein by reference at this full content all patents.

The invention also discloses the known multiple pharmaceutical composition that is used to comprise part, ophthalmic, intranasal and in ear conveying in pharmaceutical field.Pharmaceutical preparation comprises the aqueous ophthalmic solution of described reactive compound, it can exist such as the water-soluble form of eye drop, or gellan gum (gellangum) form (people such as Shedden, Clin.Ter., 440-50 (2001)) or form of hydrogels (people such as Mayer 23 (3):, Ophthalmologica, 210 (2): 101-3 (1996)); Ophthalmic ointment; The eye suspensoid, for example microgranule, be suspended in the little polymer particles (Joshi that comprises medicine in the liquid carrier medium, A.1994 J Ocul Pharmacol 10:29-45), fat-soluble preparation (people such as Alm, Prog Clin.Biol.Res., 312:447-58 (1989)) and microsphere (Mordenti, Toxicol.Sci., 52 (1): 101-6 (1999)); And ocular inserts.Be incorporated herein by reference at this full content all above-mentioned lists of references.For stability and comfortableness, it is aseptic, isoosmotic and buffered that these suitable pharmaceutical preparatioies the most often and preferably are formulated into.Pharmaceutical composition also comprises drop and spray, it is prepared into usually simulates nasal discharge in many aspects to guarantee keeping of normal ciliary action.As Remington ' s Pharmaceutical Science (MackPublishing, 18 ^ThEdition) disclosed and those skilled in the art is known in (its full content being introduced by the mode of reference) at this, appropriate formulation is the most frequent and be preferably isoosmotic, slight buffered keeping pH5.5-6.5, and the most frequent and preferably include antimicrobial preservative and suitable medicine stabilizing agent.The pharmaceutical preparation that is used for the in ear conveying is included in the suspensoid and the ointment of in ear topical application.The general solvent that is used for this class aural preparations comprises G ﹠ W.

When as anticancer, antiinflammatory or antimicrobial chemical compound, for example, described general formula I-V chemical compound or comprise that the compositions of general formula I-V can pass through oral or non-oral administration.When oral administration, it can be with capsule, tablet, granule, spray, syrup or other this class dosage form administration.When non-oral administration, it can be used as aqueous suspension, Oily preparation or similar type administration, or as drop, suppository, ointment, ointment or similar type administration, by subcutaneous, intraperitoneal, intravenous, intramuscular or similar fashion drug administration by injection.

In one embodiment, described anticarcinogen, antiinflammatory or antimicrobial can mix with other material to strengthen their effects.In one embodiment, described antimicrobial and other antimicrobial combination.In another embodiment, medicine that described antimicrobial is useful with aligning the patient that takes antimicrobial or medicament associating.

Medication

In optional embodiment, disclosed chemical compound and disclosed pharmaceutical composition pass through the specific method administration as antimicrobial.These class methods include but not limited to the administration of (a) by oral route, and this administering mode comprises with capsule, tablet, granule, spray, syrup or other this class dosage form administration; (b) by non-oral administration, this administering mode comprises as waterborne suspension, Oily preparation or similar dosage form or as drop, suppository, ointment, ointment or similar dosage form administration; By subcutaneous, intraperitoneal, intravenous, intramuscular, Intradermal or similar fashion with drug administration by injection; And the mode that those of skill in the art think fit carries out (c) topical, (d) per rectum administration, or (e) intravaginal administration, thus the described chemical compound of the present embodiment is contacted with living tissue; And (f) by controlled release preparation, bank (depot) preparation and infusion pump conveying administration.As the other example of this class administering mode with as other disclosed administering mode, the invention discloses the multiple medication of described disclosed chemical compound and pharmaceutical composition, comprise by ophthalmic, intranasal and the administering mode in ear approach.

Comprise described chemical compound, comprise general formula I-type (comprising the people) that the medicine effective dose of VI compound compositions depends on route of administration, treated animal as what dosage required, and the physical trait of particular animals all to be considered.Can adjust described dosage realizing Expected Results, but depend on following factors: for example body weight, diet, Drug therapy simultaneously and the other factors of generally acknowledging the technical staff of field of medicaments.

In the embodiment of implementing described method, use can be used or unite mutually to described product or compositions separately, or unite use with other treatment reagent or diagnostic reagent.These products can be used in vivo, usually in mammalian body, and preferably in human body, or in external application.When using in vivo, described product or compositions can adopt multiple dosage form with number of ways to the mammal administration, comprise non-intestinal, intravenous, subcutaneous, intramuscular, colon, rectum, vagina, nose or intraperitoneal administration.These methods are application testing chemism in vivo also.

Conspicuous as those skilled in the art institute, be used for the dosage of vivo medicine-feeding and concrete administering mode and will depend on mammiferous age, body weight and the kind of being treated, employed particular compound changes with the concrete purposes of using these chemical compounds.Those skilled in the art adopts conventional pharmacological method to realize that the effective dose level promptly realizes determining of the necessary dosage level of expected results.Usually, use with the human clinical who begins to carry out product than low dosage level, along with the increase of dosage level up to realizing desired effect.Selectively, adopt fixed pharmacological method, use acceptable in vitro study to set up the useful dosage and the route of administration of the compositions of this method evaluation.

In non-human animal's research, the application of potential product begins with the high dose level, along with reducing dosage up to realizing that no longer desired effect or adverse side effect disappear.Depend on desired effect and therapeutics indication, dosage range can be more wide in range.Usually, dosage can be about 10 microgram/kg body weight to 100 milligram/kg body weight, is preferably about 100 microgram/kg body weight to 10 milligram/kg body weight.Selectively, just as understood by those skilled in the art, dosage can based on calculate according to described patient's surface area.Preferably with once a day or every day twice oral administration.

Each doctor can select definite preparation, administering mode and dosage according to patient's situation.Referring to for example, the The Pharmacological Basis of Therapeutics (therapeutic pharmacological basis) of Fingl etc. for example, 1975, at this its full content is incorporated herein by reference.It is pointed out that how and when the attending doctor will know stops, interrupts or adjust administration because of toxicity or organ dysfunction disorder.On the contrary, if clinical response insufficient (eliminating toxicity), this attending doctor also will be appreciated that higher level is adjusted in treatment.Control the dosage value that adopts when paying close attention to disease will change with the seriousness and the route of administration of morbid state to be treated.For example part is estimated the seriousness of described morbid state by standard prognostic evaluation method.In addition, described dosage and possible dose frequency also change according to age, body weight and the reaction of individual patient.The scheme suitable with above-mentioned discussion scheme can be used in the veterinary.

Depend on the disease specific state of being treated, can be with this class reagent prescription and capapie or administration partly.The various technology that are used for prescription and administration can be in " Remington ' sPharmaceutical Sciences " (Lei Mingdun pharmacopedics), 18 ^ThEd., Mack Publishing Co., Easton, PA finds in (1990), at this its full content is incorporated herein by reference.That the route of administration that is fit to also comprises is oral, rectum, through skin, vagina, permeable membrane or enteral administration; Non-intestinal is carried, comprise in intramuscular, subcutaneous, intramedullary injection and the sheath, directly in the ventricle, intravenous, intraperitoneal, intranasal or intraocular injection.

For injection, can be in aqueous solution, preferably in physiological compatibility buffer such as Hanks ' solution, Ringer ' s solution or normal saline buffer solution with the reagent prescription of described embodiment.For this class permeable membrane administration, in described preparation, use the penetrating agent that is suitable for permeability barrier.This class penetrating agent is well-known in the art.The scope of described embodiment comprises that the disclosed by the invention described chemical compound that uses the medicine acceptable carrier will be used to implement described embodiment is formulated into the dosage form that is fit to the whole body administration.Select suitable carrier and suitable manufacture method, described compositions, particularly prescription disclosed by the invention are the compositions of solution, can for example pass through intravenous administration through parenterai administration.Utilize medicine acceptable carrier well known in the art can be easy to described chemical compound is formulated into the dosage form that is fit to oral administration.Described carrier can be tablet, pill, capsule, liquid, gel, syrup, unguentum, suspension or the like with the chemical compound prescription in the described embodiment, is used for patient's to be treated orally ingestible.

The reagent that can adopt those skilled in the art's technique known will be intended to administration in the cell carries out administration.For example, this class reagent encapsulation can be advanced liposome, administration as stated above then.All molecules that are present in the aqueous solution when liposome forms all are bonded in the aqueous interior.The inclusions of described liposome not only is protected and is not subjected to the influence of outside microenvironment, and because liposome and cell membrane fusion, so this inclusions is delivered in the Cytoplasm effectively.In addition, because the hydrophobicity of little organic molecule, they can directly carry out administration in the cell.

To effective dose determine it is in those skilled in the art's the limit of power, especially determine according to detailed disclosure provided by the invention.Except that described active component, these pharmaceutical compositions can comprise the suitable medicine acceptable carrier that comprises excipient and adjuvant, and it promotes described reactive compound to be processed into pharmaceutically useful preparation.The preparation that is formulated into oral administration can be tablet, coated tablet, capsule or solution form.Described pharmaceutical composition can be processed in himself known mode, for example, mixes, dissolves, granulates, makes sugar-coat agent, suspension, emulsifying, encapsulation, seals (entrap) or lyophilization by routine.

The method of application of known can be estimated chemical compound drug effect disclosed by the invention and toxicity.For example, can be by set up the toxicology of this chemical compound subclass of particular compound or shared some chemical part in the toxicity of external test pair cell system (such as mammiferous and preferred people's cell line).In the common measurable animal body of the result of this class research, as mammal or more clearly be the intravital toxicity of people.Selectively, can utilize known method to measure particular compound such as the toxicity in the animal model of mice, rabbit, Canis familiaris L. or monkey.Can use multiple art-recognized method to determine the effectiveness of particular compound, these methods are in vitro method, animal model or human clinical trial for example.Basically each class morbid state is all existed art-recognized external model, described morbid state comprises the morbid state by described compounds for reducing disclosed by the invention, comprises cancer, cardiovascular disease and panimmunity dysfunction and infectious disease.Similarly, acceptable animal model can be used for setting up the effectiveness of the chemicals for the treatment of described morbid state.When selecting model when determining effectiveness, those skilled in the art select proper model, dosage and route of administration and scheme under can instructing in the knowledge of this area.Certainly, the human clinical trial also can be used to measure chemical compound at the intravital effectiveness of people.

When as antimicrobial, anticarcinogen or antiinflammatory, described chemical compound disclosed by the invention can pass through oral or non-oral administration.When oral administration, it can be with capsule, tablet, granule, spray, syrup or other this class dosage form administration.When non-oral administration, it can be used as waterborne suspension, Oily preparation or similar type, or as drop, suppository, ointment, ointment or similar type administration, when drug administration by injection, by subcutaneous, intraperitoneal, intravenous, intramuscular, Intradermal or similar type administration.Same consideration controlled release preparation, depot formulations and infusion pump are carried.

Compositions disclosed by the invention in the pharmaceutical composition also can comprise the medicine acceptable carrier.This based composition preparation can be used to store and administration subsequently.The acceptable carrier or the diluent that are used for the treatment of purposes are known at pharmaceutical field, and for example, at " Lei Mingdun pharmacopedics ", describe in Mack publishing company (A.R.Gennaro the edits 1985) book.For example, can be with this based composition prescription and with the tablet that acts on oral administration, capsule or solution; The suppository that is used for rectum or vagina administration; The sterile solution or the suspension that are used for drug administration by injection.Injection can be prepared into following conventionally form: as solution or suspension, before injection, be adapted at making in the liquid solid dosage forms of solution or suspension, or as Emulsion.The excipient that is fit to includes but not limited to saline, glucose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride or the like.In addition, if desired, described injecting drug use pharmaceutical composition can comprise a spot of avirulence adminicle, for example wetting agent, pH buffer agent or the like.If desired, also can use absorption to strengthen preparation (for example, liposome).

To depend on route of administration, the type of animal of being treated and the physical trait of particular animals as the medicine effective quantity of the desired described compositions of dosage all will consider.Can adjust described dosage and realize Expected Results, but depend on following factors: for example body weight, diet, Drug therapy simultaneously and the other factors of generally acknowledging the technical staff of field of medicaments.

Use can be used or unite mutually to the product of above-mentioned described embodiment or compositions separately, or unite use with other treatment reagent or diagnostic reagent.These products can be in vivo or external utilization.Described useful dosage and the most useful mode of administration will depend on the particular compound of age, body weight and the animal of being treated, use and use the concrete purposes of these compositionss or multiple compositions and change.The dosage value that adopts in the processing of concrete disease or treatment will change with the seriousness and the route of administration of morbid state to be treated, and depends on described morbid state and their seriousness, can be with described compositions prescription and whole body or topical.The various technology that are used for prescription and administration can be at " Lei Mingdun pharmacopedics ", Mack publishing company, and Easton finds in PA (1990) one books.

For the chemical compound prescription with general formula I-VI is antimicrobial, anticarcinogen or antiinflammatory, can use acceptable film forming matter of known surfactant, excipient, smoothing preparation, suspending agent and medicine and coating adjuvant etc.Alcohols, esters, sulphated fatty alcohol class or the like can preferably be used as surfactant; Sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid salt, magnesium aluminate, aluminic acid methane-siliconic acid magnesium, synthetic aluminium silicate, calcium carbonate, sodium bicarbonate, calcium hydrogen phosphate, carboxymethylcellulose calcium or the like can be used as excipient; Magnesium stearate, Talcum, fixed oil or the like can be used as smoothing preparation; Oleum Cocois, olive oil, Oleum sesami, Oleum Arachidis hypogaeae semen, soybean oil can be used as suspending agent or lubricant; As cellulose acetate phthalate, or can be used as suspending agent as the methyl acetate-methacrylic acid copolymer of poly derivant such as the derivant of carbohydrates such as cellulose or sugar; And the plasticizer such as phthalic acid ester or the like can be used as suspending agent.Except aforementioned preferred composition, in the drug-delivery preparation of the chemical compound that the method by described embodiment that sweeting agent, aromatic, coloring agent, antiseptic or the like can be joined produces, particularly described chemical compound is an oral administration.

Described chemical compound and compositions can be carried out oral or non-oral administration to human patients by following dosage: about 0.001mg/kg/ days to about 10,000mg/kg/ days described active component, and more preferably about 0.1mg/kg/ days to about 100mg/kg/ days active compositions of imitating, preferably, more less preferred with administration every day above twice to about ten times with administration once a day.Selectively and also preferably, the described chemical compound that produces by described embodiment can be preferably by intravenous drip for example with certain amount successive administration.Therefore, for the patient of 70 kilograms of body weight for example, described preferred daily dose active or the infection composition is about 0.07mg/ days to about 700g/ days, and more preferably 7mg/ days to about 7g/ days.But, those skilled in the art can understand, may need dosage to surpass in some cases or even considerably beyond anticancer, the antiinflammatory or the anti-infective compounds of the described embodiment of above-mentioned dosage range, to treat specific terminal cancer or infectious disease effectively and energetically.

The antimicrobial that produces when the method for using described embodiment is during as biochemical test reagent, when the chemical compound that will the method by described embodiment produces is dissolved in organic solvent or the water-containing organic solvent and when being directly used in any different cultured cell system, it suppresses the progress of described disease.Available organic solvent comprises, for example, and methanol, dimethyl sulfoxide etc.Described preparation can be, for example, and powder, granule or other solid inhibitor, or with an organic solvent or the liquid inhibitor of water-containing organic solvent preparation.Although the chemical compound that is produced by the method for described embodiment is during as Antimicrobe compound, its preferred concentration is generally about 1 μ g/ml to about 100 μ g/ml, will change according to the type and the application target of cultured cell system but those skilled in the art can understand optimal use amount.In some applications, the dosage that those skilled in the art may need or preferably use surpasses above-mentioned scope.

In one embodiment, chemical compound is related to as the method for antimicrobial, anticarcinogen or antiinflammatory any general formula I-VI chemical compound or these compound compositions of effective dose are carried out administration.In preferred embodiments, described method comprises to the chemical compound that general formula I I is represented to the patient's administration that needs antimicrobial, need be reduced effectively or more preferably eliminates up to this.

It is appreciated for those skilled in the art that " needs " are not absolute terms and only contain the meaning that described patient can be benefited from the treatment of described antimicrobial, anticarcinogen or antiinflammatory using.The connotation of " patient " is the organism of being benefited from the use of described antimicrobial, anticarcinogen or antiinflammatory.For example, any organism of suffering from anthrax bacillus, plasmodium, leishmania, trypanosomicide or the like can be present in the intravital microbial biomass of patient from application of antimicrobial reagents, this antimicrobial and then minimizing and is benefited.As another example, suffering from any organism of cancer can be benefited from use the amount that anticarcinogen, this anticarcinogen and then minimizing be present in the intravital cancer of described patient, and described cancer is colorectal carcinoma, carcinoma of prostate, breast carcinoma, nonsmall-cell lung cancer, ovarian cancer, multiple myeloma, melanoma or the like for example.In addition, thereby any organism of suffering from the diseases associated with inflammation state can be benefited from using antiinflammatory, this antiinflammatory minimizing to be present in the relevant cell quantity of the intravital and described inflammatory response of described patient, and described diseases associated with inflammation state is rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, apoplexy, reperfusion injury, myocardial infarction or the like for example.In one embodiment, described patient's health status can not need antimicrobial, anticarcinogen or the antiinflammatory of administration, but, this patient can be still obtains some benefits from the minimizing of the level that is present in the intravital microorganism of patient, cancerous cell or inflammatory cell, thereby needs this antimicrobial, anticarcinogen or antiinflammatory.In one embodiment, described antimicrobial or anticarcinogen are effectively anti-one type microorganism or cancer, but the microorganism or the cancer of anti-other type are invalid; Therefore allow the high selectivity in described patient's treatment.In other embodiments, described antiinflammatory is effective to the diseases associated with inflammation state that is characterized by the different cells relevant with described inflammation.In selecting this class antimicrobial, anticarcinogen or antiinflammatory, can use disclosed method and result in an embodiment.In selectable embodiment, described antimicrobial is effectively to the broad-spectrum micro-organisms in the host organisms, is preferably the broad-spectrum alien bacteria, and noxious bacteria more preferably.In embodiments, described anticarcinogen and/or antiinflammatory are effective to broad-spectrum cancer and diseases associated with inflammation state/cell/material.In other embodiments, described antimicrobial is to all microorganisms, or even the born microorganism of host is effective.The microorganism example of target that can be antimicrobial is including, but not limited to anthrax bacillus, plasmodium, leishmania, trypanosomicide etc.In other embodiments, described anticarcinogen is effective to broad-spectrum cancer or all cancers.Described chemical compound is that effective cancer example comprises colorectal carcinoma, carcinoma of prostate, breast carcinoma, nonsmall-cell lung cancer, ovarian cancer, multiple myeloma, melanoma or the like to it.Described reagent is that effective exemplary diseases associated with inflammation state comprises rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, apoplexy, myocardial infarction or the like to it.

" treatment effective dose ", " medicine effective quantity " or similar term instruct the biology of just seeking or the described medicine of medical response or the amount of medicinal reagent that causes cell, tissue, whole body, animal or people.In preferred embodiments, described medical response is the reaction of being sought by research worker, veterinary, the doctor of medicine or other clinicist.

" antimicrobial " refers to survival probability or blocking-up that reduces microorganism or the chemical compound that alleviates the illeffects of microorganism.In one embodiment, described survival probability is defined as the function of individual microorganism; Therefore, described antimicrobial will increase the probability of individual microbial death.In one embodiment, described survival probability is defined as the function of micropopulation, therefore described antimicrobial will increase the probability of described micropopulation minimizing.In one embodiment, antimicrobial refers to antibiotic or other similar term.This class antimicrobial can be blocked microbial growth or the breeding of illeffects, destruction or inhibition such as antibacterial.For example, these class antibacterials or other antimicrobial are at " Antibiotics; Chemotherapeutics and Antibacterial Agents for Disease the Control " (antibiotics that is used for disease control, chemotherapy and antibacterial) (M.Grayson, editor, 1982) and people such as E.Gale " The Molecular Basis of Antibiotic Action " (molecular basis of antibiotics effect), be described in second edition (1981) book.In another embodiment, antimicrobial does not change the probability of survival, but change described microorganism in a certain mode to the deleterious probability of host.For example, if described microorganism secretion to the deleterious material of host, then described antimicrobial can act on this microorganism and make it stop secretion or neutralization or block this illeffects.In one embodiment, though antimicrobial has increased the probability of microbial death, its to around, non-microorganism, cell be that minimally is deleterious.In selectable embodiment, described antimicrobial to around, non-microorganism, cell be harmful as where be unessential, as long as it reduces the survival probability of described microorganism.

" anticarcinogen " refers to the chemical compound that reduces cancerous cell survival probability or comprises this compound compositions.In one embodiment, described survival probability is defined as the function of individual cancerous cell; Therefore, described anticarcinogen will increase the probability of this individuality cancer cell death.In one embodiment, described survival probability is defined as the function of cancer cell population, the therefore described anticancer probability that will increase this cancer cell population minimizing.In one embodiment, the anticarcinogen meaning is chemotherapeutant or other similar term.

" chemotherapeutant " is useful chemical compound in such as the treatment of the neoplastic disease of cancer.The example of chemotherapeutant comprises alkylating agent, as chlormethine, aziridine and methylmelamine, the antimetabolite of alkyl sulfonic ester, nitroso ureas and triazenes, folic acid antagonists, nucleotide metabolism, antibiotic, pyrimidine analogue, 5-fluorouracil, cisplatin, purine nucleosides, amine, aminoacid, ribavirin, 17-hydroxy-11-dehydrocorticosterone, natural product, for example antibody of vinca alkaloids, etoposide, antibiotic, enzyme, taxane and biological response modifier or biological response modifier or other reagent; Miscellaneous reagent, for example carbamide of platinum coordination complex, anthraquinone, anthracycline antibiotics, replacement, methyl hydrazine derivant or adrenocortical inhibitor; Perhaps hormone or antagonist, for example adrenocortical steroid, Progesterone, estrogen, estrogen antagonist agent, androgen, antiandrogen or promoting sexual gland hormone (gouadotropin)-releasing hormone analog.Concrete example comprises amycin, doxorubicin, 5-fluorouracil, cytosine arabinoside (" Ara-C "), cyclophosphamide, plug is for group, busulfan, cytotoxin (Cytoxin), paclitaxel, taxotere (Toxotere), methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, ametycin, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, the demethyl daunorubicin, aminopterin-induced syndrome, actinomycin D, mitomycin, enediyne class (Esperamicin), the chlormethine that melphalan is relevant with other.In this definition, also comprise the hormone reagent of regulating or suppressing the hormonal action on the tumor, for example zitazonium and onapristone.

Described anticarcinogen can directly act on cancerous cell to kill this cell, bring out this cell death, to stop this cell division or the like.Selectively, described anticarcinogen can for example act on described cancerous cell by limiting nutrition or the blood of supplying with this cell indirectly.This class anticarcinogen can destroy or suppress the growth of described cancerous cell or the ability of duplicating, and this cancerous cell is colorectal carcinoma, carcinoma of prostate, breast carcinoma, nonsmall-cell lung cancer, ovarian cancer, multiple myeloma, melanoma or the like for example.

" neoplastic disease " or " tumor " refers to the colony that shows excrescent cell or cell by than the more cell proliferation of normal structure, comprises tumor or tissue (comprising such as the suspension of medullary cell with such as the fluid of blood or serum).Tumor can be benign or virulent.

" diseases associated with inflammation state " comprises, for example, ischemia, septic shock, autoimmune disease, rheumatoid arthritis, inflammatory bowel, systemic lupus erythematosus (sle), multiple sclerosis, asthma, osteoarthritis, osteoporosis, fibrotic disease, dermatosis, comprise psoriasis, the skin lesion that atoipc dermatitis and ultraviolet radiation (UV) bring out, psoriatic arthritis, ankylosing spondylitis, tissue and organ rejection, Alzheimer (family name) disease, apoplexy, atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer, Hodgkin, cachexia, the inflammation relevant with some viral infection with infection, comprise acquired immune deficiency syndrome (AIDS) (AIDS), adult respiratory distress syndrome and ataxia.

In one embodiment, described chemical compound is preferably the chemical compound that contains general formula I-VI, comprises the chemical compound that the present invention describes, if this chemical compound can influence 10% described microorganism, cancerous cell or inflammatory cell, just think that it is effective antimicrobial, anticarcinogen or antiinflammatory.In a more preferred embodiment, if described chemical compound can influence described microorganism, cancerous cell or the inflammatory cell of 10%-50%, then this chemical compound is effective.In addition preferred embodiment in, if described chemical compound can influence described microorganism, cancerous cell or the inflammatory cell of 50%-80%, then this chemical compound is effective.In addition preferred embodiment in, if described chemical compound can influence described microorganism, cancerous cell or the inflammatory cell of 80%-95%, then this chemical compound is effective.In addition preferred embodiment in, if described chemical compound can influence described microorganism, cancerous cell or the inflammatory cell of 95%-99%, then this chemical compound is effective.Mechanism of action definition " influence " by each chemical compound.Therefore, for example, if chemical compound stops duplicating of described microorganism, then influence is to measure to stop to duplicate.Similarly, if the chemical compound destroy microorganisms, then influence is to measure microbial death.Also for example, if chemical compound stops the cancerous cell division, then influence is to measure to stop the cancerous cell division.Additionally, for example, if chemical compound stops the propagation of inflammatory cell, then influence is to measure to stop inflammatory cell propagation.Be not that all mechanism of action needs identical effect percentage ratio.In selectable embodiment, if the lower expenditure of doing is remedied by the other factorses such as specificity such as this chemical compound, then low effect percentage ratio can be suitable.Therefore, for example, chemical compound has only 10% effect, but show host or harmless microorganism or cell is not had any harmful side effect, still can consider the effectiveness of this chemical compound.

In one embodiment, the chemical compound that the present invention describes simply administration and need not administration removing microorganism, cancerous cell or inflammatory cell to the patient.For example, in some situation that microorganism goes wrong, for example in food, the chemical compound that the present invention describes can directly apply to this food to reduce the danger of microorganism in this food.Selectively, described chemical compound can be used for reducing the microorganism level that is present in such as in the environment around the operating surface etc.As another example, in the body of can the be first external back of described chemical compound (ex vivo) to such as cell sample administrations such as bone marrow or stem cell transplantations, to guarantee having only non-cancerous cells to be introduced into the receptor.Behind described compound administration, they can randomly be removed.This is particularly suitable in some cases, and this situation is that operating surface or food can contact with other surface or organism by described chemical compound destruction risk are arranged.In selectable embodiment, consider more protection, can be retained in described chemical compound in the described food or on the operating surface.Whether carry out this selection, then depend on the relative needs of situation and the risk relevant with described chemical compound, part is determined among this embodiment that narrates below.

Following non-limiting examples is the description to the preferred embodiment of described method.Those skilled in the art should firmly believe the variant of details that has specific implementation method and the precise chemical structure compositions that is obtained to be true.

Embodiment

Embodiment 1

Adopt bacterial strain CNB476 fermentation production I-7, II-16, II-17, II-20, II-24C, II-26 Chemical compound with II-28

Bacterial strain CNB476 is grown in the 500ml flask that contains the 100ml Nutrient medium, and this Nutrient medium is grouped into by the following one-tenth in every liter of deionized water: glucose, 4g; The Bacto tryptone, 3g; Bacto cheese peptone, 5g; With synthetic sea salt (Instant Ocean, AquariumSystems), 30g.Under 28 ℃, first inoculum was hatched 3 days in the gyrate shaker with the 250rpm running.The first per 5 milliliters inoculum is seeded in three 500ml flasks that contain the described Nutrient medium of 100ml.Under 28 ℃, second inoculum was hatched 2 days in the gyrate shaker with the 250rpm running.The second per 5 milliliters inoculum is seeded in 35 500ml flasks that contain the described Nutrient medium of 100ml.Under 28 ℃, the third sub-culture was hatched 2 days in the gyrate shaker with the 250rpm running.The third per 5 milliliters sub-culture is seeded in 400 500ml flasks that contain 100ml production culture medium A, and this production culture medium A is grouped into by the following one-tenth in every liter of deionized water: starch, 10g; Yeast extract, 4g; Hy-Soy, 4g; Iron sulfate, 40mg; Potassium bromide, 100mg; Calcium carbonate, 1g; With synthetic sea salt (Instant Ocean, AquariumSystems), 30g.To produce culture down at 28 ℃ hatched 1 day in the gyrate shaker with the 250rpm running.The aseptic Amberlite XAD-7 resin of about 2-3g is added in this production culture.Under 28 ℃, this production culture was further hatched 5 days in the gyrate shaker with the 250rpm running, obtain the Compound I I-16 of about 200mg/L titer.Filter this cultivation and fermentation liquid with cheese cloth and reclaim this Amberlite XAD-7 resin.With this resin of 6 liters ethyl acetate extractions 2 times, then with 1.5 liters ethyl acetate extraction 1 time.This mixed extract of vacuum drying.This exsiccant extract that will contain 3.8g formula II-16 chemical compound and more a spot of II-20 and II-24C chemical compound is then handled, with recovery type I-7, II-16, II-20, II-24C, II-26 and II-28 chemical compound.

Embodiment 2

Adopt bacterial strain NPS21184 fermentation production I-7, II-16, II-17, II-20, II-24C, II-26 With the II-28 chemical compound

Bacterial strain NPS21184 is grown in the 500ml flask that contains the 100ml Nutrient medium, and this Nutrient medium is grouped into by the following one-tenth in every liter of deionized water: glucose, 8g; Yeast extract, 6g; Hy-Soy, 6g; With synthetic sea salt (Instant Ocean, AquariumSystems), 30g.Under 28 ℃, first inoculum was hatched 3 days in the gyrate shaker with the 250rpm running.5 milliliters first inoculums are seeded in the 500ml flask that contains the described Nutrient medium of 100ml.Under 28 ℃, second inoculum was hatched 2 days in the gyrate shaker with the 250rpm running.The second per 5 milliliters inoculum is seeded in the 500ml flask that contains the described Nutrient medium of 100ml.Under 28 ℃, the third sub-culture was hatched 2 days in the gyrate shaker with the 250rpm running.The third per 5 milliliters sub-culture is seeded in the 500ml flask that contains 100ml production culture medium B, and this production culture medium B is grouped into by the following one-tenth in every liter of deionized water: starch, 20g; Yeast extract, 4g; Hy-Soy, 8g; Iron sulfate, 40mg; Potassium bromide, 100mg; Calcium carbonate, 1g; With synthetic sea salt (Instant Ocean, Aquarium Systems), 30g.To produce culture down at 28 ℃ hatched 1 day in the gyrate shaker with the 250rpm running.The aseptic Amberlite XAD-7 resin of about 2-3g is added in this production culture.Under 28 ℃, this production culture was further hatched 4 days in the gyrate shaker with the 250rpm running, obtain the Compound I I-16 of 350-400mg/L titer.

Selectively, adopt bacterial strain NPS21184 in 42L fermentation tank system, to finish the generation of described chemical compound.Bacterial strain NPS21184 is grown in the 500ml flask that contains the 100ml Nutrient medium, and this Nutrient medium is grouped into by the following one-tenth in every liter of deionized water: glucose, 8g; Yeast extract, 6g; Hy-Soy, 6g; With synthetic sea salt (Instant Ocean, AquariumSystems), 30g.Under 28 ℃, first inoculum was hatched 3 days in the gyrate shaker with the 250rpm running.5 milliliters first inoculums are seeded in the 500ml flask that contains the described Nutrient medium of 100ml.Under 28 ℃, second inoculum was hatched 2 days in the gyrate shaker with the 250rpm running.The second per 20 milliliters inoculum is seeded in the 2.8L Fernbach flask that contains the described Nutrient medium of 400ml.Under 28 ℃, the third sub-culture was hatched 2 days in the gyrate shaker with the 250rpm running.The third sub-culture of 1.2L is seeded in the 42L fermentation tank that contains 26L production culture medium A, also can uses production culture medium B and growth medium C with following composition.The production culture medium C is grouped into by the following one-tenth in every liter of deionized water: starch, 15g; Yeast extract, 6g; Hy-Soy, 6g; Iron sulfate, 40mg; Potassium bromide, 100mg; Calcium carbonate, 1g; With synthetic sea salt (Instant Ocean, Aquarium Systems), 30g.This fermentor cultivation thing is operated under following parameter: temperature, 28 ℃; Stir 200rpm; Ventilation, 13L/min and back-pressure, 4.5psi.When 36-44 hour of production cycle, the aseptic Amberlite XAD-7 of about 600g resin is added in this culture tank fermented product.Should produce culture and further under the aforesaid operations parameter, hatch, up to the 4th day of production cycle.Ventilation Rate is reduced to 8L/min.When the 5th day of production cycle, the Compound I I-16 of the about 300mg/L titer of this fermentor cultivation deposits yields.Filter this cultivation and fermentation liquid with cheese cloth and reclaim this AmberliteXAD-7 resin.With this resin of 4.5 liters ethyl acetate extractions 2 times, then with 1.5 liters ethyl acetate extraction 1 time.This mixed extract of vacuum drying.Then dried extract is handled, with recovery type I-7, II-16, II-17, II-20, II-24C, II-26 and II-28 chemical compound.

Embodiment 3

The purification of formula I-7, II-16, II-20, II-24C, II-26 and II-28 chemical compound The purification of 3A: formula II-16, II-20, II-24C, II-26 and II-28 chemical compound

Obtain the chemical compound of pure compound II-16 and formula II-20, II-24C, II-26 and II-28 by flash chromatography and HPLC subsequently.(KP-Sil Silica, 32-63 μ m 90g), handle the 8g crude extract that contains 3.8g Compound I I-16 and more a spot of II-20, II-24C, II-26 and II-28 by flash chromatography to adopt Biotage Flash40i system and Flash 40M post.This flash chromatography carries out eluting by following discontinuous gradient:

1. hexane (1L)

2. the hexane (1L) that contains 10% ethyl acetate

3. the hexane that contains 20% ethyl acetate, first eluting (1L)

4. the hexane that contains 20% ethyl acetate, second eluting (1L)

5. the hexane that contains 20% ethyl acetate, the 3rd eluting (1L)

6. the hexane (1L) that contains 25% ethyl acetate

7. the hexane (1L) that contains 50% ethyl acetate

8. ethyl acetate (1L)

To confirm to merge with the part that contains Compound I I-16 more than or equal to 70%UV purity through HPLC, and through HPLC purification as described below, obtain II-16 and II-20 and II-24C, each be pure compound.

Chromatographic column	Phenomenex Luna 10μ Silica
Chromatographic column	Phenomenex Luna 10μ Silica	Size	25cm×21.2mm ID
Flow velocity	25ml/min	Size	25cm×21.2mm ID
Flow velocity	25ml/min	Detector	ELSD
Solvent	Gradient is 24% ethyl acetate/hexane 19min, in 1 minute from 24% ethyl acetate/hexane to 100% ethyl acetate, 100% ethyl acetate then continues 4min	Detector	ELSD

With the part of enrichment Compound I I-16 (as mentioned above; Pure for Compound I I-16 about 70%) be dissolved in (60mg/ml) in the acetone.The part of this solution (950 μ l) is injected on the positive HPLC post that adopts above-mentioned condition.Compound I I-16 is eluting after 14 minutes usually, and Compound I I-24C and II-26 in the time of 11 minutes as unimodal co-elute.When the maternal sample that contains Compound I I-17, II-20 and II-28 was handled, in 100% eluent ethyl acetate process, Compound I I-17 is eluting in the time of 22 minutes, and II-20 and II-28 co-elute in the time of 23 minutes.Based on the composition of the chemical compound that exists, the part that will contain Compound I I-16 and a small amount of analog merges, and under reduced pressure evaporates on rotary evaporator.This process produces pure compound A, and the separating part that contains a spot of Compound I I-20, II-24C, II-26 and II-28, as described below this separating part is further carried out purification.

Adopt following anti-phase preparation HPLC, further separate the sample that contains II-24C and II-26 that from said process, produces.The sample (70mg) that will contain II-24C is dissolved in the acetonitrile with the concentration of 10mg/ml, then 500 μ l solution are loaded in size 21mm i.d., 15cm long, contain on the HPLC post of Eclipse XDB-C18 carrier.The solvent gradient was increased to 100% acetonitrile with the 14.5ml/min flow velocity from 15% acetonitrile/85% water linearly in 23 minutes.This solvent compositions was kept 3 minutes with 100% acetonitrile before turning back to initial solvent mixture.Compound I I-26 eluting in the time of 17.5 minutes under these conditions, and Compound I I-24C eluting in the time of 19 minutes.

Adopt the diffusion of vapor method to obtain crystal II-26.Compound I I-26 (15mg) is dissolved in the 100 μ l acetone in HPLC phial at the bottom of the 1.5ml v-.Then this phial is placed the container of the bigger sealing that contains the 1ml pentane.Under 4 ℃, after hatching 48 hours, observe the crystal that is fit to the experiment of X-radiocrystallography along the wall and the bottom of this inner tubular bottle.In UCSD crystallography laboratory, at Bruker SMART APEX CCD x-ray diffractometer (F (000)=2656, Mo _{K α}Ray, λ=0.71073 μ=0.264mm ^-1, T=100K) go up to collect crystallographic data, and employed modification method is at F ²On the complete matrix least square.Crystallization data NPI-2065:C ₁₅H ₂₀ClNO ₄, MW=313.77, tetragonal system, space group P4 (1) 2 (1) 2, a=b=11.4901 (3)

C=46.444 (2)

α=β=γ=90 °, vol=6131.6 (3)

Z=16, ρ _Calcd=1.360g cm ^-3, crystal size, 0.30 * 0.15 * 0.07mm ³, the θ scope, 1.75-26.00 °, gather 35367 reflections, 6025 independent reflection (R _Int=0.0480), final R index (I＞2 σ (I)): R ₁=0.0369, wR ₂=0.0794, GOF=1.060.

In order from II-20, to separate II-28, adopt anti-phase equal strength (isocratic) method.The sample (69.2mg) that will contain two kinds of chemical compounds be dissolved in the acetonitrile to concentration be 10mg/ml, per injection is loaded in 500 μ l on the reversed-phase HPLC post (ACE 5 C18-HL, 15cm * 21mm ID).The equal strength solvent system of 27% acetonitrile/63% water is used for separating compound II-28 and II-20 with the flow velocity of 14.5ml/min, and they are eluting after 14 minutes and 16 minutes respectively.When room temperature, under reduced pressure, the part that will contain compound of interest is evaporated on rotary evaporator immediately.Then sample is loaded on the little silicagel column, and with 70% hexane/30% acetone eluting of 10ml, to remove other impurity.

Also available 100%EtOAc grinds by what above-mentioned preparation positive HPLC method produced and contains II-20 but do not contain the sample of II-28, to remove a spot of lipotropy impurity.

Formula II-16 chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm.Low resolution mass spectrum (lowRes.Mass): m/z 314 (M+H), 336 (M+Na).

Formula II-20 chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm.Low resolution mass spectrum: m/z 226 (M+H); HRMS (ESI), m/z 266.1396 (M+H), Δ _Calc=1.2ppm.Fig. 7 has described chemical compound with formula II-20 structure ¹H NMR collection of illustrative plates.

Formula II-24C chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm.

Low resolution mass spectrum: m/z 328 (M+H), 350 (M+Na); HRMS (ESI), m/z328.1309 (M+H), Δ _Calc=-2.0ppm, C ₁₆H ₂₃NO ₄Cl.Fig. 8 has described chemical compound with formula II-24C structure ¹H NMR collection of illustrative plates.

Formula II-26 chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm; HRMS (ESI), m/z314.1158 (M+H), Δ _Calc=-0.4ppm, C ₁₅H ₂₁NO ₄Cl; Figure 51 described have formula II-26 structure chemical compound at DMSO-d ₆In ¹H NMR collection of illustrative plates.Figure 52 has described the ORTEP figure of the computer generation of the chemical compound with formula II-26 structure.

Formula II-28 chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm; HRMS (ESI), m/z266.1388 (M+H), Δ _Calc=-1.8ppm, C ₁₄H ₂₀NO ₄Figure 54 described have formula II-28 structure chemical compound at DMSO-d ₆In ¹H NMR collection of illustrative plates.

3B: the purification of formula I-7 chemical compound

Biotage Flash 75Li system and Flash 75L KP-Sil post are used to handle the filtering crude extract (10.0g) that is rich in Compound I I-16 and contains formula I-7 chemical compound.With this crude extract be dissolved in the acetone to concentration be 107mg/ml, and directly be loaded on this post.Then with following solvent discontinuous gradient with the flow velocity of 235ml/min to 250ml/min this post of flowing through:

1. the normal heptane (3.2L) that contains 10%EtOAc

2. the normal heptane (16L) that contains 25%EtOAc

3. the normal heptane (5.4L) that contains 30%EtOAc

With being rich in the part merging of Compound I I-16 and concentrating, up to total mixed solvent volume of residue about 5% by Rotary Evaporators.Solvent is removed, stayed white solid.

Then by sample (4.56g) is dissolved in 1: 1 acetone: in the normal heptane (910ml), this solid is carried out crystallization.Use Rotary Evaporators that solvent is evaporated lentamente, up to solvent be reduced to its initial volume about 43% till.Remove solution (supernatant) and it is concentrated (598mg).

This supernatant is dissolved in the acetone (80mg/ml).Employing is used for the above-mentioned condition of positive purifying compounds II-16, II-24C, II-26 and II-28, with this injection of solution of equal portions to positive HPLC post.Formula I-7 chemical compound is as pure compound eluting 7.5 minutes the time.

Formula I-7 chemical compound (Figure 58): UV (acetonitrile/water) λ _Max225 (sh) nm.Low resolution mass spectrum: m/z 298 (M+H), 320 (M+Na).

Embodiment 4

Fermentation production II-17, II-18 and II-27 chemical compound

Bacterial strain CNB476 is grown in the 500ml flask that contains 100ml first Nutrient medium, and this first Nutrient medium is grouped into by the following one-tenth in every liter of deionized water: glucose, 4g; The Bacto tryptone, 3g; Bacto cheese peptone, 5g; With synthetic sea salt (Instant Ocean, Aquarium Systems), 30g.Under 28 ℃, first inoculum was hatched 3 days in the gyrate shaker with the 250rpm running.5 milliliters first inoculums are seeded in the 500ml flask that contains 100ml second Nutrient medium, and this second Nutrient medium is grouped into by the following one-tenth in every liter of deionized water: starch, 10g; Yeast extract, 4g; Peptone, 2g; Iron sulfate, 40mg; Potassium bromide, 100mg; Calcium carbonate, 1g; And sodium bromide, 30g.Under 28 ℃, second inoculum was hatched 7 days in the gyrate shaker with the 250rpm running.The aseptic Amberlite XAD-7 resin of about 2-3g is added in second inoculum.Under 28 ℃, second inoculum was further hatched 2 days in the gyrate shaker with the 250rpm running.5 milliliters second inoculums are seeded in the 500ml flask that contains 100ml second Nutrient medium.Under 28 ℃, the third sub-culture was hatched 1 day in the gyrate shaker with the 250rpm running.The aseptic AmberliteXAD-7 resin of about 2-3g is added in the third sub-culture.Under 28 ℃, the third sub-culture was further hatched 2 days in the gyrate shaker with the 250rpm running.The third sub-culture of 5 milliliters is seeded in the 500ml flask that contains 100ml second Nutrient medium.Under 28 ℃, the 4th inoculum was hatched 1 day in the gyrate shaker with the 250rpm running.The aseptic Amberlite XAD-7 resin of about 2-3g is added in the 4th inoculum.Under 28 ℃, the 4th inoculum was further hatched 1 day in the gyrate shaker with the 250rpm running.The 4th per 5 milliliters inoculum is seeded in ten 500ml flasks that contain 100ml second Nutrient medium.Under 28 ℃, the 5th inoculum was hatched 1 day in the gyrate shaker with the 250rpm running.The aseptic AmberliteXAD-7 resin of about 2-3g is added in the 5th inoculum.Under 28 ℃, the 5th inoculum was further hatched 3 days in the gyrate shaker with the 250rpm running.The 5th per 4 milliliters inoculum is seeded in 150 500ml flasks of production culture medium of the same second Nutrient medium same composition that contains 100ml.Aseptic Amberlite XAD-7 resin that also will about 2-3g adds in this production culture.Under 28 ℃, this production culture was hatched 6 days in the gyrate shaker with the 250rpm running.Filter this cultivation and fermentation liquid with cheese cloth and reclaim this Amberlite XAD-7 resin.With this resin of 3 liters of ethyl acetate extractions 2 times, then with 1 liter ethyl acetate extraction 1 time.This mixed extract of vacuum drying.Then the extract of the dried 0.42g of containing formula II-17 chemical compound and 0.16g formula II-18 chemical compound is handled, to reclaim described chemical compound.

Embodiment 5

The purification of formula II-17, II-18 and II-27 chemical compound

Obtain pure compound II-17 and II-18 by reversed-phase HPLC as described below

Chromatographic column	ACE	5 C18-HL
Chromatographic column	ACE	5 C18-HL	Size	15cm×21mm ID
Flow velocity	14.5ml/min		Size	15cm×21mm ID
Flow velocity	14.5ml/min	Detector	214nm
Solvent	Gradient is that 35% acetonitrile in 15 minutes/65% water is to 90% acetonitrile/10% water	Detector	214nm

(100mg) is dissolved in the acetonitrile of 15ml with crude extract.The part (900 μ l) of this solution is injected the reversed-phase HPLC chromatographic column, adopt above-mentioned condition.Compound I I-17 and II-18 respectively when 7.5 minutes and 9 minutes by eluting.At first adopt nitrogen to concentrate the part that contains described pure compound and remove organic solvent.Then that this surplus solution is freezing extremely dry with lyophilizing.

Developed and selectablely be used for method with Compound I I-17 and II-18 purification to be used for fairly large purification, this method relates on positive VLC post and separates crude extract.Under these conditions, the multiple less important metabolite of the q.s that comprises Compound I I-27 is differentiated.Described crude extract (2.4g) is dissolved in the acetone (10ml), and by drying under the vacuum with this solution absorbs on silica gel (10cc).Adsorbed thick extract is loaded in (250cc silica gel, column dimension on the purification on normal-phase silica gel VLC post, diameter 2.5cm and length 15cm), discontinuous gradient with hexane/EtOAc washes, and the gradient with 5% increases the percentage ratio (every gradient 100ml solvent) of hexane.Most compounds II-16 is eluting in 60% hexane/40%EtOAc flushing, and most compounds II-17 eluting in 50% hexane/50%EtOAc flushing.Employing is by 35%ACN/65%H ₂The permanent solvent system that O forms adopts C18 HPLC chromatograph (ACE5 μ C 18-HL, 150mm * 21mm ID), finishes the last separation of described chemical compound.Under these conditions, Compound I I-27 is eluting in the time of 11 minutes, and Compound I I-17 is eluting in the time of 12.00 minutes, and the Compound I I-16 of trace is eluting in the time of 23.5 minutes, and Compound I I-18 eluting in the time of 25.5 minutes.Need not heat, in a vacuum with resulting sample drying, to remove the aqueous solvent mixture.The spectroscopic data of finding Compound I I-16 and these samples of Compound I I-18 is consistent with the spectroscopic data of the sample that is prepared by previous purification process.The sample of finding Compound I I-18 contains 8% lactone hydrolyzate, and by washing positive silica filler (diameter 1cm, height 2cm) and adopting the solvent mixture eluting of 20%EtOAc/80% hexane (25ml) to be further purified.Find that resulting sample contains pure compound II-18.

Employing increased to the solvent gradient of 100%EtOAc through 20 minutes from 100% hexane with the flow velocity of 4ml/min, adopted positive partly to prepare HPLC (Phenomenex Luna Si 10 μ, 100

250 * 10mm id), be further purified the above-mentioned part that contains Compound I I-27.Compound I I-27 as pure compound after 11.5 minutes by eluting (0.8mg is 0.03% based on the separation yield of dry extract weight).

Initial compounds II-17:UV (acetonitrile/water) λ _Max225 (sh) nm.High resolution mass spec (APCI): m/z 280.156 (M+H), Δ _Calc=2.2ppm, C ₁₅H ₂₂NO ₄Figure 49 has described chemical compound with formula II-17 structure ¹H NMR collection of illustrative plates.

Compound I I-18:UV (acetonitrile/water) λ _Max225 (sh) nm.High resolution mass spec (APCI): m/z 358.065 (M+H), Δ _Calc=-1.9ppm, C ₁₅H ₂₁NO ₄Br.Figure 50 described have formula II-18 structure chemical compound at DMSO-d ⁶In ¹H NMR collection of illustrative plates.

Compound I I-27:UV (acetonitrile/water) λ _Max225 (sh) nm; MS (HR-ESI), m/z280.1556 (M+H) Δ _Calc=2.7ppm (C ₁₅H ₂₂NO ₄); ¹H NMR (DMSO-d ₆) referring to Figure 53.

Embodiment 6

By II-16 preparation formula II-19 chemical compound

The sample (250mg) of Compound I I-16 is added in the acetone soln (10ml contains 1.5g) of sodium iodide and and stirred 6 days resulting mixture.Then described solution is filtered with 0.45 micron syringe filter and the solution of 0.95ml part is injected directly on the purification on normal-phase silica gel HPLC chromatographic column (Phenomenex Luna 10u Silica, 25cm * 21.2mm).The HPLC condition of separate type II-19 chemical compound adopts the equal strength eluting HPLC method of being made up of 24% ethyl acetate and 76% hexane from unreacted II-16, and most in the method Compound I I-19 is 2.5 minutes eluting before Compound I I-16.The part of equity in the injection each time of 10 injections is merged, generate 35mg Compound I I-19.Compound I I-19:UV (acetonitrile/water) 225 (sh), 255 (sh) nm; ESMS, m/z 406.0 (M+H); HRMS (ESI), m/z406.0513[M+H] ⁺, Δ _Calc=-0.5ppm, C ₁₅H ₂₁NO ₄I; At DMSO-d ₆In ¹HNMR (referring to Fig. 9).

Embodiment 7

Synthesizing of formula II-2, II-3 and II-4 chemical compound

Can be by catalytic hydrogenation respectively by formula II-16, II-17 and II-18 compounds accepted way of doing sth II-2, II-3 and II-4 chemical compound.

Synthetic exemplary illustration

II-16：R＝Cl II-2：R＝Cl

II-17：R＝H II-3：R＝H

II-18：R＝Br II-4；R＝Br

Embodiment 7A: the catalytic hydrogenation of formula II-16 chemical compound

Formula II-16 chemical compound (10mg) is dissolved in the acetone (5mL) in the scintillation vial (20mL), and Xiang Guanzhong adds 10% (w/w) Pd/C (1-2mg) and magnetic stir bar.At room temperature in nitrogen atmosphere, stirred this reactant mixture about 15 hours.Filter this reactant mixture with the 3cc silicagel column, and use washing with acetone.Reuse 0.2 μ m Gelman Acrodisc filters this filtrate to remove the catalyst of any trace.Under reduced pressure steam the solvent that removes in this filtrate, generate the formula II-2 chemical compound of pure white powder.UV (acetonitrile/water) 225 (sh), 255 (sh) nm.Figure 10 has described formula II-2 chemical compound at DMSO-d ₆In the NMR collection of illustrative plates.Figure 11 has described the low resolution mass spectrum of formula II-2 chemical compound: m/z 316 (M+H), 338 (M+Na).

Embodiment 7B: the catalytic hydrogenation of formula II-17 chemical compound

Formula II-17 chemical compound (5mg) is dissolved in the interior acetone (3ml) of scintillation vial (20mL), and Xiang Guanzhong adds 10% (w/w) Pd/C (about 1mg) and magnetic stir bar.At room temperature in nitrogen atmosphere, stirred this reactant mixture about 15 hours.Filter this reactant mixture to remove catalyst with 0.2 μ m Gelman Acrodisc.Steam the solvent that removes in this filtrate, generate the formula II-3 chemical compound of white powder, this chemical compound adopts following condition to carry out purification by positive HPLC:

Chromatographic column: Phenomenex Luna 10 μ Silica

Size: 25cm * 21.2mm ID

Flow velocity: 14.5ml/min

Detector: ELSD

Solvent: 5% to the 60%EtOAc/ hexane in 19 minutes; 60% arrives 100%EtOAc in 1 minute; 100%EtOAc continues 4 minutes then

Formula II-3 chemical compound in the time of 22.5 minutes as pure compound by eluting.UV (acetonitrile/water) 225 (sh), 255 (sh) nm.Figure 12 has described formula II-3 chemical compound at DMSO-d ₆In the NMR collection of illustrative plates.Figure 13 has described the low resolution mass spectrum of formula II-3 chemical compound: m/z 282 (M+H), 304 (M+Na).

Embodiment 7C: the catalytic hydrogenation of formula II-18 chemical compound

3.2mg formula II-18 chemical compound is dissolved in the interior acetone (3ml) of scintillation vial (20mL), and Xiang Guanzhong adds 10% (w/w) Pd/C (about 1mg) and magnetic stir bar.At room temperature in nitrogen atmosphere, stirred this reactant mixture about 15 hours.Filter this reactant mixture to remove catalyst with 0.2 μ m Gelman Acrodisc.Steam the solvent that removes in this filtrate, generate the formula II-4 chemical compound of white powder, this chemical compound adopts following condition to carry out purification by positive HPLC:

Chromatographic column: Phenomenex Luna 10 μ Silica

Size: 25cm * 21.2mm ID

Flow velocity: 14.5ml/min

Detector: ELSD

Solvent: 5% to the 80%EtOAc/ hexane in 19 minutes; 80% arrives 100%EtOAc in 1 minute; 100%EtOAc continues 4 minutes then

Formula II-4 chemical compound in the time of 16.5 minutes as pure compound by eluting.UV (acetonitrile/water) 225 (sh), 255 (sh) nm.Figure 14 has described formula II-4 chemical compound at DMSO-d ₆In the NMR collection of illustrative plates.Figure 15 has described the low resolution mass spectrum of formula II-4 chemical compound: m/z 360 (M+H), 382 (M+Na).

In addition, the high resolution mass spec data of Compound I I-2, II-3 and II-4 have been obtained.Compound I I-2:HRMS (ESI), m/z 316.1305[M+H] ⁺, Δ _Calc=-3.5ppm, C ₁₅H ₂₃NO ₄Cl.Compound I I-3:HRMS (ESI), m/z 282.1706[M+H] ⁺, Δ _Calc=-0.3ppm, C ₁₅H ₂₄NO ₄Compound I I-4:HRMS (ESI), m/z 360.0798[M+H] ⁺, Δ _Calc=-3.4ppm, C ₁₅H ₂₃NO ₄Br.

Embodiment 8

Synthesizing of formula II-5A and II-5B chemical compound

Can be by adopting the epoxidation of mCPBA, by formula II-16 compounds accepted way of doing sth II-5A and II-5B chemical compound.

Formula II-16 chemical compound (101mg, 0.32 mM) is dissolved in the interior dichloromethane (30mL) of 100ml round-bottomed flask, in this flask, adds metachloroperbenzoic acid (mCPBA) and the magnetic stir bar of 79mg (0.46 mM).At room temperature stirred described reactant mixture about 18 hours.This reactant mixture is poured over 20cc silica gel dodges 100% the eluent ethyl acetate of using 40ml in the post and with 1: 1 ethyl acetate/hexane of 120ml dichloromethane, 75ml, at last.This 1: 1 ethyl acetate/hexane partly produces the mixture of the diastereomer of epoxides formula II-5A and formula II-5B, and II-5A adopts following condition to separate with formula II-5B by positive HPLC:

Chromatographic column	Phenomenex Luna 10μ Silica
Chromatographic column	Phenomenex Luna 10μ Silica	Size	25cm×21.2mm ID
Flow velocity	14.5ml/min	Size	25cm×21.2mm ID
Flow velocity	14.5ml/min	Detector	ELSD
Solvent	25% to the 80%EtOAc/ hexane in the 19min; 80% arrives 100%EtOAc in the 1min; 100%EtOAc continues 5min then	Detector	ELSD

Chemical compound formula II-5A (principal product) and II-5B (inferior product) respectively in the time of 21.5 and 19 minutes as pure compound by eluting.Compound I I-5B is further dodged the enterprising circumstances in which people get things ready for a trip spectrum of post at 3cc silica gel to be separated to remove the chlorobenzoic acid reagent of trace.

Chemical constitution:

Structure is identified:

Formula II-5A:UV (acetonitrile/water) λ _Max225 (sh) nm.Low resolution mass spectrum: m/z 330 (M+H), 352 (M+Na); HRMS (ESI), m/z 330.1099[M+H] ⁺, Δ _Calc=-2.9ppm, C ₁₅H ₂₁NO ₅Cl.Figure 16-17 has described formula II-5A's respectively ¹The mass spectrum of H NMR collection of illustrative plates and formula II-5A.

Formula II-5B:UV (acetonitrile/water) λ _Max225 (sh) nm.Low resolution mass spectrum: m/z 330 (M+H), 352 (M+Na); HRMS (ESI), m/z 330.1105[M+H] ⁺, Δ _Calc=-0.9ppm, C ₁₅H ₂₁NO ₅Cl.Figure 18-19 has described II-5B's respectively ¹The mass spectrum of H NMR collection of illustrative plates and II-5B.

Embodiment 9

Synthesizing of formula IV-1, IV-2, IV-3 and IV-4 chemical compound

Synthetic (the formula IV-2) of alkylene glycol deriv

Can use AD mix-α and AD mix-β, by Sharpless dihydroxy synthesis of dibasic alcohol: AD mix-α is the premix of four reagent: K ₂OsO ₂(OH) ₄, K ₂CO ₃, K ₃Fe (CN) ₆, (DHQ) ₂-PHAL[1, two (the 9-O-dihydro quinine) phthalazines of 4-]; AD mix-β is K ₂OsO ₂(OH) ₄, K ₂CO ₃, K ₃Fe (CN) ₆, (DHQD) ₂-PHAL[1, two (9-O-dihydro chinidine) phthalazines of 4-] premix, described reagent obtains from Aldrich is commercial.Also can pass through the acidity or the basic hydrolysis synthesis of dibasic alcohol of epoxide (formula II-5A and II-5B), the spatial chemistry that the product that this method obtains is compared at the carbon atom place with hydroxyl with the product that obtains in the Sharpless dihydroxy is different.

The Sharpless dihydroxy of Compound I I-16, II-17 and II-18

Formula II-16, any in II-17 and the II-18 chemical compound can be used as initial compounds.In the following embodiments, use formula II-16 chemical compound.Described initial compounds is dissolved in the butanol/water in the round-bottomed flask, in bottle, adds AD mix-α or AD mix-β and magnetic stir bar.Monitor this reaction by silica gel tlc and mass spectrograph.Carry out purification by conventional post processing and by flash chromatography or HPLC, obtain pure dihydroxylic alcohols.Determine this structure by nuclear magnetic resonance spectroscopy and mass spectral analysis.Two hydroxyls are in an identical side in the method.

The nucleophilic ring opening of epoxide (II-5):

Use such as NaCN, NaN ₃, NaOAc, HBr, HCl or the like multiple nucleopilic reagent with described epoxide ring open loop, to be created in the multiple substituent group on this cyclohexane ring, comprise hydroxyl substituent.

For example:

Formula II-5 formula IV-1

With HCl with described epoxy addition, production IV-3:

Formula II-5 formula IV-3

Formula II-5A chemical compound (3.3mg) is dissolved in the acetonitrile (0.5ml) in the phial of 1 dram, in bottle, adds 5%HCl (500 μ l) and magnetic stir bar.At room temperature stirred described reactant mixture about 1 hour.Monitor this reaction by mass spectral analysis.Directly this reactant mixture is injected positive HPLC to obtain the pure compound of formula IV-3C without any post processing.The HPLC condition that is used for purification is as follows: 25% to the 80%EtOAc/ hexane in the Phenomenex Luna 10 μ Silica posts (25cm * 21.2mm ID), 19min; 80% arrives 100%EtOAc in the 1min; 100%EtOAc stops the solvent gradient of 5min, the flow velocity of 14.5ml/min then.Monitor this purge process with ELSD.Formula IV-3C chemical compound is eluting (2.2mg) in the time of about 18 minutes.Formula IV-3C chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm; ESMS, m/z366 (M+H), 388 (M+Na); HRMS (ESI), m/z 366.0875[M+H] ⁺, Δ _Calc=0.0ppm, C ₁₅H ₂₂NO ₅Cl ₂At DMSO-d ₆In ¹H NMR (Figure 20).According to 1: 1C ₆D ₆/ DMSO-d ₆In on cyclohexane ring observed coupling constant determine the spatial chemistry (Figure 21) of formula IV-3C chemical compound.

The reproducibility open loop of epoxide (II-5): use such as BH ₃The metal hydride of-THF complex is handled this formula chemical compound, the chemical compound of production IV-4.

Formula II-5 formula IV-4

Embodiment 10

Synthesizing of formula II-13C and II-8C chemical compound

Compound I I-16 (30mg) is dissolved in the interior CH of scintillation vial (20ml) ₂Cl ₂(6ml), in this pipe, add Dess-Martin Periodinane (122mg) and magnetic stir bar.At room temperature stirred described reactant mixture about 2 hours.Monitor the process of this reaction with TLC (hexane: EtOAc, 6: 4) and analysis HPLC.In this reactant mixture, solvent volume is reduced to 1/3rd, be adsorbed on the silica gel, be poured over 20cc silica gel and dodge on the post and adopt the gradient of from 10% to 100% hexane/ethyl acetate to carry out eluting with the part of 20ml.Containing proportional with the part of the hexane eluting that contains 30%EtOAc is the mixture of rotamer of 1.5: 8.5 formula II-13C.Use following condition to be further purified this mixture: PhenomenexLuna 10 μ Silica posts (25cm * 21.2mm ID) by positive HPLC, 25% to the 80%EtOAc/ hexane in the 19min; 80% arrives 100%EtOAc in the 1min; 100%EtOAc stops the solvent gradient of 5min, the flow velocity of 14.5ml/min.Monitor this purge process with ELSD.Formula II-13C chemical compound is mixture eluting (7mg) in the time of 13.0 and 13.2 minutes of 1.5: 8.5 rotamer with ratio.Formula II-13C:UV (acetonitrile/water) λ _Max226 (sh) ﹠amp; 330 (sh) nm; ESMS, m/z 312 (M+H) ⁺, 334 (M+Na) ⁺HRMS (ESI), m/z 312.1017[M+H] ⁺, Δ _Calc=4.5ppm, C ₁₅H ₁₉NO ₄Cl; At DMSO-d ₆In ¹H NMR (seeing Figure 22).

The rotamer mixture (4mg) of formula II-13C is dissolved in the interior acetone (1ml) of scintillation vial (20ml), in this pipe, adds 10% (w/w) Pd/C and magnetic stir bar of catalytic amount (0.5mg).At room temperature in hydrogen atmosphere, stirred described reactant mixture about 15 hours.Filter this reactant mixture to remove this catalyst with 0.2 μ mGelman Acrodisc.Solvent steamed from this filtrate remove, generate colourless gelationus formula II-8C chemical compound, adopt following condition by positive HPLC this chemical compound to be further purified: 25% to the 80%EtOAc/ hexane in the Phenomenex Luna 10 μ Silica posts (25cm * 21.2mm ID), 19min; 80% arrives 100%EtOAc in the 1min; 100%EtOAc stops the solvent gradient of 5min, the flow velocity of 14.5ml/min.Monitor this purge process with ELSD.Formula II-8C chemical compound (1mg) in the time of 13.5 minutes as pure compound by eluting.Formula II-8C:UV (acetonitrile/water) λ _Max225 (sh) nm; ESMS, m/z314 (M+H) ⁺, 336 (M+Na) ⁺HRMS (ESI), m/z 314.1149[M+H] ⁺, Δ _Calc=3.3ppm, C ₁₅H ₂₁NO ₄Cl; At DMSO-d ₆In ¹H NMR (seeing Figure 23).

Embodiment 11

By formula II-13C synthesis type II-25 chemical compound

The rotamer mixture (5mg) of formula II-13C is dissolved in the interior dimethoxy-ethane (monoglyme of scintillation vial (20ml); 1.5ml) in, in this pipe, add entry (15 μ l (whole solution concentration is 1%)) and magnetic stir bar.Above-mentioned solution is cooled to-78 ℃ in dry ice-propanone is bathed, and dropwise adds sodium borohydride solution and (contain 3.7mg NaBH ₄0.5ml dimethoxy-ethane (consider slow adding and prepare)).Stirred described reactant mixture about 14 minutes down at-78 ℃.With this reactant mixture of 4%HCl acidified aqueous solution of 2ml and use CH ₂Cl ₂Extract.Evaporate this organic layer, the generation ratio is 9.5: 0.5 the formula II-25 of white solid and the mixture of II-16 chemical compound, adopts Phenomenex Luna 10 μ Silica posts (25cm * 21.2mm ID), is further purified this mixture by positive HPLC.Mobile phase is to keep equicohesive 24%EtOAc/76% hexane in the 19min, and then 24% to 100%EtOAc linear gradient and 100%EtOAc continue 3min in the 1min; Flow velocity is 25ml/min.Monitor this purge process with ELSD.Formula II-25 chemical compound (1.5mg) in the time of 11.64 minutes as pure compound by eluting.Formula II-25 chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm; ESMS, m/z 314 (M+H) ⁺, 336 (M+Na) ⁺HRMS (ESI), m/z 314.1154[M+H] ⁺, Δ _Calc=-0.6ppm, C ₁₅H ₂₁NO ₄Cl; At DMSO-d ₆In ¹H NMR (seeing Figure 24).

Embodiment 12

By II-13C synthesis type II-31, II-32 and II-49 chemical compound; And close by II-31 and II-32 Accepted way of doing sth II-33, II-34, II-35 and II-36 chemical compound

The rotamer mixture (5mg) of formula II-13C chemical compound is dissolved in the interior acetone (4ml) of scintillation vial (20ml), in this pipe, adds 10% (w/w) Pd/C and magnetic stir bar of catalytic amount (3mg).At room temperature stirred described reactant mixture about 15 hours.Filter this reactant mixture to remove this catalyst with 0.2 μ m GelmanAcrodisc.Solvent steamed from this filtrate remove, non-enantiomer mixture of the hydroxy derivatives of production II-31 and II-32 (1: 1) and less important Compound I I-49, adopt following condition by reversed-phase HPLC it to be separated: 90% arrives 30%H in the Ace 5u C18 post (150cm * 22mm ID), 15min ₂70% to 100% acetonitrile in the O/ acetonitrile, 5min, 100% acetonitrile stops the solvent gradient of 4min, the flow velocity of 14.5ml/min.Detect this purge process with diode array detector.Compound I I-31 (2mg), II-32 (2mg) and II-49 (0.2mg) respectively when 10.6 minutes, 10.8 minutes and 11.54 minutes as pure compound by eluting.II-31:UV (acetonitrile/water) λ _Max250 (sh) nm; ESMS, m/z328.1 (M+H) ⁺﹠amp; 350.0 (M+Na) ⁺II-32:UV (acetonitrile/water) λ _Max250 (sh) nm; ESMS, m/z 328.1 (M+H) ⁺﹠amp; 350.0 (M+Na) ⁺II-49:UV (acetonitrile/water) λ _Max250 (sh) and 320nm; ESMS, m/z 326.0 (M+H) ⁺, 343.1 (M+H ₂O) ⁺﹠amp; 348.0 (M+Na) ⁺

In selectable method, (25cm * 21.2mmID), 10% to 100% hexane/EtOAc in the 24min by positive HPLC separating compound II-31, II-32 and II-49:Phenomenex Luna 10 μ Silica posts to adopt following condition; 100%EtOAc continues the solvent gradient of 3min, flow velocity 14.5ml/min.Detect this purge process with ELSD.

Under 0 ℃ to-10 ℃, in the dimethoxy-ethane solvent, can the ketonic compound of formula II-31 and II-32 be reduced about 14 minutes by using sodium borohydride.Available 4%HCl acidified aqueous solution reactant mixture, and use CH ₂Cl ₂Extract.With the organic layer evaporation, generation can be by the mixture of the isolating formula II-33 of chromatographic process, II-34, II-35 and II-36 chemical compound.

Embodiment 13

By formula II-19 synthesis type II-21 chemical compound

Acetone (7.5ml) is mixed tempestuously with 5 N NaOH (3ml), and with gained mixture vaporising under vacuum to minimum volume.100 μ l samples of this solution are mixed with formula II-19 chemical compound (6.2mg) in acetone (1ml), resulting biphase mixture whirlpool was stirred 2 minutes.Immediately with the C18 HPLC of this reaction solution through preparation.The condition that is used for purification comprises in 17 minutes from 10% acetonitrile/90% water to 90% acetonitrile/linear gradient of 10% water and use the long Ace 5 μ C18 HPLC posts of size 22mm id 150mm.Formula II-21 chemical compound eluting in the time of 9.1 minutes produces the 0.55mg chemical compound under these conditions.Formula II-21 chemical compound: UV (acetonitrile/water) λ _Max225 (sh) nm; ESMS, m/z 296.1 (M+H); At DMSO-d ₆In ¹HNMR collection of illustrative plates (seeing Figure 25).

Embodiment 14

By formula II-19 synthesis type II-22 chemical compound

60mg sodium propionate sample is added in DMSO (1ml) solution of formula II-19 chemical compound (5.3mg), and this mixture of sonication 5 minutes, though sodium propionate can not dissolve fully.After 45 minutes, filter this solution with the syringe filter of 0.45 μ, and directly carry out purification with HPLC.The condition that is used for this purification comprises in 17 minutes from 10% acetonitrile/90% water to 90% acetonitrile/linear gradient of 10% water and use the long Ace 5 μ C18HPLC posts of size 22mm id 150mm.Formula II-22 chemical compound eluting in the time of 12.3 minutes generates 0.7mg chemical compound (15% separation yield) under these conditions.UV (acetonitrile/water) λ _Max225 (sh) nm; ESMS, m/z352.2 (M+H); HRMS (ESI), m/z 352.1762[M+H] ⁺, Δ _Calc=0.6ppm; C ₁₈H ₂₆NO ₆At DMSO-d ₆In ¹H NMR (seeing Figure 26).

Embodiment 15

By II-19 synthesis type II-29 chemical compound

With NaN ₃(80mg) sample is dissolved among the DMSO (1ml), and is transferred in the phial that contains Compound I I-19 (6.2mg), and Compound I I-19 is mixed with about 10% contaminant compound II-16.This solution was at room temperature hatched 1 hour, adopt then in 17 minutes by 10% acetonitrile/90%H ₂O to 90% acetonitrile/10%H ₂The solvent gradient of O is carried out purification on C18 HPLC (ACE 5 μ C18-HL, 150mm * 21mm ID).Adopt the method, desired azido derivative I I-29 in the time of 12.5 minutes in Compound I I-16 co-elute.By 35% acetonitrile/65%H ₂The equal strength solvent gradient that O forms is with additional C 18 HPLC chromatographs (ACE 5 μ C18-HL, 150mm * 21mm ID)) be further purified the Compound I I-29 of 2.4mg part.Under these conditions, Compound I I-29 is eluting after 20 minutes, and Compound I I-16 eluting after 21.5 minutes.The resulting sample of being made up of 1.1mg Compound I I-29 is used for the sign of bioanalysis to be identified.

Compound I I-29:UV (acetonitrile/water) λ _Max225 (sh) nm, ESMS, m/z321.1 (M+H); In DMSO-d6 ¹H NMR (seeing Figure 55).

Embodiment 16

By II-19 synthesis type II-37 and II-38 chemical compound

Can pass through cyano group-dehalogenation or thiocyano-dehalogenation chemical compound respectively by formula II-19 compound formula II-37 and II-38.Available NaCN or KCN handle Compound I I-19, obtain Compound I I-37.Selectively, available NaSCN or KSCN handle Compound I I-19, obtain Compound I I-38.

Synthesis type II-38 chemical compound from II-19

With formula II-19 chemical compound (10.6mg .02616mmol) is dissolved in the 1.5ml acetone in the scintillation vial (20ml), in this pipe, add sodium rhodanate (10.0mg, 0.1234mmol), triethylamine (5 μ l .03597mmol) and magnetic stir bar.At room temperature stirred described reactant mixture about 72 hours.Under vacuum, this reactant mixture is concentrated, produce Compound I I-38.Adopt following condition by this Compound I of positive HPLC purification I-38:Phenomenex Luna 10 μ Silica posts (25cm * 21.2mm ID), in 21 minutes from 0% to 95%H ₂The solvent gradient of O/ acetonitrile, flow velocity 14.5ml/min.Monitor this purge process with diode array detector.Compound I I-38 (3.0mg, yield 34%) in the time of 18.0 minutes as purification thing eluting.II-38:UV (acetonitrile/water) λ _Max203 (sh) nm; ESMS, m/z 337.1 (M+H) ⁺﹠amp; 359.1 (M+Na) ⁺

Embodiment 17

By the synthetic general formula I I-39 chemical compound of II-19

But the dehalogenation of through type II-19 chemical compound, mercaptan and the thioether of formation general formula I I-39.For example, can form mercaptan (R=H) by handling Compound I I-19, yet can form thioether (R=alkyl), perhaps selectively by handling Compound I I-19 with the salt of mercaptan with NaSH, by in the presence of DBU, in benzene, reacting, by handling to form this thioether with mercaptan self.

Embodiment 18

By the synthetic general formula I I-40 chemical compound of II-39

Can for example adopt hydrogen peroxide or other oxidising agent, the oxidation of the thioether by general formula I I-39 forms sulfoxide (n=1) and the sulfone (n=2) of general formula I I-40.

Embodiment 19

By II-21 synthesis type II-41 chemical compound

Formula II-41 chemical compound can be by handling formula II-21 chemical compound (or the protected derivant of II-21 with mesyl chloride (mesyl chlorine) in pyridine; wherein for example the NH of the alcohol of C-5 position or lactams is protected) prepare, perhaps for example prepare by handling with mesyl chlorine when existing at triethylamine (triethylaminde).Can prepare other sulphonic acid ester similarly.

Embodiment 20

By II-19 or II-41 synthesis type II-46 chemical compound

But the dehydroiodination of through type II-19 chemical compound, or the alkene of the elimination hydrogen of through type II-41 chemical compound-mesyl oxygen base preparation formula II-46 are for example by realizing with alkali treatment.

Embodiment 21

Synthesis type II-42A chemical compound

Can finish the synthetic of boric acid or its ester (for example formula II-42A chemical compound) by the method that outlines in the following retrosynthesis scheme.The hydroboration of the alkene among the formula II-46 produces corresponding alkyl borane, and this alkyl borane can be converted into corresponding boric acid or ester, for example, and formula II-42A chemical compound.

Embodiment 22

Synthesizing of formula II-43A chemical compound

Formula II-43A chemical compound can prepare by the following method: handle formula II-19 chemical compound with triphenylphosphine, produce phosphorus ylide, this phosphorus ylide can be used such as the multiple aldehyde of glyoxalic acid methylester and handle, and obtains formula II-43A.

Embodiment 23

By II-19 synthesis type II-30 chemical compound

A CuI (100mg) is placed 25ml pyriform bottle, and washed 30 minutes with the Ar air-blowing.The Ar air-flow remains by this flask in this course of reaction.This container is cooled to-78 ℃ before adding exsiccant THF (5ml), under vigorous stirring, dropwise add immediately subsequently lithium methide dry THF solution (5.0ml, 1.6M).The dry THF solution (12mg Compound I I-19,1ml THF) that slowly adds Compound I I-19 in transparent dialkyl group cuprate solution stirred resulting mixture 1 hour down at-78 ℃.By making described THF flow of solution cross silicagel column (diameter 1cm, long 2cm) and further using 50%EtOAc/50% hexane solution (50ml) washing, stop this reaction.Blended silica gel eluent is dry under vacuum, and adopt by 35%ACN/65%H ₂The equal strength solvent gradient that O forms is infused in the enterprising single step purification of advancing of C18 HPLC (ACE5 μ C18-HL, 150mm * 21mm ID) through 2 times.Under these conditions, Compound I I-30 is eluting in the time of 23.5 minutes, and produces the 2.4mg material (separation yield 27%) that HPLC by analysis measures 90.8% purity.Selectable positive purification process can adopt Phenomenex Luna 10 μ Silica posts (25cm * 21.2mm ID), uses the solvent gradient of forming from 100% hexane/ethyl acetate to 0% hexane in 20 minutes.Compound I I-30 eluting in the time of 16.5 minutes under these conditions, and produce the 3.0mg material (separation yield 41%) of 97.1% purity that HPLC by analysis measures.

Compound I I-30:UV (acetonitrile/water) 225 (sh), ESMS, m/z 294.1 (M+H); HRMS (ESI), m/z 294.1696[M+H] ⁺, Δ _Calc=-3.2ppm; C ₁₆H ₂₄NO ₄At DMSO-d ₆In ¹H NMR (seeing Figure 56).

Embodiment 24

By II-16 synthesis type II-44 and VI-1A chemical compound

(30mg 0.096mmol) is dissolved in the interior CH of scintillation vial (20ml) with formula II-16 chemical compound ₂Cl ₂(9ml), in this pipe, add triethylamine (40 μ l, 0.29mmol), 3-mercapto-propionate (mercaptan, 250 μ l) and magnetic stir bar.At room temperature stirred described reactant mixture about 4 hours.Steam from this reactant mixture and desolventize, the mixture (19: 1) of production II-44 and VI-1A chemical compound adopts following condition to separate this mixture by reversed-phase HPLC: Ace 5u C18 post (150mm * 22mm ID); 35% to 90%H in 17 minutes ₂The O/ acetonitrile, 90% to 100% acetonitrile in 1 minute, 100% acetonitrile continues 1 minute solvent gradient; 14.5ml/min flow velocity.Monitor this purge process with diode array detector.Compound I I-44 (20mg) and VI-IA (1mg) respectively when 11.68 minutes and 10.88 minutes as pure compound by eluting.Compound I I-44:UV (acetonitrile/water) λ ^Max240 (sh) nm; ESMS, m/z 434.0 (M+H) ⁺﹠amp; 456.0 (M+Na) ⁺Compound VI-1A:UV (acetonitrile/water) λ _Max220 (sh) nm; ESMS, m/z 398.0 (M+H) ⁺﹠amp; 420.0 (M+Na) ⁺

Embodiment 25

The oxidation of the secondary hydroxyl in formula II-16, II-17 and the II-18 chemical compound and with hydroxylamine or The reaction of methoxyl group amine

Arbitrary chemical compound in formula II-16, II-17 and the II-18 chemical compound can be used as initial compounds.With the secondary hydroxyl in the described initial compounds of any one reagent oxidation of following reagent: pyridinium dichromate (PDC), pyridinium chlorochromate (PCC), Dess-Martin periodinane or oxalyl chloride (Swern oxidation) (list of references: Organic Syntheses, collective volumesI-VIII).Preferably, Dess-Martin periodinane can be used as the reagent that is used for this reaction (list of references: Fenteany G.et al.Science, 1995,268,726-73).The ketonic compound of handling gained with hydroxylamine or methoxyl group amine is to generate oxime.

Embodiment:

Embodiment 26

The reduction amination of ketone derivatives

In the presence of multiple alkali, use sodium cyanoborohydride (NaBH ₃CN) handle the amine derivative that all ketone derivatives suc as formula II-8 and II-13 produce described initial compounds, this derivant is subsequently by 10%Pd/C, H ₂The two keys in the cyclohexene ring are reduced in hydrogenation.

Embodiment 27

The cyclohexene open loop

Any chemical compound among formula II-16, II-17 and the II-18 can be used as initial compounds.Can be for example alcohol and/or in lactams the position of nitrogen protect this initial compounds, and at THF-H ₂Use OsO in the O solution ₄And NaIO ₄Handle generating two aldehyde derivatives, use NaBH ₄This two aldehyde derivatives is reduced into alcohol.Can the suitable stage in this reaction sequence remove described protecting group, to produce II-7 or II-6.

Embodiment:

Embodiment 28

Dehydration of alcohols at the engagement of loops place of lactone-lactams then forms aldehyde

For example, perhaps, the initial compounds shown in any formula II-16, II-17 or the II-18 is dewatered for example by processing with Burgess reagent or other dehydrated reagent by when alkali exists, handling with mesyl chloride.Resulting anhydro compounds OsO ₄, use NaIO subsequently ₄Handle, perhaps selectively decompose the aldehyde radical at the engagement of loops place that is created on lactone-lactams by ozone.

Embodiment 29

The oxidation of cyclohexene ring is to produce cyclohexadiene or phenyl ring

Handle the initial compounds of all ketone suc as formula II-13C with Pd/C, produce Cyclohexadiene derivatives.New two keys can be in cyclohexene ring any position.Can be for example with the described ketone of sodium borohydride reduction to obtain corresponding secondary alcohol.Perhaps, can for example further handle this Cyclohexadiene derivatives, so that this cyclophane is changed into phenyl with DDQ.Similarly, can for example obtain corresponding secondary alcohol with this ketone of sodium borohydride reduction.

As selectable method, can for example handle all initial compounds suc as formula the II-49 chemical compound with TMSC1, produce Cyclohexadiene derivatives.Can for example further handle this Cyclohexadiene derivatives, so that this cyclophane is changed into phenyl with DDQ.Can be for example remove OTMS on this phenyl with acid or alkali.Similarly, can for example obtain corresponding secondary alcohol with the described ketone of sodium borohydride reduction.

Embodiment 30

Multiple reaction on aldehyde derivatives I-1

Use multiple phosphorus ylide [for example (triphenylphosphoranylidene) ethane] on aldehyde radical, to carry out the Wittig reaction to generate alkene.Two keys in this side chain are reduced by catalytic hydrogenation.

Example:

Use multiple alkali (NH for example ₃) and sodium cyanoborohydride carry out reduction amination on described aldehyde radical, generate amine derivative.Selectively, use NaBH ₄This aldehyde reduction is to be formed on the alcohol in this side chain.

Example:

Can carry out the organic metal additive reaction to generate the secondary alcohol of multiple replacement to described aldehyde carbonyl.For example:

Embodiment 31

By II-17 synthesis type II-47 chemical compound

(25mg 0.0896mmol) is dissolved in the interior CH of scintillation vial (20ml) with formula II-17 chemical compound ₂Cl ₂(9ml), in this pipe, add triethylamine (38 μ l, 0.27mmol), 3-mercapto-propionate (mercaptan, 250 μ l) and magnetic stir bar.At room temperature stirred described reactant mixture about 4 hours.Steam from this reactant mixture and desolventize, production II-47 chemical compound adopts following condition to be further purified this chemical compound by positive HPLC: Phenomenex Luna 10uSilica post (25cm * 21.2mm ID); 10% to 100% hexane/EtOAc in 24 minutes, and 100%EtOAc continues 3 minutes solvent gradient; 14.5ml/min flow velocity.Monitor this purge process with ELSD.Compound I I-47 (15mg) in the time of 10.98 minutes as pure compound by eluting.Compound I I-47:UV (acetonitrile/water) λ _Max240 (sh) nm; ESMS, m/z 400.1 (M+H) ⁺﹠amp; 422.1 (M+Na) ⁺

Embodiment 32

By II-16 synthesis type II-48 and VI-1B chemical compound

With formula II-16 chemical compound (15mg 0.048mmol) is dissolved among 1: 1 the ACN/DMSO (8ml) in the scintillation vial (20ml), in this pipe, add triethylamine (40 μ l, 0.29mmol), glutathion (44.2mg, 0.144mmol) and magnetic stir bar.At room temperature stirred described reactant mixture about 3 hours.Steam from this reactant mixture and desolventize, production II-48 chemical compound adopts following condition by this chemical compound of reversed-phase HPLC purification: Ace 5u C18 post (150mm * 22mm ID); 10% to 70%H in 15 minutes ₂The O/ acetonitrile, 70% to 100% acetonitrile in 5 minutes, 100% acetonitrile continues 4 minutes solvent gradient; Flow velocity 14.5ml/min.Monitor this purge process with diode array detector.Compound I I-48 (10mg) in the time of 8.255 minutes as pure compound by eluting.Compound I I-48:UV (acetonitrile/water) λ _Max235 (sh) nm; ESMS, m/z621.0 (M+H) ⁺Compound I I-48 is unstable in solution, and is converted into compound VI-1B, exists with the mixture of 7: 3 ratios as II-48 and VI-1B.Compound VI-1B:UV (acetonitrile/water) λ _Max235 (sh) nm; ESMS, m/z 585.2 (M+H) ⁺

Embodiment 33

By II-16 synthesis type II-50 and VI-1C chemical compound

(10mg 0.032mmol) is dissolved in the interior CH of scintillation vial (20ml) with formula II-16 chemical compound ₂Cl ₂(9ml), in this pipe, add triethylamine (26.5 μ l, 0.192mmol), N-acetyl group-L-acthiol-J (17mg, 0.096mmol) and magnetic stir bar.At room temperature stirred described reactant mixture about 4 hours.Steam from this reactant mixture and desolventize, the mixture of production II-50 and VI-1C chemical compound adopts following condition to be further purified this mixture by positive HPLC: Phenomenex Luna 10uSilica post (25cm * 21.2mm ID); 10% to 100% hexane/EtOAc in 24 minutes, and 100%EtOAc continues 3 minutes solvent gradient; Flow velocity 14.5ml/min.Monitor this purge process with ELSD.Compound I I-50 (2mg) and VI-IC (0.2mg) respectively 10.39 minutes and 10.57 o'clock as pure compound by eluting.Compound I I-50:UV (acetonitrile/water) λ _Max230 (sh) nm; ESMS, m/z 491.1 (M+H) ⁺﹠amp; 513.0 (M+Na) ⁺Compound VI-1C:UV (acetonitrile/water) λ _Max215 (sh) nm; ESMS, m/z 455.1 (M+H) ⁺﹠amp; 577.0 (M+Na) ⁺

Embodiment 34

Extracorporeal biology

National Cancer Institute (NCI) the examination group of being made up of 60 strain human tumor cell lines is used in the initial research that is also referred to as the formula II-16 chemical compound of Salinosporamide A, and this tumor cell line is represented leukemia, melanoma and pulmonary carcinoma, colon cancer, the brain cancer, ovarian cancer, breast carcinoma, carcinoma of prostate and renal carcinoma.The detailed description of this screening procedure can be found (http: //) " dtp.nci.nih.gov/branches/btb/ivclsp.html. " in the hypertext transfer scheme

In brief, the human tumor cell line with each described 60 strain is grown in the RPMI RPMI-1640 of adding 5% hyclone and 2mM L-glutaminate.Cell is seeded in the titer plate in 96-hole and 5%CO at 37 ℃ with its proper density ₂, hatch in 95% air and 100% relative humidity.After 24 hours, multiple 10 times of serial dilutions 100 μ L, Salinosporamide A are joined in the appropriate hole of containing 100 μ L cells, causing final Salinosporamide A concentration is that 10nM is to 100 μ M.Again cell was hatched in addition 48 hours and estimated the viability or the growth of cell with sulforhodamine B analysis of protein.

Three kinds of dose response parameters of following calculating:

GI ₅₀Expression suppresses the concentration of 50% growth

TGI represents to suppress all concentration of growth

LC ₅₀The concentration of representing 50% cell-lethal

In the table 1 below the example of the research of evaluation Salinosporamide A is shown in the examination of described NCI.

Data show the average GI of Salinosporamide A ₅₀Value is less than 10nM.Observe average T GI value and average LC for tumor cell line the most responsive and that tolerate most ₅₀Wider range (＞1000 times differences) of value, this shows that Salinosporamide A shows good selectivity and do not show general toxicity.In addition, average T GI data show that Salinosporamide A shows preferred specificity to melanoma and breast cancer cell line.Repeat this analysis and show similar result.

The result of nci tumor examination shows Salinosporamide A:(1) it is compounds effective, average GI ₅₀Value is less than 10nM, and (2) its average T GI value and average LC between tumor cell line the most responsive and tolerance ₅₀The value aspect demonstrates the good tumor-selective greater than 1000 times of differences.

Table 1:NCI 60 strain human tumor cell lines are to the relative sensitivity of Salinosporamide A

Embodiment 35

The growth inhibited of tumor cell line

With B16-F10 (ATCC; CRL-6475), DU 145 (ATCC; HTB-81), HEK293 (ATCC; CRL-1573), HT-29 (ATCC; HTB-38), LoVo (ATCC; CCL-229), MDA-MB-231 (ATCC; HTB-26), MIA PaCa-2 (ATCC; CRL-1420), NCI-H292 (ATCC; CRL-1848), and OVCAR-3 (ATCC, HTB-161), PANC-1 (ATCC; CRL-1469), PC-3 (ATCC; CRL-1435), RPMI 8226 (ATCC; CCL-155) and U266 (ATCC; TIB-196) maintain in the suitable culture medium.With described cell under 37 ℃, containing 5%CO ₂With cultivate in the incubator of 95% humid air.

Analyze for the cell growth inhibited, with B16-F10, DU 145, HEK293, and HT-29, LoVo, MDA-MB-231, MIA PaCa-2, NCI-H292, OVCAR-3, PANC-1, PC-3, RPMI8226 and U266 cell are respectively with 1.25 * 10 ³, 5 * 10 ³, 1.5 * 10 ⁴, 5 * 10 ³, 5 * 10 ³, 1 * 10 ⁴, 2 * 10 ³, 4 * 10 ³, 1 * 10 ⁴, 7.5 * 10 ³, 5 * 10 ³, 2 * 10 ⁴, 2.5 * 10 ⁴Cells/well is seeded in Corning3904 black wall, in the 90 μ l complete mediums in the tissue culturing plate of clear bottom.The 20mM stock solution of preparation formula II-16 chemical compound in 100% DMSO is divided into equal portions and is stored in-80 ℃.Serial dilution formula II-16 chemical compound also joins in the instrument connection, and triplicate, causing final concentration is that 20 μ M are to 0.2pM.This culture plate is put back in this incubator cultivated 48 hours.The DMSO final concentration is 0.25% in whole samples.

After the drug exposure 48 hours, in each hole, add the no Mg that 10 μ l contain 0.2mg/ml "diazoresorcinol" (resazurin) (obtaining from Sigma-Aldrich chemical company) ²⁺, Ca ²⁺Phosphate buffered saline (PBS), and described culture plate be put back in this incubator cultivate 3-6 hour.Because living cells metabolism "diazoresorcinol" is used Fusion microtest plate exometer (PackardBioscience), use λ _Ex=535nm and λ _EmThe optical filter of=590nm is measured the fluorescence of the reduzate of "diazoresorcinol"."diazoresorcinol" dyestuff in the not celliferous culture medium is used for determining background, it is deducted from the data of all experimental ports.This data standard is turned to the mean fluorecence degree of the cell of handling with culture medium+0.25%DMSO (growth of 100% cell), and (this rule is by XLfit 3.0 with the S shape dose-effect curve match rule of standard, ID Business SolutionsLtd or Prism 3.0, GraphPad Software Inc produces) determine EC ₅₀Value (observing the drug level that 50% maximum growth suppresses).

Data in the table 2 general introduction formula II-16 is to the growth inhibitory effect of the different tumor cell lines that comprise 12 kinds of people and a kind of mice.

Table 2

Formula II-16 is to the average EC of kinds of tumor cells system ₅₀ Value

Cell line	The source	EC ₅₀(nM), meansigma methods ± SD ^*	n
Cell line	The source	EC ₅₀(nM), meansigma methods ± SD ^*	n	B16-F10	Mice, melanoma	47±20	12
DU145	The people, carcinoma of prostate	37±10	3	B16-F10	Mice, melanoma	47±20	12
DU145	The people, carcinoma of prostate	37±10	3	HEK293	The people, embryonic kidney	47	2
HT-29	The people, colorectum adenocarcinoma	40±26	5	HEK293	The people, embryonic kidney	47	2
HT-29	The people, colorectum adenocarcinoma	40±26	5	LoVo	The people, colorectum adenocarcinoma	70±8	3
MDA-MB-231	The people, breast carcinoma	87±40	12	LoVo	The people, colorectum adenocarcinoma	70±8	3
MDA-MB-231	The people, breast carcinoma	87±40	12	MIAPaCa-2	The people, cancer of pancreas	46	2
NCI-H292	The people, nonsmall-cell lung cancer	66±29	12	MIAPaCa-2	The people, cancer of pancreas	46	2
NCI-H292	The people, nonsmall-cell lung cancer	66±29	12	OVCAR-3	The people, ovarian cancer	49±31	6
PANC-1	The people, cancer of pancreas	60	2	OVCAR-3	The people, ovarian cancer	49±31	6
PANC-1	The people, cancer of pancreas	60	2	PC-3	The people, carcinoma of prostate	64±26	19
RPMI 8226	The people, multiple myeloma	8.6±1.9	26	PC-3	The people, carcinoma of prostate	64±26	19
RPMI 8226	The people, multiple myeloma	8.6±1.9	26	U266	The people, multiple myeloma	4.7±0.7	6

^*N (independent experiment number of times)=2 o'clock, demonstration be meansigma methods

Described EC ₅₀Value shows formula II-16 to B16-F10, and DU 145, HEK293, and HT-29, LoVo, MDA-MB-231, MIA PaCa-2, NCI-H292, OVCAR-3, PANC-1, PC-3, RPMI8226 and U266 cell have cytotoxicity.

Embodiment 36

At external formula I-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, II-44, VI-1A and II-47 chemical compound are to the inhibition of proteasome activity

In DMSO, be the 20mM stock solution, and be stored in-80 ℃ with little equal portions with all described compound.From CalBiochem or Boston Biochem, obtain the rabbit flesh 20S proteasome of purification.In order to strengthen the chymotrypsin-like activity of this proteasome, analysis buffer (20mM HEPES, pH7.3,0.5mM EDTA and 0.05%Triton * 100) has been added SDS, making final SDS concentration is 0.035%.The substrate that uses is suc-LLVY-AMC, and is a kind of by the fluorescence peptide substrates of the active special cutting of the chymotrypsin-like of described proteasome.In 96 hole Costar microtitration plates, in the whole volume of 200 μ l, analyze with the protease concentration of 1 μ g/ml.Formula II-2, II-4, II-16, II-17, II-18, II-19, II-21, II-22 and II-44 test with the final concentration of 500nM to 158pM with 8 dose point effect curves.Formula I-7, II-5A, II-5B, II-20, II-29, II-30 and II-38 test with the concentration of 1 μ M to 0.32nM.Formula II-3 and VI-1A test with the final concentration of 10 μ M to 3.2nM with eight dose point effect curves.Formula II-47 tests with the concentration of 5 μ M to 1.6nM, and formula II-8C, II-13C, II-24C, II-25, II-26, II-27, II-28, II-31, II-32 and IV-3C are with the final concentration test of 20 μ M to 6.3nM.In that (Thermo Electron, Waltham MA) are hatched 5 minutes in the Fluoroskan of temperature control Ascent 96 hole microtest plate readers with described sample under 37 ℃.In this preincubate step process, this substrate is diluted 25 times in containing the analysis buffer of SDS.At this preincubate after date, the substrate of this dilution by adding 10 μ l causes described reaction, and this culture plate is put back in this culture plate reader.The final concentration of substrate is 20 μ M in this reaction.At λ _Ex=390nm and λ _Em=460nm measures the fluorescence of the peptide substrates of cutting down.All data were gathered once in per 5 minutes, continue to surpass 1.5 hours, and these data are drawn according to the meansigma methods at three number strong points.Adopt S shape dose response, variable inclined-plane model, calculate EC by Prism (GraphPad software) ₅₀Value (drug level of the maximum relative fluorescence of inhibition 50%).In order to estimate of the Caspase sample active effect of described chemical compound, except Z-LLE-AMC is used as peptide substrates, react as mentioned above this 20S proteasome.Formula II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-17, II-18, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, IV-3C, II-44 and VI-1A test with the concentration of 20 μ M to 6.3nM.Formula II-2 tests with the concentration of 10 μ M to 3.2nM, and formula II-16 and II-19 test with the concentration of 5 μ M to 1.58nM.In order to estimate described chemical compound, from described analysis buffer, omit SDS, and Boc-LRR-AMC is used as peptide substrates the active effect of the trypsin-like of described proteasome.Formula II-20 tests with the concentration of 5 μ M to 1.6nM.Formula II-3, II-8C, II-13C, II-17, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, IV-3C and VI-1A test with the concentration of 20 μ M to 6.3nM.For formula II-2 and II-5B, test concentrations is 10 μ M to 3.2nM, and formula II-4, II-5A, II-16, II-18 and II-19 test with the concentration of 1 μ M to 0.32nM.Formula II-44 tests with the concentration of 2 μ M to 632pM.

Result (EC ₅₀Value) is presented in the table 3 and shows in the chemical compound of being tested, formula II-5A, II-16, II-18, II-19, II-20, II-21, II-22, II-29, II-38 and II-44 are the active effective inhibitors of the chymotrypsin-like of described 20S proteasome, its EC ₅₀Value is 2.2nM to 11nM.Formula I-7, II-2, II-4, II-5B, II-17, II-30 and II-47 suppress the chymotrypsin-like activity of this proteasome, its EC ₅₀Value is 13nM to 88nM, and the EC of formula II-3, II-26 and VI-1A ₅₀Value is 207nM to 964nM.Formula II-13C, II-24C, II-27, II-28 and IV-3C suppress this chymotrypsin-like activity, its EC ₅₀Value is 1.4 μ M to 10.6 μ M.The EC of formula II-8C, II-25, II-31 and II-32 ₅₀Value is greater than 20 μ M.Under described test condition, formula II-2, II-3, II-4, II-5A, II-5B, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-26, II-29, II-30, II-44 and VI-1A can suppress the trypsin-like activity of described 20S proteasome.Formula II-4, II-5A, II-16, II-18, II-19 and II-29 suppress Caspase sample activity, its EC ₅₀Value is 213nM to 850nM, and formula II-2, II-5B, II-17, II-20, II-21, II-22, II-30, II-44 and VI-1A have the EC of 956nM to 8.7 μ M ₅₀Value.

Table 3

Formula I-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, II-44, VI-1A and II-47 are to pure The active effect of plurality of enzymes of the rabbit 20S proteasome of changing

^*Show the EC of independent experiment once or twice ₅₀Value.When n 〉=3, show average EC ₅₀Value ± standard deviation, ^*N=3, inapplicable standard deviation.

The ND=undetermined

The results are shown among Figure 46 and show formula II-2 and the chymotrypsin-like activity of formula II-4 Profilin enzyme body, EC of the representative test of bounds evaluation II-2, formula II-3 and formula II-4 ₅₀Value is respectively 18.5nM and 15nM.Formula II-3 has activity, EC in this analyzes ₅₀Value is 890nM respectively.From independent experiment, obtain similar result.

The results are shown among Figure 47 and show formula II-5A and the chymotrypsin-like activity of formula II-5B Profilin enzyme body, EC of the representative test of bounds evaluation II-5A and formula II-5B ₅₀Value is respectively 6nM and 88nM.From independent experiment, obtain similar result.

Embodiment 37

Salinosporamide A (II-16) suppresses the chymotrypsin-like activity of rabbit flesh 20S proteasome

Utilization detects the effect of Salinosporamide A (II-16) to proteasome from the commercial test kit that obtains of Calbiochem (catalog number (Cat.No.) 539158), and this test kit is measured the activity (Calbiochem 20S Proteasome Kit) of rabbit flesh 20S proteasome with the peptide substrates of fluorescence.This peptide substrates is special to the chymotrypsin-like activity of described proteasome.

The Omuralide stock solution of preparation 10mM in DMSO, and be stored in-80 ℃ with the equal portions of 5 μ l.The Salinosporamide A solution of preparation 25.5mM and be stored in-80 ℃ in DMSO with equal portions.This assay determination Suc-LLVY-AMC is hydrolyzed into Suc-LLVY and AMC.By using λ _Ex=390nm and λ _EmThe coumarin (AMC) that=460nm fluoremetry is discharged.This analysis is implemented in titer plate (Corning 3904), and dynamically per subsequently five minutes measure once.The instrument that uses is Thermo Lab Systems Fluoroskan, and camera incubata is set in 37 ℃.This analysis is carried out according to the scheme of manufacturer, and following change is arranged.Described proteasome activates with SDS as describing ground, and remains in the ice before carrying out this analysis.In analysis buffer, dilute Salinosporamide A and Omuralide continuously to prepare 8 dose point effect curves.In described analysis plates, add each dosage of 10 μ l, triplicate, and add activatory proteasome of 190 μ l and mixing.Under 37 ℃ with described sample preincubate 5 minutes in described Fluoroskan.Add substrate, detect the kinetics of AMC subsequently, and continue 1 hour.Gather all data and draw with the meansigma methods at three number strong points.This data standard is changed into the response data that carries out when not having Salinosporamide A, and with the dose response of S shape, variable inclined-plane carries out mould and builds in Prism.

To obtain result's similar (table 2) in the external cytotoxicity, referring to Feling, et al., SngewChem I7lt Ed Engl 42:355 (2003), the EC in described 20S proteasome is analyzed ₅₀Value shows that Salinosporamide A is more effective than Omuralide, and its effect approximately is 40 times, and meansigma methods is respectively that 1.3nM is to 49nM (Figure 27).Repeat this experiment and average EC in twice analysis ₅₀Being defined as Salinosporamide A is that 2nM and Omuralide are 52nM.

Salinosporamide A is the active effective inhibitor of the chymotrypsin-like of proteasome.Cytotoxic EC ₅₀Value shows that Salinosporamide A is most of at least owing to the inhibition to proteasome in the ability of bringing out aspect the cell death in the 10-200nM scope.These data show that Salinosporamide A is the effective micromolecular inhibitor of proteasome.

Embodiment 38

Salinosporamide A (II-16) is to the active inhibition of PGPH of rabbit flesh 20S proteasome

But PGPH (the being also referred to as the Caspase sample) activity of Omuralide Profilin enzyme body; Therefore estimate the active ability of PGPH that Salinosporamide A suppresses the rabbit flesh 20S proteasome of purification.The fluorogenic substrate that use can get the active special commerce of PGPH replaces the chymotrypsin protein zymolyte supplied with in the above-mentioned protease assay kit.

The 20mM solution of preparation Salinosporamide A (II-16) and be stored in-80 ℃ in DMSO with little equal portions.The 20mM stock solution of preparation substrate Z-LLE-AMC and being stored in-20 ℃ in DMSO.The source of described proteasome is the test kit that Calbiochem (Cat.#539158) commerce can get.Because with described chymotrypsin protein zymolyte, proteasome can cut into Z-LLE-AMC Z-LLE and free AMC.Determine this activity (λ by the fluorescence of measuring the AMC that is discharged then _Ex=390nm and λ _Em=460nm).Activate this proteasome with SDS, and remain on ice according to manufacturer's recommendation.In DMSO, Salinosporamide A is diluted to produce 400 times of spissated 8 dilution series.Be used to detect the active proteasome of chymotrypsin-like and carry out preincubate with 20 times of this serial dilutions and with described with analysis buffer.After adding substrate, under 37 ℃, hatch described sample, and in exometer, monitor the release of the AMC of fluorescence.Collect all data and draw according to the meansigma methods of three points.These the experiment in, according to standardized activity with this EC ₅₀Carry out mould and build in Prism, wherein, the scale of the AMC that discharges when not having Salinosporamide A shows 100% activity.As before, the model of selection is the dose response of S shape and variable inclined-plane.

Data show Salinosporamide A suppresses the PGPH activity in the rabbit flesh 20S proteasome, its EC ₅₀Be 350nM (Figure 28).Carry out repeated experiment, produce prediction EC ₅₀Be 610nM.These results show the PGPH activity of the rabbit flesh 20S protease of the certain extracorporeal blocking purification of Salinosporamide A, though have than viewed the lower effectiveness of the active inhibition of chymotrypsin-like.

Embodiment 39

The active inhibition of the chymotrypsin-like of human red blood cell 20S proteasome

In-vitro evaluation Salinosporamide A (II-16) suppresses the active ability of chymotrypsin-like of human red blood cell 20S protease.The EC that calculates ₅₀Be worth about 3nM (Figure 29).The inhibitory action of these data show Salinosporamide A is not limited to rabbit skeletal muscleprotease body.

The 20mM solution of preparation Salinosporamide A and be stored under-80 ℃ in DMSO with little equal portions.The 20mM solution of the described substrate suc-LLVY-AMC of preparation and storage in-20 ℃ in DMSO.Obtain human red blood cell 20S protease from BIOMOL (Cat.#SE-221).This protease can cut suc-LLVY-AMC and become suc-LLVY and free AMC and determine this activity (λ by the fluorescence of measuring the AMC that is discharged _Ex=390nm and λ _Em=460nm).As the experiment of adopting rabbit myopsin body, activate this proteasome and be stored on ice with SDS.Dilution Salinosporamide A is to produce 400 times of spissated 8 dilution series in DMSO.Use the proteasome preincubate with analysis buffer down with 20 times of this serial dilutions and at 37 ℃ then.With substrate initiation reaction and the release of mensuration AMC in the little culture plate exometer of Fluoroskan subsequently.Collect data and draw according to 3 meansigma methods.Dynamically collect data and continue 3 hours, and show that these are reflected at the kinetics that shows linearity in this time period.These data standards are changed into the reaction of carrying out when not having Salinosporamide A, and in Prism, carry out mould with S shape dosage effect, variable inclined-plane and build.

Utilize dissimilar human red blood cell protease to carry out repeated experiments, produce EC ₅₀Be worth about 4nM.These results show that the chymotrypsin-like activity of external human red blood cell 20S proteasome is responsive to Salinosporamide A.

Formula II-16 also shows trypsin-like and the Caspase sample activity that suppresses the human red blood cell proteasome.For trypsin-like, studies show that EC ₅₀Be worth about 9nM, and for Caspase sample, EC ₅₀About 390nM.In addition about the active EC that studies show that of the chymotrypsin-like of human red blood cell ₅₀About 250pM.In addition, studies show that formula II-16 has specificity to proteasome, other proteolytic enzyme is shown effect seldom or do not have effect.For example, when testing the inhibition of formula II-16 to chymase, cathepsin B and thrombin, the EC of formula II-16 respectively ₅₀Value is respectively 18,000nM,＞200,000nm and＞200,000Nm.

Embodiment 40

The specificity of Salinosporamide A (II-16)

The possible mechanism of Salinosporamide A Profilin enzyme body is the reaction of the avtive spot threonine of β-lactone functional group by Salinosporamide A and this proteasome.The covalent modification of this proteasome will be blocked this avtive spot, because this residue is essential to the catalytic activity of this proteasome.Fenteany，et al.，J Biol Chem 273：8545(1998)。The chemical compound lactacystin of being correlated with on the structure, also demonstrate inhibition of histone enzyme A (Ostrowska, et al., Int J Biochem Cell Biol 32:747 (2000), Kozlowski, et al., Tumour Biol 22:211 (2001), Ostrowska, et al., Biochem Biophys Res Cornrraun 234:729 (1997)) and TPPII (Geier, et al., Science 283:978 (1999)) still do not suppress trypsin, chymase, caricin, calpain, (Fenteany, et al., Science 268:726 (1995)), thrombin and plasminogen activator (Omura, et al., JAntibiot (Tokyo) 44:113 (1991)).Similarly research suppresses prototype (prototypical) serine protease by estimating Salinosporamide A, and the ability of the catalytic activity of chymase begins to explore the specificity of Salinosporamide A to proteasome.

The 20mM solution of preparation Salinosporamide A and be stored under-80 ℃ in DMSO with little equal portions.In DMSO the preparation described substrate suc-LLVY-AMC 20mM solution and under-20 ℃, store.By protease or chymase the Proteolytic enzyme of this substrate is discharged described fluorescence-causing substance AMC, this AMC (λ of monitoring in exometer _Ex=390nm and λ _Em=460nm).From Sigma (Cat.# C-4129), obtain the chymase of Pancreas Bovis seu Bubali, and every day, (0.05%Triton X-100 was formulated as 5mg/ml solution in pH7.5) for 10mM HEPES, 0.5mM EDTA in analysis buffer.Before analysis, be diluted to 1 μ g/ml (0.2 μ g/ hole) and remain on ice at the neutral chymase soon of buffering analytic liquid.Dilution Salinosporamide A is to produce 8 dose point effect curves in DMSO.The high final concentration that acquisition suppresses the required Salinosporamide A of chymase fully need directly join the enzyme of dilution in the diluted chemical compound series.Related 1%DMSO (final concentration of the solvent in described instrument connection) does not have remarkable influence at chymase aspect this substrate active in this reaction.Cause this reaction this reactant of 37 ℃ of following preincubates 5 minutes and by adding substrate.Under 37 ℃, in Fluoroskan, dynamically collected data 1 hour and draw according to the meansigma methods at three number strong points.Described data standard is turned to the reaction of carrying out when not having Salinosporamide A, and with S shape dosage effect, variable inclined-plane carries out mould and builds in Prism.In same figure, comprise the active standardized data of chymotrypsin-like that suppresses rabbit 20S proteasome from Salinosporamide A.

Using Salinosporamide A chymase to be carried out observed average inhibition is 17.5 μ M (Figure 30 shows representative experiment) in pretreated twice experiment.These data show that the active vitro inhibition of the chymotrypsin-like to proteasome of Salinosporamide A mediation has precedence over the inhibition to the catalytic activity of chymase.

Therefore, the chymotrypsin-like of Salinosporamide A Profilin enzyme body and PGPH activity.The preliminary also trypsin-like activity of Profilin enzyme body of Salinosporamide A, its EC of studies show that ₅₀Value is about 10nM (not video data).

Embodiment 41

Formula II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-44, VI-1A and IV-3C are to the inhibition of the luciferase activity of NF-κ B mediation; HEK293 NF-κ B/ luciferase reporting cell line

HEK293 NF-κ B/ luciferase reporting cell line is human embryonic kidney cell line (ATCC; CRL-1573) derivant and carry the luciferase reporter gene of being regulated by 5X NF-κ B binding site.Described reporting cell line maintained routinely in the complete DMEM culture medium of adding 250 μ g/ml G418 (DMEM+10% (v/v) hyclone, 2mM L-glutaminate, 10mM HEPES and be respectively 100IU/ml and the penicillin/streptomycin of 100 μ g/ml).When carrying out described luciferase when analyzing, replace this DMEM basal medium and omit this G418 with no phenol red DMEM basal medium.Under 37 ℃, containing 5%CO ₂With cultivate described cell in the incubator of 95% humid air.

In order to carry out the luciferase analysis of NF-κ B mediation, with HEK293 NF-κ B/ luciferase cell with 1.5 * 10 ⁴The 90 μ l that cells/well is seeded in the Corning tissue culturing plate at the 3917 white light tight ends do not have in the phenol red DMEM complete medium.For formula II-2, II-4, II-5A and II-18, the initial dilution of preparation 400 μ M in 100%DMSO, and this dilution is used to produce 8 half-log series.This dilution series is further diluted 40 times in proper culture medium, and the equal portions of 10 μ l are joined in this instrument connection, triplicate, causing final test concentration is 1 μ M to 320pM.For formula II-3, II-5B, II-8C, II-13C, II-17, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, VI-1A and IV-3C, the initial dilution of preparation 8mM in 100%DMSO, and carry out above-mentioned same operation subsequently, causing final test concentration is 20 μ M to 6.3nM.For formula II-16, II-19 and II-44, the initial dilution of preparation 127 μ M in 100%DMSO, and final test concentration is 317nM to 0.1nM.For formula II-20, the starting soln of preparation 2.5mM or 8mM in 100%DMSO, and final test concentration is respectively 6.3 μ M to 2.0nM or 20 μ M to 6.3nM.Described culture plate is put back in the incubator cultivated 1 hour.After pretreatment in 1 hour, be added in the 50ng/ml TNF-α solution of the 10 μ l that prepare in the no phenol red DMEM culture medium, and this culture plate was hatched again 6 hours in addition.The final concentration of DMSO is 0.25% in all samples.

After finishing TNF-α stimulation, the Steady Lite HTS luciferase reagent (Packard Bioscience) of 100 μ l is joined in each hole, and before measuring this luciferase activity, described culture plate was at room temperature placed 10 minutes undisturbedly.Measure relative luciferase unit (RLU) by using Fusion microtest plate exometer (Packard Bioscience).Adopt S shape dosage effect, variable inclined-plane model in Prism (GraphPad software), to calculate EC ₅₀Value (maximal phase of inhibition 50% is to the drug level of luciferase activity).

Formula II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-44, VI-1A and IV-3C are to the inhibition of NF-kB activation

NF-κ B regulates a large amount of expression of gene that plays a significant role in inflammation, apoptosis, tumor generation and autoimmune disease.Therefore, for example, the chemical compound that can regulate or influence the NF-kB activity is used for the treatment of the disease relevant with inflammation, cancer or autoimmune disease.In its inactivation form, NF-κ B and I κ B are compound in endochylema, and when stimulating, I κ B phosphorylation, ubiquitinization are also degraded by proteasome subsequently.The degraded of I κ B causes the activation of NF-κ B and transposition thereof to nucleus.The luciferase activity of NF-κ B mediation in HEK293 NF-κ B/Luc cell is come bounds evaluation II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-44, VI-1A and the IV-3C effect to the NF-kB activation when stimulating by assessment TNF-α.

The dose dependent of luciferase activity reduces when causing TNF-α to stimulate with formula II-2, II-4, II-5A, II-5B, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-26, II-29, II-30 and II-44 to the pretreatment of NF-κ B/Luc 293 cells.The EC that suppresses the luciferase activity of NF-κ B mediation ₅₀Value is shown in the table 4, and is illustrated in this analysis Chinese style II-2 based on cell, II-4, II-5A, II-5B, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-26, II-29, II-30 and II-44 chemical compound inhibition NF-kB activity.

Table 4

Formula II-2, the II-3 that analyzes from the luciferase reporter gene of NF-κ B mediation, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-27, II-28, II-29, II-30, II-44, VI-1A and IV-3C EC ₅₀ Value

Chemical compound	EC ₅₀ ^＊
Chemical compound	EC ₅₀ ^＊	Formula II-2	71±20nM
Formula II-3	＞20μM ＞20μM	Formula II-2	71±20nM
Formula II-3	＞20μM ＞20μM	Formula II-4	67nM 88nM
Formula II-5A	33nM 30nM	Formula II-4	67nM 88nM
Formula II-5A	33nM 30nM	Formula II-5B	279nM 261nM
Formula II-8C	＞20μM ＞20μM	Formula II-5B	279nM 261nM
Formula II-8C	＞20μM ＞20μM	Formula II-13C	＞20μM ＞20μM
Formula II-16	11±3nM	Formula II-13C	＞20μM ＞20μM
Formula II-16	11±3nM	Formula II-17	960±210nM
Formula II-18	9nM 11nM	Formula II-17	960±210nM
Formula II-18	9nM 11nM	Formula II-19	7nM 10nM
Formula II-20	849±225nM ^**	Formula II-19	7nM 10nM
Formula II-20	849±225nM ^**	Formula II-21	3.2μM 2.7μM
Formula II-22	1μM 728nM	Formula II-21	3.2μM 2.7μM
Formula II-22	1μM 728nM	Formula II-24C	5.3μM 3.2μM
Formula II-25	＞20μM ＞20μM	Formula II-24C	5.3μM 3.2μM
Formula II-25	＞20μM ＞20μM	Formula II-26	4.3μM 4.1μM

Formula II-27	＞20μM ＞20μM
Formula II-27	＞20μM ＞20μM	Formula II-28	＞20μM ＞20μM
Formula II-29	1.2μM 1.4μM	Formula II-28	＞20μM ＞20μM
Formula II-29	1.2μM 1.4μM	Formula II-30	2.2μM 2.2μM
Formula II-44	17±4nM	Formula II-30	2.2μM 2.2μM
Formula II-44	17±4nM	Formula VI-1A	＞20μM ＞20μM
Formula IV-3C	＞20μM ＞20μM	Formula VI-1A	＞20μM ＞20μM

^*The EC that has shown twice independent experiment ₅₀Value.When n 〉=3, shown average EC ₅₀Value ± standard deviation. ^*Also this analysis, EC have as a result been carried out with Compound I I-20 ₅₀Value is 154nM, and this value is not included in average EC ₅₀Within the calculating of value.

When representative result of experiment (Figure 44) demonstration of bounds evaluation II-2, formula II-3 and formula II-4 caused TNF-α to stimulate with II-2 and II-4 pretreatment, the dose dependent of luciferase activity reduced in NF-κ B/Luc 293 cells.In this experiment, suppress the EC of the calculating of the derivable luciferase activity of NF-κ B ₅₀For being 73nM for formula II-2, and the EC of formula II-4 ₅₀Value is 67nM.In repeated experiments, observe similar data.

The results are shown among Figure 45 and show formula II-5A and formula II-5B suppresses the derivable luciferase activity of NF-κ B, EC of the representative test of bounds evaluation II-5A and formula II-5B ₅₀Value is respectively 30nM and 261nM.In repeated experiments, obtain similar result.

Embodiment 42

Salinosporamide A is to the effect of NF-κ B signal transduction path

Carried out the experiment of research Salinosporamide A to the effect of NF-κ B signal transduction path.The stable HEK293 clone (NF-κ B/Luc 293) of the luciferase reporter gene of being regulated by 5X NF-κ B binding site is carried in generation.This cell line that stimulates with TNF-α causes strengthening because of the NF-kB activation causes luciferase activity.

With 8 logarithms series dilution thirty (from 1 μ M to 317pM) pretreatment NF-κ B/Luc 293 cells of Salinosporamide A 1 hour, use TNF-α (10ng/mL) to stimulate subsequently 6 hours.The derivable luciferase activity of test NF-κ B in the time of the 6th hour.As previously mentioned, after 24 hours, estimate the viability of NF-κ B/Luc293 cell with Salinosporamide A processing by adding the "diazoresorcinol" dyestuff.

The dose dependent of luciferase activity reduces (Figure 31, right side y axle) when causing TNF-α to stimulate with Salinosporamide A to the pretreatment of NF-κ B/Luc 293 cells.For the inhibition of NF-κ B/ luciferase activity, the EC of calculating ₅₀Be about 7nM.The Salinosporamide A that implements cytotoxicity analysis simultaneously and demonstrate this concentration does not influence cell viability (Figure 31, left side y axle).These representative datas show by Salinosporamide A to be handled, and the reduction of viewed luciferase activity is mainly owing to the signal conduction incident of NF-κ B mediation rather than the death of cell.

Embodiment 40

Except that NF-κ B luciferase reporter gene is analyzed, estimate the effect of Salinosporamide A to I κ B α and total I κ B alpha levels of phosphorylation by the Western blotting.In HEK293 cell and described NF-κ B/Luc 293 reporter genes clone, estimate the endogenous protein level.

With Salinosporamide A pretreatment cell 1 hour under indication concentration, then the TNF-α with 10ng/mL stimulated 30 minutes.The complete form of anti-I κ B α and the antibody of phosphorylation form are used to measure each proteinic endogenous level, and microtubulin-resisting antibody is used for determining sample on the proteic equivalent.

Shown in figure 32, with the Salinosporamide A of 50nM and 500nM two kinds of cell lines are handled, this processing not only reduces the degraded of total I κ B α when stimulating with TNF-α, and keeps the level of phosphoric acid-I κ B α.These results support the mechanism of action of Salinosporamide A as proteasome inhibitor firmly, the degraded of the I κ B α of phosphorylation when it prevents TNF-α stimulation.

Embodiment 41

Salinosporamide A cell cycle is regulated proteic effect

Ubiquitin-Proteasome Pathway by regulating cyclin degraded and such as cell cycle protein dependent kinase (Cdk) inhibitor of p21 and p27, be the Proteolytic enzyme system that participates in necessity of cell cycle control.Pagano，et al.，Science 269：682(1995)，Kisselev，et al.，Chem Viol 8：739(2001)，King，et al.，Science 274：1652(1996)。In addition, p21 and p27 protein level increased when the existing of protease inhibitor.Fukuchi，et al.，BiochimBiophys Acta 1451：206(1999)，Takeuchi，et al.，Jpn J Cancer Res 93：774(2002)。Therefore, adopt HEK293 cell and described HEK293NF-κ B/ luciferase reporter gene clone, implement the western engram analysis to estimate the effect of Salinosporamide A processing to the endogenous level of p21 and p27.

Western trace shown in Figure 33 is to utilize the antibody of anti-p21 and p27 to survey to determine each proteic endogenous level, and microtubulin-resisting antibody is used for determining sample on the proteic equivalent.

Shown in Figure 33 A and 33B, preliminary result shows that the protein level of p21 and p27 improves when the Salinosporamide A with multiple concentration handles two kinds of cell lines.Data show Salinosporamide A is by Profilin enzyme body activity, thereby the inductive NF-kB activation of prevention TNF-α works.In addition, the inhibition of this proteasome causes described Cdk inhibitor, and p21 and p27 accumulate, and this reports in the apoptosis of sensitized cell.Pagano，etal.，supra(1995)，King，et al.，supra(1996)。

Embodiment 42

Salinosporamide A (II-16) is to the activation of Caspase-3

In order to verify whether Salinosporamide A brings out apoptosis, use Jurkat cell (American Type Culture Collection (ATCC) TIB-152, the acute T chronic myeloid leukemia of people) to estimate Salinosporamide A to inducing the active effect of Caspase-3.

With the Jurkat cell with 2 * 10 ⁶Cell/3mL/ hole is seeded in 6 well culture plates and at 37 ℃, 5% (v/v) CO ₂With hatch in 95% (v/v) humidity.In DMSO, prepare the stock solution of Salinosporamide A and mitoxantrone hydrochloride (Sigma, St.Louis, MO.Cat # M6545) respectively with the concentration of 20mM and 40mM.Mitoxantrone hydrochloride is a chemotherapeutics, and it is synthetic and repair in division and nondividing cell and bring out apoptosis by suppressing DNA, and in being included in as positive control.Bhalla，et al.，Blood 82：3133(1993)。Before evaluation Caspase-3 activity, use EC ₅₀Concentration is handled (table 5) cell and was hatched 19 hours.The cell of handling with 0.25%DMSO is used as negative control.By centrifugalize and remove culture medium and collect described cell., the cell precipitation thing handled so that described in the scheme of manufacturer, carry out Caspase-3 activity analysis (from the EnzChek Caspase-3 assay kit (E-13183 of Molecular Probes; The G that sees Appendix, it is the part of this application book and can obtains in the hypertext transfer scheme on world's network " probes.com/media/pis/mpl3183.pdf ").In brief, the cell precipitation thing is carried out cracking on ice, in 96 hole flat boards, mix, hatched in the dark then 30 minutes, use Packard Fusion at last, use λ with EnzChek Caspase-3 component _Ex=485nm and λ _Em=530nm light filter is read the fluorescence of the benzyloxycarbonyl group-DEVD-AMC of cutting.Measure the protein concentration of lysate with BCA analysis of protein test kit (Pierce), and these numerical value are used for standardization.

Data in the representative experiment show Salinosporamide A to the processing of Jurkat cell cause cytotoxicity and Caspase-3 activation (table 5, Figure 34)

Table 5:Salinosporamide A and mitoxantrone hydrochloride are to the Jurkat cell Cytotoxicity and EC ₅₀ Value

Embodiment 43

Salinosporamide A causes the PARP cutting in the Jurkat cell

In order to estimate the ability of Salinosporamide A cell death inducing in the Jurkat cell, the cutting of poly-(ADP-ribose) polymerase (PARP) of monitoring.PARP is a 116kDa nucleoprotein, and it is one of interior target of main cell of Caspase-3.Decker，et al.，J Biol Chem 275：9043(2000)，Nicholson，D.W，Nat Biotechnol 14：297(1996)。The cutting of PARP produces stable 89kDa product, and this process can be monitored by the western blotting.Caspase is apoptotic sign to the cutting of PARP, can be used as the excellent marker of this process equally.

Before experimentizing, with the Jurkat cell with (every milliliter 2 * 10 of low-density ⁵Cell) maintains among the RPMI that adds 10% hyclone (FBS).Be suspended in again in the culture fluid to per 3 milliliter 1 * 10 by centrifugal collection cell and with it ⁵Cell.Handle this cell suspension of 20 milliliters with the Salinosporamide A of 100nM (at the DMSO stock solutions of-80 ℃ of 20mM that store down), and take out 3 milliliters of parts and be placed on and be T on ice ₀Sample.The cell suspension that will add 3 milliliters of equal portions of Salinosporamide A places 6 orifice plates and puts back to incubator.As the positive control of PARP cutting, with the staurosporine (Staurosporine) of 350nM, a kind of known apoptosis initiator (Sigma S5921 is at the DMSO stock solution of-20 ℃ of 700 μ M that store down) is handled identical cell suspension.Under the cell situation that Salinosporamide A handles, the 2nd, 4, take out sample in the time of 6,8 and 24 hours, and in the time of 4 hours, be used for the staurosporine contrast.For each time point,, wash this cell with the PBS of 400 μ L, and precipitate this cell once more by simple centrifugal recovery cell.After removing PBS, before SDS PAGE, this precipitate is stored under-20 ℃.Each cell precipitation thing is suspended in the NuPAGE sample buffer (Invitrogen 46-5030) of 100 μ l again, and each sample of 10 μ l is separated on 10%NuPAGEBIS-Tris gel (Invitrogen NB302).To nitrocellulose, survey thin film at electrotransfer, survey (Jackson 11-055-045) with the link coupled second antibody of the alkali phosphatase of the anti-rabbit of goat subsequently with the tame rabbit polyclonal antibody (Cell Signaling 9542) of anti-PARP.Detect bonded antibody with BCIP/NBT (Roche 1681451) colorimetric analysis.

The western trace that manifests among Figure 35 shows that PARP is cut with the time dependence form in described Jurkat cell.Be exposed to the form that in the cell of handling, occurs this cutting behind the Salinosporamide A between 2 to 4 hours and (use asterisk ^*Represent), and the PARP of most remainder was cut before 24 hours.The cell (St) that staurosporine is handled shows the quick cracking of PARP, and most of albumen were cut in 4 hours.These data show consumingly Salinosporamide A can be in the Jurkat cell cell death inducing.

Embodiment 44

Anti-anthrax activity

Stop the ability that exposes the cell death that causes because of LeTx in order to analyze Salinosporamide A or other chemical compound, as described below, RAW264.7 macrophage and reorganization LF and PA lethal toxin component are as the toxic extracorporeal model system of analysis of cells.

Under 37 ℃, at the 5%CO that is containing humidity ₂Incubator in, RAW264.7 cell (ATCC # TIB-71) adapted to and maintains added 10% hyclone (ADMEM, Mediatech, Herndon, VA) the improved Eagle culture medium of senior Dulbecco ' s (Invitrogen, Carlsbad, CA) in.Under 37 ℃, containing moist 5%CO ₂Incubator in cell is seeded among the ADMEM that adds 5%FBS in 96 orifice plates with the concentration of 50000 cells/well, spend the night.Selectively, also can use in the DMEM that adds 10% hyclone cultured cells and find that this cell is applicable to this analysis.Removed culture medium and used the ADMEM of serum-free to replace in the second day morning, this ADMEM contains or does not contain the Salinosporamide A or the Omuralide that are used for 8 dose point effects of dosage range from 1 μ M to 0.5nM.From the DMSO stock solution of 1mg/mL, prepare described chemical compound and in ADMEM, be diluted to final concentration.Behind 15 minutes preincubates, with 200ng/mL LF or 400ng/mL PA separately or jointly (LeTx) add in the cell.Obtain reorganization LF and PA and described from the List biology laboratory, under-80 ℃, be stored in the sterilized water that contains 1mg/ml BSA with the 1mg/mL stock solution according to manufacturer.37 ℃ of following incubated cells 6 hours, follow adding "diazoresorcinol" as discussed previously.Before estimating cell viability, again culture plate was hatched 6 hours in addition by measurement fluorescence.Described data are three experiments, each experiment repeats 3 to 6 times summary and data representation is survival percentage ratio, use following formula, contrast (male) with this data normalization: viability %=100 * (observed OD-positive control)/(negative control-positive control) with DMSO contrast (feminine gender) and LeTx.

Data shown in Figure 36 show that the processing of carrying out with Salinosporamide A can be at the cell death of the inductive macrophage-like RAW264.7 of external prevention LeTx cell.Separately with LF or PA or seldom reduce cell viability with the RAW cell of Salinosporamide A processing separately, yet cause about 0.27% cell viability compared with the control with the processing that LeTx carries out.Salinosporamide A may be by degraded that suppresses differential protein and the survival rate that strengthens macrophage of synthesizing that reduces cytokine, and this finally causes suppressing in the body lethal effect of anthrax toxin.

Though the individual processing of Salinosporamide A when 100nM and above concentration thereof produces the very cytotoxicity of appropriateness, but in the cell that LeTx handles, RAW 264.7 cell viabilities have significant increase (Figure 36) with processes and displays lower, that relative avirulence level is carried out.For example, when with 12nM Salinosporamide A pretreatment, Salinosporamide A+LeTx processed group shows 82% cell viablity, and this concentration shows the concentration of 96% viability for Salinosporamide A individual processing the time.The average EC of Salinosporamide A in these researchs ₅₀Be 3.6nM.On the contrary, Omuralide pair cell viability shows that relative effect seldom is till it reaches 1 μ M concentration.Have only 37% viability even Omuralide under the situation of high concentration, observes, this shows that Salinosporamide A is the more effective inhibitor that suppresses the inductive RAW264.7 cell death of LeTx.With these data consistents, Tang et.al., Infect Immun 67:3055 (1999) find in described LeTx analyzes, the EC of MG132 and lactacystin (precursor of Omuralide) ₅₀Concentration is 3 μ M.In sum, these data further show with any other chemical compound of present description to be compared, and Salinosporamide A suppresses the inductive Cytotoxic more effective inhibitor of LeTx.

When the existing of LeTx, Salinosporamide A promotes the survival of RAW264.7 cell, and this shows that this chemical compound or derivatives thereof is the valuable clinical treatment agent to anthrax.In addition, it should be noted that Salinosporamide A compares many tumor cells to RAW 264.7 cells and has more lower cytotoxicity.

Embodiment 45

Salinosporamide A is to the activity of multiple myeloma and prostate cancer cell line

NF-κ B is vital to tumor growth and anti-apoptotic in multiple myeloma and has reported it constitutive activity (Hideshima T etal.2002, Shimada K et al.2002 and Palayoor ST et al.1999) is also arranged in multiple prostate cancer cell line.Degrade by the proteasome of its inhibitor I κ B α and to regulate the activity of NF-κ B.Because Salinosporamide A has demonstrated the vitro inhibition proteasome and disturbed the signal transduction path of NF-κ B, therefore estimating Salinosporamide A is the activity of RPMI 8226 and prostate cancer cell line PC-3 and DU 145 to multiple myeloma cells.

In the standard growth inhibition analysis, utilize the drug exposure of "diazoresorcinol" dyestuff and 48 hours to determine EC ₅₀Value.Result's (table 6) of 2-5 independent experiment shows the EC of Salinosporamide A to RPMI 8226 and prostate cancer cell line ₅₀The value scope is 10-37nM.

Table 6:Salinosporamide IA (II-16) is to multiple myeloma and prostate tumor cells The EC of system ₅₀ Value

Use the western engram analysis, estimate Salinosporamide A by the cutting of monitoring PARP and preceding Caspase 3 and in RPMI 8226 and PC-3 cell, bring out apoptotic ability.Simply say, handle PC-3 and RPMI 8226

cells

0,8 or 24 hours with 100nM Salinosporamide A (2345R01).Preparation total protein lysate and dissolve this lysate of 20 μ g under reduction/degeneration condition, transfer printing is on nitrocellulose then.Survey this trace with the antibody of anti-PARP or Caspase 3 then, then with anti-actin antibody desorbing and detection again.

These result of experiment show that Salinosporamide A causes the cutting (Figure 37) in the time dependence mode of PARP and preceding Caspase 3 to the processing of RPMI 8226 cells.As if RPMI 8226 cells are compared more responsive to Salinosporamide A with the PC-3 cell, induce PARP cutting and finished before 24 hours because can be observed in the time of the 8th hour.On the contrary, in the PC-3 cell, in the time of 24 hours, observe the cutting of PARP, however the cutting (Figure 37) of Caspase 3 before in this experiment, not detecting.

The Salinosporamide A that RPMI 8226 cells are used to estimate with multiple concentration handles 8 hours effect of cell.Simply say, handled RPMI 8226 cells 8 hours and made the protein cleavage thing with the Salinosporamide A (2345R01) of multiple concentration.This lysate with 25 μ g under reduction/degeneration condition dissolves also transfer printing on nitrocellulose.Survey this trace with the antibody of anti-PARP or anti-Caspase 3 then, then with anti-actin antibody desorbing and detection again.Figure 38 proves that Salinosporamide A all brings out the dose dependent cutting of PARP and preceding-Caspase 3.

Embodiment 46

Formula I-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, II-44, VI-1A, II-47 and II-50 are to the people The growth inhibited of multiple myeloma RPMI 8226 and U266 cell

Just people's multiple myeloma cells is PRMI 8226 (ATCC; CCL-155) and U266 (ATCC; TIB-196) maintain in the suitable culture medium.Under 37 ℃, containing 5%CO ₂With cultivate this cell in the incubator of 95% humid air.

In order to carry out the analysis of cell growth inhibited, with RPMI 8226 and U266 cell respectively with 2 * 10 ⁴With 2.5 * 10 ⁴Cells/well is seeded in the 96 μ l complete mediums in the tissue culturing plate of Corning 3904 black walls, clear bottom.The 20mM stock solution of the described chemical compound of preparation in 100%DMSO, and be divided into equal portions and be stored in-80 ℃.This chemical compound is diluted continuously and join in the instrument connection, triplicate.The final concentration of formula I-7, II-3, II-8C, II-5B, II-13C, II-17, II-20, II-21, II-22, II-24C, II-25, II-26, II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, VI-1A and II-47 is 20 μ M to 6.32nM.The final concentration of formula II-16, II-18, II-19, II-44 and II-50 is 632nM to 200pM.The final concentration of formula II-2, II-4 and II-5A is 2 μ M to 632pM.The DMSO final concentration is 0.25% in whole samples.

After the drug exposure 48 hours, in each hole, add the no Mg that 10 μ l contain 0.2mg/ml "diazoresorcinol" (obtaining from Sigma-Aldrich chemical company) ²⁺, Ca ²⁺Phosphate buffered saline (PBS), and described culture plate be put back in this incubator cultivate 3-6 hour.Because living cells metabolism "diazoresorcinol" is used Fusion microtest plate exometer (Packard Bioscience), use λ _Ex=535nm and λ _EmThe light filter of=590nm is measured the fluorescence of the reduzate of "diazoresorcinol"."diazoresorcinol" dyestuff in the not celliferous culture medium is used for determining background, it is deducted from the data of all experimental ports.This data standard is turned to the mean fluorecence degree of the cell of handling with culture medium+0.25%DMSO (growth of 100% cell), and the S shape dose effect curve match rule (producing ID Business Solutions Ltd by XLfit 3.0 or XLfit 4.0) with standard is determined EC ₅₀Value (observing the drug level that 50% maximum growth suppresses).Data are summarized in table 12 and the table 13.

Embodiment 47

Salinosporamide A (II-16) is to the medicine-resistant cell line retentive activity

Determine the EC of Salinosporamide A to people's sarcoma of uterus MES-SA cell line and multi-drug resistant derivant MES-SA/Dx5 thereof ₅₀Value is so that estimate Salinosporamide A to the cell line of undue express P-glycoprotein efflux pump retentive activity whether.The Paclitaxel (Paclitaxel) that comprises known substrate in contrast, P-glycoprotein pump.

Table 7:Salinosporamide A is to MES-SA and drug resistance derivant MES-SA/Dx5 EC ₅₀ Value

Result's (table 7) during these growth inhibited are analyzed shows that as what expect, Paclitaxel does not keep its activity to the MES-SA/Dx5 cell, and this also passes through EC ₅₀408 times growth of value reflects.Salinosporamide A is to MES-SA with to the EC of MES-SA/Dx5 ₅₀Be worth similar.As if this shows that Salinosporamide A can suppress the growth of multi-drug resistant cell line MES-SA/Dx5, and this expression Salinosporamide A is not the substrate of P-glycoprotein efflux pump.

In addition, also estimate Salinosporamide A to HL-60/MX2, the ability of the drug resistance derivant of human leukemia cell line HL-60, wherein HL-60/MX2 has the active feature of topoisomerase II of minimizing and is considered to have atypical multi-drug resistant.Determine the growth inhibiting EC of Salinosporamide A to HL-60 and HL-60/MX2 ₅₀Value.The bonding agent mitoxantrone hydrochloride (Mitoxantrone) that comprises DNA in contrast is because the HL-60/MX2 cell has drug resistance (Harker W.G.et al.1989) to this chemotherapeutics according to reports.

Table 8:Salinosporamide A is to the EC of HL-60 and drug resistance derivant HL-60/MX2 ₅₀ Value

Data show in table 8 is with respect to the HL-60 cell, and Salinosporamide A can keep its activity to the HL-60/MX2 cell, and this shows that Salinosporamide A is effective in the active cell of the topoisomerase II of expression decreased.On the contrary, mitoxantrone hydrochloride has about 1/29th activity to the HL-60/MX2 cell.

Salinosporamide A also shows has activity to drug resistance multiple myeloma cells cording.For example, Salinosporamide A has activity to the Dox-40 cell line of anti-MM.1R and doxorubicin.In addition, Salinosporamide A has activity to the cell line that obtains in the people's multiple myeloma patients that recurs behind the multiple therapy for treating with dexamethasone, bortezomib and thalidomide before.Therefore, Salinosporamide A has activity to the drug resistance multiple myeloma, and this drug resistance multiple myeloma comprises doxorubicin, dexamethasone, bortezomib and thalidomide are shown chemical sproof multiple myeloma.Similarly, other chemical compound disclosed herein shows that to doxorubicin, dexamethasone, bortezomib and thalidomide the drug resistance multiple myeloma of chemical sproof multiple myeloma has activity to comprising.

Embodiment 48

Salinosporamide A and several analog thereof: structure activity relationship

For the structure activity relationship (SAR) at initial stage of determining Salinosporamide A, estimating a series of Salinosporamide category-A is the ability of RPMI8226 to multiple myeloma cells like thing.In standardized growth inhibited is analyzed, determine EC with the drug exposure of "diazoresorcinol" dyestuff and 48 hours ₅₀Value.

As if result's (table 9) demonstration of the initial stage series of SAR added halogen group to and increased cytotoxicity in the ethyl.

The initial stage SAR series of table 9:Salinosporamide A

When n＞2, determine meansigma methods ± standard deviation

Embodiment 49

Biology in the body

Determining of maximum tolerated dose (MTD)

Research design is for determining the MTD of Salinosporamide A when to female BALB/c mouse intravenous administration SalinosporamideA in the body.

The BALB/c that weighs be mice and with the Salinosporamide A (from 0.01mg/kg to 0.5mg/kg) of multiple concentration with single dose intravenous administration (qdx1) or continuous 5 days of intravenous administration every day (qdx5).Observe the clinical indication of animal every day and twice of the animal of weighing individually weekly finished (mostly being 14 days most after the last day in administration) up to experiment.The results are shown in the table 10 and show Salinosporamide A the single intravenously administrable dosage until 0.25mg/kg tolerate.When administration every day in the time of continuous 5 days, the concentration of Salinosporamide A is until 0.1mg/kg is well tolerable.Do not notice that in experimentation habit changes.

Table 10 Salinosporamide A is the mensuration of the MTD in the mice at female BALB/c

Embodiment 50

The absorption of Salinosporamide A, distribution, metabolism and elimination (ADME) The preliminary assessment of feature

Implement the ADME feature of research with preliminary assessment Salinosporamide A.These researchs are by dissolubility evaluation, LogD ^7.4Determine and the inhibiting preliminary examination composition of detection cytochrome P 450 enzymes.The result of these researchs shows that the dissolubility of the estimation of Salinosporamide A in PBS (pH 7.4) is 9.6 μ M (3g/ml) and LogD ^7.4Value is 2.4.This LogD ^7.4Value be with the compatible limit accepted of drug development in (LogD ^7.4＜5.0) and show that oral availability is arranged.Preliminary P450 suppresses the examination result and shows Salinosporamide A, when testing with 10 μ M, all P450 isoforms are shown do not have or low inhibition: CYP1A2, CYP2C9 and CYP3A4 suppress 3% respectively, 6% and 6%, and CYP2D6 and CYP2C19 suppress 19% and 22% respectively.

Embodiment 51

Salinosporamide A and in vivo to the effect of whole blood proteasome activity

Salinosporamide A before shown external be the effective and special inhibitor of proteasome, to the active IC of chymotrypsin-like of the 20S protease of purification ₅₀Value is 2nM.In order to monitor Salinosporamide A activity in vivo, developed fast and repeatably and analyzed (reorganization is from Lightcap et al.2000) to estimate the proteasome activity in whole blood.

In brief, in order to pass through the described cell of hypotonic shock cracking, thawing in ice with refrigerated whole blood sample also was suspended in the EDTA of the ice-cold 5Mm of 700 μ l in one hour again, among the pH8.0.The volume of the complete blood cell of this expression hematocrit is approximately 2-3 doubly.Allow cracking 1 hour, and by removing cell debris in centrifugal 10 minutes with 14,000 Xg.With supernatant ( PAcked WHole BLood LYsate (the whole blood lysate of hematocrit) PWBL) transfers in the clean test tube, and discards pellet.As standard, analyze the protein concentration that (Pierce) determines this PWBL with BSA by BCA.About 80% sample has the total protein concentration between 800 to the 1200 μ g/mL.

By measuring the activity of the hydrolysis of the special fluorogenic substrate (suc-LLVY-AMC, Bachem Cat.1-1395) of the chymotrypsin-like activity of proteasome being determined proteasome.Control experiment shows in these extracts＞this proteic hydrolysis of 98% mediates by proteasome.Analyze by will in Costar 3904 flat boards, mixing to set up from the analysis buffer (20mM HEPES, 0.5mM EDTA, 0.05%Triton X-100,0.05%SDS, pH7.3) of PWBL with the 185 μ l of 5 μ l in the animal body.If titrimetry experiment show in this is analyzed protein concentration at 200 μ g between the 1000 μ g, then between protein concentration and hydrolysis rate, linear relationship is arranged.Cause described reaction by the 0.4mM suc-LLVY-AMC (by preparing) that adds 10 μ l with the DMSO solution of analysis buffer with this peptide of 1: 25 dilution proportion 10mM, and under 37 ℃, in exometer (Labsystems Fluoroskan), hatch.The hydrolysis of substrate causes the release of free AMC, by using λ _Ex=390nm and λ _EmThis AMC of=460nm fluoremetry.Hydrolysis rate was linear in 1 hour at least in this system.Hydrolysis rate with each sample is standardized into every milligram of proteic RFU/mg of relative fluorescence unit then).

In order to explore the activity in vivo of Salinosporamide A, handle male Swiss-Webster mice (5 every group, body weight is 20-25g) with the Salinosporamide A of multiple concentration.With Salinosporamide A intravenously administrable and suppose its LogD ^7.4Value is 2.4, hints oral availability.Also with Salinosporamide A oral administration.Before administration, by diluting Salinosporamide A stock solution (100%DMSO) with 10%solutol, causing final concentration is the drug solns of giving that 2%DMSO produces Salinosporamide A at once.The medium contrast is formed by containing 2%DMSO among the 10%solutol.In order to determine the baseline of proteasome activity, all do not give medium and Salinosporamide A to a treated animal.Salinosporamide A or medium are carried out administration with 10mL/kg, and after administration, extracted blood with this Animal Anesthesia and by cardiac puncture in 90 minutes.By the complete blood cell of centrifugal collection hematocrit, separate with PBS washing and recentrifuge.Before estimating proteasome activity, all samples are stored under-80 ℃.

For a hydrolysis activity owing to described proteasome of determining observed substrate in these experiments, use high specific proteasome inhibitor Epoxomicin carries out the dose-effect experiment about described extract.The PWBL lysate is diluted with analysis buffer with 1: 40 ratio, and this solution of 180 μ l is joined in Costar 3904 flat boards.Dilute Epoxomicin (Calbochem Cat.324800) continuously to produce 8 dose point effect curves in DMSO, the ratio with 1: 50 in analysis buffer is diluted, and this solution of 10 μ l is joined among the PWBL of dilution, and is triplicate.Under 37 ℃ with described sample preincubate 5 minutes, and with above-mentioned substrate initiation reaction.Adopt the dosage effect of S shape, and be that model is analyzed this dose effect curve in Prism with variable inclined-plane.

Figure 40 is a scatterplot, and this is presented at the standardized proteasome activity among the PWBL that derives from indivedual mices (every group of 5 mices).In each group, horizontal line is represented average standardized activity.These data show Salinosporamide A causes the remarkable reduction of proteasome activity in PWBL, and this inhibitory action is a dose dependent.In addition, these data show Salinosporamide A is effective when oral administration.

By detecting known proteasome inhibitor Epoxomicin has shown this analysis to the effect of the hydrolysis of described peptide substrates specificity.Epoxomicin is the peptide epoxide, and described proteasome is demonstrated high specificity, and other known protein enzyme is not suppressed active (Meng et al., 1999).Hatch from the lysate of medium contrast and the lysate of the animal of 0.1mg/kg Salinosporamide A intravenous injection (i.v.) processing of using by oneself with the Epoxomicin of multiple concentration, and definite IC ₅₀Value Palayoor et al., Oncogene 18:7389-94 (1999).As what show among the figure .41, Epoxomicin causes the dose-dependent inhibition effect in the hydrolysis of described proteasome substrate.The IC that in these experiments, obtains ₅₀Value with well match in the viewed 10nM value of the 20S of external use purification proteasome (not demonstration).These data show that also this is owing to described proteasome, rather than some other protease to the described substrate retentive activity in these lysates that prepare in the animal of handling with 0.1mg/kgSalinosporamide A.The residual activity of seeing in the extract of handling with the Epoxomicin of high dose is less than whole signals of 2%, and this shows with suc-LLVY-AMC as viewed 98% the activity of surpassing of substrate owing to the activity that is present in the described proteasome among the PWBL.

Estimated also that inherence in the baseline activity changes and the active ability of Salinosporamide A Profilin enzyme body between comparison.In Figure 42, shown the result of the separate analysis in several weeks of separately turning round, Qureshi, et al., J.Immunol.171 (3): 1515-25 (2003).For clear, the dosage result of a display medium contrast and coupling.Though the proteasome activity that derives from the matched group among the PWBL of individual animal exists some to change, the whole meansigma methodss between two groups are very similar.The animal of handling with Salinosporamide A (0.1mg/kg i.v.) has also shown closely similar residual activity and average inhibitory action.This shows that the result between analyzing can compare with confidence level.

Embodiment 52

Salinosporamide A suppresses the inductive TNF of LPS in vivo

Studies show that proteasome is working aspect the activation of many signaling molecules, comprise that the inhibitor (I κ B α) by albumen degraded NF-κ B is working aspect the activation of transcription factor NF-KB.The LPS signal causes many short scorching gene expressions such as TNF, IL-6 and IL-1 β by TLR4 receptor activation NF-κ B and other transcriptional regulator.The continuous expression of proinflammatory cytokine has been accredited as principal element in numerous disease.The inhibitor of TNF and IL-1 β shows effectiveness in many inflammatory models, comprise the animal model of LPS mouse model and rheumatoid arthritis and inflammatory bowel.The nearest Profilin enzyme body that studies show that can stop the inductive TNF secretion of LPS-(Qureshi et al., 2003).These data show Salinosporamide A, novel effective protein proteins enzyme body inhibitor can stop the secretion of TNF in the body in high dose LPS mouse model.

In order to estimate the ability that suppresses the inductive Plasma TNF levels of LPS of mice in the Salinosporamide A body, at BolderBioPATH, Inc.in Boulder, CO. begin to carry out research in the body.Following method has been summarized and has been the experimental program of these research design.

Inject male Swiss Webster mice (12 is a group, and body weight is 20-25g) with LPS (2mg/kg) by the intraperitoneal approach.After 30 minutes, under thermolamp after about 5 minutes, with Salinosporamide A with 2.5mg/kg dosage to mouse mainline (tail vein).Behind lps injection 90 minutes, this mice is used isoflurane anesthesia, and bleed by cardiac puncture and to obtain blood plasma.Remaining blood precipitate is suspended in again among the 500 μ l PBS with flush away residual serum albumin and recentrifuge then.Remove supernatant and freezing blood precipitate, so that the proteasome in the whole blood lysate of analysis hematocrit suppresses.

Table 11

Time

Group name	Group number	n＝	0 minute	+ 30 minutes
Group name	Group number	n＝	0 minute	+ 30 minutes	Injection/baseline not	1	5
Saline+solutol medium	2	5	Saline		Injection/baseline not	1	5

Saline+solutol medium	3	5	Saline	Solutol/DMSO
Saline+solutol medium	3	5	Saline	Solutol/DMSO	LPSi.p./medium is (30min)	4	12	LPS
LPSi.p./medium (+30m)	5	12	LPS	Solutol/DMSO	LPSi.p./medium is (30min)	4	12	LPS
LPSi.p./medium (+30m)	5	12	LPS	Solutol/DMSO	Saline/Salinosporamide A (30min) 0.25mg/kg	6	12	Saline
Saline/Salinosporamide A (+30m) 0.25mg/kg	7	12	Saline	0.25mg/kg	Saline/Salinosporamide A (30min) 0.25mg/kg	6	12	Saline
Saline/Salinosporamide A (+30m) 0.25mg/kg	7	12	Saline	0.25mg/kg	LPS/Salinosporamide A(-30min)0.25mg/kg	8	12	LPS
LPS/Salinosporamide A(+30m)0.25mg/kg	9	12	LPS	0.25mg/kg	LPS/Salinosporamide A(-30min)0.25mg/kg	8	12	LPS

100%DMSO stock solution with the Salinosporamide A of 10mg/mL prepares to drug solns.Prepare 10%solutol solution by diluting with w/w, and make 1: 160 diluent with the Salinosporamide A stock solution of 10mg/ml with no endotoxic water.With the dosage of 4ml/kg to the animal intravenous administration.Also by 1: 160 diluent of the identical 100%DMSO of preparation in 10% solutol solution, making final concentration is to contain 9.375% solutol and DMSO in the water 0.625% to prepare the medium contrast solution.According to the technical instruction of manufacturer, with Biosource mTNF Cytoset test kit (Biosource Intl., Camarillo, CA; Catalog number (Cat.No.) #CMC3014) measurement of enforcement Plasma TNF levels.With diluted sample 1: 60 to be used for this analysis.

With every group have at least 10 data that repeat to obtain in twice independent experiment that animal carries out only show with 0.125 or the processing body that carries out of the Salinosporamide A of 0.25mg/kg in reduce the inductive TNF secretion of LPS.Representative experiment is shown among Figure 43.Behind these data show injection 2mg/kg LPS 30 minutes, the processing that reuse 0.25mg/kg Salinosporamide A carries out animal caused the remarkable reduction of serum TNF level.The elder generation of the whole blood sample intravital proteasome in external back of also having analyzed hematocrit suppresses, and shows that the animal of handling with 0.125mg/kg Salinosporamide A has 70 ± 3% inhibition and with the animal of 0.25mg/kg Salinosporamide A processing 94 ± 3% inhibition arranged.In the animal of usefulness or LPS of no use processing, do not see the significant difference that suppresses about proteasome.After LPS handles 30 minutes, with 0.125 or the dosage of 0.25mg/kg when the mouse mainline administration, Salinosporamide A has reduced about 65% the inductive Plasma TNF levels of LPS.

Embodiment 53

The chemical sensitization effect that Salinosporamide A is external

Can activate transcription factor nuclear factor-kappa B (NF-κ B) in the CCL188 that comprises the LoVo cell such as the chemotherapeutant of CPT-11 (Irinotecan), cause these cells to stand the reduction of apoptotic ability, Cusack, et al., Cancer Res 61:3535 (2001).In stimulated cells not, NF-κ B is arranged in Cytoplasm with the inert complexes form with repressible protein I κ B (inhibitor of NF-κ B).Multiple stimulation can cause I κ B phosphorylation by the I kappa b kinase, then I κ B ubiquitination and by proteasome degraded I κ B.After the I κ B degraded, NF-κ B transposition to nucleus and regulator gene expressed, and influences many cell processes, thereby the rise that comprises survival genes suppresses apoptosis.

Generally acknowledged recently proteasome inhibitor, Velcade ^TM(PS-341; Millennium drugmaker) cancerous cell there is direct toxicity, and can degrade by the inductive I κ of Profilin enzyme body B and strengthen the cytotoxicity of CPT-11 the LoVo cell in the external and LoVo xenograft grafting model, Adams, J., Eur J Haematol 70:265 (2003).In addition, find Velcade ^TM(proangiogenic) chemotactic factor/cytokine GRO-α of angiogenic and the expression of VEGF before suppressing in the squamous cell carcinoma, the chances are realizes Sunwoo, et al., Clin Cancer Res 7:1419 (2001) by suppressing the NF-kB pathway.These data show that the inhibition of proteasome not only reduces the survival rate and the growth of tumor cell, and reduce angiogenesis.

Embodiment 54

Colon cancer, carcinoma of prostate, breast carcinoma, pulmonary carcinoma, ovarian cancer, multiple myeloma and black The growth inhibited of plain tumor

Human colon adenocarcinoma (HT-29; HTB-38), carcinoma of prostate (PC-3; CRL-1435), breast carcinoma (MDA-MB-231; HTB-26), nonsmall-cell lung cancer (NCI-H292; CRL-1848) adenocarcinoma ovaries (OVCAR-3; HTB-161), (RPMI 8226 for multiple myeloma; CCL-155), multiple myeloma (U266; TDB-196) and Mus melanoma (B16-F10; CRL-6475) cell is all available from ATCC and maintain in the suitable culture medium.Under 37 ℃, containing 5%CO ₂With cultivate described cell in the incubator of 95% humid air.

In order to carry out the analysis of cell growth inhibited, with HT-29, PC-3, MDA-MB-231, NCI-H292, OVCAR-3 and B16-F10 cell respectively with 5 * 10 ³, 5 * 10 ³, 1 * 10 ⁴, 4 * 10 ³, 1 * 10 ⁴With 1.25 * 10 ³Cells/well is seeded in the 90 μ l complete mediums in the tissue culturing plate of 96 holes (Corning 3904) black wall, clear bottom, and with this culture plate overnight incubation so that exponential phase is set up and entered to cell.RPMI 8226 and U266 cell are being analyzed the same day respectively with 2 * 10 ⁴With 2.5 * 10 ⁴Cells/well is seeded in the 96 interior μ l complete mediums of 96 orifice plates.The 20mM stock solution of the described chemical compound of preparation in 100%DMSO, and be stored in-80 ℃.This chemical compound is diluted continuously and join in the instrument connection, triplicate.The test concentrations of II-2 and II-4 is 6.32 μ M to 632pM.II-3 and II-17 test with the concentration of 20 μ M to 6.32nM.Formula II-18 and II-19 test with the concentration of 2 μ M to 200pM.Formula II-5A and formula II-5B test with the final concentration of 2 μ M to 632pM and the final concentration of 20 μ M to 6.32nM respectively.This culture plate is put back in this incubator cultivated 48 hours.The DMSO final concentration is 0.25% in whole samples.

After the drug exposure 48 hours, in each hole, add the no Mg that 10 μ l contain 0.2mg/ml "diazoresorcinol" (obtaining from Sigma-Aldrich chemical company) ²⁺, Ca ²⁺Phosphate buffered saline (PBS), and described culture plate be put back in this incubator cultivate 3-6 hour.Because living cells metabolism "diazoresorcinol" is used Fusion microtest plate exometer (Packard Bioscience), use λ _Ex=535nm and λ _EmThe light filter of=590nm is measured the fluorescence of the reduzate of "diazoresorcinol"."diazoresorcinol" dyestuff in the not celliferous culture medium is used for determining background, it is deducted from the data of all experimental ports.This data standard is turned to the mean fluorecence degree of the cell of handling with culture medium+0.25%DMSO (growth of 100% cell), and determine EC with the S shape dose effect curve match rule (XLfit 3.0, ID Business Solutions Ltd) of standard ₅₀Value (observing the drug level that 50% maximum growth suppresses).Wherein the maximum of cell growth suppressed less than 50% o'clock, did not then decide EC ₅₀Value.

Data in the table 12 have been summed up formula II-2, II-3, II-4, II-5A, II-5B, II-17, II-18 and II-19 to human colorectal cancer, HT-29, human prostata cancer, PC-3, human breast carcinoma, MDA-MB-231, people's nonsmall-cell lung cancer, NCI-H292, human ovarian cancer, OVCAR-3, people's multiple myeloma, the growth inhibited effect of RPMI 8226 and U266 and Mus melanoma b16-F10 cell line.

Table 12

Formula II-2, II-3, II-4, II-5A, II-5B, II-17, II-18 and II-19 are to the EC of kinds of tumor cells system ₅₀Value

^*Wherein n=3 has shown mean+SD

Described EC ₅₀Value shows that formula II-2, II-4, II-5A, II-5B, II-18 and II-19 have cytotoxicity to HT-29, PC-3, MDA-MB-231, NCI-H292, RPMI 8226, U266 and B16-F10 tumor cell line.II-2, II-5A, II-5B and II-19 also have cytotoxicity to the OVCAR-3 tumor cell.Formula II-17 has cytotoxicity to MDA-MB-231, RPMI 8226, U266 and B16-F10 tumor cell line.

It is the growth inhibited effect of RPMI 8226 and U266 to people's multiple myeloma cells that data in the table 13 have been summed up formula I-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, II-44, VI-1A, II-47 and II-50.

Table 13

Formula I-7, II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25, II-26, II-28, II-29, II-30, II-31, II-32, II-38, IV-3C, II-44, VI-1A, II-47 and II-50 are to the EC of RPMI8226 and U266 cell ₅₀Value

Wherein n 〉=3 have shown mean+SD; ^*N=3, inapplicable standard deviation; The ND=undetermined

Described EC ₅₀Value shows that formula II-2, II-4, II-5A, II-5B, II-16, II-17, II-18, II-19, II-20, II-22, II-24C, II-26, II-29 and II-30 have cytotoxicity to RPMI 8226 and U266 cell.Formula I-7, II-38, II-44, VI-1A, II-47 and II-50 have cytotoxicity to the RPMI8226 cell.Formula II-21 and IV-3C have cytotoxicity to the U266 cell.

Embodiment 55

To MES-SA, MES-SA/Dx5, HL-60 and HL-60/MX2 tumor The growth inhibited of cell line

People's sarcoma of uterus (MES-SA; CRL-1976), its multi-drug resistant derivant (MES-SA/Dx5; CRL-1977), people's acute promyelocytic leukemia cell (HL-60; CCL-240) and its multi-drug resistant derivant (HL-60/MX2; CRL-2257) available from ATCC and maintain in the suitable culture medium.Under 37 ℃, containing 5%CO ₂With cultivate this cell in the incubator of 95% humid air.

In order to carry out the analysis of cell growth inhibited, with MES-SA and MES-SA/Dx5 cell all with 3 * 10 ³Cells/well is seeded in 96 hole (Corning; 3904) in the 90 μ l complete mediums in the tissue culturing plate of black wall, clear bottom, and with this culture plate overnight incubation so that exponential phase is set up and entered to cell.HL-60 and HL-60/MX2 cell are adding chemical compound same day all with 5 * 10 ⁴Cells/well is seeded in the 90 interior μ l complete mediums of 96 well culture plates.The 20mM stock solution of the described chemical compound of preparation and storage in-80 ℃ in 100%DMSO.Described chemical compound is diluted continuously and join in this instrument connection, triplicate.The test concentrations of II-2 and II-4 is 6.32 μ M to 2nM.II-3 and II-17 test under 20 μ M to 6.32nM concentration.Compound I I-18 tests under 2 μ M to 632pM concentration.Described culture plate is put back into again in this incubator cultivated 48 hours.The final concentration of DMSO is 0.25% in whole samples.

After the drug exposure 48 hours, in each hole, add the no Mg that contains 0.2mg/ml "diazoresorcinol" (obtaining) of 10 μ l from Sigma-Aldrich Chemical Co. ²⁺, Ca ²⁺Phosphate buffered saline (PBS), and described culture plate be put back in the described incubator cultivated 3-6 hour.Because living cells metabolism "diazoresorcinol", λ is used in the exometer (PackardBioscience) of using the Fusion microtest plate _Ex=535nm and λ _EmThe light filter of=590nm is measured the fluorescence of the reduzate of "diazoresorcinol"."diazoresorcinol" dyestuff in the not celliferous culture medium is used for determining background, it is deducted from the data of all experimental ports.This data standard is changed into the mean fluorecence degree of the cell of handling with culture medium+0.25%DMSO (growth of 100% cell), and determine EC with the S shape dose effect curve match rule (XLfit 3.0, ID Business Solutions Ltd) of standard ₅₀Value (observing the drug level that 50% maximum growth suppresses).Wherein the maximum of cell growth suppresses less than 50%, does not then determine EC ₅₀Value.

Described multi-drug resistant MES-SA/Dx5 tumor cell line comes from people's sarcoma of uterus MES-SA tumor cell line and expresses the P-glycoprotein (P-gp) that raises, ATP dependency outflow pump.Data in table 14 have been summarized formula II-2, II-3, II-4, II-17 and the II-18 growth inhibited effect to MES-SA and multi-drug resistant derivant MES-SA/Dx5 thereof.The Paclitaxel (Paclitaxel) that comprises known substrate in contrast, the P-gp pump.

Table 14

Formula II-2, II-3, II-4, II-17 and II-18 are swollen to MES-SA and MES-SA/Dx5 The EC of oncocyte system ₅₀ Value

^*Change multiple=EC ₅₀The ratio (MES-SA/Dx5:MES-SA) of value

EC ₅₀Value shows that II-2, II-4, II-17 and II-18 all have cytotoxic activity to MES-SA and MES-SA/Dx5 tumor cell line.Drug-fast MES-SA/Dx5 cell is had reduce about 800 times activity and determined described multi-drug resistant phenotype by observing Paclitaxel.

HL-60/MX2 is the topoisomerase II activity that comes from the multi-drug resistant tumor cell line of human promyelocytic leukemia cell line HL-60 and be expressed as minimizing.The data that are displayed in Table 15 have been summarized the growth inhibited effect to HL-60 and multi-drug resistant derivant HL-60/MX2 thereof of formula II-2, II-3, II-4, II-17 and II-18 chemical compound.Comprise mitoxantrone in contrast, the topoisomerase II targeting agent.

Table 15

Formula II-2, II-3, II-4, II-17 and II-18 are thin to HL-60 and HL-60/MX2 tumor The EC of born of the same parents system ₅₀ Value

^*Change multiple=EC ₅₀The ratio (HL-60/MX2:HL-60) of value

Described EC ₅₀Value shows that II-2, II-4 and II-18 all keep cytotoxic activity to HL-60 and HL-60/MX2 tumor cell line.Drug-fast HL-60/MX2 cell is had reduce about 30 times activity and determined this multi-drug resistant phenotype by observing mitoxantrone.

Embodiment 56

Formula II-16, formula II-17, formula II-20 and Omuralide are to 20S egg in PRMI 8226 cells The active effect of chymotrypsin-like of white enzyme body

Under 37 ℃, at 5%CO ₂In 95% malaria, with people's multiple myeloma cells is RPMI 8226 (ATCC, CCL-155) cultivate in RPMI 1640 culture medium, this culture medium has been added 2mM L-glutaminate, 100 units/ml penicillin, 100 μ g/ml streptomycins, 1mM Sodium Pyruvate and 10% heat-inactivated hyclone.In order to estimate the active inhibitory action of the chymotrypsin-like of described 20S proteasome, the test compounds that will prepare in DMSO is suitably dilution in culture medium, and joins 2.5 * 10 ⁵In RMPI 8226 cells of/ml.For formula II-16, final test concentration is 1nM to 100nM.(CA), final test concentration is 1nM to 10 μ M for Calbiochem, San Diego for formula II-17, II-20 and Omuralide.DMSO contrasts as medium with 0.1% final concentration.Hatching RMPI 8226 cells with described chemical compound after 1 hour, at room temperature, by with 2,000rpm made cell precipitation in centrifugal 10 seconds, and with ice-cold 1X Dulbecco ' s phosphate buffered saline (PBS) (DPBS, Mediatech, Herndon VA) washs this cell 3 times.With the cell of DPBS washing added protease inhibitor cocktail (Roche Diagnostics, Indianapolis, lysis buffer IN) (20mM HEPES, 0.5mM EDTA, 0.05%Triton X-100, pH7.3) in cracking on ice 15 minutes.Under 4 ℃, by with 14,000rpm made the cell debris precipitation in centrifugal 10 minutes, and supernatant (=cell lysate) is transferred in the new test tube.(Pierce Biotechnology, Rockford IL) measure protein concentration with BCA analysis of protein test kit.Containing proteasome analysis buffer (the 20mM HEPES that final concentration is 0.035%SDS, 0.5mMEDTA, pH8.0) in, by using Suc-LLVY-AMC fluorescence peptide substrates (BostonBiochem, Cambridge, MA) the chymotrypsin-like activity of the described 20S proteasome of mensuration.Join initiation reaction in this cell pyrolysis liquid of 190 μ l by 0.4mM Suc-LLVY-AMC (by preparing) with the DMSO solution of analysis buffer with this peptide of 1: 25 dilution proportion 10mM with 10 μ l, and under 37 ℃, in Thermo Lab SystemsFluoroskan culture plate reader, hatch.By using λ _Ex=390nm and λ _EmThe coumarin (AMC) that=460nm fluorescence measurement discharges.In microtitration plate (Corning 3904), implement described analysis, measured once in dynamically per subsequently five minutes, continue 2 hours.Being used for each proteic total amount of analyzing is 20 μ g.The final concentration of Suc-LLVY-AMC and DMSO is respectively 20 μ M and 0.2%.The result is shown as with respect to the DMSO contrast the active inhibition percentage ratio of this 20S proteasome chymotrypsin-like.

Result in the table 16 shows the active inhibition of chymotrypsin-like that RPMI 8226 cellular exposure is caused described 20S proteasome in formula II-16, formula II-17, formula II-20 and Omuralide.In the middle of them, formula II-16 has suppressed the chymotrypsin-like activity of 85 ± 7% 20S proteasome when 5nM.When 100nM, formula II-16 can suppress the chymotrypsin-like activity of 20S proteasome fully.When 100nM, formula II-17, formula II-20 and Omuralide are merely able to suppress respectively this chymotrypsin-like activity of 30 ± 4%, 66 ± 3% and 32 ± 8%.

Table 16

Measure the chymotrypsin-like activity of 20S proteasome of RMPI 8226 cells of use by oneself formula II-16, formula II-17, formula II-20 and Omuralide processing

To the active inhibition of the chymotrypsin-like % chemical compound of 20S proteasome in RMPI 8226 cell lysates (meansigma methods+SD, n=3)

10,000nM 1,000nM 500nM 100nM 50nM 10nM 5nM 1nM

II-16 ND ND ND 98±1 97±0 94±3 85±7 30±7

II-17 65±5 46±4 39±3 30±4 26±5 6±6 10±5 6±6

II-20 87±4 73±2 71±2 66±3 64±3 37±3 31±9 3±10

Omuralide 93±1 80±8 68±11 32±8 17±11 4±9 8±9 5±9

ND: undetermined

Embodiment 57

Formula II-16, formula II-17, formula II-20 and Omuralide are to 20S proteasome in the PC-3 cell The active effect of chymotrypsin-like

Under 37 ℃, at 5%CO ₂In 95% malaria, with PC-3 PC-3 (ATCC, CRL-1435) cultivate in the F12K culture medium, this culture medium has been added 2mM L-glutaminate, 100 units/ml penicillin, 100 μ g/ml streptomycins and 10% heat-inactivated hyclone.In order to estimate the active inhibitory action of the chymotrypsin-like of described 20S proteasome, the test compounds that will prepare in DMSO is suitably dilution in culture medium, and joins 1.25 * 10 ⁵In the PC-3 cell of/ml.For formula II-16, final test concentration is 1nM to 50nM.(CA), final test concentration is 1nM to 10 μ M for Calbiochem, SanDiego for formula II-17, II-20 and Omuralide.DMSO contrasts as medium with 0.1% final concentration.Hatching the PC-3 cell after 1 hour with described chemical compound, (Herndon VA) washs this cell 3 times for DPBS, Mediatech with ice-cold 1X Dulbecco ' s phosphate buffered saline (PBS).With the cell of DPBS washing added protease inhibitor cocktail (RocheDiagnostics, Indianapolis, lysis buffer IN) (20mM HEPES, 0.5mMEDTA, 0.05%Triton X-100, pH7.3) in cracking on ice 15 minutes.Under 4 ℃, by with 14,000rpm made the cell debris precipitation in centrifugal 10 minutes, and supernatant (=cell lysate) is transferred in the new test tube.(Pierce Biotechnology, Rockford IL) measure protein concentration with BCA analysis of protein test kit.Containing proteasome analysis buffer (the 20mM HEPES that final concentration is 0.035% SDS, 0.5mM EDTA, pH8.0) in, by using Suc-LLVY-AMC fluorescence peptide substrates (Boston Biochem, Cambridge, MA) the chymotrypsin-like activity of the described 20S proteasome of mensuration.Join initiation reaction in this cell lysate of 190 μ l by 0.4mMSuc-LLVY-AMC (by preparing) with the DMSO solution of analysis buffer with this peptide of 1: 25 dilution proportion 10mM with 10 μ l, and under 37 ℃, in Thermo Lab Systems Fluoroskan culture plate reader, hatch.By using λ _Ex=390nm and λ _EmThe coumarin (AMC) that=460nm fluorescence measurement discharges.In microtitration plate (Corning 3904), implement described analysis, measured once in dynamically per subsequently five minutes, continue 2 hours.Being used for each proteic total amount of analyzing is 20 μ g.The final concentration of Suc-LLVY-AMC and DMSO is respectively 20 μ M and 0.2%.The result is shown as with respect to the DMSO contrast the active inhibition percentage ratio of this 20S proteasome chymotrypsin-like.

Result in the table 17 shows the PC-3 cellular exposure is caused the active inhibition of chymotrypsin-like of 20S proteasome in formula II-16, formula II-17, formula II-20 and Omuralide, and is similar to the result who obtains in the experiment based on RPMI 8226 cells.Formula II-16 has suppressed the chymotrypsin-like activity of 69% 20S proteasome when 5nM.When 50nM, formula II-16 can suppress the chymotrypsin-like activity of 20S proteasome fully.When 100nM, formula II-17, formula II-20 and Omuralide have suppressed this chymotrypsin-like activity of 26%, 57% and 36% respectively.

Table 17

Mensuration derives from the chymotrypsin-like activity of the 20S proteasome of the PC-3 cell of handling with formula II-16, formula II-17, formula II-20 and Omuralide

The active inhibition of the chymotrypsin-like of 20S proteasome % in the PC-3 cell lysate

Chemical compound

10,000 1,000 100nM 50nM 10nM 5nM 1nM

nM nM

II-16 ND ND ND 98 ND 69 19

II-17 79 49 26 ND 16 ND ND

II-20 90 71 57 ND 38 ND ND

Omuralide 90 80 36 ND 18 ND ND

ND: undetermined

Embodiment 58

People's multiple myeloma PRMI 8226, people's colon gland in the culture medium that contains 1% or 10% serum The growth inhibited of cancer HT-29 and Mus melanoma b16-F10 cell

When existing, measured formula II-16, II-17 and II-18 growth inhibitory activity at 1% or 10% hyclone (FBS) to people's multiple myeloma RPMI 8226, human colon adenocarcinoma HT-29 and Mus melanoma b16-F10 cell.

RPMI 8226 (CCL-155), HT-29 (HTB-38) and B16-F10 (CRL-6475) cell are available from ATCC.RPMI 8226 cells are maintained in PRMI 1640 culture medium, and this culture medium is added 10% (v/v) FBS, 2mM L-glutaminate, 1mM Sodium Pyruvate and is respectively 100IU/ml and the penicillin/streptomycin of 100 μ g/ml.The HT-29 cell is maintained among the McCoys 5A, and this McCoys 5A adds 10% (v/v) FBS, 2mM L-glutaminate, the nonessential aminoacid of 1mM Sodium Pyruvate, 1% (v/v), 10mM HEPES and is respectively 100IU/ml and the penicillin/streptomycin of 100 μ g/ml.The B16-F10 cell is maintained among the DMEM, and this DMEM adds 10% (v/v) FBS, 2mM L-glutaminate, 10mMHEPES and is respectively 100IU/ml and the penicillin/streptomycin of 100 μ g/ml.Under 37 ℃, described cell is being contained 5%CO ₂With cultivate in the incubator of 95% humid air.

In order to carry out the analysis of cell growth inhibited, with HT-29 and B16-F10 respectively with 5 * 10 ³With 1.25 * 10 ³Cells/well is seeded in 96 hole (Corning; 3904) in the 90 μ l culture medium that contain 10% (v/v) FBS or 1% (v/v) FBS in black wall, the tissue culturing plate of clear bottom.With this culture plate overnight incubation so that the logarithmic (log) phase growth is set up and entered to cell.With RPMI 8226 cells with 2 * 10 ⁴Cells/well is seeded in 96 hole (Corning; 3904) in the 90 μ l culture medium that contain 10% (v/v) FBS or 1% (v/v) FBS in black wall, the tissue culturing plate of clear bottom.The 20mM stock solution of preparation formula II-16, II-17 and II-18 in 100%DMSO is divided into equal portions and is stored in-80 ℃.With formula II-16, II-17 and II-18 contain 1% or the culture medium of 10%FBS in dilution continuously, and it is joined in the instrument connection, triplicate.The final concentration of formula II-16 is 2 μ M to 200pM.The final concentration of formula II-17 is 20 μ M to 6.3nM.The final concentration of formula II-18 is 2 μ M to 630pM.This culture plate is put back in the described incubator cultivated 48 hours.The final concentration of DMSO is 0.25% in whole samples.

After the drug exposure 48 hours, in each hole, add the no Mg that 10 μ l contain 0.2mg/ml "diazoresorcinol" (obtaining from Sigma-Aldrich chemical company) ²⁺, Ca ²⁺Phosphate buffered saline (PBS), and described culture plate be put back in the incubator cultivate 3-6 hour.Because living cells metabolism "diazoresorcinol" is used Fusion microtest plate exometer (Packard Bioscience), use λ _Ex=535nm and λ _EmThe light filter of=590nm is measured the fluorescence of the reduzate of "diazoresorcinol"."diazoresorcinol" dyestuff in the not celliferous culture medium is used for determining background, it is deducted from the data of all experimental ports.This data standard is turned to the mean fluorecence degree of the cell of handling with culture medium+0.25%DMSO (growth of 100% cell), and determine EC with the S shape dose effect curve match rule (by XLfit 3.0 generations, ID Business Solutions Ltd) of standard ₅₀Value (observing the drug level that 50% maximum growth suppresses).

Data in the table 17 summarized formula II-16, II-17 and II-18 to contain 1% or the culture medium of 10%FBS in people's multiple myeloma cells be the growth inhibited effect of RPMI 8226.

Table 17

Formula II-16, II-17 and II-18 to contain 1% or the culture medium of 10%FBS in the EC of RPMI8226 cell ₅₀Value

Chemical compound 1%FBS, EC ₅₀(nM) 10%FBS, EC ₅₀(nM)

II-16 6.2 12

6.8 9.6

Meansigma methods 6.5 11

II-17 1100 3000

1300 2300

Meansigma methods 1,200 2700

II-18 15 20

13 20

Meansigma methods 14 20

Described EC ₅₀Value show formula II-16, II-17 and II-18 to contain 1% or the culture medium of 10%FBS in RPMI 8226 cells cytotoxicity is arranged.In the culture medium that contains 1%FBS, when in containing the culture medium of 10%FBS, testing, the average EC of formula II-16, II-17 and II-18 ₅₀There is the attenuating less than 3 times in value.

Data in the table 18 summarized formula II-16 to contain 1% or the culture medium of 10%FBS in human colon adenocarcinoma HT-29 and the growth inhibited effect of Mus melanoma b16-F19 cell line.

Table 18

Formula II-16 to contain 1% or the culture medium of 10%FBS in HT-29 and the average EC of B16-F19 cell ₅₀Value

Compound H T-29, EC ₅₀(nM) meansigma methods ± SD B16-F19, EC ₅₀(nM) meansigma methods ± SD

1％FBS 10％FBS 1％FBS 1％FBS

II-16 16±5 23±10 18±9 13±1

Meansigma methods EC50 value show formula II-16 to contain 1% or the culture medium of 10%FBS in HT-29 and B16-F10 cell cytotoxicity is arranged.In the culture medium that contains 1%FBS, when in containing the culture medium of 10%FBS, testing, the average EC of formula II-16 ₅₀There is the attenuating less than 2 times in value.In sum, these data show are about the cell in vitro cytotoxic activity to tumor cell line, 1% or 10%FBS exist up-to-date style II-16, II-17 and II-18 to keep similar biological activity.

Embodiment 59

Suppress the anthrax lethal toxin

Anthrax toxin is to form the related indication reason of anthrax.In this disease, the anthrax bacillus spore is inhaled into and lives with in the lung, and they are taken in by macrophage there.In this macrophage, spore germinates, duplicates, causes this cell to be killed.But before killing this cell, the macrophage migration of infection is in lymph node, and when dead there, they discharge their inclusions, and this organism is entered in the blood system, further duplicate the justacrine lethal toxin.

(LF, two kinds of albumen 90kDa) play an important role in the pathogeny of anthrax to be called as protection antigen (PA 83kDa) and lethal factor.These albumen jointly are called as lethal toxin (LeTx).When they made up, when in the intravenous injection precession object, PA and LF caused death.Lethal toxin is cultivated in the system in a few cell of macrophage also activity, causes cell death in several hrs.LeTx can bring out necrosis and apoptosis during extracorporeal treatment in mouse macrophage sample RAW264.7 cell.

The external analysis of the Cytotoxic inhibitor of lethal toxin mediation based on cell

Under 37 ℃, at the 5%CO that is containing humidity ₂Incubator in, RAW264.7 cell (obtaining from American type culture collection) adapted to and maintain the RPMI-1640 culture medium (complete medium) of adding 10% hyclone, 2mM L-glutaminate and 1% penicillin/streptomycin.In order to carry out described analysis, cell is seeded in the complete medium in 96 orifice plates with the concentration of 50,000 cells/well, spend the night.Removed culture medium at second day and replace with the complete medium of serum-free, this complete medium contain or do not contain with 330nM be initial concentration and be 8 dose point effects and with ¹/ ₂The formula II-2 of the variable concentrations that logarithm dilutes at interval, II-3, II-4, II-5A, II-5B, II-13C, II-17, II-18 and IV-3C.Behind 45 minutes preincubates, with 1 μ g/ml LF and 1 μ g/ml PA separately or jointly (LF:PA is also referred to as lethal toxin (LeTx)) add in the cell.Obtain reorganization LF and PA from the List biology laboratory.Comprise other culture plate in contrast, that do not add LeTx.Cell is hatched 6 hours then, then is added in no Mg++, the PBS of Ca++ (Mediatech, Herndon, VA) in the preparation 0.02mg/ml "diazoresorcinol" dyestuff (Molecular Probes, Eugene, OR).Before the assessment cell viability, culture plate was hatched again 1.5 hours in addition.Because living cells metabolism "diazoresorcinol", excite and measure fluorescence with the light filters of 590 emissions and estimate cytotoxicity or cell viability by using 530.Use following formula, data representation is contrasted the survival percentage ratio that (low) makes comparisons separately for contrasting (height) and LeTx separately with DMSO: survival percentage ratio=100* (the low contrast of the OD-of measurement)/(high contrast-low contrast).

The cytotoxicity that suppresses anthrax lethal toxin mediation in RAW 264.7 cells

Data general introduction formula II-2, formula II-3 among Figure 48 and formula II-4 are to the Cytotoxic effect of the LeTx mediation of RAW 264.7 mouse macrophage like cells system.With formula II-2 and formula II-4 the processing of RAW 264.7 cells is caused the viability of the cell handled through LeTx to increase its EC ₅₀Value is 14nM (Figure 48).Under the concentration of test, do not determine the EC of formula II-3 to the LeTx protection ₅₀Value (EC ₅₀＞330nM, the Cmax of evaluation).Data show formula II-5A, II-5B, II-13C, II-17, II-18 and IV-3C in table 19 is to the Cytotoxic effect of the LeTx mediation of RAW 264.7 mouse macrophage like cells system.With formula II-5A and II-18 to the processes and displays of RAW264.7 cell the viability of RAW 264.7 cells handled through LeTx increase its EC ₅₀Value is respectively 3nM and 4nM.Cause the viability of the cell handled through LeTx to increase its EC with the processing of formula II-17 and formula II-5B ₅₀Value is respectively 42nM and 45nM.Under the concentration of test, can't determine formula II-13C and IV-3C EC to the LeTx protection ₅₀Value (EC ₅₀＞330nM, the Cmax of evaluation).

Table 19: to EC by the inhibition of RAW 264.7 cell cytotoxicities of anthrax lethal toxin mediation ₅₀Value

Chemical compound	EC ₅₀(nM)
Chemical compound	EC ₅₀(nM)	Formula II-17	42
Formula II-18	4	Formula II-17	42
Formula II-18	4	Formula II-5A	3
Formula II-5B	45	Formula II-5A	3

Formula II-13C	＞330nM
Formula II-13C	＞330nM	Formula IV-3C	＞330nM

Embodiment 60

R ₁ The structure activity relationship of side chain

Have the relative activity of the multiple chemical compound of following general formula by analysis, infer disclosed chemical compound and particularly general formula (I), (II), (III), (IV) and (V) R of chemical compound ₁The structure activity relationship of side chain:

Analyze the cytotoxicity of these chemical compounds to the RPMI cell, and to chymotrypsin-like, trypsin-like and the active inhibition of Caspase sample of NF-κ B and 20S proteasome.The results are shown in the table 20 of 6 representative compounds

Table 20: have different R ₁The EC of the chemical compound of group ₅₀Value

Chemical compound	R ₁	Cytotoxicity RPMI EC ₅₀(nM)	NF-κB EC ₅₀ (nM)	Proteasome chymotrypsin-like EC ₅₀(nM)	Proteasome trypsin-like EC ₅₀(nM)	Proteasome Caspase sample EC ₅₀(nM)
Chemical compound	R ₁	Cytotoxicity RPMI EC ₅₀(nM)	NF-κB EC ₅₀ (nM)	Proteasome chymotrypsin-like EC ₅₀(nM)	Proteasome trypsin-like EC ₅₀(nM)	Proteasome Caspase sample EC ₅₀(nM)	II-16	CH ₂CH ₂Cl	8.6±1.9	11±3	2.6±0.2	21±2.6	427±61
II-18	CH ₂CH ₂Br	6.3，6.3	11，9	2.3，2	14，14	286，213	II-16	CH ₂CH ₂Cl	8.6±1.9	11±3	2.6±0.2	21±2.6	427±61
II-18	CH ₂CH ₂Br	6.3，6.3	11，9	2.3，2	14，14	286，213	II-19	CH ₂CH ₂I	6，7	10，7	3，3	13，15	573，739
II-17	CH ₂CH ₃	6150，3460	960±210	26±6.7	573，602	1247，1206	II-19	CH ₂CH ₂I	6，7	10，7	3，3	13，15	573，739
II-17	CH ₂CH ₃	6150，3460	960±210	26±6.7	573，602	1247，1206	II-20	CH ₃	8510±3260	849±225	7.7±3.0	318，321	1425，1420
II-21	CH ₂CH ₂OH	＞20000，＞20000	3172，2707	7，8	720，879	2585，2328	II-20	CH ₃	8510±3260	849±225	7.7±3.0	318，321	1425，1420

The result of above-mentioned analysis can be interpreted as show R ₁Group is that the chemical compound of chloroethyl, bromoethyl or iodine ethyl is effective protein proteins enzyme body inhibitor and shows very effective cytotoxicity.On the contrary, R ₁Group is that the chemical compound of methyl, ethyl or ethoxy shows relatively low cytotoxicity (tire and reduced by 3 logarithms), lower NF-κ B suppresses (tire and reduced by 3 logarithms) and lower Caspase sample (tire and reduced by 2 to 10 times) and trypsin-like (tire and reduced by 20 to 50 times) proteasome suppresses.

Be not subjected to any special theory, the applicant notices that The above results supported following hypothesis: at R ₁The increase that contains the compound activity of Cl, Br or I in the group is because halogen is the characteristic of good leaving group.The lactonic ring open loop of observed Compound I I-16 has also been supported this hypothesis with the fact that forms cyclic ether by nucleophilic displacement of fluorine, is wherein replaced according to following reaction chlorine:

Presumablely be, such as Compound I I-16, II-18 and II-19 etc. at R ₁Have in the side chain in the chemical compound of good leaving group, described proteasome forms cyclic ether with the mode nucleophilic addition similar in appearance to above-mentioned reaction to β-lactonic ring.Guess that this cyclic ether advantageously interacts with this proteasome.

Not fettered by any particular theory, the applicant notices that The above results also supported another hypothesis: another nucleophilic group on described proteasome has been replaced described leaving group, therefore formation 2-point covalency adduct between described chemical compound and described enzyme.In any case, at R ₁Leaving group functional group on the side chain promotes the interaction between this chemical compound and this enzyme to increase, thereby promotes active increasing.Therefore, can be expected at R ₁The chemical compound performance high activity that has other leaving group on the side chain.

Not fettered by any particular theory, the applicant notices that The above results also supports the hypothesis of single-point leaving group.As an example, at R ₁The existence of halogen or other leaving group promotes the conveying of described chemical compound to its target in the side chain, as intracellular target or other biological target, thereby strengthens therapeutical effect.In figure below exemplary illustration the example of single-point leaving group.

Single-point covalency medicine-enzyme adduct has the metathetical single 2-point covalency medicine of intramolecularly Cl-enzyme adduct

Point covalency medicine-enzyme adduct

Used " leaving group " of the present invention is meant can be by another atom or metathetical any atom of part or part in chemical reaction.More particularly, in some embodiments, " leaving group " refers in nucleophilic substitution by metathetical atom or group.In some embodiments, " leaving group " refers to any atom or the part as the conjugate base of strong acid.The non-limiting feature of leaving group and example can referring to, for example, Organic Chemistry, 2d ed., FrancisCarey (1992), 328-331 page or leaf; Introduction to Organic Chemistry, 2d ed., Andrew Streitwieser and Clayton Heathcock (1981), 169-171 page or leaf; With Organic Chemistry, 5 ^ThEd., John McMurry (2000), the 398th and 408 page; At this its full content is incorporated herein by reference.

Embodiment 61

Structure activity relationship

The many embodiment preferred of illustrating in the above listed table of data instance explanation.About general formula I I, at R ₁It is preferred having the substituent chemical compound of halogenation, and this compounds is equivalent in above-mentioned analysis usually.Most preferably at R ₁Has the n-halogenated ethyl.

In addition, most preferably at E ₅The position has the chemical compound of oh group, and the carbon that is connected is S configuration (chemical compound that for example, has the stereochemical structure of Compound I I-18).Hydroxyl is more less preferred to the oxidation of ketone.

In a preferred embodiment, at R ₄The position preferred substituted is a cyclohexene.In another embodiment preferred, this cyclohexene is oxidized to epoxide.The chemical compound that substituent pair of key of more less preferred cyclohexene is hydrogenated.

In addition in some embodiments, preferably, R ₃Be methyl, more less preferred ethyl.

Embodiment 62

The inhibition of angiogenesis

Angiogenesis is the important physical process, does not have angiogenesis then fetal development and wound healing can not take place.But over-drastic and unsuitable angiogenesis and multiple disease, morbid state are relevant with disadvantageous therapeutic outcome.Comprise diseases associated with inflammation with the disease type of excessive angiogenesis and the example of morbid state, as immunity and non-immunity inflammation, rheumatoid arthritis, beaevais' disease and psoriasis; The disease relevant with unsuitable or untimely vascular invasion for example is blood capillary proliferation and the osteoporosis in diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, degeneration of macula, corneal graft rejection, Terry's sign disease, RI, the atheromatous plaque; And the disease relevant with cancer, for example comprise solid tumor, tumor metastasis, as leukemic haematogenous tumor, fibrohemangioma, Kaposi sarcoma, as angiomatous benign tumor, acoustic neuroma, neurofibroma, trachoma and botryomycosis hominis, and other needs neovascularization to keep the cancer of tumor growth.The other example of angiogenesis-dependence disease comprises, for example, and the Osler-Webber syndrome; The angiogenesis of cardiac muscle; The speckle neovascularization; Telangiectasis; Bleeder's joint and wound granulation form.In addition, too much angiogenesis is also with relevant as the clinical problem of a biology and mechanical graft (tissue/organ graft, support an or the like) part.Compositions of the present invention can be used for suppressing angiogenesis, therefore can be used for treating this class morbid state.That angiogenesis works and can to adopt other disease of chemical compound of the present invention and compositions be conventionally known to one of skill in the art.

About can be referring to Folkman J. (1985) Tumor angiogenesis (tumor-blood-vessel growth) .Adv Cancer Res.1985 as the special discussion of the angiogenesis in the relevant diease occurrences such as degeneration of macula, endometriosis and obesity of cancer, rheumatoid arthritis, diabetic retinopathy, the age morbid state of science; 43:175-203.; Folkman, J. (2001) .Angiogenesis-dependent diseases (angiogenesis-dependence disease) .Semin Oncol, 28,536-42.; Grosios, K., Wood, J., Esser, R., Raychaudhuri, A.﹠amp; Dawson, J. (2004) .Angiogenesis inhibition by the novel VEGF receptortyrosine kinase inhibitor, PTK787/ZK222584, causes significantanti-arthritic effects in models of rheumatoid arthritis (new vegf receptor tyrosine kinase inhibitor PTK787/ZK222584 causes significant arthritis effect to being suppressed at of angiogenesis in the rheumatoid arthritis model) .Inflamm Res, 53,133-42; Hull, M.L., Charnock-Jones, D.S., Chan, C.L., Bruner-Tran, K.L., Osteen, K.G., Tom, B.D., Fan, T.P.﹠amp; Smith, S.K. (2003); Antiangiogenic agents areeffective inhibitors of endometriosis (anti-angiogenic agent is endometriotic effective inhibitor) .J Clin Endocrinol Metab, 88,2889-99; Liu, L.﹠amp; Meydani, M. (2003); Angiogenesis inhibitors may regulate adiposity (angiogenesis inhibitor scalable obesity) .Nutr Rev, 61,384-7; Mousa, S.A.﹠amp; Mousa, A.S. (2004); Angiogenesis inhibitors:current ﹠amp; Future directions (angiogenesis inhibitor: current with direction future) .Curr Pharm Des, 10,1-9.Be incorporated herein by reference at this full content each above-mentioned list of references.

Chemical compound disclosed by the invention suppresses angiogenesis.This is for example confirmed by formula II-16 chemical compound that this chemical compound is the inductive multiple myeloma cells migration of vegf blocker (VEGF) in striding hole (transwell) migration analysis.Other chemical compound disclosed by the invention is tested in striding hole migration analysis, and suppresses migration.

Chemical compound disclosed by the invention is in multiple other angiogenesis test and analyze in any one of (comprise following one or more) and show angiogenesis inhibiting activity.

Chemical compound disclosed by the invention shows anti-angiogenesis activity in multiple other external and body inner analysis.Some examples comprise: the analyzed in vitro of estimating anti-angiogenic compounds comprises, 1) the Boyden chamber of improvement is analyzed, endothelial cell migration (the Alessandri G of angiogenesis factor (pro-angiogenicfactor) before its assessment response, Raju K, Gullino PM. (1983) " Mobilization of capillary endothelium in vitro induced by effectors ofangiogenesis in vivo " (" by moving of the intravital inductive external capillary endothelium of angiogenic effect ") Cancer Res.43 (4): 1790-7.), 2) fractional analysis, for example Matrigel analyzes, wherein analyzing endotheliocyte adheres to, move and be divided into tubule (Lawley TJ, Kubota Y. (1989) .Induction of morphologic differentiation of endothelial cells inculture (morphology of endotheliocyte differentiation in the inducing culture thing) .J Invest Dermatol.Aug; 93 (2 Suppl): 59S-61S) with 3) organ culture analyzes, wherein monitor the endotheliocyte growth of (with other cell) (Nicosia RF, Ottinetti A. (1990) .Growth of microvessels inserum-free matrix culture of rat aorta (the blood capillary growth in the serum-free substrate culture of rat aorta) .A quantitative assay of angiogenesis in vitro (the external quantitative analysis of angiogenesis) .Lab Invest.Jul; 63 (1): 115-22.)。Some body inner analysis of estimating angiogenesis inhibitor are 1) sponge implantation analysis, to contain in this analysis that the sponge of cell and/or angiogenesis factor and test substances are subcutaneous to be implanted to (Plunkett ML, Hailey JA. (1990) in the animal body that is used for research angiogenesis in the body.An in vivo quantitativeangiogenesis model using tumor cells entrapped in alginate (use is embedded in the interior quantitatively angiogenesis model of body of the tumor cell in the alginate) .Lab Invest.1990Apr; 62 (4): 510-7), 2) the chorioallantois film analysis of chicken inserts test compounds in this analysis and passes window, inserts eggshell.The shortage of maturation immunity system allows angiogenesis (Folkman J. (1985) Tumor angiogenesis (tumor-blood-vessel growth) the .Adv Cancer Res.1985 of research tumor promotion in 7-8 days instar chicken embryos; 43:175-203.) and 3) the kinds of tumors model, special histologic analysis can be used for checking the effect to blood vessel in this model, as vessel density (CD31/CD34 dyeing), blood flow and association neoplasm necrosis/apoptosis (TUNEL dyeing).The example of analyzed in vitro comprises endotheliocyte test (HUVEC (Human umbilical vein endothelial cells), aorta, blood capillary); Endothelial cell proliferation is analyzed; Endotheliocyte DNA synthesis analysis; The endothelial cell growth thing is analyzed (aortic annulus); Endothelial cell migration is analyzed (above-mentioned; Chemokinesis phenomenon (aurosol), chemotaxis (Boyden chamber)); The endotheliocyte pipe forms to be analyzed; Endothelial cell apoptosis is analyzed; The endotheliocyte viability is analyzed (trypan blue); The endothelial cell line of angiogenesis factor-transfection; With the magnetization microballon on endotheliocyte.Be incorporated herein by reference at this full content each list of references in this section.

The example of body inner analysis comprises transparent chambers test (for example, rabbit ear, hamster cheek, cranium window and dorsal part skin); Substrate implant (for example, using subcutaneous injection, subcutaneous disk (polyethylene implant), rat dorsal part alveolar, the sponge implant of sodium alginate); For example the cornea crystallite lattice (cornea micropocket) in rabbit and other rodent are analyzed; Preceding eye/iris chamber is implanted analysis, mice and is knocked out analysis; At pig and the intravital ameroid constriction of Canis familiaris L. (heart); Family's rabbit hind leg ischemia test; In-house vascularization (intradermal vaccination, contain the implant peritoneal cavity/nethike embrane; The tumor implant is for example in rabbit, mice or rat body.

In addition, use disclosed chemical compound to implement earlier external back body inner analysis.Example comprises CAM (chicken chorioallantois film analysis) and the vertical CAM that utilizes polymer gel.Immunoassay for example is serum analysis, urinalysis, cerebrospinal fluid analysis and histogenic immunity tissue chemical analysis.

Some that described in following paper in the above-mentioned analysis are analyzed, and are incorporated herein by reference at this full content with each paper.People such as Grant, In Vitro Cell Dev.Biol.27A:327-336 (1991); People such as Min, Cancer Res.56:2428-2433 (1996); People such as Schnaper, J.Cell.Physiol.165:107-118 (1995); People such as Schnaper, J.Cell.Physiol.165:107-118 (1995); People such as Oikawa, Cancer Lett.59:57-66 (1991).

Embodiment related to chemical compound of the present invention and compositions individually or with other reagent use in conjunction, to suppress angiogenesis and treatment or alleviation disease and the morbid state relevant with excessive or unsuitable angiogenesis.Preferably, suppress relevant, and this vascularization is relevant with the disease relevant with angiogenesis with vascularization, this disease for example be cancer or above-mentioned other disease with the known disease of those those skilled in that art in any.Described chemical compound and compositions can be carried with suitable amount of suppression.Amount of suppression is meant when to tissue, animal or individual administration, makes degree, quantity or the speed aspect of neovascularization weaken the amount of needed chemical compound or compositions.The effective required chemical compound or the dosage of compositions depend in the treatment, for example, angiogenesis-dependent disease, route of administration and form to be treated, by the effectiveness of the molecule of administration and big active half-life (big-active half-life), tissue, animal or individual weight and state and before or collaborative therapy.Those skilled in the art can adopt guidance provided by the invention to measure the suitable application quantity of described method.For example, can know described amount by inference from angiogenesis analysis in the above-mentioned external or body.Those skilled in the art generally acknowledges in the process of treatment needs to monitor from start to finish patient's state, thereby can adjust the amount of the compositions of administration.

Chemical compound of the present invention and compositions can be and also can be used in combination with other angiogenesis inhibitor.Angiogenesis inhibitor is well known in the art, and can prepare by known method.For example, angiogenesis inhibitor comprises that integrin suppresses chemical compound, and ([α V β 3] integrin suppresses antibody, and cell adhesion protein or its comprise the function fragment of cell adhesion binding sequence as α-V-β-3.Other angiogenesis inhibitor comprises, for example, angiostatin (angiostatin), the function fragment of angiostatin, endostatin, fibroblast growth factor (FGF) inhibitor, the FGF acceptor inhibitor, the VEGF inhibitor, the vegf receptor inhibitor, vascular permeability factor (VPF) inhibitor, the VPF acceptor inhibitor, thrombospondin, platelet factor 4, interferon-' alpha ', interferon-, interferon inducible protein 10, interleukin 12, the 16kDa N-terminal fragment of gro-β and prolactin antagonist, other mechanism of Thalidomide and inhibition angiogenesis.

Therefore, described method can comprise chemical compound or compositions to the step of suffering from the animals administer of the morbid state that excessively angiogenesis is relevant.Described method also can comprise chemical compound of the present invention or compositions and another anti-angiogenic medicaments or the together administration of other therapies (for example, with chemotherapy or immunotherapy treatment cancer) of the morbid state that is used for being treated.

Described chemical compound or compositions can anyly be suitable for disease and/or patient's mode and carry.Example comprises, mode such as intravenous, oral, intramuscular, ophthalmic, intranasal, intraperitoneal is carried.

Provide about being used for the other textbook of use, administration and analytical method that angiogenesis suppresses below with reference to document: J.Clifford MurrayAngiogenesis Protocols (angiogenesis treatment scheme) (Methods in Molecular Medicine) (molecular medicine method), Humana Press (March 15 calendar year 2001) ISBN:0896036987; R.J.Bicknell. Claire E.Lewis, Napoleone FeUmour Angiogenesis (tumor-blood-vessel growth), Oxford University Press (on JIUYUE 1st, 1997) ISBN:0198549377; With Gabor M.RubanviAngiogenesis in Health and Disease:Basic Mechanismsand Clinical Applications (angiogenesis in the healthy and disease: mechanism and clinical practice substantially), Marcel Dekker (on November 1st, 1999) ISBN:0824781023.Be incorporated herein by reference at this full content every book.Especially be incorporated herein therapeutic scheme and method.

Embodiment 63

The preparation that is used for oral administration or its similar type

To fully be mixed into mixture by the method acquisition of described embodiment and 1g chemical compound, 98g lactose and the 1g hydroxypropyl cellulose of purification, this mixture be made granule by conventional method.With this granule finish-drying and screening granular preparation to obtain to be suitable for to bottle or seal.Depend on symptom, as the suitable dose that those of ordinary skill is thought in the cancerous tumour field in the treatment human body, with the granular preparation of gained with about 100ml/ days about 1000ml/ days oral administrations extremely.

Embodiment 64

Treatment is to the Gleevec sensitivity and anti-Gleevec

Tumor

Chronic myelocytic leukemia (CML) cell and acute lymphoblastic leukemia (ALL) cell in most of the cases are shared in undiscovered chromosomal abnormality in the normal cell.Be called t (9; 22) this is the mutual transposition between

chromosome

9 and 22 unusually.This transposition causes chromosome 9 longer and chromosome 22 is shorter than normal chromosome than normal chromosome.The fracture of chromosome 22 occurs in the middle of the gene that is called BCR.Resulting chromosome 22, i.e. Philadelphia chromosome (Ph ¹) have a BCR part that merges with the proto-oncogene of the most c-ABL of being called.The fragment of this fusion forms unusual " bcr-abl " oncogene, the unusual tyrosine kinase of its coding continuous activation, irritation cell division and anti-apoptotic.

Gleevec

(STI-571, signal transduction inhibitor 571, or imatinib mesylate) is the selective depressant of BCR-ABL tyrosine kinase, the origin cause of formation sudden change that this BCR-ABL tyrosine kinase is CML.Gleevec

Or the inhibitor of the receptor tyrosine kinase of stem cell factor (SCF), c-KIT, and suppress platelet-derived somatomedin (PGDF) receptor and the cytohistology variation of SCF-mediation.In clinical research, Gleevec

In 60% quilt treatment patient, show and reduce gastrointestinal stromal tumor (GIST).In CML, seen similarly significant effect.But, in all patients, all produce Gleevec by the mechanism of multiple proposition

Drug resistance.Be not subjected to the constraint of any particular theory, to Gleevec

Possible resistance mechanism may be because of cross the expressing of bcr-abl, Gleevec

The sudden change in the bonded abl-kinase domain or the activation of other carcinogenic approach cause.

Chemical compound disclosed herein is used for the treatment of Gleevec

Responsive and anti-Gleevec

Tumor.This is confirmed that by for example formula II-16 chemical compound this chemical compound has been proved at CML, Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph ⁺ALL; Comprise new isolating Ph from the patient ⁺The ALL cell) cell death inducing and in acute myeloblastic leukemia (AML) cell line.Formula II-16 chemical compound can be used as independent reagent and uses or and Gleevec

The associating use.Especially, formula II-16 chemical compound treatment responsive or drug-fast Philadelphia chromosome-positive leukemia in can be used as independent reagent and use or and Gleevec

The associating use.

Formula II-16 chemical compound or any other chemical compound that comprises formula I, II, III, IV, V and VI chemical compound can be used for treatment to Gleevec

Responsive tumor and anti-Gleevec

Tumor.

The above-mentioned embodiment that provides is only in order to help to understand described embodiment.Therefore, it be appreciated for those skilled in the art that described method also can provide the derivant of chemical compound.

Technical staff under this area understands easily, and the present invention is fit to realize described purpose and reach described target and advantage well, and other inherent inherent character.Method that the present invention describes and operation only are to represent preferred embodiment, and are exemplary, can not be interpreted as limitation of the scope of the invention.Those skilled in the art can change and other application of the present invention making, and this all is included in the spirit of the present invention.

Those skilled in the art be it is evident that do not departing under the scope and spirit of the present invention, can make different substitutions and modifications described embodiment disclosed by the invention.

The technical staff's that all patents mentioned in this description and publication are indicating the technical field of the invention level.At this all patents and publication are incorporated herein by reference, point out to be introduced into as a reference clearly and individually as each independent patent.

The present invention of the exemplary description of this paper can be suitable for not implementing when the present invention has disclosed especially any key element or various key element, any restriction or various restriction not to exist.Used term and expression way all are used for illustration purpose and unrestricted purpose, and do not have purpose to show that this term of use and expression way mean eliminating equivalent shown or described feature or its part.Be recognized that within the scope of the invention and can carry out various modifications.Therefore, though should understand the present invention is undertaken specifically open by embodiment preferred and optional feature, but those skilled in the art can adopt the modification and the conversion of notion disclosed by the invention, and this modification and conversion should be thought to belong in the scope of the present invention.

Claims

1. acceptable salt of chemical compound and medicine thereof and esters prodrug are used for handling the purposes of the medicine of mdr cell in preparation, and wherein said chemical compound has the general formula I structure:

General formula I

Wherein dotted line is represented singly-bound or two key, wherein R ₁Be independently selected from single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl; wherein n equals 1 or 2; if and n equals 2, then R ₁Can be identical or different;

R wherein ₂Be selected from single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl;

R wherein ₃Be selected from single replacement of halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and the haloalkyl that comprises multi-haloalkyl;

Each E wherein ₁, E ₂, E ₃And E ₄For replacing or unsubstituted hetero atom; And

Wherein said mdr cell is the cancerous cell that is selected from leukemia, ovarian cancer, carcinoma of ureter, bladder cancer, carcinoma of prostate, rectal cancer, gastric cancer, lymphoma, multiple myeloma, cancer of pancreas, hepatocarcinoma, renal carcinoma, endocrine cancer, sarcoma, skin carcinoma, hemangioma and the brain cancer.

2. purposes as claimed in claim 1, wherein said chemical compound are general formula I I structure, with and acceptable salt of medicine and esters prodrug:

R wherein ₃Be independently selected from single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl;

R wherein ₄Be independently selected from single replacement of hydrogen, halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl; cyano group and the haloalkyl that comprises multi-haloalkyl; wherein m equals 1 or 2; if and m equals 2, then R ₄Can be identical or different; And

Each E wherein ₁, E ₂, E ₃, E ₄And E ₅For replacing or unsubstituted hetero atom.

3. purposes as claimed in claim 1, wherein said chemical compound are the general formula III structure, with and acceptable salt of medicine and esters prodrug:

Wherein dotted line is represented singly-bound or two key, wherein R ₁Be independently selected from hydrogen, halogen, single replacement of following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl; wherein n equals 1 or 2; if and n equals 2, then R ₁Can be identical or different;

R wherein ₄Be independently selected from single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl; wherein m equals 1 or 2; if and m equals 2, then R ₄Can be identical or different; And

Each E wherein ₁, E ₂, E ₃, E ₄And E ₅Be to replace or unsubstituted hetero atom.

4. purposes as claimed in claim 1, wherein said chemical compound are general formula I V structure, with and acceptable salt of medicine and esters prodrug:

Wherein dotted line is represented singly-bound or two key, wherein R ₁Be independently selected from single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro; azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl; wherein n equals 1 or 2; if and n equals 2, then R ₁Can be identical or different;

R wherein ₃Be independently selected from single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl;

R wherein ₅Be independently selected from single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro; azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl; wherein m equals 0,1,2,3,4,5,6,7,8,9,10 or 11; if and m is greater than 1, then R ₅Can be identical or different; And

Each E wherein ₁, E ₂, E ₃, E ₄And E ₅Be that replace or unsubstituted hetero atom.

5. purposes as claimed in claim 1, wherein said chemical compound are general formula V structure, with and acceptable salt of medicine and esters prodrug:

6. purposes as claimed in claim 1, wherein said chemical compound are general formula VI structure, with and acceptable salt of medicine and esters prodrug:

General formula VI

R wherein ₁Can be independently selected from single replacement of following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, phenyl, cycloalkyl acyl group, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, sulfo-, sulfoxide, sulfone borate and the haloalkyl that comprises multi-haloalkyl; n equals 1 or 2; if and n equals 2, then R ₁Can be identical or different;

R wherein ₃Can be selected from single replacement of halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, the ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, the cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group, sulfo-, sulfoxide, sulfone, sulphonic acid ester, thiocyano, boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl; Each E wherein ₁, E ₂, E ₃And E ₄Can be to replace or unsubstituted hetero atom.

7. as the described purposes of arbitrary claim among the claim 1-6, wherein said chemical compound is:

R wherein ₈Be selected from H, F, Cl, Br and I.

8. purposes as claimed in claim 7, wherein said chemical compound are the structure of formula II-16, with and acceptable salt of medicine and esters prodrug:

9. as the described purposes of arbitrary claim among the claim 1-8, wherein said mdr cell is chronic myelocytic leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), sarcoma or multiple myeloma.

10. as the described purposes of arbitrary claim among the claim 1-9, wherein said mdr cell is MES-SA/Dx5, HL-60/MX2, MM.1R multiple myeloma or the Dox-40 of anti-doxorubicin multiple myeloma.

11. as the described purposes of arbitrary claim among the claim 1-10, wherein said cell to its drug-fast medicine be bortezomib,

Mitoxantrone hydrochloride, doxorubicin, daunorubicin, actinomycin D, vincristine, paclitaxel, colchicine, ametycin, Rituximab, dexamethasone, Thalidomide, Paclitaxel or American and French human relations.

12. suppress the method for mdr cell growth, comprise described cell is contacted with the esters prodrug with chemical compound with general formula I structure and the acceptable salt of medicine thereof:

General formula I

Each E wherein ₁, E ₂, E ₃And E ₄Be replacement or unsubstituted hetero atom,

Wherein said cancer is leukemia, ovarian cancer, carcinoma of ureter, bladder cancer, carcinoma of prostate, rectal cancer, gastric cancer, lymphoma, multiple myeloma, cancer of pancreas, hepatocarcinoma, renal carcinoma, endocrine cancer, sarcoma, skin carcinoma, hemangioma or the brain cancer.

13. method as claimed in claim 12, wherein said chemical compound are general formula I I structure, with and acceptable salt of medicine and esters prodrug:

14. method as claimed in claim 12, wherein said chemical compound are the general formula III structure, with and acceptable salt of medicine and esters prodrug:

15. method as claimed in claim 12, wherein said chemical compound are general formula I V structure, with and acceptable salt of medicine and esters prodrug:

R wherein ₃Be independently selected from single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkanes acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl;

R wherein ₅Be independently selected from single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro; azido, phenyl, cycloalkanes acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl; wherein m equals 0,1,2,3,4,5,6,7,8,9,10 or 11; if and m is greater than 1, then R ₅Can be identical or different; And

16. method as claimed in claim 12, wherein said chemical compound are general formula V structure, with and acceptable salt of medicine and esters prodrug:

17. method as claimed in claim 12, wherein said chemical compound are general formula VI structure, with and acceptable salt of medicine and esters prodrug:

General formula VI

18. as the described method of arbitrary claim among the claim 12-17, wherein said chemical compound is:

R wherein ₈Be selected from H, F, Cl, Br and I.

19. method as claimed in claim 18, wherein said chemical compound are the structure of formula II-16, with and acceptable salt of medicine and esters prodrug:

20. as the described method of arbitrary claim among the claim 12-19, wherein said mdr cell is chronic myelocytic leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), sarcoma or multiple myeloma.

21. as the described method of arbitrary claim among the claim 12-20, wherein said mdr cell is MES-SA/Dx5, HL-60/MX2, MM.1R multiple myeloma or the Dox-40 of anti-doxorubicin multiple myeloma.

22. as the described method of arbitrary claim among the claim 12-21, wherein said cell to its drug-fast medicine be bortezomib,

Mitoxantrone hydrochloride, doxorubicin, actinomycin D, daunorubicin, vincristine, paclitaxel, colchicine, ametycin, Rituximab, dexamethasone, Thalidomide, Paclitaxel or American and French human relations.

23. acceptable salt of chemical compound and medicine thereof and esters prodrug thereof are used for the treatment of purposes in the medicine of the disease with excessive or unsuitable angiogenesis feature in preparation, wherein said chemical compound has the general formula I structure:

General formula I

24. purposes as claimed in claim 23, wherein said chemical compound are general formula I I structure, with and acceptable salt of medicine and esters prodrug:

25. purposes as claimed in claim 23, wherein said chemical compound are the general formula III structure, with and acceptable salt of medicine and esters prodrug:

26. purposes as claimed in claim 23, wherein said chemical compound are general formula I V structure, with and acceptable salt of medicine and esters prodrug:

27. purposes as claimed in claim 23, wherein said chemical compound are general formula V structure, with and acceptable salt of medicine and esters prodrug:

28. purposes as claimed in claim 23, wherein said chemical compound are general formula VI structure, with and acceptable salt of medicine and esters prodrug:

General formula VI

29. as the described purposes of arbitrary claim among the claim 23-28, wherein said chemical compound is:

R wherein ₈Be selected from H, F, Cl, Br and I.

30. purposes as claimed in claim 29, wherein said chemical compound are the structure of formula II-16, with and acceptable salt of medicine and esters prodrug:

31. as the described purposes of arbitrary claim among the claim 23-30, wherein said disease is the inflammatory diseases state.

32. purposes as claimed in claim 31, wherein said inflammatory diseases state are immunity and non-immunity inflammation, rheumatoid arthritis, beaevais' disease and psoriasis.

33. as the described purposes of arbitrary claim among the claim 23-32, wherein said disease has the feature of inappropriate or untimely vascular invasion.

34. purposes as claimed in claim 33, wherein said disease are blood capillary proliferation, osteoporosis, leukemia, fibrohemangioma, Kaposi sarcoma, hemangioma, acoustic neuroma, neurofibroma, trachoma and botryomycosis hominis in diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, degeneration of macula, corneal graft rejection, Terry's sign disease, red stain, the atheromatous plaque.

35., also comprise the use of anti-angiogenic agent as the described purposes of arbitrary claim among the claim 23-34.

36. being α-V-β-3 (α V β 3) integrin, purposes as claimed in claim 35, wherein said anti-angiogenic agent suppress antibody, cell adhesion protein, the function fragment that comprises the cell adhesion protein of cell adhesion binding sequence, angiostatin, the function fragment of angiostatin, endostatin, fibroblast growth factor (FGF) inhibitor, the FGF acceptor inhibitor, the VEGF inhibitor, the vegf receptor inhibitor, vascular permeability factor (VPF) inhibitor, the VPF acceptor inhibitor, thrombospondin, platelet factor 4, interferon-' alpha ', interferon-, interferon-induced protein 10, interleukin 12, the 16kDa N-terminal fragment and the Thalidomide of gro-β and prolactin antagonist.

37. as the described purposes of arbitrary claim among the claim 23-36, wherein said animal is a mammal.

38. as the described purposes of arbitrary claim among the claim 23-36, wherein said animal is behaved.

39. as the described purposes of arbitrary claim among the claim 23-36, wherein said animal is a rodent.

40., also comprise the steps: as the described purposes of arbitrary claim among the claim 23-36

The individuality that the meeting of discriminating is benefited from the administration of the reagent of inhibition angiogenesis;

To the described individual described method of implementing.

41. suppress the method for angiogenesis, comprise and will have the chemical compound of general formula I structure and the acceptable salt of medicine thereof and esters prodrug to animals administer:

General formula I

42. method as claimed in claim 41, wherein said chemical compound are general formula I I structure, with and acceptable salt of medicine and esters prodrug:

43. method as claimed in claim 41, wherein said chemical compound are the general formula III structure, with and acceptable salt of medicine and esters prodrug:

44. method as claimed in claim 41, wherein said chemical compound are general formula I V structure, with and acceptable salt of medicine and esters prodrug:

45. method as claimed in claim 41, wherein said chemical compound are general formula V structure, with and acceptable salt of medicine and esters prodrug:

46. method as claimed in claim 41, wherein said chemical compound are general formula VI structure, with and acceptable salt of medicine and esters prodrug:

General formula VI

R wherein ₃Can be selected from single replacement of halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂₄Alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, the ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, the cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group, sulfo-, sulfoxide, sulfone, sulphonic acid ester, thiocyano, boric acid and ester thereof and the haloalkyl that comprises multi-haloalkyl; Each E wherein ₁, E ₂, E ₃And E ₄For replacing or unsubstituted hetero atom.

47. as the described method of arbitrary claim among the claim 41-46, wherein said chemical compound is:

R wherein ₈Be selected from H, F, Cl, Br and I.

48. method as claimed in claim 47, wherein said chemical compound are the structure of formula II-16, with and acceptable salt of medicine and esters prodrug:

49. as the described method of arbitrary claim among the claim 41-48, wherein said animal suffers from disease, wherein said disease is the inflammatory diseases state.

50. method as claimed in claim 49, wherein said inflammatory diseases state are immunity and non-immunity inflammation, rheumatoid arthritis, beaevais' disease and psoriasis.

51. as the described method of claim 49-50, wherein said disease has the feature of inappropriate or untimely vascular invasion.

52. method as claimed in claim 51, wherein said disease are blood capillary proliferation, osteoporosis, leukemia, fibrohemangioma, Kaposi sarcoma, hemangioma, acoustic neuroma, neurofibroma, trachoma and botryomycosis hominis in diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, degeneration of macula, corneal graft rejection, Terry's sign disease, red stain, the atheromatous plaque.

53., also comprise the use of anti-angiogenic agent as the described method of arbitrary claim among the claim 41-52.

54. being α-V-β-3 (α V β 3) integrin, method as claimed in claim 53, wherein said anti-angiogenic agent suppress antibody, cell adhesion protein, the function fragment that comprises the cell adhesion protein of cell adhesion binding sequence, angiostatin, the function fragment of angiostatin, endostatin, fibroblast growth factor (FGF) inhibitor, the FGF acceptor inhibitor, the VEGF inhibitor, the vegf receptor inhibitor, vascular permeability factor (VPF) inhibitor, the VPF acceptor inhibitor, thrombospondin, platelet factor 4, interferon-' alpha ', interferon-, interferon-induced protein 10, interleukin 12, the 16kDa N-terminal fragment and the Thalidomide of gro-β and prolactin antagonist.

55. as the described method of arbitrary claim among the claim 41-54, wherein said animal is a mammal.

56. as the described method of arbitrary claim among the claim 41-55, wherein said animal is behaved.

57. as the described method of arbitrary claim among the claim 41-56, wherein said animal is a rodent.

58., also comprise the steps: as the described method of arbitrary claim among the claim 41-58

To the described individual described method of implementing.

59. it is right to treat

Responsive or anti-

Method for cancer, comprise and will have the chemical compound of general formula I structure and the acceptable salt of medicine thereof and esters prodrug to animals administer:

General formula I

R wherein ₃Be selected from single replacement of halogen and following residue, polysubstituted or unsubstituted variant: saturated C ₁-C ₂4 alkyl, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro, azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and the haloalkyl that comprises multi-haloalkyl;

60. method as claimed in claim 59, wherein said chemical compound are general formula I I structure, with and acceptable salt of medicine and esters prodrug:

61. method as claimed in claim 59, wherein said chemical compound are the general formula III structure, with and acceptable salt of medicine and esters prodrug:

62. method as claimed in claim 59, wherein said chemical compound are general formula I V structure, with and acceptable salt of medicine and esters prodrug:

Wherein dotted line is represented singly-bound or two key, wherein R ₁Be independently selected from single replacement of hydrogen, halogen, following residue, polysubstituted or unsubstituted variant: saturated C ₁-C _{24 alkane}Basic, undersaturated C ₂-C ₂₄Thiazolinyl or C ₂-C ₂₄Alkynyl, acyl group, acyloxy, oxyl ketonic oxygen base, aryloxycarbonyl oxygen base, cycloalkyl, cycloalkenyl group, oxyl, ring oxyl, aryl, heteroaryl, aryl oxyl carbonyl, oxyl carbonyl acyl group, amino, amino carbonyl, amino carbonyl oxygen base, nitro; azido, phenyl, cycloalkyl acyl group, hydroxyl, alkylthio group, arylthio, oxygen base sulfonyl, carboxyl, cyano group and comprise the haloalkyl of multi-haloalkyl; wherein n equals 1 or 2; if and n equals 2, then R ₁Can be identical or different;

63. method as claimed in claim 59, wherein said chemical compound are general formula V structure, with and acceptable salt of medicine and esters prodrug:

64. method as claimed in claim 59, wherein said chemical compound are general formula VI structure, with and acceptable salt of medicine and esters prodrug:

General formula VI

65. as the described method of arbitrary claim among the claim 59-64, wherein said chemical compound is:

R wherein ₈Be selected from H, F, Cl, Br and I.

66. as the described method of claim 65, wherein said chemical compound is the structure of formula II-16, with and acceptable salt of medicine and esters prodrug:

67. as the described method of arbitrary claim among the claim 59-66, wherein said cancer is chronic myelocytic leukemia, Philadelphia chromosome-positive acute lymphoblastic leukemia and acute myeloblastic leukemia.

68., also comprise as the described method of arbitrary claim among the claim 59-66

Use.

69. as the described method of arbitrary claim among the claim 59-66, wherein said animal is a mammal.

70. as the described method of arbitrary claim among the claim 59-66, wherein said animal is behaved.

71. as the described method of arbitrary claim among the claim 59-66, wherein said animal is a rodent.

72. the treatment method for cancer comprises and will have the chemical compound of general formula VI structure and the acceptable salt of medicine thereof and esters prodrug to animals administer:

General formula VI

73. as the described method of claim 72, wherein said cancer is a multiple myeloma.

74. as the described method of claim 72, wherein said cancer is a carcinoma of prostate.

75. as the described method of claim 72, wherein said cancer is an ovarian cancer.

76. as the described method of claim 72, wherein said cancer is a cancer of pancreas.

77., also comprise the use of chemotherapeutant as the described method of claim 72.

78. as the described method of arbitrary claim among the claim 72-77, wherein said chemotherapeutant is an amycin, doxorubicin, 5-fluorouracil, cytosine arabinoside (" Ara-C "), cyclophosphamide, plug is for group, busulfan, cytotoxin, paclitaxel, taxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, ametycin, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunorubicin, carminomycin, aminopterin-induced syndrome, actinomycin D, mitomycin, the enediyne class, melphalan, zitazonium or onapristone.

79. as the described method of claim 72, wherein said cancer is drug-fast cancer.

80. as the described method of claim 79, wherein said drug-fast cancer is sarcoma, multiple myeloma or leukemia.

81. as the described method of claim 80, wherein said drug-fast cancer shows at least a of following situation: the level of P-glycoprotein outflow pump improves, by expression increase, the drug absorption of the multi-drug resistant associated protein 1 of MRP1 coding reduce, the change of drug targets or to the reparation enhancing of drug-induced DNA damage, the change of apoptotic pathways and the activation of cytochrome P 450 enzymes.

82. as the described method of claim 72, wherein said animal is a mammal.

83. as the described method of claim 72, wherein said animal is behaved.

84. as the described method of claim 72, wherein said animal is a rodent.

85., also comprise the steps: as the described method of claim 72

Differentiate the individuality that from the administration of anticarcinogen, to be benefited;

To the described individual described method of implementing.