CN101899084A - (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid kyrine conjugate, preparation method and application thereof - Google Patents
(3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid kyrine conjugate, preparation method and application thereof Download PDFInfo
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- CN101899084A CN101899084A CN2009100853249A CN200910085324A CN101899084A CN 101899084 A CN101899084 A CN 101899084A CN 2009100853249 A CN2009100853249 A CN 2009100853249A CN 200910085324 A CN200910085324 A CN 200910085324A CN 101899084 A CN101899084 A CN 101899084A
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- Prior art keywords
- tetrahydroisoquinoline
- boc
- arg
- obzl
- gly
- Prior art date
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Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 43
- BWKMGYQJPOAASG-VIFPVBQESA-N (3s)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Chemical compound C1=CC=C2CN[C@H](C(=O)O)CC2=C1 BWKMGYQJPOAASG-VIFPVBQESA-N 0.000 title claims abstract description 11
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 14
- 230000002785 anti-thrombosis Effects 0.000 claims abstract description 12
- 230000008878 coupling Effects 0.000 claims abstract description 8
- 238000010168 coupling process Methods 0.000 claims abstract description 8
- 238000005859 coupling reaction Methods 0.000 claims abstract description 8
- 125000006239 protecting group Chemical group 0.000 claims abstract description 5
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 12
- -1 dicyclohexyl carbonyl diimine Chemical compound 0.000 claims description 11
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 9
- 229910000071 diazene Inorganic materials 0.000 claims description 7
- 239000000863 peptide conjugate Substances 0.000 claims description 7
- HFPVZPNLMJDJFB-LBPRGKRZSA-N (3s)-2-[(2-methylpropan-2-yl)oxycarbonyl]-3,4-dihydro-1h-isoquinoline-3-carboxylic acid Chemical compound C1=CC=C2C[C@@H](C(O)=O)N(C(=O)OC(C)(C)C)CC2=C1 HFPVZPNLMJDJFB-LBPRGKRZSA-N 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 13
- 239000002245 particle Substances 0.000 abstract description 10
- 229960004676 antithrombotic agent Drugs 0.000 abstract description 3
- 239000002105 nanoparticle Substances 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 description 49
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 42
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 42
- 239000007864 aqueous solution Substances 0.000 description 31
- 239000007787 solid Substances 0.000 description 27
- 239000000243 solution Substances 0.000 description 26
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 23
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 20
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 20
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 18
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 17
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000000562 conjugate Substances 0.000 description 12
- 239000011734 sodium Substances 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 11
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 9
- 229960001701 chloroform Drugs 0.000 description 9
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 230000006837 decompression Effects 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 208000007536 Thrombosis Diseases 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000003208 petroleum Substances 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 239000004471 Glycine Substances 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 229960005261 aspartic acid Drugs 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- MVEAAGBEUOMFRX-UHFFFAOYSA-N ethyl acetate;hydrochloride Chemical compound Cl.CCOC(C)=O MVEAAGBEUOMFRX-UHFFFAOYSA-N 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 208000005189 Embolism Diseases 0.000 description 2
- OPINTGHFESTVAX-BQBZGAKWSA-N Gln-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N OPINTGHFESTVAX-BQBZGAKWSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960001008 heparin sodium Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- VRPJIFMKZZEXLR-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(O)=O VRPJIFMKZZEXLR-UHFFFAOYSA-N 0.000 description 1
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 1
- 241000304531 Allium macrostemon Species 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- DWKPPFQULDPWHX-VKHMYHEASA-N l-alanyl ester Chemical compound COC(=O)[C@H](C)N DWKPPFQULDPWHX-VKHMYHEASA-N 0.000 description 1
- 208000020442 loss of weight Diseases 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- GBDRMPRTNVKBAD-BYPYZUCNSA-N methyl (2s)-2,5-diamino-5-oxopentanoate Chemical class COC(=O)[C@@H](N)CCC(N)=O GBDRMPRTNVKBAD-BYPYZUCNSA-N 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000012454 non-polar solvent Substances 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 235000008729 phenylalanine Nutrition 0.000 description 1
- 150000002994 phenylalanines Chemical class 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- CHKVPAROMQMJNQ-UHFFFAOYSA-M potassium bisulfate Chemical compound [K+].OS([O-])(=O)=O CHKVPAROMQMJNQ-UHFFFAOYSA-M 0.000 description 1
- 229910000343 potassium bisulfate Inorganic materials 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
Images
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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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid kyrine conjugate, a preparation method and application thereof. The preparation method of the conjugate comprises the following steps of: preparing N-[(3S)-N-Boc-1,2,3,4- tetrahydroisoquinoline-3-formoxyl]-AA1-AA2-Arg (NO2)-OBzl by coupling N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formoxyl]-AA1 and AA2-Arg (NO2)-OBzl; and detracting a protecting group, wherein AAl is selected from Gly, Asp or Gln, and AA2 is selected from Gly, Asp or Gln. The conjugate has excellent antithrombotic activity, can be clinically used as an antithrombotic agent, can be automatically assembled into nano particles in a water solution and is stable in the water solution; and the particle diameter is mostly from 200nm to 600nm.
Description
Invention field
The present invention relates to the conjugate that a class has antithrombotic acitivity, relate in particular to by (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and tripeptides coupling and conjugate, the invention still further relates to the preparation method of this conjugate and this conjugate as the application of antithrombotic agent, belong to biomedicine field.
Background technology
Vessel embolism is to the negative most important responsibility of the high mortality of cardiovascular and cerebrovascular diseases.Thrombosis is the most important cause of disease of vessel embolism morbidity.Seeking antithrombotic reagent is one of focus of new drug research.The contriver notices, 1,2,3, and 4-tetrahydrochysene-β-Ka Lin-3-S-carboxylic acid is a kind of composition in the Chinese medicine Longstamen Onion Bulb, has platelet aggregation inhibitory activity (Yao new life etc., Chinese pharmaceutical chemistry magazine, 1995,5,134).The contriver thinks that (3S)-1,2,3, the reasonable step that 4-tetrahydrochysene-β-Ka Lin-3-carboxylic acid structure is simplified should be to remove pyrrole ring, simplifies (3S)-1,2,3 that obtains, and 4-tetrahydroisoquinoline-3-carboxylic acid also has antithrombotic acitivity.But (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid solubleness in polar solvent and non-polar solvent is all bad, and bioavailability is low, has to be overcome.
Summary of the invention
At (3S)-1,2,3, though 4-tetrahydroisoquinoline-3-carboxylic acid has antithrombotic acitivity, there are defectives such as water-soluble degree is little, bioavailability is low, the present invention is with (3S)-1,2,3, the structure of 4-tetrahydroisoquinoline-3-carboxylic acid is modified, under the prerequisite that guarantees its antithrombotic acitivity, improve (3S)-1,2,3, the absorption of 4-tetrahydroisoquinoline-3-carboxylic acid improves (3S)-1,2,3, the bioavailability of 4-tetrahydroisoquinoline-3-carboxylic acid.
Technical problem to be solved by this invention is achieved through the following technical solutions:
(3S)-1,2,3 with antithrombotic acitivity, 4-tetrahydroisoquinoline-3-carboxylic acid three peptide conjugates, its structural formula is shown in the general formula I:
Wherein, AA1 is selected from Gly, Asp or Gln; AA2 is selected from Gly, Asp or Gln.
The present invention also provides the intermediate of compound of Formula I, and the structure of this intermediate is shown in the general formula I I:
Wherein, AA1 is selected from Gly, Asp or Gln; AA2 is selected from Gly, Asp or Gln.
The present invention also provides a kind of method for preparing above-mentioned general formula I conjugate, may further comprise the steps:
(1) in the presence of DCC and NMM, with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1With AA
2-Arg (NO
2)-OBzl coupling prepares N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-AA
2-Arg (NO
2)-OBzl; AA wherein
1Be selected from Gly, Asp or Gln; AA
2Be selected from Gly, Asp or Gln;
(2) with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-AA
2-Arg (NO
2)-OBzl sloughs protecting group, promptly.
Preferably, in the step (2) in the presence of trifluoracetic acid and trifluoromethanesulfonic acid, with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-AA
2-Arg (NO
2)-OBzl sloughs protecting group; Wherein, the volume ratio of trifluoracetic acid and trifluoromethanesulfonic acid is 4: 1.
Wherein, described N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1Prepare with reference to following method: (1) preparation (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; (2) at (Boc)
2With (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was converted into (3S)-N-Boc-1 under O existed, and 2,3,4-tetrahydroisoquinoline-3-carboxylic acid; (3) in the presence of dicyclohexyl carbonyl diimine (DCC) and N-methylmorpholine with (3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and L-AA
1-OMe coupling, preparation N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-OMe; (4) with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-OMe hydrolysis prepares N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1AA wherein
1Be selected from Gly, Asp or Gln.
Described AA
2-Arg (NO
2)-Obzl can prepare with reference to following method:
(1) in the presence of dicyclohexyl carbonyl diimine (DCC) and N-methylmorpholine with Boc-AA
2With NH
2-Arg (NO
2)-OBzl coupling prepares Boc-AA
2-Arg (NO
2)-OBzl; (2) with Boc-AA
2-Arg (NO
2)-OBzl takes off Boc and prepares NH
2-AA
2-Arg (NO
2)-OBzl; Wherein, AA
2Be selected from Gly, Asp or Gln;
Adopt the antithrombotic acitivity of having estimated conjugate of the present invention on the rat suppository model, test-results shows that conjugate of the present invention has outstanding antithrombotic acitivity, can use as antithrombotic agent.
Conjugate of the present invention can be self-assembled into nano particle in the aqueous solution, stable in the aqueous solution (can in the aqueous solution stable existence 8 days), and the particle diameter of conjugate of the present invention is mostly between 200～600nm.
Another purpose of the present invention provides the medicinal compositions of the above-mentioned general formula compound of a kind of the present invention of containing, and this medicinal compositions is gone up effective dose by treatment conjugate of the present invention is with pharmaceutically acceptable excipient or assist and add agent and form; That is: with the conjugate of the present invention of significant quantity with after pharmaceutically acceptable carrier or thinner cooperate, by the formulation method of this area routine it is prepared into any one appropriate drug composition.Usually said composition is suitable for oral administration and drug administration by injection, also is fit to other medication.Said composition can be liquid preparation forms such as tablet, capsule, pulvis, granule, lozenge, suppository, or oral liquid.According to different medications, pharmaceutical composition of the present invention can contain 0.1%-99% weight, the conjugate of the present invention of preferred 10-60% weight.
Description of drawings
Fig. 1 N-1,2,3,4-tetrahydroisoquinoline-3-S-formyl radical-AA
1-AA
2The synthetic route chart .I of-Arg) formaldehyde and concentrated hydrochloric acid; II) (Boc)
2O, 1N sodium hydroxide, tetrahydrofuran (THF); III) L-AA
1-OMe, DCC, HOBt and NMM, (AA
1=Gly, Asp, Gln); IV) the 2N NaOH aqueous solution; V) NH
2-Arg (NO
2) OBzl, DCC, HOBt and NMM (AA
2=Gly, Asp, Gln); VI) 4N hydrogenchloride-ethyl acetate solution; (AA
2=Gly, Asp, Gln); VII) DCC, HOBt and NMM; VIII) trifluoracetic acid and trifluoromethanesulfonic acid. at 1a, AA among the 2a
1=Gly, AA
2=Gly, 1b, AA among the 2b
1=Gly, AA
2=Asp, 1c, AA among the 2c
1=Gly, AA
2=Gln, 1d, AA among the 2d
1=Asp, AA
2=Gly, 1e, AA among the 2e
1=Asp, AA
2=Asp, 1f, AA among the 2f
1=Asp, AA
2=Gln, 1g, AA among the 2g
1=Gln, AA
2=Gly, 1h, AA among the 2h
1=Gln, AA
2=Asp, 1i, AA among the 2i
1=Gln, AA
2=Cln.
The compound 2a-i (10 that Fig. 2 laser particle size analyzer is measured
-3M) in the stability of the nanometer ball of water assembling.
The compound 2a-i (10 that Fig. 3 laser particle size analyzer is measured
-5M) in the stability of the nanometer ball of water assembling.
The compound 2a-i (10 that Fig. 4 laser particle size analyzer is measured
-7M) in the stability of the nanometer ball of water assembling.
Embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
In 4.0g (24.2mmol) L-phenylalanine, dropwise add 21.6ml formaldehyde earlier, dropwise add 36ml 35% concentrated hydrochloric acid again.The suspension oil bath that obtains is heated to 2 two hours phenylalanines of 90-100 ℃ of stirring and dissolves fully, reacts after 2.5 hours, and beginning adularescent precipitation generates, and reacts TLC (CHCl 7 hours
3/ CH
3OH=5/1) show that the L-phenylalanine disappears, suction filtration gets the 4.2g faint yellow solid, it is dried, then the yolk yellow solid of gained is joined in the 86ml ethanol (80%) 80 ℃ of oil baths and be heated to the colorless solid dissolving, be cooled to room temperature, slowly drip the 2mol/mlNaOH potassium hydroxide solution, have colourless precipitation to separate out, to the precipitation filter the most for a long time 4.17g (97.5%) title compound, be colorless solid.Mp.302.1～302.5 ℃, ESI-MS (m/e) 176[M-1]
-.[α]
D 25=-68 (c=0.6 methyl alcohol).
1HNMR (300MHz, DMSO): δ/ppm=2.712～2.919 (m, J=5.6Hz, 2H); 4.136～4.528 (m, J=4.5Hz, 3H), 7.228 (t, J=7.7Hz, 7.3Hz, 1H); 7.22 (t, J=8.2Hz, 7.2Hz, 1H); 7.44 (d, J=8.0Hz, 1H); 7.563 (d, J=7.5Hz, 1H); 8.91 (d, J=6.2Hz, 1H); 12.9 (s, 1H);
13CNMR (300MHZ, DMSO): 30.2,46.4,56.6,35.5,125.6,126.8,128.4,127.2,135.9,173.2.
The following 2.49g of ice bath (62.15mmol) sodium hydroxide dissolves in 62.2ml water, adds 10g (56.49mmol) then (3S)-1,2,3, and 4-tetrahydroisoquinoline-3-carboxylic acid is made suspension.With 40ml tetrahydrofuran (THF) dissolving 14.8g (67.8mmol) (Boc)
2O adds in the suspension.Reaction mixture stirred 24 hours, constantly extracted the CO that reaction generates in the reaction process
2, when the solution becomes clarification, TLC (methyl alcohol/chloroform: 1: 10) shows (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid disappears, and stops reaction.The reaction mixture concentrating under reduced pressure is removed tetrahydrofuran (THF), the oily matter acetic acid ethyl dissolution that obtains.The solution that obtains is used 5%KHSO successively
4Wash with the saturated NaCl aqueous solution.Organic layer is concentrated into dried with anhydrous sodium sulfate drying, filtration, filtrate decompression, the oily matter that obtains is done recrystallization with ether dissolution, and the colorless solid that obtains filters, and obtains 14.84g (95%) title compound.ESI-MS (m/e) 278.2[M+1]
+, [α]
D 25=-6.78 (c=0.5 methyl alcohol)
Ice bath down toward 0.256g (0.924mmol) (3S)-N-Boc-1,2,3, add 0.151g (1.109mmol) N-hydroxy benzo triazole (HOBt) in the solution of 4-tetrahydroisoquinoline-3-carboxylic acid and the anhydrous THF of 10ml, stir and add 0.228g (1.08mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes, obtain reaction solution (I).0.136g (0.102mmol) HClGly-OMe is suspended among the anhydrous THF of 4ml, adds 1mlN-methylmorpholine (NMM) and transfer pH value 8～9, stir and obtain reaction solution (II).(I) added in (II), stirring at room 12h, TLC (ethyl acetate/petroleum ether, 1: 3) shows (3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid disappears.Leach dicyclohexylurea (DCU) (DCU), filtrate decompression is concentrated into dried, residue 50ml acetic acid ethyl dissolution.The solution that obtains is used 5%NaHCO successively
3The aqueous solution is washed 3 times, the saturated NaCl aqueous solution and is washed 5%KHSO 3 times
4The aqueous solution is washed 3 times, and the saturated NaCl aqueous solution is washed 3 times, saturated NaHCO
3The aqueous solution is washed 3 times, and the saturated NaCl aqueous solution is washed till neutrality.The ethyl acetate layer anhydrous Na
2SO
4Drying, filtration, filtrate decompression concentrate 0.31g (97%) title compound, be colorless solid.TLC (ethyl acetate/petroleum ether, 1: 3, Rf=0.17), Mp:125.7～125.9 ℃, ESI-MS (m/e) 349.2[M+1]
+, 249.2[M+1-Boc]
+, [α]
D 25=-15.83 (c=0.7 methyl alcohol).
According to the operation of embodiment 3, from 0.6g (2.186mmol) (3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and 0.512g (2.8mmol) HClAsp-(OMe)
2Make 0.8g (96%) title compound, be oily matter.TLC (ethyl acetate/petroleum ether, 1: 3, Rf=0.18), Mp:105.6～106.9 ℃, ESI-MS (m/e) 407.3[M+1]
+, 307.3[M+1-Boc]
+, [α]
D 25=-15.83 (c=0.7 methyl alcohol).
According to the operation of embodiment 3, from 0.6g (2.186mmol) (3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and 0.534g (2.8mmol) HClGln-OMe make 0.84g (98%) title compound, are oily matter.TLC (ethyl acetate/petroleum ether, 1: 3, Rf=0.30); Mp:100.1～101.5 ℃, ESI-MS (m/e) 421.3[M+2]
2+, 321.2[M+1-Boc]
2+, [α]
D 25=-28.78 (c=0.6 methyl alcohol).
With 1.04g (3mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-the L-glycine methyl ester is dissolved in 4ml methyl alcohol, splashes into 10ml NaOH (2N) aqueous solution under the ice bath in the solution that obtains., solution fades to yellow, and TLC after 5 hours (ethyl acetate/petroleum ether, 1: 3) shows N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-disappearance of L-alanine methyl ester, concentrating under reduced pressure boils off solvent, and residue adds the saturated KHSO of 30ml
4The accent pH value is 1-2, uses ethyl acetate extraction 3 times, and the ethyl acetate layer of merging is washed 1 time with saturated NaCl, uses anhydrous Na
2SO
4Dry.Filter, filtrate decompression concentrates, and gets 0.96g (96%) title compound, is colorless solid.Mp:147.2～149.3 ℃, ESI-MS (m/e) 333.2[M-1]
-, [α]
D 25=-7.22 (c=0.5 methyl alcohol).
According to the operation of embodiment 6, from 0.736g (1.75mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-L-aspartic acid methyl esters makes 0.680g (99%) title compound, is oily matter.Mp:131.1～132.9 ℃, ESI-MS (m/e) 391.1[M-1]
-, [α]
D 25=-7.92 (c=0.5 methyl alcohol).
According to the operation of embodiment 6, from 1.8g (4.3mmol) preparation N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-L-glutamine methyl esters makes 1.75g (98%) title compound, is colorless solid.Mp:153.5～156.7 ℃, ESI-MS (m/e) 404.2[M-1]
-, 809.3[2M-1]
-, [α]
D 25=-45.38 (c=0.5 methyl alcohol).
Embodiment 9 preparation Boc-Gly-Arg (NO
2) OBzl
Ice bath adds 1.21g (8.963mmol) N-hydroxy benzo triazole (HOBt) down in the solution of 1.73g (9.885mmol) Boc-Gly-OH and the anhydrous THF of 10ml, stir and add 2.21g (10.73mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes, obtain reaction solution (I).3.1g (8.973mmol) HClArg (NO
2) OBzl is suspended among the anhydrous THF of 3ml, adds 1mlN-methylmorpholine (NMM) and transfers pH value 8～9, stir and obtain reaction solution (II).(I) added in (II), stirring at room 12h, TLC (chloroform/methanol, 50: 1) shows HClArg (NO
2) the OBzl disappearance.Leach dicyclohexylurea (DCU) (DCU), filtrate decompression is concentrated into dried, and residue dissolves with the 200ml trichloromethane.The solution that obtains is used 5%NaHCO successively
3The aqueous solution is washed 3 times, the saturated NaCl aqueous solution and is washed 5%KHSO 3 times
4The aqueous solution is washed 3 times, and the saturated NaCl aqueous solution is washed 3 times, saturated NaHCO
3The aqueous solution is washed 3 times, and the saturated NaCl aqueous solution is washed till neutrality.Trichloromethane layer anhydrous Na
2SO
4Drying, filtration, filtrate decompression concentrate 3.98g (95%) title compound, be colorless solid.ESI-MS(m/e)489.3[M+23]
+，955.6[2M+23]
+；Mp：124.5～126.7℃，[α]
D 25＝-2.53(c＝0.5DMF).
Embodiment 10 preparation Boc-Asp (Arg (NO
2) OBzl)
2
According to the operation of embodiment 9, from 1.31g (5.62mmol) Boc-Asp and 4g (11.58mmol) HClArg (NO
2) 4.5g (95.3%) title compound of OBzl system, be colorless solid.ESI-MS(m/e)838.4[M+23]
+，Mp：185.2～186.2℃，[α]
D 25＝-11.38(c＝0.7DMF).
Embodiment 11 preparation Boc-Gln-Arg (NO
2) OBzl
According to the operation of embodiment 9, from 1.73g (7.032mmol) Boc-Gln and 3.2g (6.65mmol) HClArg (NO
2) 3.2g (89.6%) title compound of OBzl system, be colorless solid.ESI-MS(m/e)560.4[M+23]
+.，Mp：158.2～159.0℃，[α]
D 25＝-21.08(c＝0.5DMF).
Embodiment 12 preparation HClGly-Arg (NO
2) OBzl
With 2.33g (5mmol) Boc-Gly-Arg (NO
2) OBzl adds in the 5ml ether, adds 25ml hydrogenchloride-ethyl acetate (4N) solution again in the solution that obtains.0 ℃ is stirred 6h, and TLC (chloroform/methanol, 50/1) shows Boc-Gly-Arg (NO
2) disappearance of OBzl point.Reaction mixture is evaporated to dried, and residue stirs with the 10ml ether, is evaporated to dried again.This operation three times repeatedly.Get 1.86g (92.53%) title compound, be colorless solid.ESI-MS(m/e)367.3[M+1]
+，Mp：97.2～97.8℃，[α]
D 25＝-12.58(c＝0.6DMF).
Embodiment 13 preparation HClAsp (Arg (NO
2) OBzl)
2
According to the operation of embodiment 12, from 4.07g (5mmol) Boc-Asp (Arg (NO
2) OBzl)
23.5g (93%) title compound of system is colorless solid.ESI-MS(m/e)716.7[M+1]
+，Mp：118.0～118.2℃，[α]
D 25＝-10.38(c＝0.5DMF).
Embodiment 14 preparation HClGln-Arg (NO
2) OBzl
According to the operation of embodiment 12, from 3.2g (6mmol) Boc-Gln-Arg (NO
2) 2.8g (99.6%) title compound of OBzl system, be colorless solid.ESI-MS(m/z)438.4[M+1]
+，Mp：102.4～103.0℃，[α]
D 25＝-11.38(c＝0.7DMF).
Embodiment 15 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Gly-Arg (NO
2)-OBzl (1a)
Ice bath is down toward 1.102g (3.3mmol) N-[(3S)-N-Boc-1; 2; 3; 4-tetrahydroisoquinoline-3-formyl radical]-add 0.405g (3mmol) N-hydroxy benzo triazole (HOBt) in the solution of glycine and the anhydrous THF of 10ml; stir and add 0.742g (3.6mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes, obtain reaction solution (I).1.208g (3mmol) HClGly-Arg (NO2) OBzl is suspended in the 5ml dry DMF, adds 1mlN-methylmorpholine (NMM) and transfer pH value 8～9, stir and obtain reaction solution (II).(I) added in (II), stirring at room 24h, TLC (chloroform/methanol, 50: 1) shows HClGly-Arg (NO
2) the OBzl disappearance.Leach dicyclohexylurea (DCU) (DCU), filtrate decompression is concentrated into dried, and residue dissolves with the 200ml trichloromethane.The solution that obtains is washed 3 times, the saturated NaCl aqueous solution with the 5%NaHCO3 aqueous solution successively and is washed 3 times, and the 5%KHSO4 aqueous solution is washed 3 times, and the saturated NaCl aqueous solution is washed 3 times, and the saturated NaHCO3 aqueous solution is washed 3 times, and the saturated NaCl aqueous solution is washed till neutrality.The trichloromethane layer with anhydrous Na 2SO4 drying, filtration, filtrate decompression concentrate light yellow solid, chloroform: methyl alcohol=column chromatography obtained 1.208g (59%) title compound in 60: 1, was colorless solid.Mp：98.7～99.4℃，[α]
D 25＝-15.03(c＝0.55DMF)；ESI(m/e)682[M+H]
+，705[M+Na]
+.
Embodiment 16 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Asp-[Arg (NO
2)-OBzl]
2(1b)
According to the operation of embodiment 15, from 0.367g (1.1mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-glycine and 0.751g (1mmol) HClAsp (Arg (NO
2) OBzl)
20.648g (62.58%) title compound of system is colorless solid.ESI-MS(m/e)1054[M+23]
+，Mp：102.7～102.9℃，[α]
D 25＝-15.11(c＝0.6DMF).
Embodiment 17 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Gln-Arg (NO
2)-OBzl (1c)
According to the operation of embodiment 15, from 1.052g (3.15mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-glycine and 1.42g (3mmol) HClGln-Arg (NO
2) 0.648g (62.58%) title compound of OBzl system, be colorless solid.ESI(m/e)754[M+H]
+，776[M+Na]
+，Mp：101.9～103.6℃，[α]
D 25＝-12.14(c＝0.7DMF).
Embodiment 18 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Asp-Gly-Arg (NO
2)-OBzl)
2(1d)
According to the operation of embodiment 15, from 0.58g (1.48mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical] Asp (OH)
2And 1.2g (2.96mmol) HClGly-Arg (NO
2) 0.894g (55.6%) title compound of OBzl system, be colorless solid.ESI(m/e)：1111[M+Na]
+，Mp：111.2～112.4℃，[α]
D 25＝-17.89(c＝0.6DMF).
Embodiment 19 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Asp-[Asp (Arg (NO
2)-OBzl)
2]
2(1e)
According to the operation of embodiment 15, from 0.412g (1.05mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-aspartic acid and 1.58g (2.1mmol) HClAsp[Arg (NO
2)-OBzl]
20.946g (50.4%) title compound of system is colorless solid.ESI(m/e)：1809[M+Na]
+，Mp：112.9～114.8℃，[α]
D 25＝-20.78(c＝0.85DMF).
Embodiment 20 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Asp-[Gln-Arg (NO
2)-OBzl]
2(1f)
According to the operation of embodiment 15, from 0.58g (1.48mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-aspartic acid and 1.401g (2.96mmol) HClGln-Arg (NO
2) 0.886g (53%) title compound of OBzl system, be colorless solid.ESI(m/e)：1253[M+Na]+，Mp：101.7～102.7℃，[α]
D 25＝-32.06(c＝0.7DMF).
According to the operation of embodiment 15, from 1.053g (2.6mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-glutamine and 1.098g (2.73mmol) HClGly-Arg (NO
2) 1.42g (63%) title compound of OBzl system, be colorless solid.ESI(m/e)：776[M+Na]
+，Mp：111.7～111.9℃，[α]
D 25＝-17.60(c＝0.65DMF).
Embodiment 22 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Asp[Arg (NO
2)-OBzl]
2(1h)
According to the operation of embodiment 15, from 1.276g (3.15mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-glutamine and 2.254g (3mmol) HClAsp (Arg (NO
2) OBzl) 2 2.1g (70%) title compounds of making, be colorless solid.ESI(m/e)：1103[M+H]
+，1125[M+Na]
+，Mp，108.7～109.2℃，[α]
D 25＝-20.93(c＝0.6DMF).
Embodiment 23 preparation N-[(3S)-and N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Gln-Arg (NO
2)-OBzl (1i)
According to the operation of embodiment 15, from 1.276g (3.15mmol) N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-glutamine and 1.42g (3mmol) HClGlnArg (NO
2) 1.98g (78%) title compound of OBzl system, be colorless solid.ESI(m/e)：847[M+Na]
+，Mp：101.6～102.4℃，[α]
D 25＝-21.80(c＝0.5DMF).
Embodiment 24 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Gly-Arg (2a)
With 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Gly-Arg (NO
2) OBzl usefulness 20ml trifluoracetic acid dissolving under ice bath, and then in reaction solution, add the trifluoromethanesulfonic acid of 5ml, react 40 minutes TLC monitoring raw material points and disappear, add the 150ml ether, separate out white solid in the reaction solution gradually while in reaction solution, stir, ether is toppled over, decompressing and extracting obtains brown solid, again with solid with water dissolution after, the filtering insolubles, SepdexG10 carries out desalination, obtains aqueous solution freeze-drying and obtains 310mg (94.2%) title compound.ESI(m/e)：448[M+H]
+，Mp：154.6～155.8℃，[α]
D 25＝-19.14(c＝0.85DMF).
Embodiment 25 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Asp-(Arg)
2(2b)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Asp[Arg (NO
2) OBzl]
2In obtain 298mg (93%) title compound, ESI (m/e): 662[M+H]
+, Mp:136.6～137.8 ℃, [α]
D 25=-36.86 (c=0.5DMF).
Embodiment 26 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Gln-Arg (2c)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gly-Gln-Arg (NO
2) obtain 320mg (92.7%) title compound, ESI (m/e): 519[M+H among the OBzl]
+, Mp:178.4～178.8 ℃, [α]
D 25=-15.37 (c=0.85DMF).
Embodiment 27 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Asp-(Gly-Arg)
2(2d)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]--Asp-(Gly-Arg (NO
2) OBzl)
2In obtain 298mg (93%) title compound, ESI (m/e): 719[M+H]
+, Mp:111.6～111.8 ℃, [α]
D 25=-29.69 (c=0.65DMF).
Embodiment 29 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Asp-(Asp (Arg)
2)
2(2e)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Asp-[Asp-(Arg (NO
2) OBzl)
2]
2In obtain 300mg (93.7%) title compound, ESI (m/e): 1147[M+H]
+Mp:118.6～119.2 ℃, [α]
D 25=-20.27 (c=0.5DMF).
Embodiment 30 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Asp-(Gln-Arg)
2(2f)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]--Asp-[Gln-Arg (NO
2)-OBzl]
2In obtain 330mg (94.2%) title compound, ESI (m/e): 861[M+H]
+, Mp:122.6～123.8 ℃, [α]
D 25=-41.89 (c=0.6DMF).
Embodiment 31 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Gly-Arg (2g)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Gly-Arg (NO
2Obtain 318mg (92.4%) title compound among the)-OBzl, ESI (m/e): 519[M+H]
+, Mp:108.4～108.8 ℃, [α]
D 25=-9.41 (c=0.6DMF), ESI (m/e): 519[M+H]
+.
Embodiment 32 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Asp (Arg)
2(2h)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Asp-[Arg (NO
2)-OBzl]
2In obtain 314mg (94.3%) title compound, ESI (m/e): 733[M+H]
+, Mp:116.6～117.8 ℃, [α]
D 25=-23.68 (c=0.6DMF).
Embodiment 33 preparation N-[(3S)-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Gln-Arg (2i)
According to the operation of embodiment 24, from 500mg N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-Gln-Gln-Arg (NO
2Obtain 332mg (92.4%) title compound among the)-OBzl, ESI m/e): 590[M+H]
+, Mp:142.6～143.8 ℃, [α]
D 25=-27.53 (c=0.5DMF).
The antithrombotic acitivity test of test example 1 The compounds of this invention (2a-i)
1, test compound: the compound that the embodiment of the invention is prepared is numbered 2a-i respectively;
2, test method and result: before measuring test compound 2a-I is dissolved in physiological saline.(5.0mg/ml, 3ml/kg) right carotid and left jugular vein are separated in the anesthesia back to male Wistra rat (180-220g) with vetanarcol.The silk thread that the long prior precision of a 6cm is weighed is placed in the polyethylene tube, after intubate being full of the normal saline solution (50IU/ml) of heparin sodium, one end inserts the left side vein, add quantitatively (dosage 1ml/kg) heparin sodium anti-freezing from an end, and (dosage is 10nmol/kg to add the normal saline solution of 3a-i; 3ml/kg), insert the right side artery then.Blood flow flows into the left side vein from the right side artery polyethylene tube of flowing through, and takes out the silk thread with thrombus after 15 minutes, dips in drop of blood on filter paper gently, puts into the centrifuge tube of weighing in advance, accurately weighs and record.The loss of weight method calculates the weight in wet base of thrombus.And record weight in wet base.(NS 3ml/kg) makes blank, and (dosage 12mg/kg) makes positive control with acetylsalicylic acid with physiological saline.Each medicine repeats 12 administrations.The wet weight of thrombus of each group of statistics
And be t and check.The results are shown in Table 1.
The weight (mg) of table 1 compound 2a-i effect back thrombus
N=12; A. compare with physiological saline, P＜0.01.b compares with physiological saline, P＜0.05.
C compares indifference with physiological saline
The stability test of test example 2 The compounds of this invention in the aqueous solution
1, test compound: the compound (2a-i) that the embodiment of the invention is prepared;
2, test method and result: the stability of synthetic target compound in the aqueous solution in order to measure, take by weighing a certain amount of test compound sample respectively and be dissolved in the tri-distilled water, be mixed with 10
-3The mol/ml aqueous solution dilutes 100,10000 times respectively with the solution for preparing again, is mixed with 10 respectively
-5Mol/ml and 10
-7The aqueous solution of mol/ml.With nanometer particle size-electrokinetic potential determinator the particle diameter of these three different concns self-assemblies is measured, sample was now with the current in first day, and measure after ultrasonic, later on to this sample tie-in eight days, but from second day to the 8th day mensuration process, no longer carry out ultrasonic to sample.And write down the particle size values of every day, observe its size and change in time.
By particle diameter as a result table 2 as can be known test compound can in the aqueous solution, be self-assembled into nano particle, and can be in the aqueous solution stable existence 8 days, the particle diameter of most of test compound is between 200～600nm.
Table 2 is observed the 2a-i size and is changed (10-3M) in time
Table 3 is observed the 2a-i size and is changed (10 in time
-5M)
Table 4 is observed the 2a-i size and is changed (10 in time
-7M)
Claims (8)
1. (3S)-1,2,3 that has antithrombotic acitivity, 4-tetrahydroisoquinoline-3-carboxylic acid three peptide conjugates, its structural formula is shown in the general formula I:
Wherein, AA
1Be selected from Gly, Asp or Gln; AA
2Be selected from Gly, Asp or Gln.
2. described (3S)-1,2,3 of claim 1, the intermediate of 4-tetrahydroisoquinoline-3-carboxylic acid three peptide conjugates, its structure is shown in the general formula I I:
Wherein, AA
1Be selected from Gly, Asp or Gln; AA
2Be selected from Gly, Asp or Gln.
3. one kind prepares described (3S)-1,2,3 of claim 1, and the method for 4-tetrahydroisoquinoline-3-carboxylic acid three peptide conjugates may further comprise the steps:
(1) with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1With AA
2-Arg (NO
2)-OBzl coupling prepares N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-AA
2-Arg (NO
2)-OBzl; AA wherein
1Be selected from Gly, Asp or Gln; AA
2Be selected from Gly, Asp or Gln;
(2) with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-AA
2-Arg (NO
2)-OBzl sloughs protecting group, promptly.
4. in accordance with the method for claim 3, it is characterized in that: in the step (2) in the presence of trifluoracetic acid and trifluoromethanesulfonic acid, with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-AA
2-Arg (NO
2)-OBzl sloughs protecting group; Wherein, the volume ratio of trifluoracetic acid and trifluoromethanesulfonic acid is 4: 1.
5. in accordance with the method for claim 3, it is characterized in that described N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA1 prepares in accordance with the following methods: (1) preparation (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; (2) at (Boc)
2With (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was converted into (3S)-N-Boc-1 under O existed, and 2,3,4-tetrahydroisoquinoline-3-carboxylic acid; (3) in the presence of dicyclohexyl carbonyl diimine (DCC) and N-methylmorpholine with (3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and L-AA
1-OMe coupling, preparation N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-OMe; (4) with N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1-OMe hydrolysis prepares N-[(3S)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-formyl radical]-AA
1AA wherein
1Be selected from Gly, Asp or Gln.
6. in accordance with the method for claim 3, it is characterized in that described AA
2-Arg (NO
2)-Obzl prepares in accordance with the following methods:
(1) in the presence of dicyclohexyl carbonyl diimine (DCC) and N-methylmorpholine with Boc-AA
2With NH
2-Arg (NO
2)-OBzl coupling prepares Boc-AA
2-Arg (NO
2)-OBzl; (2) with Boc-AA
2-Arg (NO
2)-OBzl takes off Boc and prepares NH
2-AA
2-Arg (NO2)-OBzl; Wherein, AA
2Be selected from Gly, Asp or Gln.
7. pharmaceutical composition is characterized in that: by described (3S)-1,2,3 of claim 1 that significant quantity is gone up in treatment or prevention, 4-tetrahydroisoquinoline-3-carboxylic acid three peptide conjugates and pharmaceutically acceptable carrier or auxiliary material are formed.
8. described (3S)-1,2,3 of claim 1, the purposes of 4-tetrahydroisoquinoline-3-carboxylic acid three peptide conjugates in the preparation antithrombotic reagent.
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