CN115677808A - Method for preparing aristolochic acid-deoxynucleoside conjugate - Google Patents
Method for preparing aristolochic acid-deoxynucleoside conjugate Download PDFInfo
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- CN115677808A CN115677808A CN202211337892.5A CN202211337892A CN115677808A CN 115677808 A CN115677808 A CN 115677808A CN 202211337892 A CN202211337892 A CN 202211337892A CN 115677808 A CN115677808 A CN 115677808A
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- 238000000034 method Methods 0.000 title claims description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 90
- 239000002904 solvent Substances 0.000 claims abstract description 58
- 230000002140 halogenating effect Effects 0.000 claims abstract description 22
- 150000002367 halogens Chemical group 0.000 claims abstract description 20
- 239000003054 catalyst Substances 0.000 claims abstract description 19
- AADCWJFQHQIXSD-UHFFFAOYSA-N Aristololactam Natural products COC1=C(OC)C=C2C3=C(OC)C(OC)=CC(C(=O)N4C)=C3C4=CC2=C1 AADCWJFQHQIXSD-UHFFFAOYSA-N 0.000 claims abstract description 15
- MXOKGWUJNGEKBH-UHFFFAOYSA-N aristololactam Chemical compound COC1=CC=CC(C2=C34)=C1C=C3NC(=O)C4=CC1=C2OCO1 MXOKGWUJNGEKBH-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 10
- 238000006443 Buchwald-Hartwig cross coupling reaction Methods 0.000 claims abstract description 9
- 241000726094 Aristolochia Species 0.000 claims abstract description 8
- 230000009471 action Effects 0.000 claims abstract description 8
- -1 aristolochia lactam Chemical class 0.000 claims abstract description 8
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 239000003513 alkali Substances 0.000 claims abstract description 6
- 229910052736 halogen Inorganic materials 0.000 claims abstract description 6
- 239000002244 precipitate Substances 0.000 claims description 125
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 64
- 239000006228 supernatant Substances 0.000 claims description 59
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 51
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 51
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 claims description 41
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 39
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 32
- 238000010438 heat treatment Methods 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 238000010992 reflux Methods 0.000 claims description 27
- 238000000926 separation method Methods 0.000 claims description 23
- 239000012043 crude product Substances 0.000 claims description 21
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 20
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 19
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 18
- 238000000746 purification Methods 0.000 claims description 18
- 238000001035 drying Methods 0.000 claims description 17
- 238000001816 cooling Methods 0.000 claims description 15
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 12
- 239000002798 polar solvent Substances 0.000 claims description 12
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 11
- 238000009210 therapy by ultrasound Methods 0.000 claims description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 239000003446 ligand Substances 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- VRLDVERQJMEPIF-UHFFFAOYSA-N dbdmh Chemical compound CC1(C)N(Br)C(=O)N(Br)C1=O VRLDVERQJMEPIF-UHFFFAOYSA-N 0.000 claims description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims description 9
- 239000001257 hydrogen Substances 0.000 claims description 9
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 claims description 5
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 claims description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 5
- 150000004820 halides Chemical class 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- SFLXUZPXEWWQNH-UHFFFAOYSA-K tetrabutylazanium;tribromide Chemical compound [Br-].[Br-].[Br-].CCCC[N+](CCCC)(CCCC)CCCC.CCCC[N+](CCCC)(CCCC)CCCC.CCCC[N+](CCCC)(CCCC)CCCC SFLXUZPXEWWQNH-UHFFFAOYSA-K 0.000 claims description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 4
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 4
- 238000005658 halogenation reaction Methods 0.000 claims description 4
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 3
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 3
- 229910052794 bromium Inorganic materials 0.000 claims description 3
- 229910052801 chlorine Inorganic materials 0.000 claims description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 239000008096 xylene Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 229920000515 polycarbonate Polymers 0.000 claims 1
- 239000004417 polycarbonate Substances 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 abstract description 13
- 230000015572 biosynthetic process Effects 0.000 abstract description 11
- 239000002994 raw material Substances 0.000 abstract description 5
- 239000013065 commercial product Substances 0.000 abstract description 4
- 238000003756 stirring Methods 0.000 description 49
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 48
- CXNIUSPIQKWYAI-UHFFFAOYSA-N xantphos Chemical compound C=12OC3=C(P(C=4C=CC=CC=4)C=4C=CC=CC=4)C=CC=C3C(C)(C)C2=CC=CC=1P(C=1C=CC=CC=1)C1=CC=CC=C1 CXNIUSPIQKWYAI-UHFFFAOYSA-N 0.000 description 33
- 150000001875 compounds Chemical class 0.000 description 26
- 229910052757 nitrogen Inorganic materials 0.000 description 24
- 239000000047 product Substances 0.000 description 19
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 13
- 230000005587 bubbling Effects 0.000 description 11
- 238000005119 centrifugation Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 239000008188 pellet Substances 0.000 description 6
- MHABMANUFPZXEB-UHFFFAOYSA-N O-demethyl-aloesaponarin I Natural products O=C1C2=CC=CC(O)=C2C(=O)C2=C1C=C(O)C(C(O)=O)=C2C MHABMANUFPZXEB-UHFFFAOYSA-N 0.000 description 4
- 150000002431 hydrogen Chemical class 0.000 description 4
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 3
- BBFQZRXNYIEMAW-UHFFFAOYSA-N aristolochic acid I Chemical compound C1=C([N+]([O-])=O)C2=C(C(O)=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-N 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 229910017489 Cu I Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 238000005893 bromination reaction Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
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- 239000013558 reference substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Saccharide Compounds (AREA)
Abstract
The invention provides a preparation method of aristolochic acid-deoxynucleoside conjugate. The preparation method comprises the following steps: step S1, under the action of a halogenating reagent, taking aristololactam as a substrate, and carrying out a halogenating reaction in a first solvent to obtain a halogen substituent of the aristololactam; and S2, taking halogen substitutes of the aristolochia lactam and deoxynucleoside as substrates, carrying out Buchwald-Hartwig cross coupling reaction in a second solvent under the action of a catalyst and alkali, and separating and purifying the obtained reaction liquid to obtain the aristolochic acid-deoxynucleoside conjugate with the structure shown in the formula III. By applying the technical scheme of the invention, the commercial product is used as the reaction raw material, the reaction route is simplified, the preparation process is easy to control, the operation is convenient, the aristolochic acid-deoxynucleoside conjugate with high purity can be obtained, and the large-scale synthesis is convenient.
Description
Technical Field
The invention relates to the technical field of organic synthesis, in particular to a preparation method of an aristolochic acid-deoxynucleoside conjugate.
Background
Aristolochic acid can be metabolized by enzyme in vivo to form active cyclic lactam ions, and then combined with exocyclic amino groups of DNA bases to generate aristolochic acid-DNA combination, so as to induce the gene to generate ectopic mutation from A: T to T: A. This transformation process is the main mechanism of oncogenic mutagenesis of aristolochic acids (sci. Trans. Med.2013,5,197).
Due to expensive monomer raw materials, poor preparation process, low yield and the like, no commercial aristolochic acid-DNA conjugate is supplied in the market at present, although reports of laboratory synthesis studies on a very small scale are available. Methods for preparing aristolochic acid-DNA conjugates have been reported as follows: zinc powder reduction, biological culture, organic synthesis, etc. However, these methods still have some problems: (1) the synthetic conversion rate is low, only 0.1-2%, the yield is extremely low, the cost is extremely high, and the prepared target product is not even enough to be weighed (J.environ.Sci.2009, 21,1769); (2) the reaction conditions are relatively harsh (J.Carcinog.1988, 9,297) and have high requirements on an oxygen-free environment and a pH value; (3) complex synthesis process, large separation loss of intermediate and product, poor effect (chem.res.toxicol.2014, 27,1236), and the like.
Due to the lack of an effective preparation method, a commercial reference substance cannot be obtained, and the research on in-vivo detection and related carcinogenic and mutagenic mechanisms in the field is greatly restricted. Currently, the use safety of aristolochic acid drugs is very important, and intensive research on the aristolochic acid-DNA conjugate as an important toxicity marker is urgently needed. Therefore, it is necessary and urgent to establish a synthetic method that is efficient, simple, and capable of mass production of aristolochic acid-deoxynucleoside conjugates.
Disclosure of Invention
The invention mainly aims to provide a preparation method of an aristolochic acid-deoxynucleoside conjugate, which aims to solve the problems that the aristolochic acid-deoxynucleoside conjugate in the prior art is difficult to prepare and difficult to obtain in batches.
In order to achieve the above object, the present invention provides a methodA method for preparing an aristolochic acid-deoxynucleoside conjugate, the method comprising the steps of: step S1, under the action of a halogenating reagent, taking aristololactam with a structure shown in a formula I as a substrate, and carrying out a halogenating reaction in a first solvent to obtain a halogen substituent of aristololactam with a structure shown in a formula II; s2, taking halogen substitutes of aristololactam with the structure shown in the formula II and deoxynucleoside as substrates, carrying out Buchwald-Hartwig cross coupling reaction in a second solvent under the action of a catalyst and alkali, and separating and purifying the obtained reaction liquid to obtain an aristolochic acid-deoxynucleoside conjugate with the structure shown in the formula III; wherein R in formula I, formula II and formula III 1 All represent hydrogen or methoxyl, X in the formula II represents any one of Cl, br and I, and R in the formula III 2 Represents any one of deoxyadenosine, deoxycytidine and deoxyguanosine.
Further, the halogenating agent is a brominating agent, preferably, the brominating agent is one or more of bromine, N-bromosuccinimide, 1,3-dibromo-5,5-dimethyl hydantoin and tetrabutyl ammonium tribromide; more preferably, the molar ratio of the brominating reagent to the aristololactam is 1:1 to 5:1.
Further, the halogenation reaction is carried out in a nitrogen atmosphere; preferably, the halogenating agent is added in two or more portions.
Further, the step S1 comprises the step S11 of adding aristolochia lactam and a halogenating reagent into the first solvent to react for 3-48 hours to obtain a halogenating reaction solution; preferably, the reaction temperature is 15 to 60 ℃, more preferably 20 to 35 ℃; s12, carrying out centrifugal separation on the halogenated reaction liquid to obtain supernatant and precipitate, collecting the precipitate, and drying to obtain a halogen substitute;
preferably, the supernatant is concentrated, and then is subjected to second centrifugal separation, and the precipitate obtained by the second centrifugal separation is purified to obtain a halogen substituent; preferably, the purification comprises adding one or more of acetic acid, n-butanol and tetrahydrofuran to the precipitate obtained by the second centrifugation, subjecting the mixture to sonication, performing a third centrifugation, and collecting the precipitate to obtain a halogen substituent.
Further, the first solvent in step S1 includes any one or more of tetrahydrofuran, N-dimethylformamide, dimethyl sulfoxide, dichloromethane, and acetic acid;
preferably, the amount of the first solvent added is 20 to 400 mL-mmol based on the substance amount of aristololactam -1 。
Further, the catalyst of step S2 is Pd 2 (dba) 3 、Pd(OAc) 2 、Pd(CH 3 CN) 2 Cl 2 And one or more of Cu I, preferably, the molar ratio of the catalyst to the halogen substituent is 0.04-1:1.
Further, the catalyst also comprises a ligand, preferably, the ligand is selected from any one or more of 4,5-bis diphenylphosphine-9,9-dimethyl xanthene and pyridine, and the preferred molar ratio of the ligand to the catalyst is 1:1-3:1.
Further, buchwald-Hartwig cross coupling reaction is carried out in nitrogen atmosphere, preferably, the reaction temperature is 80-120 ℃, and the reaction time is 5-10 h; preferably, the molar ratio of halo substituent to deoxynucleoside employed is 1:1 to 1:5.
Further, the alkali is one or more of potassium carbonate and cesium carbonate, and preferably, the molar ratio of the alkali to the halide is 1:1-3:1; and/or the second solvent is any one or more of N, N-dimethylacetamide and xylene.
Further, the separation and purification in step S2 includes: step S201, carrying out centrifugal separation on the reaction liquid, collecting supernatant, and removing the solvent to obtain a crude product of the aristolochic acid-deoxynucleoside conjugate; step S202, adding a strong polar solvent and a weak polar solvent into the aristolochic acid-deoxynucleoside conjugate crude product, heating and refluxing, preferably, the refluxing time is 0.5-2 h, and cooling to room temperature; the strong polar solvent is water, and the weak polar solvent is any one or more of acetone, n-butanol, ethyl acetate, toluene, dichloromethane and tetrahydrofuran; and step S203, performing centrifugal separation on the substance obtained in the step S202, and collecting precipitates to obtain the aristolochic acid-deoxynucleoside conjugate.
By applying the technical scheme of the invention, the commercial product is used as the reaction raw material, the reaction route is simplified, the preparation process is easy to control, the operation is convenient, the aristolochic acid-deoxynucleoside conjugate with high purity can be obtained, and the large-scale synthesis is convenient.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As analyzed in the background of the present application, in the prior art, the aristolochic acid-deoxynucleoside conjugate is difficult to prepare and synthesize in large scale, so that there is no commercial control, and research on the mechanisms of related carcinogenesis and mutagenesis of the aristolochic acid-deoxynucleoside conjugate is severely limited, and in order to solve the problem, the present application provides a preparation method of the aristolochic acid-deoxynucleoside conjugate, which comprises the following steps: step S1, under the action of a halogenating reagent, taking aristololactam with a structure shown in a formula I as a substrate, and carrying out a halogenating reaction in a first solvent to obtain a halogen substituent of aristololactam with a structure shown in a formula II; and S2, taking halogen substitutes of the aristolochia lactam with the structure shown in the formula II and deoxynucleoside as substrates, carrying out Buchwald-Hartwig cross coupling reaction in a second solvent under the action of a catalyst and alkali, and separating and purifying the obtained reaction liquid to obtain the aristolochic acid-deoxynucleoside conjugate with the structure shown in the formula III.
Wherein R in formula I, formula II and formula III 1 All represent hydrogen or methoxy, X in formula II represents any one of Cl, br and I, R in formula III 2 Represents any one of deoxyadenosine (dA), deoxycytidine (dC) and deoxyguanosine (dG).
The method adopts the commercial product as the reaction raw material, simplifies the reaction route, is easy to control the preparation process, is convenient to operate, can obtain the aristolochic acid-deoxynucleoside conjugate with high purity, and is convenient for amplification synthesis.
The synthetic route for aristolochic acid-deoxynucleoside conjugates of the present application is as follows:
the halogenating agent may be selected from the prior art, and in some embodiments, is a brominating agent, which helps to increase the yield of the halogenation reaction and Buchwald-Hartwig cross-coupling reaction. Preferably, the brominating reagent is any one or more of bromine, N-bromosuccinimide (NBS), 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) and tetrabutylammonium tribromide. The molar ratio of the brominating reagent to the aristolochia can be determined according to the stoichiometric ratio of the chemical reaction, and the preferable molar ratio of the brominating reagent to the aristolochia lactam is 1:1-5:1.
In some embodiments, the halogenation reaction described above is conducted under a nitrogen atmosphere, which advantageously increases the yield of halogenated products. The halogenating reagent can be added at one time or can be added in two times or more than two times, and the halogenating reagent is added for multiple times, so that the phenomenon that the target halogenated product yield is reduced because the halogenating reagent is generated into multiple or multiple halogenated byproducts due to overhigh concentration caused by one-time addition is reduced, meanwhile, certain difficulty is brought to subsequent separation and purification, and the improvement of the halogenated product yield is facilitated.
In some exemplary embodiments, the step S1 includes a step S11 of adding aristololactam and a halogenating agent into a first solvent to react for 3 to 48 hours to obtain a halogenating reaction solution; preferably, the reaction temperature is 15 to 60 ℃, more preferably 20 to 35 ℃; s12, carrying out centrifugal separation on the halogenated reaction liquid to obtain supernatant and precipitate, collecting the precipitate, and drying to obtain a halogen substitute; in order to further improve the purity of the halogen substituent, the supernatant is concentrated, second centrifugal separation is carried out, and the precipitate obtained by the second centrifugal separation is purified to obtain the halogen substituent; preferably, the purification comprises adding one or more of acetic acid, n-butanol and tetrahydrofuran to the precipitate obtained by the second centrifugation, subjecting the mixture to sonication, performing a third centrifugation, and collecting the precipitate to obtain a halogen-substituted compound. The purification operation may be carried out once or repeatedly.
The first solvent may be selected from the solvents available in the bromination reaction, and in some preferred embodiments, the first solvent in step S1 includes any one or more of Tetrahydrofuran (THF), N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), and acetic acid; in some embodiments, the first solvent is a mixed solvent of N, N-dimethylformamide and tetrahydrofuran, and the volume ratio of N, N-dimethylformamide to tetrahydrofuran is 4:1 to 9:1; preferably, the amount of the first solvent added is 20 to 400 mL-mmol based on the substance amount of aristololactam -1 。
The catalyst of step 2 may be selected from available catalysts for Buchwald-Hartwig cross-coupling reaction, and in some embodiments, the catalyst of step S2 is Pd 2 (dba) 3 、Pd(OAc) 2 、Pd(CH 3 CN) 2 Cl 2 And one or more of Cu I, preferably, the molar ratio of the catalyst to the halogen substituent is 0.04-1:1. In some embodiments, the catalyst further comprises a ligand, preferably, the ligand is selected from any one or more of 4,5-bis diphenylphosphine-9,9-dimethyl xanthene (xanthphos) and bipyridine (Bpy), and the molar ratio of the ligand to the catalyst is 1:1-3:1. In some typical embodiments, the combination of catalyst and ligand is Pd 2 (dba) 3 /Xantphos、Pd(OAc) 2 /Xantphos、Pd(CH 3 CN) 2 Cl 2 Xantphos or CuI/Bpy, especially using Pd 2 (dba) 3 /Xantphos、Pd(OAc) 2 In case of Xantphos, the catalytic effect is obviously improved.
In some embodiments, to further increase the yield of the reaction, the Buchwald-Hartwig cross-coupling reaction is carried out under nitrogen atmosphere, preferably at a temperature of 80-120 deg.C for a reaction time of 5-10 h. The amount of halide and deoxynucleoside used can be determined on a stoichiometric basis, and in some embodiments herein, the molar ratio of halide to deoxynucleoside used is 1:1 to 1:5 in order to increase the conversion of the halo substituent described above.
The base and the second solvent in the step S2 may be selected from the prior art, preferably, the base is any one or more of potassium carbonate and cesium carbonate, when cesium carbonate is used, the yield of the target product is particularly obviously improved, and preferably, the molar ratio of the base to the halide is 1:1-3:1. In some embodiments, the second solvent is any one or more of N, N-dimethylacetamide and xylene.
In some exemplary embodiments of the present application, the separation and purification in step S2 includes: step S201, carrying out centrifugal separation on the reaction liquid, collecting supernatant, and removing the solvent to obtain a crude product of the aristolochic acid-deoxynucleoside conjugate; step S202, adding a strong polar solvent and a weak polar solvent into the aristolochic acid-deoxynucleoside conjugate crude product, heating and refluxing, preferably, the refluxing time is 0.5-2 h, and cooling to room temperature; the strong polar solvent is water, and the weak polar solvent is any one or more of acetone, n-butanol, ethyl acetate, toluene, dichloromethane and tetrahydrofuran; and step S203, performing centrifugal separation on the substance obtained in the step S202, and collecting precipitates to obtain the aristolochic acid-deoxynucleoside conjugate. The steps S202 and S203 can be repeated to further improve the purity of the target product.
The following examples are provided to further illustrate the benefits that can be achieved by the present application.
Example 1
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, adding AL I (AL:)/(AL: (AL))Namely R 1 Aristolochia lactam, which is methoxy, 14.65mg,0.05mmol, as described below), 10mL of THF and stirring magneton, NBS (8.8mg, 0.05mmol) was added and the mixture was stirred in a water bath at 27 ℃ for 4h; NBS (8.8mg, 0.05mmol) is added, and the reaction is continued to be stirred for 20h at 27 ℃. After the reaction, the supernatant and the precipitate were centrifuged. Directly collecting the precipitate after drying, wherein the obtained precipitate is a target crude product 7-Br-AL I (B) 1 ) The purity was 89%, and the yield was 15.1%.
Example 2
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, sequentially adding AL I (14.65mg, 0.05mmol), THF/DMF 10mL (the volume ratio of THF to DMF is 9:1) and a stirring magneton into a three-necked flask, adding NBS (8.8mg, 0.05mmol), placing in a water bath at 27 ℃ and stirring for reaction for 8 hours; NBS (8.8mg, 0.05mmol) is added, and the reaction is continued to be stirred for 16h at 27 ℃. After the reaction, the supernatant and the precipitate were centrifuged. Directly collecting the precipitate after drying, wherein the obtained precipitate is a target crude product 7-Br-AL I (B) 1 ) The purity was 89%, and the yield was 30.0%.
Example 3
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, sequentially adding AL I (14.65mg, 0.05mmol), THF/DMF 10mL (the volume ratio of THF to DMF is 9:1) and a stirring magneton into a three-necked flask, adding NBS (8.8mg, 0.05mmol), placing in a water bath at 20 ℃ and stirring for reaction for 4 hours; then NBS (8.8mg, 0.05mmol) is added, and the reaction is continued to be stirred for 20h at the temperature of 20 ℃. After the reaction, the supernatant and the precipitate were separated by centrifugation. Directly collecting the precipitate after drying, wherein the obtained precipitate is a target crude product 7-Br-AL I (B) 1 ) The purity was 89% and the yield was 31.3%.
Example 4
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, into a three-necked flask, AL I (14.65mg, 0.05mmol), THF/DMF 10mL (the volume ratio of THF to DMF is 9:1) and a stirring magneton are sequentially addedNBS (8.8mg, 0.05mmol) is placed in a water bath at 27 ℃ for stirring reaction, the NBS (8.8mg, 0.05mmol) is supplemented after the reaction is carried out for 4h and 8h respectively, the NBS is added for two times, and the stirring reaction is continued at 27 ℃ until the total reaction time is 24h. After the reaction, the supernatant and the precipitate were centrifuged. Directly collecting the precipitate after drying, wherein the obtained precipitate is a target crude product 7-Br-AL I (B) 1 ) The purity was 89%, and the yield was 52.4%.
Example 5
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, sequentially adding AL I (14.65mg, 0.05mmol), THF/DMF 10mL (the volume ratio of THF to DMF is 9:1) and a stirring magneton into a three-necked bottle, adding NBS (8.8mg, 0.05mmol), placing the mixture into a water bath at 27 ℃ for stirring reaction, adding NBS (8.8mg, 0.05mmol) after 2h, 6h and 10h of reaction respectively, adding NBS (8.8mg, 0.05mmol) for three times, and continuing stirring reaction at 27 ℃ until the total reaction time is 24h. After the reaction, the supernatant and the precipitate were centrifuged. Directly collecting the precipitate after drying, wherein the obtained precipitate is a target crude product 7-Br-AL I (B) 1 ) The purity was 89%, and the yield was 35.3%.
Example 6
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, sequentially adding AL I (14.65mg, 0.05mmol), THF/DMF 10mL (8:2) and stirring magneton into a three-necked bottle, adding NBS (8.8mg, 0.05mmol), placing in a water bath at 20 ℃ and stirring for reaction for 8h; adding NBS (8.8mg, 0.05mmol), continuing stirring and reacting at 20 ℃ for 16h, heating to 30 ℃, further adding NBS (8.8mg, 0.05mmol), continuing stirring and reacting for 24h. After the reaction, the supernatant and the precipitate were separated by centrifugation. Directly collecting the precipitate after drying, wherein the obtained precipitate is a target crude product 7-Br-AL I (B) 1 ) The purity was 89%, and the yield was 53.7%.
Example 7
The difference from example 6 is that: the supernatant from the centrifugation was concentrated to about 1mL, centrifuged again, and the precipitate was collected and dried. The precipitates obtained in two times are both the target crude product 7-Br-AL I (B) 1 ) Purity of is89% and the yield thereof was 86%.
Example 8
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, into a three-necked flask, AL I (15.0 mg, 0.05mmol), DMSO (5 mL) and a stirring magneton were sequentially added, NBS (44mg, 0.25mmol) was added, and the mixture was stirred in a water bath at 20 ℃ for 10 hours. After the reaction is finished, centrifugally separating supernate and precipitate, drying the precipitate, directly collecting the precipitate, and taking the obtained precipitate as a target crude product 7-Br-AL I (B) 1 ) The purity was 89%, and the yield was 21.5%.
Example 9
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, sequentially adding AL I (14.65mg, 0.05mmol), acetic acid (10 mL) and a stirring magneton into a three-necked flask, adding NBS (8.8mg, 0.05mmol), and stirring in a water bath at 20 ℃ for reaction for 5 hours; then adding NBS (8.8mg, 0.05mmol), continuing stirring and reacting for 2h at 20 ℃, heating to 35 ℃, and stirring and reacting for 2h; finally, NBS (8.8mg, 0.05mmol) is added for reaction at 35 ℃ for 30min. After the reaction, the supernatant and the precipitate were separated by centrifugation. Directly collecting the precipitate after drying; the supernatant was concentrated to about 1mL, centrifuged again, and the pellet was collected and dried. The precipitates obtained in two times are both the target crude product 7-Br-AL I (B) 1 ) The purity was 92% and the yield was 91%.
Example 10
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Adding AL I (14.65mg, 0.05mmol), acetic acid (10 mL) and stirring magneton into a three-neck flask in sequence under nitrogen atmosphere, adding tetrabutylammonium tribromide (24.1mg, 0.05mmol), and stirring in a water bath at 20 ℃ for reaction for 5h; then adding tetrabutylammonium tribromide (24.1mg, 0.05mmol), continuing to stir at 20 ℃ for 2h, then heating to 35 ℃ to continue to react for 2h, and standing at room temperature for reacting overnight. After the reaction is finished, centrifugally separating supernate and precipitate, drying the precipitate, directly collecting the precipitate, and taking the obtained precipitate as a target crude product 7-Br-AL I (B) 1 ) The purity is 89%The yield thereof was found to be 33%.
Example 11
Compound B 1 (R 1 A structure shown as formula II with methoxyl and X being Br) synthesis
Under nitrogen atmosphere, into a three-necked flask, AL I (14.65mg, 0.05mmol), acetic acid (10 mL) and a stirring magneton were sequentially added, DBDMH (14.3mg, 0.05mmol) was added, and the mixture was stirred in a water bath at 20 ℃ for reaction for 5 hours. After the reaction, the supernatant and the precipitate were centrifuged. Directly collecting the precipitate after drying; the supernatant was concentrated to about 1mL, centrifuged again, and the pellet was collected and dried. The precipitates obtained in two times are both the target crude product 7-Br-AL I (B) 1 ) The purity was 92% and the yield was 86%.
Example 12
Compound B 1 (R 1 The structure shown as the formula II with methoxyl and X being Br) is synthesized
Under nitrogen atmosphere, sequentially adding AL I (14.65mg, 0.05mmol), acetic acid (10 mL) and a stirring magneton into a three-necked flask, adding DBDMH (14.3mg, 0.05mmol), placing in a water bath at 20 ℃ for stirring reaction for 3h, and supplementing DBDMH (14.3mg, 0.05mmol) and continuing stirring reaction at 20 ℃ for 5h. After the reaction, the supernatant and the precipitate were separated by centrifugation. Directly collecting the precipitate after drying; the supernatant was concentrated to about 1mL, centrifuged again, and the pellet was collected and dried. The precipitates obtained in two times are both the target crude product 7-Br-AL I (B) 1 ) The purity was 92% and the yield was 90%.
Example 13
Compound B 1 (R 1 A structure shown as formula II with methoxyl and X being Br) synthesis
Under nitrogen, AL I (146.5 mg, 0.50mmol), acetic acid (100 mL) and a stirring magneton were added sequentially to a three-necked flask, DBDMH (143mg, 0.50mmol) was added, and the mixture was stirred in a water bath at 20 ℃ for reaction for 9 hours. After the reaction, the supernatant and the precipitate were centrifuged. Directly collecting the precipitate after drying; the supernatant was concentrated to about 10mL, centrifuged again, and the pellet was collected and dried. The precipitates obtained in two times are both the target crude product 7-Br-AL I (B) 1 ) The purity was 92% and the yield was 90%.
Example 14
Compound B 2 (R 1 The synthesis of a structure shown as formula II) with hydrogen and X being Br
Under nitrogen atmosphere, sequentially adding AL II (namely R) into a three-mouth bottle 1 Aristolochianolamide as hydrogen, 13.15mg, 0.05mmol), acetic acid (15 mL) and stirring magneton, DBDMH (14.3mg, 0.05mmol) was added, and the mixture was stirred in a water bath at 20 ℃ for reaction for 7 hours. After the reaction, the supernatant and the precipitate were centrifuged. Directly collecting the precipitate after drying; the supernatant was concentrated to about 1mL, centrifuged again, and the pellet was collected and dried. The precipitates obtained in the two times are both the target crude product 7-Br-AL II (B) 2 ) The purity was 90% and the yield 80%.
Example 15
Compound B 2 (R 1 The synthesis of a structure shown as formula II) with hydrogen and X being Br
To a three-necked flask, AL II (131.5mg, 0.50mmol), acetic acid (150 mL) and a stirring magneton were sequentially added under nitrogen, DBDMH (143mg, 0.50mmol) was added, and the mixture was stirred in a water bath at 20 ℃ for reaction for 9 hours. After the reaction, the supernatant and the precipitate were separated by centrifugation. Directly collecting the precipitate after drying; the supernatant was concentrated to about 10mL, centrifuged again, and the pellet was collected and dried. The precipitates obtained in the two times are both the target crude product 7-Br-AL II (B) 2 ) The purity was 90% and the yield was 87%.
Example 16
The difference from example 9 is that the reaction was carried out in an air atmosphere. The purity of the target crude product 7-Br-AL I was 89%, and the yield was 80%.
Example 17
AAⅠ-dA(C 1 ，R 1 Is methoxy, R 2 (III) the structure of formula III as deoxyadenosine) and purification
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 ) (20.89 mg, purity 89%,0.05 mmol), dA (18.80mg, 0.075mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd 2 (dba) 3 4mL of (0.9 mg,2.0 mu mol Pd), xantphos (1.2 mg,2.0 mu mol), DMAc/PX (1:1) mixed solvent and stirring magneton, after bubbling for 10min with nitrogen, raising the temperature to 100 ℃ under the protection of nitrogen, and stirring for reactionAnd 9h. And cooling to room temperature. Detecting target product AA I-dA (C) in system by HPLC 1 ) The content is about 4.5%. Due to the target product AA I-dA (C) 1 ) The content is less, and the yield is difficult to accurately weigh without counting.
Example 18
AAⅠ-dA(C 1 ，R 1 Is methoxy, R 2 (III) the structure of formula III as deoxyadenosine) and purification
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 ) (20.89 mg, purity 89%,0.05 mmol), dA (25.12mg, 0.10mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd 2 (dba) 3 (13.74mg, 0.03mmol Pd), xantphos (17.36mg, 0.03mmol), 5.0mL DMAc and stirring magneton, after bubbling nitrogen for 10min, the temperature was raised to 100 ℃ under the protection of nitrogen, and the reaction was stirred for 5h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA I-dA (C) 1 ) Purity of>98% and a yield of 96%.
Example 19
AAⅠ-dA(C 1 ，R 1 Is methoxy, R 2 (III) the structure of formula III as deoxyadenosine) and purification
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 ) (20.89 mg, purity 89%,0.05 mmol), dA (18.80mg, 0.075mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd(CH 3 CN) 2 Cl 2 (7.78mg, 0.03mmol Pd), xantphos (17.36mg, 0.03mmol), DMAc 5mL, stirring magneton, bubbling nitrogen for 10min, raising temperature to 100 ℃ under the protection of nitrogen, and stirring for reaction for 7h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging to separate supernatant, and mixing with the supernatant obtained in the previous stepAnd, the solvent was evaporated to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA I-dA (C) 1 ) Purity of>98% and a yield of 70%.
Example 20
AAⅠ-dA(C 1 ，R 1 Is methoxy, R 2 Structure of formula III as deoxyadenosine) and purification thereof
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 ) (20.89 mg, purity 89%,0.05 mmol), dA (18.80mg, 0.075mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd(OAc) 2 (6.74mg, 0.03mmol Pd), xantphos (17.36mg, 0.03mmol), DMAc 5mL, stirring magneton, bubbling nitrogen for 10min, heating to 100 ℃ under the protection of nitrogen, and stirring for 5h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA I-dA (C) 1 ) Purity of>98% and a yield of 92%.
Example 21
AAⅠ-dA(C 1 ，R 1 Is methoxy, R 2 Structure of formula III as deoxyadenosine) and purification thereof
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 ) (20.89 mg, purity 89%,0.05 mmol), dA (25.12mg, 0.10mmol), and K 2 CO 3 (13.8mg，0.1mmol)、Pd(OAc) 2 (6.74mg, 0.03mmol Pd), xantphos (17.36mg, 0.03mmol), 5.0mL DMAC and stirring magneton, bubbling nitrogen for 10min, heating to 100 ℃ under the protection of nitrogen, and stirring for reaction for 5h. And cooling to room temperature. Centrifuging to separate precipitate and supernatant. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA I-dA (C) 1 ) Purity of>98% and a yield of 67%.
Example 22
AAⅠ-dA(C 1 ，R 1 Is methoxy, R 2 Structure of formula III as deoxyadenosine) and purification thereof
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 )(205.1mg，0.50mmol)、dA：(251.2mg，1.00mmol)、Cs 2 CO 3 (325.8mg，1.00mmol)、Pd 2 (dba) 3 (160.3mg, 0.35mmol Pd), xantphos (202.5mg, 0.35mmol), 30.0mL of N, N-dimethylacetamide (DMAc) and stirring magnetons, after bubbling for 10min with nitrogen, the temperature was raised to 100 ℃ under the protection of nitrogen, and the reaction was stirred for 10h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 20mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 20mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 3 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA I-dA (C) 1 ) Purity of>98% and the yield 98.7%.
Example 23
AAⅠ-dG(C 2 ，R 1 Is methoxy, R 2 Structure of formula III for deoxyguanosine) and purification
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 ) (20.89 mg, purity 89%,0.05 mmol), dG (28.53mg, 0.10mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd 2 (dba) 3 (13.74mg, 0.03mmol Pd), xantphos (17.36mg, 0.03mmol), 5.0mL DMAC andstirring the magnetons, bubbling nitrogen for 10min, raising the temperature to 100 ℃ under the protection of nitrogen, and stirring for reacting for 5h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA I-dG (C) 2 ) Purity of>98% and a yield of 86%.
Example 24
AAⅠ-dC(C 3 ，R 1 Is methoxy, R 2 Structure of formula III for deoxycytidine) and purification
To a three-necked flask, the compound 7-Br-AL I (B) was added in sequence 1 ) (20.89 mg, purity 89%,0.05 mmol), dC (30.72mg, 0.10mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd 2 (dba) 3 (13.74mg, 0.03mmol Pd), xantphos (17.36mg, 0.03mmol), 5.0mL DMAC and stirring magneton, bubbling nitrogen for 10min, raising the temperature to 100 ℃ under the protection of nitrogen, and stirring for reaction for 5h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA I-dC (C) 3 ) Purity of>98% and a yield of 90%.
Example 25
AAⅡ-dA(C 4 ，R 1 Is hydrogen, R 2 Structure of formula III as deoxyadenosine) and purification thereof
To a three-necked flask, the compound 7-Br-AL II (B) was added in sequence 2 ) (17.10 mg, purity 90%,0.05 mmol), dA (25.12mg, 0.10mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd 2 (dba) 3 (22.90mg, 0.05mmol Pd), xantphos (28.93mg, 0.05mmol), 5.0mLDMAc and stirring magneton, after bubbling for 10min with nitrogen, heating to 100 ℃ under the protection of nitrogen, and stirring for reaction for 5h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA II-dA (C) 4 ) Purity of>98% and a yield of 94%.
Example 26
AAⅡ-dG(C 5 ，R 1 Is hydrogen, R 2 Structure of formula III for deoxyguanosine) and purification
To a three-necked flask, the compound 7-Br-AL II (B) was added in sequence 2 ) (17.10 mg, purity 90%,0.05 mmol), dG (28.53mg, 0.10mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd 2 (dba) 3 (22.90mg, 0.05mmol Pd), xantphos (28.93mg, 0.05mmol), 5.0mL DMAC and stirring magneton, after bubbling nitrogen for 10min, raising the temperature to 100 ℃ under the protection of nitrogen, and stirring for reaction for 5h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, fully cooling for 2h, and performing centrifugal separation to obtain a precipitate 1 and a supernatant; adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA II-dG (C) 5 ) Purity of>98% and a yield of 84%.
Example 27
AAⅡ-dC(C 6 ，R 1 Is hydrogen, R 2 Is deoxycytidine of the formula IIIStructure shown) synthesis and purification
To a three-necked flask, the compound 7-Br-AL II (B) was added in sequence 2 ) (17.10 mg, purity 90%,0.05 mmol), dC (30.72mg, 0.10mmol), cs 2 CO 3 (32.58mg，0.1mmol)、Pd 2 (dba) 3 (22.90mg, 0.05mmol Pd), xantphos (28.93mg, 0.05mmol), 5.0mLDMAc and stirring magneton, after bubbling for 10min with nitrogen, heating to 100 ℃ under the protection of nitrogen, and stirring for reaction for 5h. And cooling to room temperature. The precipitate and supernatant were centrifuged. Adding 1mL DMAc into the precipitate, performing ultrasonic treatment, centrifuging, separating supernatant, combining with the supernatant obtained in the previous step, and volatilizing the solvent to obtain a reaction residue. Adding 10mL of water into the reaction residue after the solvent is volatilized, heating and refluxing for 2h, and centrifuging to obtain a precipitate 1; adding 10mL of toluene, DCM and THF into the precipitate 1, heating and refluxing for 2h, repeating for 2 times, and centrifuging to obtain a precipitate 2; volatilizing the solvent in the precipitate 2 to obtain the target product AA II-dC (C) 6 ) Purity of>98% and a yield of 91%.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the commercial product is used as a reaction raw material, so that the reaction route is simplified, the preparation process is easy to control, the operation is convenient, the aristolochic acid-deoxynucleoside conjugate with high purity can be obtained, and the large-scale synthesis is convenient.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for preparing an aristolochic acid-deoxynucleoside conjugate, comprising the steps of:
step S1, under the action of a halogenating reagent, taking aristololactam with a structure shown in a formula I as a substrate, and carrying out a halogenating reaction in a first solvent to obtain a halogen substituent of aristololactam with a structure shown in a formula II;
s2, taking halogen substitutes of the aristolochia lactam with the structure shown in the formula II and deoxynucleoside as substrates, carrying out Buchwald-Hartwig cross coupling reaction in a second solvent under the action of a catalyst and alkali, and separating and purifying the obtained reaction liquid to obtain an aristolochic acid-deoxynucleoside conjugate with the structure shown in the formula III;
wherein R in formula I, formula II and formula III 1 All represent hydrogen or methoxy, X in formula II represents any one of Cl, br and I, R in formula III 2 Represents any one of deoxyadenosine, deoxycytidine and deoxyguanosine.
2. The method of claim 1, wherein the halogenating agent is a brominating agent,
preferably, the brominating reagent is one or more of bromine, N-bromosuccinimide, 1,3-dibromo-5,5-dimethyl hydantoin and tetrabutylammonium tribromide;
more preferably, the molar ratio of the brominating reagent to the aristololactam is 1:1-5:1.
3. The production method according to claim 2, wherein the halogenation reaction is carried out in a nitrogen atmosphere;
preferably, the halogenating agent is added in two or more portions.
4. The method for preparing a polycarbonate according to claim 1, wherein the step S1 comprises,
step S11, adding the aristolochia lactam and a halogenating reagent into the first solvent, and reacting for 3-48 h to obtain a halogenating reaction solution; preferably, the reaction temperature is 15 to 60 ℃, more preferably 20 to 35 ℃;
s12, carrying out centrifugal separation on the halogenated reaction liquid to obtain supernatant and precipitate, collecting the precipitate, and drying to obtain the halogen substituent;
preferably, the supernatant is concentrated, and subjected to a second centrifugal separation, and the precipitate obtained by the second centrifugal separation is purified to obtain the halogen substituent;
preferably, the purification comprises adding any one or more of acetic acid, n-butanol and tetrahydrofuran to the precipitate obtained by the second centrifugal separation, performing ultrasonic treatment, performing third centrifugal separation, and collecting the precipitate to obtain the halogen substituent.
5. The method according to claim 1, wherein the first solvent in step S1 comprises any one or more of tetrahydrofuran, N-dimethylformamide, dimethyl sulfoxide, methylene chloride and acetic acid;
preferably, the amount of the first solvent added is 20 to 400 mL-mmol based on the substance amount of the aristololactam -1 。
6. The method according to claim 1, wherein the catalyst of step S2 is Pd 2 (dba) 3 、Pd(OAc) 2 、Pd(CH 3 CN) 2 Cl 2 And Cu i, preferably the molar ratio of the catalyst to the halogen substituent is from 0.04 to 1:1.
7. The preparation method of claim 1, wherein the catalyst further comprises a ligand, preferably the ligand is selected from any one or more of 4,5-bis diphenylphosphine-9,9-dimethyl xanthene and pyridine, and preferably the molar ratio of the ligand to the catalyst is 1:1-3:1.
8. The method of claim 1, wherein the Buchwald-Hartwig cross-coupling reaction is performed in a nitrogen atmosphere,
preferably, the reaction temperature is 80-120 ℃, and the reaction time is 5-10 h;
preferably, the molar ratio of the halo substituent to the deoxynucleoside is 1:1 to 1:5.
9. The preparation method of claim 1, wherein the base is any one or more of potassium carbonate and cesium carbonate, and preferably, the molar ratio of the base to the halide is 1:1-3:1;
and/or the second solvent is one or more of N, N-dimethylacetamide and xylene.
10. The method according to claim 1, wherein the separation and purification in step S2 comprises:
step S201, carrying out centrifugal separation on the reaction liquid, collecting supernatant, and removing a solvent to obtain a crude product of the aristolochic acid-deoxynucleoside conjugate;
step S202, adding a strong polar solvent and a weak polar solvent into the crude aristolochic acid-deoxynucleoside conjugate, heating and refluxing, preferably, the refluxing time is 0.5-2 h, and cooling to room temperature; the strong polar solvent is water, and the weak polar solvent is any one or more of acetone, n-butanol, ethyl acetate, toluene, dichloromethane and tetrahydrofuran; and step S203, performing centrifugal separation on the substance obtained in the step S202, and collecting precipitates to obtain the aristolochic acid-deoxynucleoside conjugate.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4782077A (en) * | 1987-08-20 | 1988-11-01 | Monoclonetics International, Inc. | Taliscanin and other aristolactams for treating neurological disorders, Parkinson's disease, Alzheimer disease and impotence |
| US20020040488A1 (en) * | 1998-09-18 | 2002-04-04 | The University Of Kentucky Research Foundation, Kentucky Corporation | Synthases |
| CN1690686A (en) * | 2004-04-19 | 2005-11-02 | 北京大学 | Method for pretreatment of blood sample containing aristolochic acid and lactam ingredients thereof |
| CN1959408A (en) * | 2005-11-01 | 2007-05-09 | 北京大学 | Method for mensurating in vivo sample of containing aristolochic acid and components of lactam categories |
| CN106198771A (en) * | 2015-05-27 | 2016-12-07 | 北京大学 | Measure the HPLC-FLD-DAD of two breeds of horses Semen Oroxyli acid-DNA adduct in tissue simultaneously and analyze method |
| CN106872247A (en) * | 2015-12-10 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of preprocess method for being enriched with aristolochic acid |
| CN110194758A (en) * | 2019-04-29 | 2019-09-03 | 山东省分析测试中心 | A method of the fast separating and purifying Aristolochic Acid compound from caulis aristologhiae manshuriensis |
| CN114624376A (en) * | 2020-12-14 | 2022-06-14 | 中国科学院上海药物研究所 | Method for detecting aristolochic acid I-DNA adduct in tissue |
-
2022
- 2022-10-28 CN CN202211337892.5A patent/CN115677808B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4782077A (en) * | 1987-08-20 | 1988-11-01 | Monoclonetics International, Inc. | Taliscanin and other aristolactams for treating neurological disorders, Parkinson's disease, Alzheimer disease and impotence |
| US20020040488A1 (en) * | 1998-09-18 | 2002-04-04 | The University Of Kentucky Research Foundation, Kentucky Corporation | Synthases |
| CN1690686A (en) * | 2004-04-19 | 2005-11-02 | 北京大学 | Method for pretreatment of blood sample containing aristolochic acid and lactam ingredients thereof |
| CN1959408A (en) * | 2005-11-01 | 2007-05-09 | 北京大学 | Method for mensurating in vivo sample of containing aristolochic acid and components of lactam categories |
| CN106198771A (en) * | 2015-05-27 | 2016-12-07 | 北京大学 | Measure the HPLC-FLD-DAD of two breeds of horses Semen Oroxyli acid-DNA adduct in tissue simultaneously and analyze method |
| CN106872247A (en) * | 2015-12-10 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of preprocess method for being enriched with aristolochic acid |
| CN110194758A (en) * | 2019-04-29 | 2019-09-03 | 山东省分析测试中心 | A method of the fast separating and purifying Aristolochic Acid compound from caulis aristologhiae manshuriensis |
| CN114624376A (en) * | 2020-12-14 | 2022-06-14 | 中国科学院上海药物研究所 | Method for detecting aristolochic acid I-DNA adduct in tissue |
Non-Patent Citations (5)
| Title |
|---|
| SIVAPRASAD ATTALURI，ET AL.: ""DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells"", 《NUCLEIC ACIDS RESEARCH》, vol. 38, no. 1, 23 October 2009 (2009-10-23), pages 339 - 352, XP055055519, DOI: 10.1093/nar/gkp815 * |
| SIVAPRASAD ATTALURI，ET AL.: ""Total Synthesis of the Aristolochic Acids, Their Major Metabolites, and Related Compounds"", 《CHEM. RES. TOXICOL.》, vol. 27, 30 May 2014 (2014-05-30), pages 1236 - 1242 * |
| 刘倩 等: ""马兜铃酸Ⅰ-DNA 加合物合成条件优选"", 《中成药》, vol. 35, no. 11, 30 November 2013 (2013-11-30), pages 2433 - 2438 * |
| 季文萱 等: ""马兜铃酸-脱氧核糖核酸加合物的质谱分析"", 《分析化学》, vol. 36, no. 7, 31 July 2008 (2008-07-31), pages 930 - 934 * |
| 杨晓芹 等: ""马兜铃酸Ⅰ-脱氧核苷结合物对照品制备方法研究"", 《中国中药杂志》, 10 March 2024 (2024-03-10), pages 1 - 9 * |
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