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CN118561953A - Macrocyclic alkynyl thioether peptide compound and preparation method and application thereof - Google Patents

Macrocyclic alkynyl thioether peptide compound and preparation method and application thereof Download PDF

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CN118561953A
CN118561953A CN202310881207.3A CN202310881207A CN118561953A CN 118561953 A CN118561953 A CN 118561953A CN 202310881207 A CN202310881207 A CN 202310881207A CN 118561953 A CN118561953 A CN 118561953A
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compound
cancer
macrocyclic
reaction
thioether peptide
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庄让笑
曹宇
席建军
张建康
何若愚
刘寿荣
黄劲松
喻剑华
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Hangzhou Xixi Hospital
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P35/02—Antineoplastic agents specific for leukemia
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention provides a macrocyclic alkynyl thioether peptide compound, a preparation method and application thereof, wherein the structural formula of the macrocyclic alkynyl thioether peptide compound is as follows:

Description

Macrocyclic alkynyl thioether peptide compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a macrocyclic alkynyl thioether peptide compound, and a preparation method and application thereof.
Background
Malignant tumor has become one of the main public health problems seriously threatening human health, and according to the results shown in the latest statistics data of journal of clinical tumor, about 1930 ten thousand cancer cases are newly added in 2020, and about 1000 ten thousand cancer death cases are caused (CA-cancer. J. Clin.2021,71, 209-249). The development of anticancer drugs has been an important task to solve human health problems in recent decades, as malignant tumors have been leading to an increasing trend of morbidity and mortality.
The proteasome is a giant protein complex in organisms, and has important functions of degrading misfolded, unwanted or damaged proteins in cells, maintaining protein homeostasis in cells, and the like. In addition to maintaining constant protein levels in the body, proteasomes are involved in a variety of vital activities including cell cycle regulation, apoptosis, gene transcription regulation, DNA repair, and antigen presentation. For tumor cells, the tumor cells mainly realize cell growth by degrading some cancer-inhibiting factors and cell cycle checkpoint inhibiting factors through the proteasome, and have certain dependence on the proteasome, so that the aim of tumor treatment can be achieved by inhibiting the function of the proteasome.
Three short peptide proteasome inhibitor small molecules are currently available in the market for the treatment of multiple myeloma. There are also several inhibitor molecules entering clinical studies that more demonstrate the effectiveness of proteasomes as tumor therapeutic targets. The proteasome inhibitor is injected, has poor compliance, develops a compound which can be orally administrated, and has important practical significance for improving the metabolic stability and the oral bioavailability.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a brand-new macrocyclic alkynyl thioether peptide proteasome inhibitor, a preparation method and application thereof, and the compounds are subjected to proteasome inhibition activity evaluation and tumor cell proliferation inhibition activity evaluation. The results show that the compounds have better proteasome inhibition activity and cell proliferation inhibition activity, are used as a novel skeleton cyclic peptide proteasome inhibitor, and are expected to become effective choices for clinical treatment of tumors.
Definition of terms:
The term "heterocyclyl" as used herein refers to a monocyclic group having 4 to 6 ring atoms in the ring, at least one or two of which are heteroatoms selected from nitrogen, oxygen, boron, the remaining ring atoms being carbon atoms. Specific heterocyclic groups may be boron ester heterocyclic groups, oxetanyl groups, and the like.
The term "aralkyl" as used herein refers to an alkyl group substituted with an aryl group, preferably a phenyl group, the alkyl group preferably having 1 to 6 carbon atoms, and specifically may be benzyl, phenethyl, benzisopropyl, and the like.
The term "halogen" as used herein means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
A macrocyclic alkynyl thioether peptide compound, the structural formula of the macrocyclic alkynyl thioether peptide compound is as follows:
wherein R is selected from one of aralkyl, halogenated aralkyl and C 1-4 alkyl-D-Ra heterocyclic aryl.
Preferably, the aralkyl group is benzyl.
Preferably, the halogenated aralkyl group is a benzyl group substituted with a chlorine atom in the ortho position on the benzene ring.
Preferably, the C 1-4 alkyl is isobutyl.
Preferably, D is selected from one of C, CCO.
Preferably, ra is selected from one of an oxetanyl group and a boroester heterocycle.
Preferably, the macrocyclic alkynyl thioether peptide compound is selected from one of the following compounds:
(3 s,6s,9 r) -9-amino-N, 6-dibenzyl-5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide;
(3 s,6s,9 r) -9-amino-6-benzyl-N- (2-chlorobenzyl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide;
(3 s,6s, 9R) -9-amino-6-benzyl-5,8,17-trioxo-N- ((R) -1,2,3, 4-tetrahydronaphthalen-1-yl) -1-oxa-11-thia-4, 7-diazepin-12-ene-3-carboxamide;
(3S, 6S, 9R) -9-amino-6-benzyl-N- ((S) -4-methyl-1- ((R) -2-methoxy-2-yl) -1-oxopentan-2-yl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide;
(3 s,6s, 9R) -9-amino-6-benzyl-N- ((R) -3-methyl-1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) butyl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazacyclohepta-12-yl-3-carboxamide.
The invention also provides a preparation method of the macrocyclic alkynyl thioether peptide compound, which comprises the following steps:
(1) Under the protection of N 2, reacting 5-hexynoic acid with NBS and AgNO 3 in acetone to obtain a compound 2;
(2) Under the action of HOBt, EDCI and DIPEA, the compound 2 reacts with N- { [ (9H-fluoren-9-ylmethyl) oxy ] carbonyl } -L-serine butyl ester to generate a compound 3;
(3) Dissolving the compound 3 in dichloromethane, adding triethylamine to react to remove Fmoc protecting groups, and obtaining a compound 4;
(4) Dissolving CTC resin 5 and Fmoc-Phe-OH in dichloromethane, adding DIPEA under the protection of N 2, stirring, washing, and removing Fmoc protecting groups by using a piperidine solution; then adding condensing agent HBTU and Boc-Cys (Trt) -OH for condensation reaction, adding a cracking buffer solution into the product after the reaction is finished, and stirring to obtain a compound 6;
(5) Condensing the compound 6 and the compound 4 under the action of TCFH and 1-methylimidazole to generate a compound 7;
(6) Dissolving the compound 7 in dichloromethane, adding TFA and triisopropylsilane, and stirring to react to generate a compound 8;
(7) Dissolving 4-tertiary butyl-2- (4-tertiary butyl-2-pyridine) pyridine and 2, 6-lutidine in acetonitrile, adding tetra-acetonitrile copper hexafluorophosphate [ Cu (MeCN) 4PF6 ] under the protection of N 2, and then adding an acetonitrile solution of a compound 8 for reaction to obtain a compound 9;
(8) Dissolving the compound 9 in HAC, and stirring for reaction to obtain a compound 10;
(9) And condensing the compound 10 with primary amine under the action of condensing agents HATU and DIPEA to obtain the macrocyclic alkynyl thioether peptide compound.
Preferably, the primary amine is selected from one of benzylamine, o-chlorobenzylamine, tetrahydronaphthylamine, S-2-amino-4-methyl-1- (R) -2-methyl oxiran-2-yl pentanone, 3-methyl-1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) butane-1-amine.
Preferably, in step (1):
The molar ratio of 5-hexynoic acid to NBS is 1: (1-1.5).
The molar ratio of 5-hexynoic acid to AgNO 3 is 1: (0.1-0.3).
The reaction temperature is room temperature and the reaction time is 1.5-3 h.
After the reaction is finished, the reaction system is extracted by methylene dichloride, an organic layer is washed by water, a saturated sodium chloride solution is washed, anhydrous sodium sulfate is dried and then is decompressed and concentrated, and a pure product compound 2 is obtained through column chromatography separation and purification.
Preferably, in step (2):
The molar ratio of compound 2 to N- { [ (9H-fluoren-9-ylmethyl) oxy ] carbonyl } -L-serine butyl ester was 1: (0.7-1.1).
The molar ratio of compound 2 to HOBt is 1: (1-1.5).
The molar ratio of compound 2 to DIPEA is 1: (1-1.5).
The molar ratio of compound 2 to EDCI is 1: (0.9-1.2).
The reaction solvent is methylene dichloride, the reaction temperature is 23-27 ℃, and the reaction time is 10-15 h.
After the reaction is finished, washing a reaction product by HCl, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence, drying by anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain a crude product of the compound 3; the obtained crude product can be directly used for the next reaction, and can also be used after being purified by column chromatography.
Preferably, in step (3):
the mol volume ratio of the compound 3 to the triethylamine is 0.7-1.1 mol/L.
The reaction temperature is 23-27 ℃ and the reaction time is 10-15 h.
After the reaction, the mixture is concentrated under reduced pressure and purified by column chromatography to obtain the compound 4.
Preferably, in step (4):
the molar ratio of CTC resin 5 to Fmoc-Phe-OH was 1: (0.8-1.2).
The molar ratio of CTC resin 5 to DIPEA was 1: (8-12).
The molar ratio of CTC resin 5 to HBTU was 1: (1.7-2.2).
The molar ratio of CTC resin 5 to Boc-Cys (Trt) -OH was 1: (0.8-1.2).
After the completion of the stirring with DIPEA, the mixture was washed 5 times with DMF.
The piperidine solution was 20% piperidine/DMF solution.
The reaction temperature of the condensation reaction is 23-27 ℃ and the reaction time is 50-70 min.
And after stirring and cracking are finished, filtering and collecting filtrate, and evaporating the solvent under reduced pressure to obtain a crude product of the compound 6.
The lysis buffer may be selected from 20% HFIP/dichloromethane solution.
DIPEA is added in two times, and part of DIPEA is added into a dichloromethane solution of CTC resin 5 and Fmoc-Phe-OH for reaction; the rest DIPEA and HBTU are added into a Boc-Cys (Trt) -OH condensation reaction system together for reaction; the addition ratio of the two parts is 3:2.
Preferably, the specific process of step (4) is as follows:
Dissolving CTC resin 5 and Fmoc-Phe-OH in dichloromethane, dropwise adding DIPEA under the protection of N 2, and stirring; methanol was added, stirred, the solution was discharged, and the solution was washed 5 times with DMF; adding piperidine solution, removing Fmoc protecting group, discharging the solution, and washing with DMF for 5 times; then adding Boc-Cys (Trt) -OH, mixing, adding HBTU and DIPEA, and carrying out reaction under the protection of N 2; during the period, the reaction process is monitored by an ninhydrin test, after the reaction is finished, DMF is used for washing 5 times, methanol is used for washing 3 times, and then vacuum drying is carried out, so that side chain protection peptide is obtained;
Adding a cleavage buffer solution into the obtained side chain protection peptide, and stirring at room temperature for cleavage; after the completion of the cleavage, the filtrate was collected by filtration, and the solvent was distilled off under reduced pressure to obtain a crude product of Compound 6.
Preferably, in step (5):
The molar ratio of compound 6 to compound 4 is 1: (1-1.5).
The molar ratio of compound 6 to TCFH is 1: (1-1.5).
The molar ratio of compound 6 to 1-methylimidazole is 1: (3-4).
The reaction temperature is 23-27 ℃, and the reaction time is 1.5-3 h.
After the reaction, the solvent was distilled off under reduced pressure, and the mixture was purified by preparative high performance liquid chromatography to give compound 7.
Preferably, in step (6):
the molar ratio of the compound 7, TFA and triisopropylsilane is 1: (4-6): (4-6).
The reaction temperature is 23-27 ℃, and the reaction time is 2.5-3.5 h.
After the reaction, the solvent was distilled off under reduced pressure to give a crude product of Compound 8.
Preferably, in step (7):
The molar ratio of 4-tert-butyl-2- (4-tert-butyl-2-pyridine) pyridine to 2, 6-lutidine is 1:
(8～12)。
the molar ratio of compound 8 to copper tetraacetonitrile hexafluorophosphate is 1: (0.05-0.15).
The molar ratio of compound 8 to 4-tert-butyl-2- (4-tert-butyl-2-pyridine) pyridine was 1: (0.1-0.3).
The reaction temperature of the acetonitrile solution added with the compound 8 is 23-27 ℃ and the reaction time is 10-15 h.
After the reaction, the mixture was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography to give compound 9.
Preferably, in step (8):
the molar volume ratio of the compound 9 to the HAC is 80-85 mmol/L.
The stirring reaction temperature is 35-45 ℃ and the reaction time is 14-18 h.
After the completion of the reaction, the mixture was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography to give compound 10.
Preferably, in step (9):
the molar ratio of compound 10 to HATU was 1: (1.2-1.9).
The molar ratio of compound 10 to DIPEA was 1: (1.5-2.5).
The molar ratio of compound 10 to primary amine was 1: (0.8-1.2).
Preferably, in step (9), compound 10 is dissolved in DCM, HATU and DIPEA are added and stirred for 0.5h, and then primary amine is added for reaction; the reaction temperature after the addition of the primary amine was 25℃and the reaction time was 3h.
After the reaction is finished, concentrating under reduced pressure, and purifying by a preparation-high performance liquid chromatography method to obtain the macrocyclic alkynyl thioether peptide compound.
The reaction process of the preparation method is as follows:
the invention also provides a pharmaceutical composition comprising an active ingredient and at least one or more pharmaceutically acceptable carriers or excipients;
Wherein the active component is one or more of stereoisomers or pharmaceutically acceptable salts of the macrocyclic alkynyl thioether peptide compounds.
Preferably, the pharmaceutical composition is administered orally, by injection, by inhalation, by implantation, or the like. Further preferred are injection administration and oral administration.
The pharmaceutical composition of the invention can be prepared into various medicinal dosage forms, such as injection, freeze-dried powder injection, tablet, capsule or granule, etc.
The pharmaceutical composition and various preparations thereof can be prepared by using conventional medicinal carriers, and the applicability is strong.
The invention also provides an application of the macrocyclic alkynyl thioether peptide compound in preparing medicaments for malignant tumors and/or immune diseases.
The invention also provides an application of the pharmaceutical composition in preparing medicines for malignant tumors and/or immune diseases.
Preferably, the malignant tumor is one of myeloma, lymphoma, leukemia, breast cancer, sarcoma, lung cancer, macroglobulinemia, prostate cancer, kidney cancer, colon cancer, rectal cancer, pancreatic cancer, glioma, neuroblastoma, head cancer, neck cancer, liver cancer, thyroid cancer, ovarian cancer, cervical cancer, vulvar cancer, endometrial cancer, bladder cancer, testicular cancer, esophageal cancer, stomach cancer, cheek cancer, nasopharyngeal cancer, oral cancer, skin cancer, and gastrointestinal stromal tumor. Further preferred is one of myeloma and leukemia.
Compared with the prior art, the invention has the beneficial effects that:
The macrocyclic alkynyl thioether peptide compound provided by the invention has good proteasome inhibition activity and good in-vitro proliferation inhibition effect on multiple myeloma cell line RPMI-8226 and acute leukemia cell line MV-4-11. The preparation method of the macrocyclic alkynyl thioether peptide compound has the advantages of easily available raw materials, reasonable route design, mild reaction conditions, high yield of each step, simple operation and suitability for industrial production.
Detailed Description
The invention is further illustrated by reference to examples, which are provided solely for the purpose of illustrating the technical solution of the invention and are not intended to limit the invention in any way.
Note that: in the following examples, phe: phenylalanine; cys: cysteine (S)
Example 1
Step 1, preparation of 6-bromo-5-hexynoic acid (2)
5-Heteroalkynoic acid (Compound 1, 40.0g, 317 mmol) was dissolved in 170mL of acetone, N 2 was protected, the temperature was controlled below 25 ℃, NBS (76.2 g,428 mmol) and AgNO 3 (12.1 g,71.3 mmol) were added respectively, and reacted at room temperature for 2h; water (200 mL) was added, extracted 3 times with dichloromethane (400 mL. Times.3), the organic layer was washed 3 times with water (200 mL. Times.3), washed with saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a yellow oil; finally, purification by silica gel column chromatography (dichloromethane: methanol=100:1 to 20:1) gave 32.0g of compound 2 as a white solid (yield 46.9%).
1HNMR(400MHz,CDCl3):δ2.51(t,J＝7.2Hz,14.4Hz,2H),2.33(t,J＝7.2Hz,14.0Hz,2H),1.86(q,J＝7.2Hz,14.0Hz,2H).
ESI-MS:m/z＝190.9[M+H]+。
Step 2 preparation of { [ (2S) -1- [ (6-bromo-1-oxohex-5-ynyl) oxy ] -5, 5-dimethyl-3-oxohex-2-yl ] amino } methanesulfonic acid-9H-fluoren-9-ylmethyl ester (3)
N- { [ (9H-fluoren-9-ylmethyl) oxy ] carbonyl } -L-serine butyl ester (14.0 g,36.5 mmol) was dissolved in 140mL of dichloromethane, and Compound 2 (7.67 g,40.2 mmol), HOBt (7.40 g,54.8 mmol), DIPEA (7.07 g,54.8 mmol), EDCI (8.10 g,42.6 mmol) were added sequentially and reacted at 25℃for 12H; washed successively with 0.5mol/L HCl (100 mL), saturated sodium bicarbonate solution (100 mL), saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 27.0g of Compound 3 as a black oil (crude).
ESI-MS:m/z＝578.1[M+Na]+。
Step 3, preparation of 6-bromo-5-hexynoic acid- (2S) -2-amino-5, 5-dimethyl-3-oxohexy-l ester (4)
Compound 3 (crude product obtained by silica gel column chromatography) (20.0 g,36.0 mmol) was dissolved in 160mL of methylene chloride, and triethylamine (40 mL) was added thereto for reaction at 25℃for 12 hours; after concentration under reduced pressure, purification by silica gel column chromatography (dichloromethane: methanol=100:1-50:1) gave 6.00g of compound 4 as a white solid (yield 50.0%).
1HNMR(400MHz,CDCl3):δ6.51(brd,J＝6.5Hz,1H),4.56(dt,J＝7.2,3.7Hz,1H),3.87-3.99(m,2H),2.40(t,J＝7.4Hz,2H),2.32(t,J＝6.9Hz,2H),1.88(q,J＝7.2Hz,2H),1.50(s,9H).
Preparation of N- (t-Butoxycarbonyl) -S-trityl-L-cysteinyl-L-phenylalanine (6)
Synthesis of the polypeptide: CTC resin 5 (50.0 mmol,56.8 g) and Fmoc-Phe-OH (19.4 g,50.0mmol,1.0 eq) were placed in a flask, 600mL of dichloromethane was added, DIPEA (300 mmol,6.0 eq) was added dropwise under N 2 protection, and stirred for 2h; methanol (57.0 mL) was added, stirred for 30min, the solution was discharged and washed 5 times with DMF; adding 20% piperidine/DMF solution, reacting for 30min to remove Fmoc protecting groups, discharging the solution, and washing with DMF for 5 times; boc-Cys (Trt) -OH (46.3 g,50.0mmol,2.0 eq) was then added, after mixing for 30s, HBTU (95 mmol,1.90 eq) and DIPEA (200 mmol,4.0 eq) were added and reacted for 1h under N 2 protection at 25 ℃. During the period, ninhydrin test is used to monitor the reaction process, after the reaction is finished, DMF is used for washing 5 times, methanol is used for washing 3 times, and then vacuum drying is carried out, so as to obtain the side chain protecting peptide (polypeptide).
Peptide cleavage and purification: putting the obtained side chain protecting peptide into a flask, adding a lysis buffer (20% HFIP/dichloromethane), and stirring for 1h at room temperature; the filtrate was collected by filtration, and the solvent was distilled off under reduced pressure to give 35.5g of crude compound 6.
Step 5, preparation of 6-bromo-5-hexynoic acid- (6R, 9S, 12S) -9-benzyl-12- (3, 3-dimethyl-1-oxobutylene) -2, 2-dimethyl-4, 7, 10-trioxymethylene-6- { [ (triphenylmethyl) thio ] methyl } -3-oxa-5, 8, 11-triazatridec-n-13-yl ester (7)
Compound 6 (crude product obtained by purification by silica gel column chromatography) (8.00 g,13.0 mmol) and Compound 4 (5.26 g,15.8 mmol) were dissolved in 160mL acetonitrile, and TCFH (4.40 g,15.7 mmol) and 1-methylimidazole (3.77 g,45.5 mmol) were added, respectively, and reacted at 25℃for 2 hours; after completion of the reaction, the solvent was distilled off under reduced pressure, and purified by preparative high performance liquid chromatography (TFA condition) to give 6.00g of compound 7 as a white solid (yield 49.6%).
1HNMR(400MHz,CDCl3):δ7.41(dd,J＝7.2,4.6Hz,8H),7.23-7.34(m,12H),7.22(br,J＝1.8Hz,3H),7.09-7.13(m,2H),5.72(dt,J＝11.9,5.9,5.9,2.4Hz,1H),4.89(q,J＝6.9Hz,1H),4.58-4.76(m,2H),3.56-3.77(m,1H),3.13-3.25(m,1H),2.95-3.11(m,2H),2.51-2.60(m,1H),2.22-2.35(m,2H),1.78-1.88(m,1H),1.29-1.55(m,18H).
ESI-MS:m/z＝948.3[M+Na]+。
Step 6 preparation of 6, 6-bromo-5-hexynoic acid- (6R, 9S, 12S) -9-benzyl-12- (3, 3-dimethyl-1-oxobutylene) -2, 2-dimethyl-4, 7, 10-trioxolidene-6- (mercaptomethyl) -3-oxa-5, 8, 11-triazatridecan-13-yl ester (8)
Compound 7 (5.00 g,5.40 mmol) was dissolved in 50.0mL of dichloromethane, TFA (3.07 g,27.0mmol,2 mL) and triisopropylsilane (4.27 g,27.0 mmol) were added, stirred at 25℃for 3h, and the solvent was distilled off under reduced pressure to give 3.5g of crude product of compound 8 as a white solid.
Preparation of { [ (3S, 6S, 9R) -6-benzyl-3- (3, 3-dimethyl-1-oxobutylene) -5,8,17-trioxo-4, 7-diaza-1-oxa-11-thia-heptadec-12-yn-9-yl ] amino } methanoic acid-2-methylpropan-2-yl ester (9)
4-Tert-butyl-2- (4-tert-butyl-2-pyridine) pyridine (274 mg,1.02 mmol) and 2, 6-lutidine (1.10 g,10.2 mmol) were dissolved in 1.30L acetonitrile, copper tetraacetonitrile hexafluorophosphate [ Cu (MeCN) 4PF6 ] (190 mg, 511. Mu. Mol) was added under N 2 protection, and then compound 8 (crude product obtained by purification by silica gel column chromatography) (3.50 g,5.11 mmol) was added to react at 25℃for 12 hours; after completion of the reaction, the mixture was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography (TFA condition) to give 1.20g of compound 9 as a white solid (yield 38.3%).
1HNMR(400MHz,CDCl3):δ7.27-7.34(m,3H),7.12-7.20(m,2H),6.90-7.01(m,1H),5.07(brs,1H),4.64-4.81(m,2H),4.52-4.59(m,1H),4.44-4.52(m,1H),4.18-4.40(m,1H),3.27-3.49(m,1H),3.13-3.25(m,1H),2.99-3.13(m,1H),2.73-2.95(m,1H),2.57(brs,1H),2.13-2.48(m,3H),1.70-2.00(m,2H),1.46-1.56(m,9H),1.43(d,J＝16.6Hz,9H).
ESI-MS:m/z＝604.3[M+H]+。
Preparation of (3S, 6S, 9R) -9-amino-6-benzyl-5,8,17-trioxo-4, 7-diaza-1-oxa-11-thia-heptadec-12-alkyne-3-carboxylic acid (10)
Compound 9 (50.0 mg, 82.8. Mu. Mol) was dissolved in 1.0mL of HAC, stirred at 40℃for 16h, concentrated under reduced pressure, and purified by preparative high performance liquid chromatography under neutral conditions to give 7.00mg of compound 10 (yield 18.9%).
1HNMR(400MHz,D2O):δ7.23-7.35(m,3H),7.14-7.21(m,1H),7.02-7.10(m,1H),4.94-5.08(m,1H),4.36-4.47(m,1H),4.16-4.33(m,1H),3.31-3.61(m,2H),2.94-3.18(m,2H),2.38-2.66(m,2H),2.14-2.29(m,1H),1.94-2.11(m,1H),1.51(brs,2H),1.06-1.43(m,2H).
ESI-MS:m/z＝448.1[M+H]+。
Preparation of (3S, 6S, 9R) -9-amino-N, 6-dibenzyl-5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide (11)
Compound 10 (50.0 mg, 111.8. Mu. Mol) was dissolved in 2.0mL of DCM, HATU (63.8 mg, 167.7. Mu. Mol) and DIPEA (28.9 mg, 223.6. Mu. Mol) were then added, and after stirring the reaction system at 25℃for 0.5h, benzylamine (11.9 mg, 111.8. Mu. Mol) was added thereto and stirring continued at 25℃for 3h until the reaction was complete; the reaction solution was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography under neutral conditions to give 10.00mg of compound 11 (yield 16.7%).
1HNMR(400MHz,D2O):δ7.24-7.32(m,3H),7.18-7.23(m,4H),7.14-7.16(m,2H),7.00-7.13(m,1H),4.89-5.04(m,1H),4.39-4.55(m,1H),4.25-4.30(d,J＝2.5Hz,2H),4.14-4.20(m,1H),3.29-3.61(m,2H),2.90-3.25(m,2H),2.43-2.71(m,2H),2.26-2.34(m,1H),1.90-2.17(m,1H),1.58(brs,2H),1.03-1.51(m,2H).
ESI-MS:m/z＝537.2[M+H]+。
Example 2
Preparation of (3S, 6S, 9R) -9-amino-6-benzyl-N- (2-chlorobenzyl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide (12)
Compound 10 was prepared according to steps 1-8 of example 1.
Compound 10 (50.0 mg, 111.8. Mu. Mol) was dissolved in 2.0mL of DCM, HATU (63.8 mg, 167.7. Mu. Mol) and DIPEA (28.9 mg, 223.6. Mu. Mol) were then added, and after stirring the reaction system at 25℃for 0.5h, o-chlorobenzylamine (15.8 mg, 111.8. Mu. Mol) was added thereto and stirring continued at room temperature for 3h until the reaction was completed; concentrated under reduced pressure, and purified by preparative high performance liquid chromatography under neutral conditions to give 7.00mg of compound 12 (yield 11.4%).
1HNMR(400MHz,D2O):δ7.59-7.68(m,1H),7.27-7.35(m,3H),7.22-7.25(m,3H),7.17-7.19(m,2H),7.03-7.15(m,1H),4.92-5.11(m,1H),4.38-4.57(m,1H),4.29-4.33(m,2H),4.16-4.25(m,1H),3.23-3.68(m,4H),2.40-2.68(m,2H),2.24-2.38(m,1H),1.92-2.18(m,1H),1.47-1.59(m,2H),1.06-1.43(m,2H).
ESI-MS:m/z＝572.1[M+H]+。
Example 3
Preparation of (3S, 6S, 9R) -9-amino-6-benzyl-5,8,17-trioxo-N- ((R) -1,2,3, 4-tetrahydronaphthalen-1-yl) -1-oxa-11-thia-4, 7-diazepin-12-ene-3-carboxamide (13)
Compound 10 was prepared according to steps 1-8 of example 1.
Compound 10 (50.0 mg, 111.8. Mu. Mol) was dissolved in 2.0mL of DCM, HATU (63.8 mg, 167.7. Mu. Mol) and DIPEA (28.9 mg, 223.6. Mu. Mol) were then added, and after stirring the reaction at 25℃for 0.5h, tetrahydronaphthylamine (16.5 mg, 111.8. Mu. Mol) was added thereto and stirring continued at room temperature for 3h until the reaction was completed; concentrated under reduced pressure, and purified by preparative high performance liquid chromatography under neutral conditions to give 12.00mg of compound 13 (yield 18.6%).
1HNMR(400MHz,D2O):δ7.27-7.33(m,3H),7.15-7.19(m,1H),6.95-7.15(m,5H),4.94-5.13(m,2H),4.38-4.54(m,1H),4.17-4.26(m,2H),3.25-3.64(m,2H),2.70-2.87(m,2H),2.40-2.68(m,2H),2.25-2.39(m,2H),1.87-1.95(m,4H),1.79-1.85(m,1H),1.45-1.62(m,2H),1.05-1.41(m,2H).
ESI-MS:m/z＝577.2[M+H]+。
Example 4
Preparation of (3S, 6S, 9R) -9-amino-6-benzyl-N- ((S) -4-methyl-1- ((R) -2-methoxy-2-yl) -1-oxopentan-2-yl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide (14)
Compound 10 was prepared according to steps 1-8 of example 1.
Compound 10 (50.0 mg, 111.8. Mu. Mol) was dissolved in 2.0mL of DCM, HATU (63.8 mg, 167.7. Mu. Mol) and DIPEA (28.9 mg, 223.6. Mu. Mol) were then added and after stirring the reaction at 25℃for 0.5h, S-2-amino-4-methyl-1- (R) -2-methylethoxy-2-yl pentanone (19.1 mg, 111.8. Mu. Mol) was added thereto and stirring was continued at room temperature for 3h until the reaction was completed; concentrated under reduced pressure, and purified by preparative high performance liquid chromatography under neutral conditions to give 15.00mg of compound 14 (yield 22.3%).
1HNMR(400MHz,D2O):δ7.20-7.36(m,3H),7.11-7.25(m,1H),7.02-7.08(m,1H),4.90-5.07(m,1H),4.37-4.51(m,1H),4.32-4.36(m,1H),4.14-4.30(m,1H),3.38-3.64(m,2H),2.94-3.18(m,4H),2.38-2.54(m,2H),2.25-2.34(m,2H),2.13-2.28(m,1H),1.87-2.10(m,3H),1.47-1.53(m,3H),1.25(s,3H),1.13-0.95(d,J＝0.8Hz,6H).
ESI-MS:m/z＝601.3[M+H]+。
Example 5
Preparation of (3 s,6s, 9R) -9-amino-6-benzyl-N- ((R) -3-methyl-1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) butyl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazacyclohepta-12-yl-3-carboxamide (15)
Compound 10 was prepared according to steps 1-8 of example 1.
Compound 10 (50.0 mg, 111.8. Mu. Mol) was dissolved in 2.0mL of DCM, HATU (63.8 mg, 167.7. Mu. Mol) and DIPEA (28.9 mg, 223.6. Mu. Mol) were then added and after stirring the reaction at 25℃for 0.5h, 3-methyl-1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) butan-1-amine (23.8 mg, 111.8. Mu. Mol) was added thereto and stirring was continued at room temperature for 3h until the reaction was complete; concentrated under reduced pressure, and purified by preparative high performance liquid chromatography under neutral conditions to give 12.00mg of compound 15 (yield 16.7%).
1HNMR(400MHz,D2O):δ7.20-7.32(m,3H),7.11-7.18(m,1H),7.03-7.12(m,1H),4.92-5.03(m,1H),4.32-4.46(m,1H),4.13-4.29(m,1H),3.31-3.61(m,4H),2.92-3.13(m,4H),2.34-2.62(m,2H),2.12-2.25(m,1H),1.94-2.11(m,1H),1.47-1.63(m,3H),1.24(s,12H),1.06-0.92(d,J＝0.8Hz,6H).
ESI-MS:m/z＝643.3[M+H]+。
Performance test:
1. the macrocyclic alkynyl thioether peptide compounds (compounds 11 to 15) prepared in examples 1 to 5 were tested for proteasome inhibition activity, respectively
The experimental method comprises the following steps: the inhibition of enzyme activity by different compounds was observed using fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC to initially evaluate the inhibition effect of the compounds. The proteasome chymotrypsin (chymotrypsin-like protease) hydrolyzes Tyr-AMC sequence in the substrate, releases AMC, can detect fluorescence absorption value of the product AMC after hydrolysis under the condition that excitation light is 355nm and emission light is 460nm, observes the inhibition condition of the compound on proteasome activity, adopts a positive reference substance of Bortezomib in the experiment, and the result is shown in Table 1.
2. Test of proliferation inhibitory Activity of the macrocyclic alkynyl thioether peptides (Compounds 11 to 15) prepared in examples 1 to 5, respectively
The experimental method comprises the following steps: cell viability was measured by the MTS method, i.e., cells grown in the logarithmic phase were digested with 0.01% pancreatin, counted, plated in 96-well plates at a cell density of 2.0X103/well at 100ml, and incubated overnight at 37℃in a 5% CO 2 incubator. Six concentration gradients were set for each compound, three duplicate wells were set for each concentration, each concentration was added to the corresponding well, and incubated in a 5% CO 2 37 ℃incubator for 72 hours, with 20ml of 5mg/ml MTS added. After incubation for 3 hours at 37℃the supernatant was pipetted, dissolved in 100ml of DMSO, the 490nm (L1) absorbance was measured using SpectraMAX 340, the reference wavelength 690nm (L2), the (L1-L2) values plotted against the different concentrations of inhibitor and the IC 50 was obtained by fitting the formula, the results of which are shown in Table 1.
TABLE 1 inhibition of proteasome CT-L by different Compounds and proliferation inhibition of multiple myeloma cells and acute leukemia cells
As can be seen from Table 1, the compounds 11 to 15 prepared in examples 1 to 5 all have strong proteasome inhibitory activity and hematological malignancy cell proliferation inhibitory activity, and in particular, the proteasome inhibitory activity and hematological malignancy cell proliferation inhibitory activity of the compound 13 are excellent.
The performance test experiment shows that the macrocyclic alkynyl thioether peptide compound has excellent antitumor application prospect and good commercial value.

Claims (10)

1. A macrocyclic alkynyl thioether peptide compound is characterized in that, the structural formula of the macrocyclic alkynyl thioether peptide compound is as follows:
wherein R is selected from one of aralkyl, halogenated aralkyl and C 1-4 alkyl-D-Ra heterocyclic aryl.
2. The macrocyclic alkynyl thioether peptide compound according to claim 1, wherein the aralkyl group is benzyl;
The halogenated aralkyl is benzyl substituted by chlorine atoms at the ortho position of the benzene ring;
C 1-4 alkyl is isobutyl;
D is selected from C, CCO;
ra is selected from one of an oxetanyl group and a boron ester heterocycle.
3. The macrocyclic alkynyl thioether peptide compound according to claim 1, wherein the macrocyclic alkynyl thioether peptide compound is selected from one of the following compounds:
(3 s,6s,9 r) -9-amino-N, 6-dibenzyl-5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide;
(3 s,6s,9 r) -9-amino-6-benzyl-N- (2-chlorobenzyl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide;
(3 s,6s, 9R) -9-amino-6-benzyl-5,8,17-trioxo-N- ((R) -1,2,3, 4-tetrahydronaphthalen-1-yl) -1-oxa-11-thia-4, 7-diazepin-12-ene-3-carboxamide;
(3S, 6S, 9R) -9-amino-6-benzyl-N- ((S) -4-methyl-1- ((R) -2-methoxy-2-yl) -1-oxopentan-2-yl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazepin-12-yl-3-carboxamide;
(3 s,6s, 9R) -9-amino-6-benzyl-N- ((R) -3-methyl-1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) butyl) -5,8,17-trioxo-1-oxa-11-thia-4, 7-diazacyclohepta-12-yl-3-carboxamide.
4. A process for the preparation of the macrocyclic alkynylsulfide peptide compound according to any one of claims 1 to 3, comprising the steps of:
(1) Under the protection of N 2, reacting 5-hexynoic acid with NBS and AgNO 3 in acetone to obtain a compound 2;
(2) Under the action of HOBt, EDCI and DIPEA, the compound 2 reacts with N- { [ (9H-fluoren-9-ylmethyl) oxy ] carbonyl } -L-serine butyl ester to generate a compound 3;
(3) Dissolving the compound 3 in dichloromethane, adding triethylamine to react to remove Fmoc protecting groups, and obtaining a compound 4;
(4) Dissolving CTC resin 5 and Fmoc-Phe-OH in dichloromethane, adding DIPEA under the protection of N 2, stirring, washing, and removing Fmoc protecting groups by using a piperidine solution; then adding condensing agent HBTU and Boc-Cys (Trt) -OH for condensation reaction, adding a cracking buffer solution into the product after the reaction is finished, and stirring to obtain a compound 6;
(5) Condensing the compound 6 and the compound 4 under the action of TCFH and 1-methylimidazole to generate a compound 7;
(6) Dissolving the compound 7 in dichloromethane, adding TFA and triisopropylsilane, and stirring to react to generate a compound 8;
(7) Dissolving 4-tertiary butyl-2- (4-tertiary butyl-2-pyridine) pyridine and 2, 6-lutidine in acetonitrile, adding copper tetraacetonitrile hexafluorophosphate under the protection of N 2, and then adding an acetonitrile solution of a compound 8 for reaction to obtain a compound 9;
(8) Dissolving the compound 9 in HAC, and stirring for reaction to obtain a compound 10;
(9) And condensing the compound 10 with primary amine under the action of condensing agents HATU and DIPEA to obtain the macrocyclic alkynyl thioether peptide compound.
5. The method for preparing the macrocyclic alkynyl thioether peptide compound according to claim 4, wherein the primary amine is selected from one of benzylamine, o-chlorobenzylamine, tetrahydronaphthylamine, S-2-amino-4-methyl-1- (R) -2-methyl oxirane-2-yl pentanone, 3-methyl-1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) butane-1-amine.
6. A pharmaceutical composition comprising an active ingredient and at least one or more pharmaceutically acceptable carriers or excipients;
wherein the active component is one or more of stereoisomers or pharmaceutically acceptable salts of the macrocyclic alkynyl thioether peptide compounds according to any one of claims 1 to 3.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is administered orally, by injection, by inhalation, or by implantation;
the pharmaceutical composition is in the form of injection, freeze-dried powder injection, tablet, capsule or granule.
8. Use of a macrocyclic alkynyl thioether peptide compound as defined in any one of claims 1 to 3 in the manufacture of a medicament for malignant and/or immunological diseases.
9. Use of a pharmaceutical composition according to any one of claims 6 to 7 for the preparation of a medicament for malignant and/or immunological diseases.
10. The use according to claim 8 or 9, wherein the malignancy is one of myeloma, lymphoma, leukemia, breast cancer, sarcoma, lung cancer, macroglobulinemia, prostate cancer, renal cancer, colon cancer, rectal cancer, pancreatic cancer, glioma, neuroblastoma, head cancer, neck cancer, liver cancer, thyroid cancer, ovarian cancer, cervical cancer, vulvar cancer, endometrial cancer, bladder cancer, testicular cancer, esophageal cancer, gastric cancer, buccal cancer, nasopharyngeal cancer, oral cancer, skin cancer, gastrointestinal stromal tumor.
CN202310881207.3A 2023-07-18 2023-07-18 Macrocyclic alkynyl thioether peptide compound and preparation method and application thereof Pending CN118561953A (en)

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