WO2017020282A1 - 有抗凝血和抗血小板活性的多靶点化合物及制法和用途 - Google Patents

有抗凝血和抗血小板活性的多靶点化合物及制法和用途 Download PDF

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WO2017020282A1
WO2017020282A1 PCT/CN2015/086173 CN2015086173W WO2017020282A1 WO 2017020282 A1 WO2017020282 A1 WO 2017020282A1 CN 2015086173 W CN2015086173 W CN 2015086173W WO 2017020282 A1 WO2017020282 A1 WO 2017020282A1
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gly
group
polypeptide
compound
peptide
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PCT/CN2015/086173
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English (en)
French (fr)
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范蔷薇
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陕西麦科奥特科技有限公司
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Priority to US15/749,622 priority Critical patent/US11643439B2/en
Priority to JP2018526284A priority patent/JP6530564B2/ja
Priority to CN202110662996.2A priority patent/CN113201049B/zh
Priority to PCT/CN2015/086173 priority patent/WO2017020282A1/zh
Priority to EP15900056.1A priority patent/EP3333177B1/en
Priority to CN202110662995.8A priority patent/CN113773369B/zh
Priority to ES15900056T priority patent/ES2886576T3/es
Priority to CN202110661682.0A priority patent/CN113201048B/zh
Priority to CN201580082185.8A priority patent/CN108137653B/zh
Publication of WO2017020282A1 publication Critical patent/WO2017020282A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof

Definitions

  • the invention relates to the field of biomedicine, in particular to a multi-target compound for anticoagulation and antagonism of platelet GP II b/IIIa receptor, a preparation method thereof and use thereof.
  • Thrombosis is an early event in the development of thrombotic diseases and runs through the beginning and the end of disease development. Platelet activation and activation of the coagulation system play an important role in the process of thrombosis, and the two are closely related in vivo. Thrombin, which is produced after activation of the coagulation system, is a potent platelet-activating factor that promotes the clotting process after platelet activation.
  • the principle of prevention and treatment of thrombotic diseases is to improve hypercoagulable state, prevent thrombus enlargement and new thrombus formation, dissolve thrombus, and then clear or rebuild blood flow pathway to prevent tissue ischemia and necrosis.
  • Therapeutic methods for thrombotic diseases include antithrombotic, thrombolytic, interventional, and surgical treatments.
  • Antithrombotic therapy including antiplatelet and anticoagulant therapy, has attracted much attention as a cornerstone for the treatment of thrombotic diseases, especially cardiovascular diseases.
  • Anticoagulant drugs prevent blood clotting by preventing clotting factors, preventing thrombosis; antiplatelet drugs inhibit thrombus formation by inhibiting platelet adhesion, aggregation, and release.
  • the clinically applied thrombin inhibitors mainly include low molecular weight heparin and bivalirudin
  • platelet GP II b/IIIa receptor antagonists mainly include lamivudine and tirofiban.
  • the above drugs have side effects such as bleeding tendency, and have certain application limitations. More importantly, all of the above drugs inhibit thrombus formation through a single target. At present, many drugs with different target targets are combined clinically, thereby achieving a clinical effect of increasing efficacy and reducing side effects. However, there are problems such as dose matching, bleeding, and coordination of effects in combination therapy, which greatly affects the clinical antithrombotic effect.
  • the present invention provides an antithrombin and a multi-target antagonist compound antagonizing a platelet GP II b/IIIa receptor having a structure represented by the formula (1): ALB-L'-C (1) Wherein A and B are binding sites to thrombin, C is a binding site for platelet GP II b/IIIa receptors, L is a first linking group, and L' is a second linking group.
  • L' is as shown in the formula (2): ((Gly) n1 - (Ser) n2 ) n3 (2)
  • n1 is 1, 2 or 3 or 4; n2 is 0 or 1; n3 is 0, 1, 2 or 3.
  • L' is as shown in the formula (3): (Glu-Ala-Ala-Ala-Lys) n1 Formula (3), wherein n1 is 0, 1, 2 or 3.
  • L' is as shown in the formula (4): (Arg-Val-Leu-Ala-Glu-Ala) n1 Formula (4), wherein n1 is 0, 1, 2 or 3.
  • A1-A2-A3-A4 Formula (5) wherein A1 is D-Phe; A2 is Pro or Pip; and A3 is Arg, Lys, Orn or Har; A4 is Pro, D-Pro or Ser.
  • B1 is a dipeptide consisting of any two acidic amino acids
  • B2 is Val, Leu, Ile, Nle or Phe
  • B3 is Hyp, Ser, Pro or an N-methyl amino acid
  • B4 is composed of any two acidic amino acids.
  • B5 is an amino acid selected from one of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, and Pro, or B5 contains Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha Or a dipeptide of at least one amino acid in Pro.
  • C is as shown in the formula (7): Cys-Har-C1-Asp-Trp-Pro-C2 Formula (7), wherein C1 is Gly or Ser, and C2 is Cys or Cys-OH-
  • C1 is Gly or Ser
  • C2 is Cys or Cys-OH-
  • a disulfide bond is formed between the two fluorenyl groups in the formula (7).
  • L1-L2-L3-L4-Gly-Asp-L5 Formula (8) wherein L1 is Gly, Ala, Val or Gly-Gly; L2 is Gly or Cys; L3 is Gly, Gly-Gly, Gly-Gly-Gly or dextrorotatory amino acid; L4 is Asn or Gln; L5 is selected from Phe, Tyr, Phe, and the benzene ring obtained by substitution is substituted with the benzene ring of Tyr. One of the derivatives obtained after the substitution.
  • A-L-B is selected from one of the polypeptide sequences shown in SEQ ID No. 1 to SEQ ID No. 3.
  • the compound has a structure represented by the formula (9): X-A-L-B-L'-C-Y (9)
  • X is selected from the group consisting of hydrogen, one or two C1-C6 alkyl groups, one or two C2-C10 acyl groups, a benzyloxycarbonyl group or a tert-butoxycarbonyl group; and Y is selected from the group consisting of OH, C1-C6.
  • X is selected from the group consisting of hydrogen, one or two C1-C6 alkyl groups, one or two C2-C10 acyl groups, a benzyloxycarbonyl group or a tert-butoxycarbonyl group
  • Y is selected from the group consisting of OH, C1-C6.
  • the compound comprises a polypeptide sequence of the polypeptide structure shown in SEQ ID No. 4 to SEQ ID No. 7.
  • the compound is selected from one of the polypeptide structures shown in SEQ ID No. 4 to SEQ ID No. 7.
  • the invention also provides a salt of the thrombin and platelet GP II b/IIIa receptor multi-target antagonist compound.
  • the salt is an acetate salt of the compound or a trifluoroacetate salt of the compound.
  • the invention also provides a method for preparing the compound of the invention, the method comprising the steps of:
  • the solid phase synthesis method is used to insert the protected amino acid or fragment from the carboxy terminus according to the polypeptide sequence to obtain the side chain fully protected amino acid polypeptide-Wang resin; (2) the side chain fully protected amino acid polypeptide with the acidolytic reagent- The Wang resin is subjected to acid hydrolysis to obtain a crude linear polypeptide; (3) the crude linear polypeptide is cyclized to form a disulfide bond, and then purified by a high pressure preparative liquid phase to obtain a polypeptide sequence.
  • the invention also provides a pharmaceutical composition, the active ingredient of which comprises a compound of the invention or a salt of a compound of the invention.
  • the pharmaceutical composition is in the form of an injection, a tablet, a capsule, a pill, a powder, a granule, a suspension or an emulsion.
  • the present invention also provides the use of a compound of the present invention or a salt of the compound of the present invention for the preparation of a medicament for preventing and treating peripheral arterial thrombosis and arteriovenous bypass thrombosis.
  • the present invention also provides the use of the compound of the present invention or a salt of the compound of the present invention for the preparation of a medicament for preventing and treating the formation of progressive ischemic stroke; in the preparation for the treatment of acute coronary syndrome, Application of percutaneous coronary intervention or PCI in the treatment of thrombosis in coronary stents; application in the preparation of drugs for the prevention and treatment of acute pulmonary embolism; preparation of thrombosis in the prevention and treatment of organ tissue transplantation Application in medicine.
  • the anti-thrombin and the multi-target antagonist compound antagonizing the platelet GP II b/IIIa receptor provided by the invention have direct, reversible and specific antithrombin function, and also have the effects of inhibiting the GP II b/IIIa receptor.
  • Anticoagulant antithrombotic effects can be achieved at lower doses. At the same time, reduce the risk of bleeding, avoid dose matching, bleeding, coordination of the effects of the combination of drugs.
  • Figure 1 shows the results of mass spectrometric detection of polypeptide 1.
  • Figure 2 shows the results of mass spectrometric detection of polypeptide 2.
  • Figure 3 shows the results of mass spectrometric detection of polypeptide 3.
  • Figure 4 shows the results of mass spectrometric detection of polypeptide 4.
  • Figure 5 is the effect of peptide 2 on Felc 3 stimulation of inferior vena cava thrombosis in rats.
  • Figure 6 is the effect of peptide 2 on FeCl 3 stimulation of common carotid artery thrombosis in rats.
  • Figure 7 is the effect of polypeptide 2 on rat APTT.
  • Figure 8 is the effect of polypeptide 2 on rat PT.
  • Figure 9 is a flow chart of an arterial thrombosis model test.
  • FIG 10 is a 2 in dry weight of thrombus of a single dose intravenous forming polypeptide rabbit femoral artery thrombosis induced by FeCl 3.
  • Figure 11 shows the effect of a single intravenous administration of peptide 2 for 15 min on the thrombus dry weight of rabbit femoral arteriovenous bypass thrombosis.
  • Figure 12 is a graph showing the effect of single intravenous administration of polypeptide 2 on platelet aggregation function in rabbits (platelet aggregation inhibition rate).
  • Figure 13 is a graph showing the effect of a single intravenous administration of polypeptide 2 on rabbit APTT.
  • Figure 14 is a graph showing the effect of a single intravenous administration of polypeptide 2 on rabbit PT.
  • Figure 15 is a graph showing the effect of a single intravenous administration of polypeptide 2 on rabbit ACT.
  • Figure 16 is a graph showing the effect of polypeptide 2 on thrombin (1500 u/kg) induced pulmonary coagulation in a mouse lung embolism model.
  • thrombin means a proteolytic enzyme formed by a thrombin precursor (essential component in plasma) which promotes blood coagulation by catalyzing the conversion of fibrinogen to fibrin.
  • the platelet GP II b/IIIa receptor is an adhesion glycoprotein on the surface of platelets and is one of the integrin families. There are approximately 50,000 to 80,000 GP II b/IIIa receptor molecules on the surface of each unactivated platelet, which is the most integrated integrin on the surface of platelets.
  • Amino acid is a generic term for a class of organic compounds containing an amino group and a carboxyl group, is a basic constituent unit of a protein, and is a basic substance constituting a protein required for animal nutrition.
  • the amino acid in the present invention is represented by a conventional abbreviation in the art.
  • Acidic amino acids refer to amino acids having an isoelectric point of less than 7, including aspartic acid (Asp) and glutamic acid (Glu).
  • the N-methyl amino acid refers to an amino acid obtained by substituting an amino group on an amino acid with a methyl group.
  • the derivative obtained after the substitution of the benzene ring means a derivative of an amino acid obtained by substituting a benzene ring of an amino acid with a linear or branched alkane, a halogen, an alkoxy group, an amide group, an acyloxy group or the like.
  • the C1-C6 alkyl group means an alkyl group having 1 to 6 carbon atoms
  • the C2-C10 acyl group means an acyl group having 2 to 10 carbon atoms.
  • One embodiment of the present invention provides an antithrombin and a multi-target antagonist compound antagonizing a platelet GP II b/IIIa receptor having a structure represented by the formula (1): ALB-L' -C Formula (1), wherein A and B are binding sites to thrombin, C is binding site to platelet GP II b/IIIa receptor, L is the first linking group, and L' is the second linkage Group. Wherein the first linking group L connects A and B to form an A-L-B structure, and the second linking group L' connects the A-L-B structure and the C structure to form a structure represented by the formula (1).
  • the A-L-B structure can be directly joined to the C structure to form a multi-target antagonist compound.
  • L' may be a structure as shown in the formula (2): ((Gly) n1 - (Ser) n2 ) n3 (2)
  • n1 is 1, 2, 3 or 4; n2 is 0 or 1; and n3 is 0, 1, 2 or 3.
  • the structure of L' is Gly-Gly-Gly-Gly-Ser or Gly-Gly-Gly-Ser.
  • L' may be a structure as shown in formula (3): (Glu-Ala-Ala-Ala-Lys) n1 (3)
  • n1 is 0, 1, 2 or 3.
  • the structure of L' is Glu-Ala-Ala-Ala-Lys.
  • L' may be a structure as shown in formula (4): (Arg-Val-Leu-Ala-Glu-Ala) n1 (4)
  • n1 is 0, 1, 2 or 3.
  • the structure of L' is Arg-Val-Leu-Ala-Glu-Ala.
  • the structure of A is as shown in the formula (5): A1-A2-A3-A4 (5)
  • Pip refers to 4-aminopiperidin-4-carboxyl, Orn refers to ornithine, and Har refers to homoarginine.
  • the structure of A is D-Phe-Pro-Arg-Pro.
  • the structure of B is as shown in the formula (6): B1-B2-B3-B4-B5 (6)
  • B1 is a dipeptide composed of any two acidic amino acids
  • B2 is Val, Leu, Ile, Nle or Phe
  • B3 is Hyp, Ser, Pro or an N-methyl amino acid
  • B4 is a composition of any two acidic amino acids a peptide
  • B5 is an amino acid selected from one of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, and Pro, or B5 contains Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, and A dipeptide of at least one amino acid in Pro.
  • Nle represents norleucine
  • Hyp represents hydroxyproline.
  • the structure of B is Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu.
  • C is as shown in the formula (7): Cys-Har-C1-Asp-Trp-Pro-C2 Formula (7), wherein C1 is Gly or Ser, and C2 is Cys or Cys-OH-
  • C1 is Gly or Ser
  • C2 is Cys or Cys-OH-
  • Har represents high arginine.
  • L1 is Gly, Ala, Val or Gly-Gly;
  • L2 is Gly or Cys;
  • L3 is Gly, Gly-Gly, Gly-Gly-Gly or right-handed amino acid;
  • L4 is Asn or Gln;
  • L5 is selected from Phe, Tyr, One of the derivatives obtained after the benzene ring of Phe is substituted and the benzene ring of Tyr is substituted.
  • L1 is preferably Gly-Gly
  • L2 is preferably Gly
  • L3 is preferably Gly
  • L4 is preferably Asn
  • L5 is preferably Phe.
  • A-L-B is selected from one of the sequences shown in SEQ ID No. 1 to SEQ ID No. 3.
  • the N-terminal phenylalanine is D-type phenylalanine.
  • the compound has a structure represented by the formula (9): X-A-L-B-L'-C-Y (9)
  • X is selected from the group consisting of hydrogen, one or two C1-C6 alkyl groups, one or two C2-C10 acyl groups, a benzyloxycarbonyl group or a t-butoxycarbonyl group; alternatively, the C1-C6 The alkyl group may be a methyl group or an ethyl group.
  • Y is selected from OH, C1-C6 alkoxy group, an amino group, one or two C1-C4 alkyl group substituted with an amino group; In one embodiment of the present invention, Y is OH or NH 2. In a particularly preferred embodiment, when the compound has the structure shown in SEQ ID No. 5, and the C-terminal Y is NH 2 , the binding time to the platelet GP II b/IIIa receptor is longer, Better inhibition of platelet function.
  • the alkyl group may be a linear structure or a branched structure
  • the acyl group may be a linear structure or a branched structure, wherein when X is two C1-C6 alkyl groups, the two C1- The types of alkyl groups of C6 may be the same or different. Wherein when X is two C2-C10 acyl groups, the two C2-C10 acyl groups may be the same or different.
  • Y is two C1-C4 alkyl-substituted amino groups, the C1-C4 alkyl-substituted amino groups may be the same or different.
  • the compound comprises a polypeptide sequence of the polypeptide structure shown in SEQ ID No. 4 to SEQ ID No. 7.
  • the N-terminal Phe is a D-configuration phenylalanine.
  • the compound is selected from one of the polypeptide structures shown in SEQ ID No. 4 to SEQ ID No. 7.
  • One aspect of the invention provides a salt of the anti-thrombin and a multi-target antagonist compound that antagonizes the platelet GP II b/IIIa receptor.
  • the salt is in the form of a pharmaceutically acceptable salt.
  • “Pharmaceutically acceptable salt” means that it is suitable for contact with tissues of a human or animal without excessive toxicity, irritation, allergies and the like.
  • Pharmaceutically acceptable salts are well known in the art. Such salts may be prepared during the final isolation and purification of the peptides of the invention, or may be prepared separately by reacting the free base or acid with a suitable organic or inorganic acid or base.
  • Representative acid addition salts include, but are not limited to, acetic acid, trifluoroacetic acid, dihexanoate, citrate, aspartate, benzoate, besylate, hydrogen sulfate, butyrate, glycol Acid salt, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate, oxalate, propionate, phosphate , glutamate, bicarbonate, p-toluenesulfonate.
  • the salt is an acetate, trifluoroacetate or hydrochloride salt of the compound, particularly preferably the salt is an acetate or a trifluoroacetate salt.
  • One aspect of the present invention provides a method for preparing the antithrombin and an antagonistic platelet GP II b/IIIa receptor multi-target antagonist compound, the method comprising the steps of: (1) using a solid phase synthesis method according to a polypeptide sequence The carboxy terminus starts to sequentially access the corresponding protective amino acid or fragment to obtain a side chain fully protected amino acid polypeptide-Wang resin; (2) acidolysis of the side chain fully protected amino acid polypeptide-Wang resin with an acidolytic reagent to obtain a linear polypeptide crude product (3) The linear polypeptide is cyclized to form a disulfide bond, and then purified by a high pressure preparative liquid phase to obtain a polypeptide sequence.
  • One aspect of the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antithrombin of the present invention and a multi-target antagonist compound or a antagonizing platelet GP II b/IIIa receptor. a salt of the compound.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in the form of an injection or a microemulsion.
  • the pharmaceutically acceptable carrier material conventionally used in the art can be selected depending on the form of the pharmaceutical composition.
  • the active ingredient is contained in an amount of not less than 85%.
  • the type of carrier material includes, but is not limited to, physiological saline.
  • the pharmaceutical composition of the present invention can be used for the treatment and/or prevention of thrombotic diseases, mainly for anticoagulation, antithrombotic and antiplatelet aggregation.
  • a drug having the antithrombin and a multi-target antagonist compound antagonizing the platelet GP II b/IIIa receptor and/or a salt of the compound as an active ingredient is administered to the subject.
  • One aspect of the present invention also provides the use of a compound of the present invention or a salt of the compound of the present invention for the preparation of a medicament for preventing and treating peripheral arterial thrombosis, arteriovenous bypass thrombosis.
  • One aspect of the present invention also provides the use of a compound of the present invention or a salt of the compound of the present invention for the preparation of a medicament for preventing and treating the formation of progressive ischemic stroke.
  • One aspect of the present invention also provides the use of a compound of the present invention or a salt of the compound of the present invention for the preparation of a medicament for preventing and treating acute pulmonary embolism.
  • the invention also provides the use of the compound of the invention or the salt of the compound of the invention for preparing a medicament for preventing and treating thrombosis in organ tissue transplantation; in preparing for treating acute coronary syndrome, percutaneous Application of coronary intervention or PCI in the treatment of thrombosis in coronary stents; in the preparation of drugs for the prevention and treatment of acute pulmonary embolism; in the preparation of drugs for the prevention and treatment of thrombosis in organ tissue transplantation Applications.
  • the use according to the invention comprises the preparation of a medicament containing a compound according to the invention, the dosage form of which includes, but is not limited to, a lyophilized powder, an injection, a microemulsion, a microsphere, a micelle or the like.
  • the antithrombotic drug includes a drug for treating or preventing a peripheral arterial occlusive disease (acting by anticoagulation, antithrombotic, antiplatelet aggregation), progressive ischemic stroke, acute coronary syndrome, and the like.
  • the antithrombotic drug may also include prevention of the formation of arteriovenous fistulas in hemodialysis patients during VAF and VAG surgery.
  • Example 1 is used to illustrate a compound according to an embodiment of the present invention and a process for its preparation.
  • Fmoc-Gly-Gly-Gly-Gly-OH Fmoc-Gly-2-Cl-Trt-resin (23.5 g, 16.5 mmol) with a substitution degree of 0.7 mmol/g was weighed and used 2.5 L 25 The %PIP/DMF solution was protected by Fmoc for 25 minutes. After filtration, the resin was washed three times with DMF and DCM, respectively, for not less than 1 minute. Fmoc-Gly-OH (14.8 g, 50 mmol) was added, and the reaction was stirred at 30 ° C for 4 hours, and the reaction end point was detected by the ninhydrin method. After the completion of the reaction, the resin was washed three times with DMF and DCM, respectively.
  • the deprotected amino acid resin was further added, and the reaction was stirred at 30 ° C for 3 hours, and the reaction end point was detected by the ninhydrin method. After the completion of the reaction, the resin was washed three times with DMF and DCM, respectively.
  • linear polypeptide 1 resin Fmoc-D-Phe-Pro-Arg(pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)- Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(OtBu)-Leu-Gly-Gly-Gly-Gly-Gly-Ser(Trt)-Cys(Trt) -Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-Wang
  • the deprotected amino acid resin was further added, and the reaction was stirred at 30 ° C for 3 hours, and the reaction end point was detected by the ninhydrin method. After the reaction was completed, the resin was washed three times with DMF and DCM, respectively.
  • the above method was used to sequentially access Fmoc-Gly-Gly-Gly-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH.
  • polypeptide 1 The structural formula of polypeptide 1 is as follows (polypeptide sequence shown in SEQ ID No. 4): D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gly Gly Gly Gly Ser Cys Har Gly Asp Trp Pro Cys. 10 g of the linear polypeptide 1 prepared in the step 4 was dissolved in 200 ml of purified water, and a 5% I 2 solution was slowly added dropwise with stirring. The reaction was detected by HPLC. After the reaction was completed, the target product was purified by high-preparation liquid chromatography, and lyophilized to obtain the final product polypeptide 1. The mass is 1g. The mass spectrometric detection results of polypeptide 1 are shown in Fig. 1.
  • Polypeptide Compound 2 was prepared in the same manner as in Example 1 except that the L' fragment was Glu Ala Ala Ala Lys to obtain polypeptide 2, and the product mass was 1.2 g.
  • the mass spectrometric detection results of polypeptide 2 are shown in Fig. 2.
  • polypeptide 2 The structural formula of the polypeptide 2 is shown below (the polypeptide sequence shown in SEQ ID No. 5): D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Glu Ala Ala Ala Lys Cys Har Gly Asp Trp Pro Cys
  • Polypeptide Compound 3 was prepared in the same manner as in Example 1 except that the Gly Gly Gly Gly Ser fragment (i.e., L' fragment) was removed to obtain polypeptide 3, and the product mass was 1.5 g.
  • the mass spectrometric detection results of polypeptide 3 are shown in FIG.
  • polypeptide 3 The structural formula of polypeptide 3 is shown below (polypeptide sequence shown in SEQ ID No. 6): D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Cys Har Gly Asp Trp Pro Cys
  • Polypeptide Compound 4 was prepared in the same manner as in Example 1 except that the L' fragment was Arg Val Leu Ala Glu Ala to obtain polypeptide 4, and the product mass was 0.86 g.
  • the mass spectrometric detection results of polypeptide 4 are shown in FIG.
  • polypeptide 4 The structural formula of polypeptide 4 is shown below (polypeptide sequence shown in SEQ ID No. 7): D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Arg Val Leu Ala Glu Ala Cys Har Gly Asp Trp Pro Cys
  • Test Examples 1-4 were used to illustrate the effects of the polypeptides 1-4 prepared in Examples 1-4 against human coagulation and anti-human platelet aggregation.
  • a healthy volunteer was given 30 ml of venous blood, and the blood was quickly injected into a plastic tube containing 3 ml of 3.8% sodium citrate, and gently mixed upside down.
  • the extracted blood was centrifuged at 810 rpm for 8 min to obtain platelet rich plasma (PRP), PRP was taken out, and the blood sample was centrifuged at 3510 rpm for 8 min to obtain platelet poor plasma (PPP).
  • PRP platelet rich plasma
  • PRP platelet poor plasma
  • Prothrombin time (PT), thrombin time (TT), and activated partial thrombin time (APTT) determination Reconstitution and preservation of reagents according to the operation requirements of the kit. Each PPP was taken and tested in 10 test cups. In addition, 10 ⁇ L of each of physiological saline, polypeptide 1, polypeptide 2, polypeptide 3, and peptide 4 were added, and then PT, TT, and APTT reagents were separately added, and the test was started immediately. The effect of polypeptides 1-4 on human coagulation function is shown in Table 3 (n represents the number of cases).
  • Table 3 show that four compounds of polypeptide 1-4 can affect human coagulation function, and prolong PT, APTT and TT, and the final concentration is 1 ⁇ 10 -6 mol/L.
  • the APTT has the largest multiple.
  • the platelet aggregation instrument was preheated for 30 minutes, PPP was adjusted, and 270 ⁇ l of PRP was added to the test cup, and 10 ⁇ l of polypeptide 1, polypeptide 2, polypeptide 3, and peptide 4 (1.0 ⁇ 10 -7 mol/L) and/or were respectively added. Or 20 ⁇ l of physiological saline in a total volume of 300 ⁇ l. After 5 minutes of warm bath, 5 ⁇ l of the inducer ADP and 5 ⁇ l of epinephrine were added to start the test. After 5 minutes, the test was completed, the maximum aggregation rate was recorded within 5 minutes, and the data and graphs were printed.
  • This test example is for explaining the effect of the polypeptide 2 prepared in Example 2 against rat thrombosis.
  • Experimental animals 160 healthy, adult SD rats, male, body weight 250g ⁇ 300g, purchased from Beijing Weitong Lihua Company. Animal production license number: SCXK (Beijing) 2012-0001.
  • Test drug peptide 2, the peptide content was 97.21%.
  • test peptide 2 solution accurately weigh 2200mg of polypeptide, make 10mg/mL mother liquor of normal saline, and take the corresponding mother liquor to prepare the required dose according to the animal's body weight before the experiment.
  • ferric chloride solution Take 35 g of ferric chloride and dissolve in 100 mL of distilled water.
  • Model control group intravenous injection of the same volume of normal saline; 2) 6 dose groups of peptide 2, starting from 1.5 mg/kg + 3.75 mg/kg/h, decreasing to 1 mg/kg + 2.5 mg/kg /h, 0.5mg/kg+1.25mg/kg/h, 0.25mg/kg+0.75mg/kg/h, 0.125mg/kg+0.375mg/kg/h, 0.041mg/kg+0.125mg/kg/h 3) Positive control enoxaparin 5 dose groups, starting from 30U/kg+45U/kg/h, up to 60U/kg+60U/kg/h, 30U/kg+120U/kg/h, 90U/kg+120U/kg/h, 60U/kg+240U/kg/h, 4) positive control, 5 doses of bivalirudin, starting from 0.5mg/kg+2mg/kg/h, decreasing It was 0.35 mg/kg+
  • the first dose of the drug was dissolved in 2mL of physiological saline in one time
  • the maintenance dose was dissolved in 9ml physiological saline, placed on a two-channel micro-injection pump, and dropped at a rate of 0.1ml/min for 90min. Finish.
  • the sham operation group gave the same volume of normal saline.
  • the rats were fasted one night before the experiment and were given free access to water.
  • the external jugular vein cannula was used for drug administration; one side of the common carotid artery was used for blood sampling to determine coagulation function PT, APTT; one side of the common carotid artery was used to establish an arterial thrombus model; and the inferior vena cava was used for a venous thrombosis model.
  • the drug was administered for 60 minutes.
  • the blood was taken before the drug was stopped to determine the activated partial thromboplastin (APTT) and the prothrombin time (PT).
  • APTT activated partial thromboplastin
  • PT prothrombin time
  • Blood samples were centrifuged at 3510 rpm for 8 min to obtain platelet poor plasma (PPP). Reconstitute and save the reagent according to the operation requirements of the kit. Each blood sample is placed in the test cup, and then the PT and APTT reagents are added respectively. The test is started immediately, and the test results are recorded.
  • vascular occlusion time After the dose was administered and maintained for 30 minutes, a 3 mm diameter, 35% FeCl 3 filter paper was placed under the common carotid artery, and a small piece of plastic film (2.5 x 2.0 cm) was placed underneath to protect the surrounding tissue. The temperature was measured at the distal end of the artery using a temperature probe, and the time required for the self-stimulation to decrease the temperature by 2.5 degrees was considered to be the vascular occlusion time.
  • Coagulation function measurement 0 min before administration, respectively, 60 min after administration, blood collection at the time of withdrawal, blood coagulation function (PT, APTT), ACT was measured by platelet coagulation factor analyzer.
  • the time from the placement of FeCl 3 to the arteries indicating a temperature drop of 2.5 ° C is the time of vascular occlusion, reflecting thrombosis.
  • the blood vessel segment forming the thrombus was cut out, and the wet weight was weighed, and the dry weight of the thrombus was measured overnight at room temperature. The thrombus inhibition rate was calculated.
  • Figure 5 shows the effect of peptide 2 on Felc 3 stimulation of inferior vena cava thrombosis in rats. Compared with the model group, *P ⁇ 0.05, **P ⁇ 0.01.
  • Figure 6 is the effect of peptide 2 on FeCl 3 stimulation of common carotid artery thrombosis in rats. Arterial occlusion time change multiples. *P ⁇ 0.05, **P ⁇ 0.01 compared to the model group.
  • Figure 8. Effect of polypeptide 2 on rat PT.
  • This test example is for explaining the pharmacodynamic experiment of the intravenous maintenance of the polypeptide 2 prepared in Example 2 for thrombosis inhibition in rabbits.
  • 3% sodium pentobarbital Accurately weigh 3 g of sodium pentobarbital and distilled water to make up to 100 ml.
  • Adrenaline Take 1.319ml of the original solution and dilute to 100ml of normal saline to prepare a 60 ⁇ M solution, store in a 4°C refrigerator, and dispense before use.
  • ADP Accurately weigh 14.3 mg of ADP dissolved in 5 ml of physiological saline to obtain 6000 ⁇ mol/L of ADP solution, and store at 4 ° C. Before the experiment, it was diluted with physiological saline to make it 600 ⁇ mol/L.
  • Aluminum foil paper cut into 2.5cm ⁇ 2.5cm with a ruler, numbered and weighed; non-absorbable surgical suture: 8cm long with a ruler, weighed.
  • 50% ferric chloride solution 50 g of ferric chloride was taken and dissolved in 100 mL of distilled water.
  • Rabbit surgery preparation After the rabbits were sent to the laboratory for 1 day, they were first screened. Blood was collected from the middle ear artery for blood routine and APTT, and the blood was normal. APTT was performed in rabbits between 16s and 28s. The test rabbits fasted the night before the experiment and were free to drink water. Intravenous injection of 3% pentobarbital sodium solution 1ml/kg anesthesia, supine position fixation, separation of one side of the common carotid artery, external jugular vein, one femoral artery, femoral vein and one side of the forelimb vein.
  • Forelimb vein cannula for drug administration; left femoral vein cannula for blood sampling for determination of platelet aggregation rate; femoral artery for arterial thrombosis model; common carotid artery and external jugular vein for arteriovenous bypass thrombosis model .
  • AT Arterial thrombosis
  • a small piece of filter paper (1 cm ⁇ 1.5 cm) with 50% FeCl 3 solution was applied to the right femoral artery while placing a small piece of plastic film (2.5 ⁇ 2.0 cm). Used to protect surrounding tissue. After 10 minutes, the filter paper strip was removed, the blood vessel and the local tissue were washed with warm physiological saline, the wrapped blood vessel was cut, the thrombus adhered to the blood vessel wall was peeled off onto the tin foil paper, and the wet weight was weighed, and the dry weight was weighed at room temperature overnight.
  • Platelet aggregation rate 0 min before administration, 30 min, 60 min, 90 min, 60 min, 120 min, 180 min, 2.7 ml of blood was taken from the femoral vein, and quickly injected into a centrifuge tube containing 0.3 ml of 3.8% sodium citrate. Mix well. Centrifuge at 810 rpm (100 g) for 8 min, take PRP, Centrifuge at 3500 rpm (1863 g) for 8 min, and take PPP for platelet aggregation.
  • the platelet aggregation rate was measured before the administration (0 min), 5 min after the administration, and 15 min after the administration, and the inhibition rate of platelet aggregation was calculated. Calculated as follows:
  • Coagulation function (PT, TT, APTT) was measured by platelet coagulation factor analyzer at 0 min before administration, 30 min, 60 min, 90 min, 60 min, 120 min, and 180 min. Blood samples were centrifuged at 3510 rpm for 8 min to obtain platelet poor plasma (PPP). Reconstitute and store the reagents according to the operation requirements of the kit. Each blood sample is placed in the test cup, and then PT, TT, and APTT reagents are added respectively. The test is started immediately, and the test results are recorded.
  • PPP platelet poor plasma
  • thrombosis weight After the end of the experiment, the blood vessel segment forming the thrombus was cut, and the wet weight was weighed. The dry weight of the thrombus was measured overnight at room temperature. The thrombus inhibition rate was calculated. The formula is as follows:
  • Group Dosage (mg/kg + mg/kg/h) n Dosing for 30 minutes 60 minutes of administration 90 minutes of administration Blank group - 2 12.1 ⁇ 17.1 12.1 ⁇ 17.1 13.1 ⁇ 18.6 Enoxaparin 50U+150U 1 26.7 0.00 35.27 Peptide 2 8.0+20.0 2 61.0 ⁇ 14.1 72.1 ⁇ 9.6 55.8 ⁇ 7.7 Group Dosage (mg/kg + mg/kg/h) n Stopping for 60 minutes Stopping for 120 minutes Stop the drug for 180min Blank group - 2 22.1 ⁇ 31.2 13.9 ⁇ 19.7 - Enoxaparin 50U+150U 1 0.00 41.9 - Peptide 2 8.0+20.0 2 14.1 ⁇ 1.5 7.5 ⁇ 1.8 47.5 ⁇ 2.5
  • Table 11-14 shows the effect of intravenous administration of peptide 2 on the coagulation function of rabbits
  • S, Table 12 shows the effect of intravenous administration of peptide 2 on coagulation function PT in rabbits
  • S, Table 13 shows the effect of intravenous administration of peptide 2 on the coagulation function of rabbits
  • S, Table 14 shows the effect of intravenous administration of peptide 2 on rabbit ACT (S, ).
  • the dosage unit is (mg/kg + mg/kg/h). The results indicate that intravenous administration of polypeptide 2 can inhibit platelet aggregation in rabbits. The inhibition of platelet aggregation gradually diminished with time after withdrawal.
  • peptide 2 (v) + instillation (vd) 8.0 mg / kg + 20.0 mg / kg 90min can inhibit platelet aggregation in rabbits, affect coagulation function of rabbits, and prolong APTT, PT, TT and ACT. Inhibition of femoral artery thrombosis.
  • peptide 2 is equivalent to enoxaparin in inhibiting femoral artery thrombosis and inhibiting platelet aggregation, enoxaparin has a longer effect on APTT than polypeptide 2, indicating that the risk of enoxaparin bleeding is greater than that of peptide 2.
  • Grouping and administration 48 rabbits. Randomly divided into 6 groups, 8 in each group.
  • Sham operation group (NS): intravenous injection of the same volume of normal saline; 2) positive control bivalirudin group (6mg/kg); 3) positive control enoxaparin group (200U/kg); 4) peptide 2 small Dose group (3.0 mg/kg); 5) Peptide 2 medium dose group (6.0 mg/kg); 6) Peptide 2 high dose group (12.0 mg/kg).
  • Peptide 2 is administered in a single intravenous dose. According to the kilogram of rabbit body weight, the drug was dissolved in 2 mL of physiological saline for one-time bolus injection. The sham operation group gave the same volume of normal saline.
  • Rabbit surgery preparation After the rabbits were sent to the laboratory for 1 day, they were first screened. Blood was collected from the middle ear artery for blood routine and APTT, and the blood was normal. APTT was performed in rabbits between 16s and 28s. The test rabbits fasted the night before the experiment and were free to drink water. Intravenous injection of 3% pentobarbital sodium solution 1ml/kg anesthesia, supine position fixation, separation of one side of the common carotid artery, external jugular vein, one femoral artery, femoral vein and one side of the forelimb vein.
  • Forelimb vein cannula for drug administration; left femoral vein cannula for blood sampling for determination of platelet aggregation rate; femoral artery for arterial thrombosis model; common carotid artery and external jugular vein for arteriovenous bypass thrombosis model .
  • arteriovenous bypass thrombosis (AVST) model arteriovenous shunt consists of two casings, the outer tube is about 8cm long, the inner diameter is 7.9mm, the inner tube is about 2.5cm long, the inner diameter is 4.8mm, and it is placed in the polyethylene tube.
  • a wire of about 8 cm was filled with 50 ⁇ /ml of sodium heparin solution, one end was inserted into the left femoral artery, and the other end was inserted into the right femoral vein. After 30s of administration, the blood flow was released, and the blood flowed from the left artery to the polyethylene tube and returned to the right vein. After 15 minutes, the blood flow was interrupted, and the silk thread was quickly taken out and placed on an aluminum foil paper (size 2.5 cm ⁇ 2.5 cm), and weighed. Heavy, room temperature overnight, weighed dry weight.
  • AT Arterial thrombosis
  • a small piece of filter paper (1 cm ⁇ 1.5 cm) with 50% FeCl 3 solution was applied to the right femoral artery while placing a small piece of plastic film (2.5 ⁇ 2.0 cm). Used to protect surrounding tissue. After 10 minutes, the filter paper strip was removed, the blood vessel and the local tissue were washed with warm physiological saline, the wrapped blood vessel was cut, the thrombus adhered to the blood vessel wall was peeled off onto the tin foil paper, and the wet weight was weighed, and the dry weight was weighed at room temperature overnight.
  • the experimental procedure is shown in Fig. 9. After the canine forelimb and the bilateral femoral arteries were separated, they were stabilized for 10 min and administration was started. At the same time of administration, the AT model was wrapped with 50% FeCl 3 filter paper, and the model was removed after 10 min. The AVST model was established and taken out after 40 min. Continuous observation 120 min after administration, the test was all over.
  • Platelet aggregation rate 2.7 ml of blood was taken from the femoral vein, and rapidly injected into a centrifuge tube containing 0.3 ml of 3.8% sodium citrate, and thoroughly mixed. After centrifugation at 810 rpm (100 g) for 8 min, PRP was taken, centrifuged at 3500 rpm (1863 g) for 8 min, and PPP was taken for platelet aggregation.
  • the platelet aggregation rate was measured before the administration (0 min), 5 min after the administration, and 15 min after the administration, and the inhibition rate of platelet aggregation was calculated. Calculated as follows:
  • Coagulation function (PT, TT, APTT) was measured by platelet coagulation factor analyzer at 0 min before administration, 5 min after administration, and 15 min. Blood samples were centrifuged at 3510 rpm for 8 min to obtain platelet poor plasma (PPP). Reconstitute and store the reagents according to the operation requirements of the kit. Each blood sample is placed in the test cup, and then PT, TT, and APTT reagents are added respectively. The test is started immediately, and the test results are recorded.
  • PPP platelet poor plasma
  • thrombosis weight After the end of the experiment, the blood vessel segment forming the thrombus was cut, and the wet weight was weighed. The dry weight of the thrombus was measured overnight at room temperature. The thrombus inhibition rate was calculated.
  • the small dose of peptide 2 in the 3.0 mg/kg group was statistically significantly different (P ⁇ 0.05), and the peptide 2 in the dose of 6.0 mg/kg and the high dose of 12.0 mg/kg was statistically significantly different ( P ⁇ 0.01), the anti-arterial thrombosis effect of the peptide group in the dose group was better than that of the bivalirudin group.
  • a single dose of different concentrations of peptide 2 can reduce the dry weight of the arteriovenous bypass thrombus in rabbits and inhibit the formation of arteriovenous bypass thrombosis in rabbits.
  • the dose of 6.0 mg/kg and the high dose of 12.0 mg/kg in the peptide 2 group was statistically significantly different (P ⁇ 0.01), and the effect of the anti-arteriovenous bypass thrombus in the peptide 2 group was significantly better.
  • Bivalirudin and enoxaparin groups were significantly better.
  • Each dose of peptide 2 was administered intravenously for 5 minutes, which inhibited platelet aggregation in rabbits to varying degrees.
  • the dose of 6.0 mg/kg and the high dose of 12.0 mg/kg in the peptide 2 group were statistically significantly different (P ⁇ 0.05), and the anti-platelet effect of the peptide 2 group was significantly better than that of bivalirudin.
  • Enoxaparin group After 15 minutes of administration, the inhibitory effect on platelet aggregation in rabbits was gradually weakened.
  • P ⁇ 0.05 there was a significant difference (P ⁇ 0.05) in the large dose of polypeptide 2 at 12.0 mg/kg.
  • rabbit APTT, PT, TT and ACT can be prolonged after administration of polypeptide 2. With the extension of time after withdrawal, the prolongation of the above coagulation index is gradually weakened.
  • Each dose of peptide 2 can prolong the APTT of rabbits to varying degrees. With the prolongation of time, the prolongation of APTT is gradually weakened by 15 min. After administration for 5 min, there was a statistically significant difference between the dose of 6.0 mg/kg and the high dose of 12.0 mg/kg in the peptide 2 group (P ⁇ 0.05). When the drug was administered for 15 minutes, compared with the saline group. There was a significant difference in the dose of 6.0 mg/kg and 12.0 mg/kg in the peptide 2 (P ⁇ 0.05).
  • the results are shown in Table 22.
  • the peptide 2 was administered to each dose group for 5 min and 15 min, and the rabbit TT was extended beyond the detection limit by 180 s.
  • the results are shown in Figure 15, Table 23 and Table 24.
  • the peptide 2 can prolong the ACT of rabbits in different degrees. With the prolongation of time, the prolongation of ACT is gradually weakened by 15 min.
  • peptide 2 Single intravenous administration of peptide 2 inhibited FeCl 3 -induced femoral artery thrombosis and dose-dependently inhibited arteriovenous bypass thrombosis in rabbits.
  • the antithrombotic effect of dose and high dose group was better than that of Bivalu.
  • Group 2; peptide 2 can inhibit platelet aggregation in rabbits in a dose-dependent manner, affecting coagulation function in rabbits, and prolonging APTT, PT, TT and ACT.
  • This test is used to illustrate the protective effect of peptide 2 on progressive ischemic stroke.
  • Experimental animals 60 healthy, adult SD rats, male, body weight 250g ⁇ 300g, purchased from the Animal Center of Xi'an Jiaotong University School of Medicine. Animal production license number: SCXK (Shaan) 2012-003.
  • Test drug peptide 2, peptide content was 97.21%; control drug: enoxaparin injection, produced by Sanofi (Beijing) Pharmaceutical Co., Ltd., batch number: 4SH69.
  • line plug method carotid artery insertion method: the neck takes the median incision to open the skin, separate the total neck, external neck, internal carotid artery, ligature of the common carotid artery at the proximal end, distal end ligation External carotid artery. Temporary clamping of the distal end of the internal carotid artery with a small blood vessel clamp, a ligature at the neck, the neck, and the internal carotid artery bifurcation, and a small opening at the proximal common carotid artery of the common carotid artery with an ophthalmic scissors.
  • the MCAO suture has a certain degree of curvature, and is inserted into the internal carotid artery along the common carotid artery.
  • the suture is bent in the direction of the operator, and slowly advances about 18 mm forward (from the bifurcation of the artery), and stops when there is resistance. Line, the reserved line is ligated at the bifurcation to fix the tying line. After the molding for 15 minutes, the first dose of the venous vein is maintained for 60 minutes. After the end of the administration, the neck wound is routinely sutured.
  • Neurobehavioral score Neurobehavioral scores were scored at 4h, 8h, and 24h after surgery, with Longa's five-pointed method as the standard: 0 points: no neurological defect; 1 point: when lifting the tail Side forelimb adduction, can not be fully extended; 2 points: to the opposite side; 3 points: to the opposite side when walking; 4 points: can not walk or coma. 1-4 is divided into effective models.
  • This test example is used to illustrate the protective effect of polypeptide 2 on thrombin-induced pulmonary embolism in mice.
  • mice Kunming mice, male, 6-8 weeks old, 20-25 g, provided by Experimental Animal Center of Xi'an Jiaotong University, animal production license number SCXK (Shaan) 2012-003.
  • mice were randomly divided into 7 groups of 10 animals each.
  • 200 ul of normal saline was injected into the tail vein, and the other groups were dosed separately.
  • mice After 2 minutes, the saline group was injected with 100 ul of normal saline in the tail vein, and the other groups were injected with 100 ul of thrombin 1500 u/kg (80-90% lethal dose). The mice were observed to respond after administration and died within 15 minutes. No dead mice were treated within 15 min.
  • mice that had not died within 15 minutes blood was taken from the eyeball, and after EDTA anticoagulation, the number of platelets was measured, and the lungs were weighed and the lungs were weighed and measured to determine the lung coefficient.
  • mice in the LPS model group died 12/13, and the mortality rate was 85%. There was a significant difference between the two groups compared with the saline group (P ⁇ 0.01). Enoxaparin 0.5mg/kg group, bivalirudin 8mg/kg and peptide 2 large dose 10.0mg/kg group died 1/10, the mortality rate was 10%, compared with the model group, the mortality rate of the two groups The difference was very significant (P ⁇ 0.01). In the case of peptide 2, the dose of 5.0 mg/kg group died 3/10, and the mortality rate was 30%. Compared with the model group, the difference of mortality between the two groups was very significant (P ⁇ 0.01). There was no significant difference in mortality between the two groups (P>0.05) compared with enoxaparin 0.5 mg/kg and bivalirudin 8 mg/kg.
  • the lung coefficient of the mice in the bivalirudin 8 mg/kg group was reduced, compared with the model group.
  • the difference of learning was significant (P ⁇ 0.05).
  • the lung coefficient of mice with large dose of 10.0 mg/kg was decreased, and the statistical difference was significant compared with the model group (P ⁇ 0.01).
  • the results are shown in Table 27, Figure 16.
  • Peptide 2 has a protective effect on thrombin-induced pulmonary embolism in mice.
  • the thrombin and platelet GP II b/IIIa receptor multi-target antagonist compound provided by the invention has direct, reversible and specific antithrombin function, and also has the function of inhibiting GP II b/IIIa receptor, which can be smaller
  • the anticoagulant antithrombotic effect is achieved at the dose. At the same time, reduce the risk of bleeding, avoid dose matching, bleeding, coordination of the effects of the combination of drugs.

Abstract

提供了一种抗凝血和拮抗血小板GPIIb/IIIa受体多靶点化合物，其通式如下：A-L-B-L'-C，其中A和B为与凝血酶结合位点，C为与血小板GPIIb/IIIa受体结合位点，L为第一连接基团，L'为第二连接基团。还提供了上述化合物的制备方法及其用途。所述化合物具有体外人凝血酶抑制活性、体外血小板GPIIb/IIIa受体抑制作用以及体外/体内抗血小板聚集、体内抗凝血和抗血栓作用。

Description

有抗凝血和抗血小板活性的多靶点化合物及制法和用途 技术领域
本发明涉及生物医药领域，具体涉及一种抗凝血和拮抗血小板GP Ⅱ b/Ⅲa受体的多靶点化合物及其制备方法和用途。
背景技术
血栓形成是血栓性疾病发生的早期事件，并贯穿于疾病发展的始末。血小板活化与凝血系统激活在血栓形成过程中起着重要的作用，两者在体内具有紧密的联系。凝血系统激活后产生的凝血酶，是一个强有力的血小板活化因子，血小板活化后又将促进凝血过程。防治血栓性疾病的原则在于改善高凝状态，防止血栓扩大及新血栓形成，溶解血栓，然后疏通或重建血流通路，以防止组织缺血、坏死。血栓性疾病的治疗方法包括抗栓、溶栓、介入疗法及手术治疗，其中抗栓治疗包括抗血小板和抗凝治疗，它作为血栓性疾病，尤其是心血管疾病治疗的基石备受关注。抗凝血药物通过影响凝血因子，从而阻止血液凝固过程，防止血栓形成；抗血小板药物通过抑制血小板黏附、聚集以及释放等功能抑制血栓形成。目前临床上应用的凝血酶抑制剂主要有低分子肝素和比伐卢定，血小板GP Ⅱ b/Ⅲa受体拮抗剂主要有拉米非班、替罗非班等。上述药物具有出血倾向等副作用，存在一定的应用局限性。更重要的是，上述药物都是通过单一靶点抑制血栓形成，目前临床上多将不同作用靶点的药物联用，从而达到增加疗效、降低副作用的临床效果。但是联合用药目前存在剂量匹配、出血、作用协调等问题，这大大影响了临床抗栓效果。
因此有必要开发一种能够通过多靶点抑制作用抑制血栓形成的技术方案，从而实现通过施用一种药物同时对不同作用靶点同时起效的效果，避免联合用药所带来的问题。
发明内容
本发明的目的在于克服现有技术中存在的缺陷，提供一种能够同时靶向作用于凝血酶和血小板GP Ⅱ b/Ⅲa受体的多靶点拮抗化合物。
本发明提供了一种抗凝血酶和拮抗血小板GP Ⅱ b/Ⅲa受体的多靶点拮抗化合物，所述化合物具有如式(1)所示的结构：A-L-B-L’-C  式(1)，其中A和B为与凝血酶结合位点，C为与血小板GP Ⅱ b/Ⅲa受体结合位点，L为第一连接基团，L’为第二连接基团。
可选的，L’的结构如式(2)所示：((Gly)n1-(Ser)n2)n3  式(2)
其中n1为1、2或3或4；n2为0或1；n3为0、1、2或3。
可选的，L’的结构如式(3)所示：(Glu-Ala-Ala-Ala-Lys)n1  式(3)，其中n1为0、1、2或3。
可选的，L’的结构如式(4)所示：(Arg-Val-Leu-Ala-Glu-Ala)n1  式(4)，其中n1为0、1、2或3。
可选的，A的结构如式(5)所示：A1-A2-A3-A4  式(5)，其中A1为D-Phe；A2为Pro或Pip；A3为Arg、Lys、Orn或Har；A4为Pro、D-Pro或Ser。
可选的，B的结构如式(6)所示：B1-B2-B3-B4-B5  式(6)
其中B1为任意两个酸性氨基酸组成的二肽；B2为Val、Leu、Ile、Nle或Phe；B3为Hyp，Ser，Pro或一种N-甲基氨基酸；B4为任意两个酸性氨基酸组成的二肽；B5为选自Tyr、Trp、Phe、Leu、Nle、Ile、Val、Cha和Pro中的一种的氨基酸，或者B5为含有Tyr、Trp、Phe、Leu、Nle、Ile、Val、Cha或Pro中的至少一种氨基酸的二肽。
可选的，C的结构如式(7)所示：Cys-Har-C1-Asp-Trp-Pro-C2  式(7)，其中C1为Gly或Ser，C2为Cys或Cys的-OH被-NH2取代后获得的结构，式(7)中的两个巯基之间形成二硫键。
可选的，L的结构如式(8)所示：L1-L2-L3-L4-Gly-Asp-L5  式(8)，其中L1为Gly、Ala、Val或Gly-Gly；L2为Gly或Cys；L3为Gly、Gly-Gly、Gly-Gly-Gly或右旋氨基酸；L4为Asn或Gln；L5选自Phe、Tyr、Phe的苯环被取代后获得的衍生物和Tyr的苯环被取代后获得的衍生物中的一种。
可选的，A-L-B选自SEQ ID No.1至SEQ ID No.3所示多肽序列中的一种。
可选的，所述化合物具有如式(9)所示的结构：X-A-L-B-L’-C-Y  式(9)
其中，X选自氢、一个或两个C1-C6的烷基、一个或两个C2-C10的酰基、苄氧羰基或叔丁氧羰基中的一种；Y选自OH、C1-C6的烷氧基、氨基、一个或两个C1-C4的烷基取代的氨基中的一种。
可选的，所述化合物包括SEQ ID No.4至SEQ ID No.7所示的多肽结构中的一种多肽序列。
可选的，所述化合物选自SEQ ID No.4至SEQ ID No.7所示的多肽结构中的一种。
本发明还提供了所述的凝血酶和血小板GP Ⅱ b/Ⅲa受体多靶点拮抗化合物的盐。
可选的，所述盐为所述化合物的醋酸盐或所述化合物的三氟乙酸盐。
本发明还提供了本发明所述的化合物的制备方法，该方法包括以下步骤：
(1)采用固相合成法按照多肽序列从羧基端开始依次接入保护型氨基酸或片段，得到侧链全保护氨基酸多肽-Wang树脂；(2)用酸解试剂对侧链全保护氨基酸多肽-Wang树脂进行酸解得到线性多肽粗品；(3)对线性多肽粗品进行环化形成二硫键，然后采用高压制备液相进行纯化，得到多肽序列。
本发明还提供了一种药物组合物，所述组合物的活性成分包括本发明所述的化合物或者本发明所述的化合物的盐。
可选的，所述药物组合物的剂型为注射剂、片剂、胶囊剂、丸剂、散剂、颗粒剂、悬浮剂或乳剂。
本发明还提供了本发明所述的化合物或者本发明所述的化合物的盐在制备预防和治疗外周动脉血栓、动静脉旁路血栓形成的药物中的应用。本发明还提供了本发明所述的化合物或者本发明所述的化合物的盐在制备预防和治疗进展性缺血性脑卒中形成的药物中的应用；在制备用于治疗急性冠脉综合征、经皮冠状动脉介入治疗或PCI放置冠脉内支架治疗中血栓形成的药物中的应用；在在制备预防和治疗急性肺栓塞的药物中的应用；在制备预防和治疗器官组织移植中血栓形成的药物中的应用。
本发明所提供的抗凝血酶和拮抗血小板GP Ⅱ b/Ⅲa受体的多靶点拮抗化合物具有直接、可逆、特异抗凝血酶功能，还具有抑制GP Ⅱ b/Ⅲa受体的作用，可以在较小的剂量下达到抗凝抗栓效果。同时降低出血风险、避免联合用药带来的剂量匹配、出血、作用协调等问题。
附图说明
图1为多肽1的质谱检测结果。
图2为多肽2的质谱检测结果。
图3为多肽3的质谱检测结果。
图4为多肽4的质谱检测结果。
图5为多肽2对Fecl3刺激大鼠下腔静脉血栓形成的影响。
图6为多肽2对Fecl3刺激大鼠颈总动脉血栓形成的影响。
图7为多肽2对大鼠APTT的影响。
图8为多肽2对大鼠PT的影响。
图9为动脉血栓模型试验流程图。
图10为多肽2静脉单次给药对FeCl3诱导的家兔股动脉血栓形成血栓干重的影响。
图11为多肽2静脉单次给药15min对家兔股动静脉旁路血栓形成血栓干重的影响。
图12为多肽2静脉单次给药对家兔血小板聚集功能的影响(血小板聚集抑制率)。
图13为多肽2静脉单次给药对家兔APTT的影响。
图14为多肽2静脉单次给药对家兔PT的影响。
图15为多肽2静脉单次给药对家兔ACT的影响。
图16为多肽2对凝血酶(1500u/kg)诱导的小鼠肺栓塞模型肺系数的影响。
具体实施方式
下面将通过具体实施方式对本发明进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
在本发明中，凝血酶表示一种由凝血酶前体(血浆中的必要成分)形成的蛋白质水解酶，能通过催化纤维蛋白原变成纤维蛋白而促使血液凝固。血小板GP Ⅱ b/Ⅲa受体为血小板表面的粘附性糖蛋白，为整合素家族中的一种。每个未激活的血小板表面大约有50000到80000个GP Ⅱ b/Ⅲa受体分子，是血小板表面数量最多的整合素。氨基酸指含有氨基和羧基的一类有机化合物的通称，是蛋白质的基本组成单位，是构成动物营养所需蛋白质的基本物质，本发明中氨基酸用本领域常规的缩写表示。酸性氨基酸指等电点小于7的氨基酸，包括天冬氨酸(Asp)和谷氨酸(Glu)。N-甲基氨基酸指氨基酸上的氨基被甲基取代所获得的氨基酸。苯环取代后获得的衍生物指氨基酸上的苯环被直链或支链烷烃、卤素、烷氧基、酰胺基、酰氧基等取代所获得的氨基酸的衍生物。C1-C6的烷基指具有1-6个碳原子的烷基，C2-C10的酰基指具有2-10个碳原子的酰基。
本发明的一种实施方式提供了一种抗凝血酶和拮抗血小板GP Ⅱ b/Ⅲa受体的多靶点拮抗化合物，所述化合物具有如式(1)所示的结构：A-L-B-L’-C  式(1)，其中，其中A和B为与凝血酶结合位点，C为与血小板GP Ⅱ b/Ⅲa受体结合位点，L为第一连接基团，L’为第二连接基团。其中，所述第一连接基团L将A和B连接起来从而形成A-L-B结构，第二连接集团L’将A-L-B结构与C结构连接起来从而形成式(1)所示的结构。在本发明的一种实施方式中，A-L-B结构可以直接与C结构连接起来形成多靶点拮抗化合物。
在本发明的一种实施方式中，L’可以为如式(2)所示的结构：((Gly)n1-(Ser)n2)n3  式(2)
在式(2)中n1为1、2、3或4；n2为0或1；n3为0、1、2或3。
在一种优选的实施方式中，L’的结构为Gly-Gly-Gly-Gly-Ser或Gly-Gly-Gly-Ser。
在本发明的一种实施方式中，L’可以为如式(3)所示的结构：(Glu-Ala-Ala-Ala-Lys)n1  式(3)
其中n1为0、1、2或3。在一种优选的实施方式中，L’的结构为Glu-Ala-Ala-Ala-Lys。
在本发明的一种实施方式中，L’可以为如式(4)所示的结构：(Arg-Val-Leu-Ala-Glu-Ala)n1  式(4)
其中n1为0、1、2或3。在一种优选的实施方式中，L’的结构为Arg-Val-Leu-Ala-Glu-Ala。
在本发明的一种实施方式中，A的结构如式(5)所示：A1-A2-A3-A4  式(5)
其中A1为D-Phe；A2为Pro或Pip；A3为Arg、Lys、Orn或Har；A4为Pro、D-Pro或Ser。其中，Pip指4-氨基哌啶-4-羧基，Orn指鸟氨酸，Har指高精氨酸。
在一种优选的实施方式中，A的结构为D-Phe-Pro-Arg-Pro。
在本发明的一种实施方式中，B的结构如式(6)所示：B1-B2-B3-B4-B5  式(6)
B1为任意两个酸性氨基酸组成的二肽；B2为Val、Leu、Ile、Nle或Phe；B3为Hyp，Ser，Pro或一种N-甲基氨基酸；B4为任意两个酸性氨基酸组成的二肽；B5为选自Tyr、Trp、Phe、Leu、Nle、Ile、Val、Cha和Pro中的一种的氨基酸，或者B5为含有Tyr、Trp、Phe、Leu、Nle、Ile、Val、Cha和Pro中的至少一种氨基酸的二肽。其中，Nle表示正亮氨酸，Hyp表示羟脯氨酸。
在本发明的一种优选的实施方式中，B的结构为Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu。
可选的，C的结构如式(7)所示：Cys-Har-C1-Asp-Trp-Pro-C2  式(7)，其中C1为Gly或Ser，C2为Cys或Cys的-OH被-NH2取代后获得的结构，式(7)中的两个巯基之间形成二硫键，其中，Har表示高精氨酸。
可选的，L的结构如式(8)所示：L1-L2-L3-L4-Gly-Asp-L5  式(8)
其中L1为Gly、Ala、Val或Gly-Gly；L2为Gly或Cys；L3为Gly、Gly-Gly、Gly-Gly-Gly或右旋氨基酸；L4为Asn或Gln；L5选自Phe、Tyr、Phe的苯环被取代后获得的衍生物和Tyr的苯环被取代后获得的衍生物中的一种。其中，L1优选为Gly-Gly，L2优选为Gly，L3优选为Gly，L4优选为Asn，L5优选为Phe。
在优选的情况下，A-L-B选自SEQ ID No.1至SEQ ID No.3所示序列中的一种。在SEQ ID No.1至SEQ ID No.3所示的序列中，N端的苯丙氨酸均为D-型苯丙氨酸。
SEQ ID No.1:D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Tyr Glu Asp Ile Pro-Glu Glu Tyr Leu
SEQ ID No.2:D-Phe Pro Arg Ser Gly Gly Gly Gly Asn Gly Asp Phe Glu Asp Ile Pro Glu Glu Tyr Leu
SEQ ID No.3:D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro-Glu Glu Tyr Leu
在本发明的一种实施方式中，所述化合物具有如式(9)所示的结构：X-A-L-B-L’-C-Y  式(9)
其中，X选自氢、一个或两个C1-C6的烷基、一个或两个C2-C10的酰基、苄氧羰基或叔丁氧羰基中的一种；可选的，所述C1-C6的烷基可以为甲基或乙基。Y选自OH、C1-C6的烷氧基、氨基、一个或两个C1-C4的烷基取代的氨基中的一种；在本发明的一种实施方式中，Y为OH或NH2。在一种特别优选的实施方式中，当所述化合物具有SEQ ID No.5所示的结构，且C末端的Y为NH2时，与血小板GP Ⅱ b/Ⅲa受体结合时间较长，有较好的抑制血小板功能。
其中，所述烷基可以为直链结构或支链结构，所述酰基可以为直链结构或支链结构，其中当X为两个C1-C6的烷基时，所述的两个C1-C6的烷基的种类可以相同或不同。其中当X为两个C2-C10的酰基时，所述的两个C2-C10的酰基的种类可以相同或不同。当Y为两个C1-C4的烷基取代的氨基时，所述C1-C4的烷基取代的氨基的种类可以相同或不同。
可选的，所述化合物包括SEQ ID No.4至SEQ ID No.7所示的多肽结构中的一种多肽序列。在SEQ ID No.4至SEQ ID No.7中，N末端的Phe为D-构型苯丙氨酸。
SEQ ID No.4:D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gly  Gly Gly Gly Ser Cys Har Gly Asp Trp Pro Cys
SEQ ID No.5:D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Glu Ala Ala Ala Lys Cys Har Gly Asp Trp Pro Cys
SEQ ID No.6:D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Cys Har Gly Asp Trp Pro Cys
SEQ ID No.7:D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Arg Val Leu Ala Glu Ala Cys Har Gly Asp Trp Pro Cys
在本发明的一种优选的实施方式中，所述化合物选自SEQ ID No.4至SEQ ID No.7所示的多肽结构中的一种。
本发明的一个方面提供了所述抗凝血酶和拮抗血小板GP Ⅱ b/Ⅲa受体的多靶点拮抗化合物的盐。
在本发明中，所述的盐为药物可接受的盐形式。“药物可接受的盐”指适合于与人或动物的组织接触，无过多的毒性、刺激、变态反应等。药物可接受的盐是本领域熟知的。这种盐可以在本发明的化合物肽的最终分离和纯化的过程中制备，也可以将游离的碱或酸与适当的有机或无机酸或碱反应单独制备。代表性酸加成盐包括但不限于醋酸、三氟乙酸、二己酸盐、柠檬酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、丁酸盐、庚酸盐、己酸盐、富马酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、乳酸盐、马来酸盐、甲磺酸盐、草酸盐、丙酸盐、磷酸盐、谷氨酸盐、碳酸氢盐、对甲苯磺酸盐。优选的，所述盐为所述化合物的醋酸盐、三氟乙酸盐或盐酸盐，特别优选的，所述盐为醋酸盐或三氟乙酸盐。
本发明的一个方面提供了所述抗凝血酶和拮抗血小板GP Ⅱ b/Ⅲa受体多靶点拮抗化合物的制备方法，该方法包括以下步骤：(1)采用固相合成法按照多肽序列从羧基端开始依次接入相对应的保护型氨基酸或片段，得到侧链全保护氨基酸多肽-Wang树脂；(2)用酸解试剂对侧链全保护氨基酸多肽-Wang树脂进行酸解得到线性多肽粗品；(3)对线性多肽粗品进行环化形成二硫键，然后采用高压制备液相进行纯化，得到多肽序列。
本发明的一个方面还提供了一种药物组合物，所述药物组合物的活性成分包括本发明所述的抗凝血酶和拮抗血小板GP Ⅱ b/Ⅲa受体的多靶点拮抗化合物或者所述化合物的盐。可选的，所述药物组合物还包括药物可接受的载体。所述药物组合物的形式可以为注射液或微乳液。可以根据药物组合物的形式选择使用本领域常用的药物可接受的载体物质。在所述药物组合物中，活性成分的含量不低于85％。所述载体物质的种类包括但不限于生理盐水。本发明所述的药物组合物可以用于治疗和/或预防血栓性疾病，主要作用为抗凝、抗血栓和抗血小板聚集。将以所述抗凝血酶和拮抗血小板GP Ⅱ b/Ⅲa受体的多靶点拮抗化合物和/或所述化合物的盐为活性成分的药物施用于受试者。
本发明的一个方面还提供了本发明所述的化合物或者本发明所述的化合物的盐在制备预防和治疗外周动脉血栓、动静脉旁路血栓形成的药物中的应用。本发明的一个方面还提供了本发明所述的化合物或者本发明所述的化合物的盐在制备预防和治疗进展性缺血性脑卒中形成的药物中的应用。本发明的一个方面还提供了本发明所述的化合物或者本发明所述的化合物的盐在制备预防和治疗急性肺栓塞的药物中的应用。本发明还提供了本发明所述的化合物或者本发明所述的化合物的盐在制备预防和治疗器官组织移植中血栓形成的药物中的应用；在制备用于治疗急性冠脉综合征、经皮冠状动脉介入治疗或PCI放置冠脉内支架治疗中血栓形成的药物中的应用；在在制备预防和治疗急性肺栓塞的药物中的应用；在制备预防和治疗器官组织移植中血栓形成的药物中的应用。本发明所述的应用包括制备含有本发明所述的化合物的药物，所述药物的剂型包括但不限于冻干粉针、注射液、微乳、微球、胶束等剂型。所述抗血栓形成的药物包括用于治疗或预防外周动脉闭塞性疾病(通过抗凝、抗血栓、抗血小板聚集发挥作用)、进展性缺血性脑卒中、急性冠脉综合症等疾病的药物。此外所述抗血栓形成药物也可以包括预防血液透析患者在VAF和VAG手术中动静脉瘘血栓的形成。
下面通过实施例对本发明的具体实施方式进行进一步说明，以下实施例中所涉及的仪器和试剂均可以通过购买市售产品的方式获得。
以下实施例中部分实验材料见表1，仪器设备见表2。
表1
名称 生产厂家 规格 批号
枸橼酸钠 天津市百世化工有限公司 500g/瓶 20050318
0.9％Nacl注射液 西安京西双鹤药业有限公司 250mL/瓶 11806471
PT测定试剂盒 陕西方舟生物科技有限公司 10×2ml 20140913
APPT测定试剂盒 陕西方舟生物科技有限公司 10×2ml 20141012
表2
名称 生产厂家 型号
电子天平 赛多利斯科学仪器(北京)有限公司 BSA124S-CW
离心机 安徽中科中佳科学仪器有限公司 SC-3610
精密微量加样器 日本Nichipet产品 5μl,100μl,200μl,1000μl
血小板聚集凝血因子分析仪 北京中勤世帝科学仪器有限公司 LG-PABER-I
出血时间测定器 江苏康健医疗用品有限公司 5mm宽1mm深
温度测定仪 自制  
实施例1
实施例1用于说明根据本发明的一种实施方式的化合物及其制备过程。
1、片段Ⅰ Fmoc-Gly-Gly-Gly-Gly-OH的制备：称取替代度为0.7mmol/g的Fmoc-Gly-2-Cl-Trt-树脂(23.5g，16.5mmol)采用2.5L 25％PIP/DMF溶液去Fmoc保护25分钟，过滤后树脂分别用DMF，DCM交替洗涤3次，每次不少于1分钟。加入Fmoc-Gly-OH(14.8g，50mmol)，30℃下搅拌反应4小时，以茚三酮法检测反应终点，反应完成后，过滤后树脂分别用DMF、DCM交替洗涤3次。
重复上述步骤2次，连接另外2个Gly，最终得到Fmoc-Gly-Gly-Gly-Gly-2-Cl-Trt-树脂。将得到的树脂溶于5L的30％六氟异丙醇/DCM溶液，搅拌反应2小时，过滤收集滤液，30℃真空干燥10小时，得到Fmoc-Gly-Gly-Gly-Gly-OH 7.2g，收率为96％，纯度95.6％，MS m/z:469(M+1)。
2、片段Ⅱ Fmoc-Cys(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-Wang树脂的制备
称取Fmoc-Cys(Trt)-Wang树脂10g(荷载量1.0mmol/g,10mmol)置于固相反应器中，加入25％的PIP/DMF溶液150ml脱保护，25℃下搅拌30分钟，反应完毕后，将树脂分别用DMF，DCM交替洗涤3次。将Fmoc-Pro-OH(10.1g，30mmol)，HOBt(4.05g，30mmol)溶于150ml DMF中，加入反应器中，在0-5℃下向反应液中缓慢加入DIC(3.78g，30mmol)，再加入脱保护后的氨基酸树脂，30℃下搅拌反应3小时，以茚三酮法检测反应终点，反应完成后，过滤后树脂分别用DMF、DCM交替洗涤3次。重复上述步骤，按照肽序列依次偶联Fmoc-Trp-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Gly-OH，Fmoc-Har-OH和Fmoc-Cys(Trt)-OH得到Fmoc-Cys(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-Wang树脂，检测替代度为0.6mmol/g。
3、线性多肽1树脂的合成：线性多肽1树脂为：Fmoc-D-Phe-Pro-Arg(pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(OtBu)-Leu-Gly-Gly-Gly-Gly-Ser(Trt)-Cys(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-Wang
取Fmoc-Cys(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-Wang树脂20g(替代度0.6mmol/g，12mmol)，加入25％的PIP/DMF溶液300ml脱保护，25℃下搅拌30分钟，反应完毕后，将树脂分别用DMF，DCM交替洗涤3次。将Fmoc-Ser(Trt)-OH(20.5g，36mmol)，HOBt(4.86g，36mmol)溶于150ml DMF中，加入反应器中，在0-5℃下向反应液中缓慢加入DIC(4.53g，36mmol)，再加入脱保护后的氨基酸树脂，30℃下搅拌反应3小时，以茚三酮法检测反应终点，反应完成后，过滤后树脂分别用DMF、DCM交替洗涤3次。采用上述方法依次接入Fmoc-Gly-Gly-Gly-Gly-OH，Fmoc-Leu-OH，Fmoc-Tyr(OtBu)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Pro-OH，Fmoc-Ile-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Phe-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Gly-OH，Fmoc-Asn(Trt)-OH，Fmoc-Gly-Gly-Gly-Gly-OH，Fmoc-Pro-OH，Fmoc-Arg(pbf)-OH，Fmoc-Pro-OH，Fmoc-D-Phe-OH得到侧链全保护氨基酸多肽1-Wang树脂。
4、线性多肽1粗品的制备：将步骤3制备的侧链全保护氨基酸多肽1-Wang树脂20g加入到酸解剂(三氟乙酸：三异丙基硅烷：水＝190ml：5ml：5ml)中，在25℃下反应2小时，过滤树脂，用少量三氟乙酸洗涤树脂，合并滤液。在剧烈搅拌下将滤液缓慢加入1.1L预冷却的乙醚内，出现白色沉淀，静置1小时后，抽滤，并用 冰乙醚洗涤滤饼5次，真空干燥得到粗品6g。
5、多肽1的二硫键的形成与纯化：多肽1结构式如下所示(SEQ ID No.4所示的多肽序列)：D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gly Gly Gly Gly Ser Cys Har Gly Asp Trp Pro Cys。将10g步骤4制备的线性多肽1溶于200ml纯净水中，搅拌下缓慢滴加5％I2溶液，HPLC检测反应，反应完全后采用高效制备液相纯化目标产物，冻干得到最终产品多肽1，质量为1g。多肽1的质谱检测结果如图1所示。
实施例2
按照与实施例1相同的方法制备多肽化合物2，区别仅在于L’片段为Glu Ala Ala Ala Lys获得多肽2，产品质量为1.2g。多肽2的质谱检测结果如图2所示。
多肽2结构式如下所示(SEQ ID No.5所示的多肽序列)：D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Glu Ala Ala Ala Lys Cys Har Gly Asp Trp Pro Cys
实施例3
按照与实施例1相同的方法制备多肽化合物3，区别仅在于去掉Gly Gly Gly Gly Ser片段(即L’片段)获得多肽3，产品质量为1.5g。多肽3的质谱检测结果如图3所示。
多肽3结构式如下所示(SEQ ID No.6所示的多肽序列)：D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Cys Har Gly Asp Trp Pro Cys
实施例4
按照与实施例1相同的方法制备多肽化合物4，区别仅在于L’片段为Arg Val Leu Ala Glu Ala获得多肽4，产品质量为0.86g。多肽4的质谱检测结果如图4所示。
多肽4结构式如下所示(SEQ ID No.7所示的多肽序列)：D-Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Arg Val Leu Ala Glu Ala Cys Har Gly Asp Trp Pro Cys
测试例1-4
测试例1-4用于说明实施例1-4中制备的多肽1-4抗人凝血、抗人血小板聚集的作用。抽健康志愿者静脉血30ml，将血液迅速注入含有3ml 3.8％枸橼酸钠的塑料管中，轻轻上下颠倒混匀。将抽取的血液810rpm离心8min得到富血小板血浆(platelet rich plasma,PRP)，取出PRP，再将血样3510rpm离心8min得到贫血小板血浆(platelet poor plasma，PPP)。1、凝血酶原时间(PT)、凝血酶时间(TT)、活化部分凝血酶时间(APTT)测定：按照试剂盒操作要求进行试剂重建及保存，每个PPP各取受检10份于测试杯中，分别加入生理盐水、多肽1、多肽2、多肽3、多肽4各10μL，再分别加入PT、TT、APTT试剂，立即开始测试。多肽1-4对人体外凝血功能的影响见表3(n代表例数)。
表3多肽1-4对人体外凝血功能的影响
受试品 浓度(mol/L) PT(S) PT延长倍数 APTT(S) APTT延长倍数 TT(S)
生理盐水 0 11.22 — 31.10 — 14.07
多肽1(n＝16) 1×10-6 25.86 2.30 87.51 2.87 >180
多肽2(n＝7) 1×10-6 34.78 3.04 104.14 3.35 >180
多肽3(n＝3) 1×10-6 25.67 2.14 101.97 2.94 >180
多肽4(n＝3) 1×10-6 58.93 4.92 98.85 2.57 >180
表3的结果显示：多肽1-4四个化合物都可以影响人体外凝血功能，不同程度延长PT、APTT和TT，终浓度为1×10-6mol/L的各个化合物中，多肽2延长PT、APTT的倍数最大。
2、血小板聚集测定：血小板聚集仪开机预热30min，PPP校正，测试杯中加入270μl PRP，分别加入10μl多肽1、多肽2、多肽3、多肽4(1.0×10-7mol/L)和/或20μl的生理盐水，总体积300μl。温浴5min后，再加入诱导剂ADP 5μl、肾上腺素5μl，开始测试。5min后测试完毕，记录5min内最大聚集率，并打印数据及图。根据测试结果根据计算公式1计算不同浓度的多肽1-4对人血小板聚集的抑制率，多肽1-4对ADP诱导人体外血小板聚集功能的影响列于表4。计算公式如下：
Figure PCTCN2015086173-appb-000001
表4多肽1-4对ADP诱导人体外血小板聚集功能的影响
受试品 浓度(mol/L) 血小板聚集率(％) 血小板聚集抑制率(％)
生理盐水 — 73.39 —
多肽1(n＝8) 3×10-6 39.10 47.07
多肽2(n＝7) 3×10-6 26.12 66.70
多肽3(n＝3) 3×10-6 57.85 26.35
多肽4(n＝3) 3×10-6 61.28 18.88
多肽1(n＝8) 1×10-5 4.90 92.04
多肽2(n＝7) 1×10-5 0 100
多肽3(n＝3) 1×10-5 35.40 47.07
多肽4(n＝3) 1×10-5 54.80 27.97
表4的结果显示：多肽1、多肽2、多肽3、多肽4四个化合物都可以影响ADP诱导的人体外血小板聚集，终浓度为3×10-6mol/L的依替巴肽可完全抑制ADP诱导的人体外血小板聚集，抑制率为100％，而终浓度为3×10-6mol/L、1×10-5mol/L的多肽1、多肽2、多肽3、多肽4四个化合物中，多肽2抑制ADP诱导的人体外血小板聚集作用最强。
测试例5
本测试例用于说明实施例2中制备的多肽2抗大鼠血栓形成的效果。
1.实验动物：健康、成年SD大鼠160只，雄性，体质量250g～300g，购自北京维通利华公司。动物生产许可证号：SCXK(京)2012-0001。
2.受试药品：多肽2，肽含量为97.21％。依诺肝素钠注射液Aventis Intercontinental，赛诺菲(北京)制药有限公司，分装批准文号：国药准字J20090094。批号：2SN76，0.4ml:4000AxaIU×2支。注射用比伐卢定，西安新通药物研究有限公司，分子量2180.2，纯度96.55％，含量69.44％。
3.实验方法
3.1试剂的配制及材料的制备方法
3.1.1试剂的配制
3.1.1.1受试品多肽2溶液配制：精确称取多肽2200mg，生理盐水制成10mg/mL的母液，每天实验前根据动物体重，取相应的母液配制成需要的剂量。
3.1.1.2 20％乌拉坦：精密称取乌拉坦20g加蒸馏水定容至100ml。
3.1.1.3 3.8％枸橼酸钠：精密称取二水枸橼酸钠4.33g加生理盐水定容至100ml配制成3.8％的溶液备用。
3.1.2材料的制备
3.1.2.1铝箔纸：用直尺测量剪成2.5cm×2.5cm，编号并称重。
3.1.2.2 35％三氯化铁溶液：取三氯化铁35g，溶于100mL蒸馏水中。
3.2.分组及给药
大鼠160只。随机分组，每组5-10只。1)模型对照组：静脉注射同体积生理盐水；2)多肽2的6个剂量组，以1.5mg/kg+3.75mg/kg/h为起始，向下降为1mg/kg+2.5mg/kg/h，0.5mg/kg+1.25mg/kg/h，0.25mg/kg+0.75mg/kg/h，0.125mg/kg+0.375mg/kg/h，0.041mg/kg+0.125mg/kg/h，3)阳性对照依诺肝素5个剂量组，以30U/kg+45U/kg/h为起始，向上加为60U/kg+60U/kg/h，30U/kg+120U/kg/h，90U/kg+120U/kg/h，60U/kg+240U/kg/h，4)阳性对照比伐卢定5个剂量组，以0.5mg/kg+2mg/kg/h为起始，向下降为0.35mg/kg+1.3mg/kg/h，0.17mg/kg+0.65mg/kg/h，0.08mg/kg+0.325mg/kg/h，实验前12小时动物禁食不禁水。(如表5所示)。
按大鼠公斤体重计量，将首剂量药物溶于2mL生理盐水中一次性推注，维持剂量溶于9ml生理盐水中，置于双道微量注射泵上，以0.1ml/min滴速90min内滴完。假手术组给同样体积生理盐水。
4.实验操作
4.1大鼠手术准备
大鼠于实验前一晚禁食，自由饮水。静脉注射20％乌拉坦溶液5ml/kg麻醉，仰卧位固定，分离两侧颈总动脉、颈外静脉和下腔静脉。颈外静脉插管用于给药；一侧颈总动脉插管用于取血测定凝血功能PT，APTT；一侧颈总动脉用于建立动脉血栓模型；下腔静脉用于静脉血栓模型。
4.2凝血功能测定
分别于给药前，给药60min，停药前取血测定活化部分凝血活酶时间(activated partial thromboplastin tim，APTT)，凝血酶原时间(prothrombin time，PT)。血样3510rpm离心8min得到贫血小板血浆(platelet poor plasma,PPP)。按照试剂盒操作要求进行试剂重建及保存，每个血样于测试杯中，再分别加入PT、APTT试剂，立即开始测试，测试结束记录结果。
4.3动脉血栓模型
负荷量给药、维持给药30min后，将直径3mm，浸泡35％FeCl3滤纸敷于颈总动脉下，其下置小片塑料薄膜(2.5×2.0cm)，用于保护周围组织。在动脉远心端用温度探头检测温度，考察自刺激开始至温度下降2.5度所需要的时间为血管阻塞时间。
4.4静脉脉血栓模型
负荷量给药、维持给药30min后，将直径3mm、吸有35％FeCl3溶液的小片滤纸敷于下腔静脉下，其下置小片塑料薄膜(2.5×2.0cm)，用于保护周围组织。15min后去掉滤纸条。再观察45min后用温生理盐水冲洗血管及局部组织，将被包裹的血管剪下，将血管壁粘附的血栓剥离到锡箔纸上称湿重，室温过夜称干重。持续给药至实验结束。
5.检测指标
5.1凝血功能测定：分别于给药前0min，给药60min，停药时采血用血小板凝血因子分析仪测定凝血功能(PT，APTT)，ACT。
5.2阻塞时间
从放置FeCl3开始到动脉表明温度下降2.5℃的时间为血管阻塞时间，反映血栓形成情况。
5.3血栓重量
实验结束后，剪下形成血栓的血管段，称其湿重，室温下过夜测得血栓干重。计算血栓形成抑制率。
表5多肽2和对照药剂量分组
编号 组别 剂量(mg/kg+mg/kg/h) n
1 模型 —— 10
2 依诺肝素一组 30+45u 6
3 依诺肝素二组 60+60u 5
4 依诺肝素三组 30+120u 5
5 依诺肝素四组 90+120u 6
6 依诺肝素五组 60+240u 10
7 比伐卢定一组 0.08+0.325 6
8 比伐卢定二组 0.17+0.65 6
9 比伐卢定三组 0.35+1.3 8
10 比伐卢定四组 0.5+2 5
11 多肽2一组 0.041+0.125 10
12 多肽2二组 0.125+0.375 10
13 多肽2三组 0.25+0.75 9
14 多肽2四组 0.5+1.25 10
15 多肽2五组 1+2.5 9
16 多肽2六组 1.5+3.75 10
表6不同剂量多肽2和对照药静脉维持给药对FeCl3诱导的大鼠血栓形成的影响
Figure PCTCN2015086173-appb-000002
Figure PCTCN2015086173-appb-000003
Figure PCTCN2015086173-appb-000004
表7不同剂量多肽2和对照药静脉维持给药对大鼠凝血功能的影响结果
Figure PCTCN2015086173-appb-000005
Figure PCTCN2015086173-appb-000006
由表6和表7的结果可知，多肽2可剂量依赖性地抑制FeCl3诱导的大鼠下腔静脉血栓形成，抑制FeCl3诱导的大鼠颈总动脉血栓形成，延长大鼠APTT和PT。
图5为多肽2对Fecl3刺激大鼠下腔静脉血栓形成的影响，与模型组相比，*P<0.05，**P<0.01。图6为多肽2对Fecl3刺激大鼠颈总动脉血栓形成的影响。动脉阻塞时间变化倍数。与模型组相比，*P<0.05，**P<0.01。图7多肽2对大鼠APTT的影响。图8多肽2对大鼠PT的影响。
测试例6
本测试例用于说明实施例2中制备的多肽2静脉维持给药对家兔血栓抑制的药效学实验。
1.实验动物和材料
1.1实验动物：健康成年家兔10只，雄性，体重2～2.5kg，由西安市迪乐普生物资源开发有限公司提供，动物生产许可证号：SCXK(陕)2006-001。受试药品：多肽2，肽含量为97.21％。阳性对照品：依诺肝素钠注射液Aventis Intercontinental，赛诺菲(北京)制药有限公司，分装批准文号：国药准字J20090094。批号：2SN76，0.4ml:4000AxaIU×2支。
1.2实验材料，见表8。
表8
名称 生产厂家 规格 批号
戊巴比妥钠 美国sigma公司 25g/瓶 090205
枸橼酸钠 天津市百世化工有限公司 — 20080418
0.9％Nacl注射液 西安京西双鹤药业有限公司 250mL/瓶 11806471
肝素钠注射液 天津生物化学制药有限公司 2mL 12500IΜ 20120202
磷酸二腺苷 SIGMA 1g/瓶 购于2009.12.10
盐酸肾上腺注射液 上海禾丰制药有限公司 1mg/mL 20090701
PT测定试剂盒 陕西方舟生物科技有限公司 10×2ml 20140703
TT测定试剂盒 陕西方舟生物科技有限公司 10×2ml 20140702
APPT测定试剂盒 陕西方舟生物科技有限公司 10×2ml 20140702
1.3受试品多肽2溶液配制：取多肽2，电子天平精密称取200mg，生理盐水制成10mg/mL的母液。充分混匀，每5mL分装，-20℃保存。试验时按家兔的体重计算取适量稀释使用。
3％戊巴比妥钠：精密称取戊巴比妥钠3g加蒸馏水定容至100ml。
3.8％枸橼酸钠：精密称取二水枸橼酸钠4.33g加生理盐水定容至100ml配制成3.8％的溶液备用。
肾上腺素：取1.319ml原液定容至100ml的生理盐水中配制成60μM的溶液，4℃冰箱保存，用前分装。
ADP：精密称取ADP 14.3mg溶于5ml的生理盐水中得到6000μmol/L的ADP溶液，4℃保存，实验前加生理盐水将其稀释成600μmol/L备用。
1.4材料的制备
铝箔纸：用直尺测量剪成2.5cm×2.5cm，编号并称重；非吸收性外科缝线：用直尺量取8cm长，称重。
50％三氯化铁溶液：取三氯化铁50g，溶于100mL蒸馏水中。
2.分组及给药：家兔10只。随机分为3组，每组2-3只。1)假手术组(NS)：静脉注射同体积生理盐水，手术方法同上；2)多肽2给药组8.0mg/kg+20.0mg/kg/h；3)阳性对照依诺肝素组50U/kg+150U/kg/h。按家兔公斤体重计量，将首剂量药物溶于2mL生理盐水中一次性推注，维持剂量溶于90ml生理盐水中，置于双道微量注射泵上，以1ml/min滴速90min内滴完。假手术组给同样体积生理盐水。
3.实验操作
3.1家兔手术准备：家兔送到实验室适应1天后，首先进行筛选。耳中动脉采血进行血常规及APTT测定，选择血象正常，APTT在16s～28s之间的家兔进行实验。试验家兔于实验前一晚禁食，自由饮水。静脉注射3％戊巴比妥钠溶液1ml/kg麻醉，仰卧位固定，分离一侧颈总动脉、颈外静脉，一侧股动脉、股静脉及一侧前肢静脉。前肢静脉插管用于给药；左侧股静脉插管用于取血，以备测定血小板聚集率；股动脉用于动脉血栓模型；颈总动脉、颈外静脉用于动静脉旁路血栓模型。
3.2动脉血栓(AT)模型：给药的同时将吸有50％FeCl3溶液的小片滤纸(1cm×1.5cm)敷于右侧股动脉上，其下置小片塑料薄膜(2.5×2.0cm)，用于保护周围组织。10min后去掉滤纸条，用温生理盐水冲洗血管及局部组织，将被包裹的血管剪下，将血管壁粘附的血栓剥离到锡箔纸上称湿重，室温过夜称干重。
3.3实验流程：将家兔前肢和两侧股动脉分离后，稳定10min，开始给药。给药的同时用50％FeCl3滤纸包裹股动脉建立AT模型，包裹15min后去除。连续观察给药后120min，试验全部结束。
4.检测指标
4.1血小板聚集率：给药前0min，给药30min，60min，90min，停药60min，120min，180min从股静脉取血2.7ml，迅速注入含有0.3ml 3.8％枸橼酸钠的离心管中，充分混匀。810rpm(100g)离心8min，取PRP， 3500rpm(1863g)离心8min，取PPP做血小板聚集。
分别于给药前(0min)，给药后5min，15min采血测定血小板聚集率，计算血小板聚集抑制率。计算公式如下：
Figure PCTCN2015086173-appb-000007
4.2凝血功能测定：分别于给药前0min，给药30min，60min,90min,停药60min,120min,180min采血用血小板凝血因子分析仪测定凝血功能(PT,TT,APTT)。血样3510rpm离心8min得到贫血小板血浆(platelet poor plasma,PPP)。按照试剂盒操作要求进行试剂重建及保存，每个血样于测试杯中，再分别加入PT、TT、APTT试剂，立即开始测试，测试结束记录结果。
4.3 ACT的测定：给药前0min，给药5min，15min取全血，加白陶土，37℃水浴锅内，记录全血凝固时间。
4.4血栓重量：实验结束后，剪下形成血栓的血管段，称其湿重，室温下过夜测得血栓干重。计算血栓形成抑制率。公式如下：
Figure PCTCN2015086173-appb-000008
5.试验结果：
5.1多肽2静脉维持给药对FeCl3诱导的家兔股动脉血栓形成的影响
结果表明，多肽28.0mg/kg+20.0mg/kg/h给药，对FeCl3诱导的家兔股动脉血栓形成的影响，依诺肝素50U/kg+150U/kg/h给药血栓抑制率低于多肽2，与生理盐水组相比，无统计学差异(P>0.05)。结果见表9。
表9多肽2静脉维持给药对家兔股动脉血栓形成的影响
Figure PCTCN2015086173-appb-000009
Figure PCTCN2015086173-appb-000010
5.2多肽2静脉维持给药对家兔血小板聚集功能的影响
结果表明，多肽2静脉维持给药可以抑制家兔血小板聚集。停药后随着时间的延长，对血小板聚集的抑制作用逐渐减弱。依诺肝素50U/kg+150U/kg/h给药血小板聚集抑制率低于多肽2。结果见表10。
表10多肽2静脉单次给药对家兔血小板聚集功能的影响(血小板聚集抑制率，％，
Figure PCTCN2015086173-appb-000011
)
组别 剂量(mg/kg+mg/kg/h) n 给药30min 给药60min 给药90min
空白组 - 2 12.1±17.1 12.1±17.1 13.1±18.6
依诺肝素 50U+150U 1 26.7 0.00 35.27
多肽2 8.0+20.0 2 61.0±14.1 72.1±9.6 55.8±7.7
组别 剂量(mg/kg+mg/kg/h) n 停药60min 停药120min 停药180min
空白组 - 2 22.1±31.2 13.9±19.7 -
依诺肝素 50U+150U 1 0.00 41.9 -
多肽2 8.0+20.0 2 14.1±1.5 7.5±1.8 47.5±2.5
5.3多肽2静脉维持给药对家兔凝血功能的影响
结果见表11-14，表11为多肽2静脉维持给药对家兔凝血功能APTT的影响(S，
Figure PCTCN2015086173-appb-000012
)；表12为多肽2静脉维持给药对家兔凝血功能PT的影响(S，
Figure PCTCN2015086173-appb-000013
)；表13为多肽2静脉维持给药对家兔凝血功能TT的影响(S，
Figure PCTCN2015086173-appb-000014
)；表14为多肽2静脉维持给药对家兔ACT的影响(S，
Figure PCTCN2015086173-appb-000015
)。表11-14中，剂量单位为(mg/kg+mg/kg/h)。结果表明，多肽2静脉维持给药可以抑制家兔血小板聚集。停药后随着时间的延长，对血小板聚集的抑制作用逐渐减弱。
表11
Figure PCTCN2015086173-appb-000016
表12
Figure PCTCN2015086173-appb-000017
表13
Figure PCTCN2015086173-appb-000018
表14
Figure PCTCN2015086173-appb-000019
以上结果说明多肽2推注(iv)+滴注(vd)8.0mg/kg+20.0mg/kg 90min可以抑制家兔血小板聚集，影响家兔凝血功能，延长APTT、PT、TT和ACT。抑制股动脉血栓形成。多肽2与依诺肝素抑制股动脉血栓和抑制血小板聚集作用相当时，依诺肝素对APTT延长作用大于多肽2，说明依诺肝素出血风险大于多肽2。
测试例7
本测试例用于说明静脉单次给药家兔体体内抗血栓形成暨抗凝抗血小板聚集实验。实验动物：健康成年家兔48只，雄性，体重2-2.5kg，由西安市迪乐普生物资源开发有限公司提供，动物合格证号：SCXK(陕)2006-001。受试药品：多肽2，肽含量为97.21％。阳性药：依诺肝素与比伐卢定。
【实验方法】
1.分组及给药：家兔48只。随机分为6组，每组8只。1)假手术组(NS)：静脉注射同体积生理盐水；2)阳性对照比伐卢定组(6mg/kg)；3)阳性对照依诺肝素组(200U/kg)；4)多肽2小剂量组(3.0mg/kg)；5)多肽2中剂量组(6.0mg/kg)；6)多肽2大剂量组(12.0mg/kg)。多肽2采取静脉单次给药方式。按家兔公斤体重计量，将药物溶于2mL生理盐水中一次性推注。假手术组给同样体积生理盐水。
2.实验操作
2.1家兔手术准备：家兔送到实验室适应1天后，首先进行筛选。耳中动脉采血进行血常规及APTT测定，选择血象正常，APTT在16s～28s之间的家兔进行实验。试验家兔于实验前一晚禁食，自由饮水。静脉注射3％戊巴比妥钠溶液1ml/kg麻醉，仰卧位固定，分离一侧颈总动脉、颈外静脉，一侧股动脉、股静脉及一侧前肢静脉。前肢静脉插管用于给药；左侧股静脉插管用于取血，以备测定血小板聚集率；股动脉用于动脉血栓模型；颈总动脉、颈外静脉用于动静脉旁路血栓模型。
2.2动静脉旁路血栓(AVST)模型：动静脉分流器由两个套管组成，外管长约8cm，内径7.9mm，内管长约2.5cm，内径4.8mm，在聚乙烯管内放入长约8cm的丝线，充满50Μ/ml的肝素钠溶液，将一头插入左侧股动脉，另一头插入右侧股静脉。给药30s放开血流，血液从左侧动脉流至聚乙烯管内，返回右侧静脉；15min后中断血流，迅速取出丝线放于铝箔纸(大小为2.5cm×2.5cm)上，称湿重，室温过夜，称干重。
2.3动脉血栓(AT)模型：给药的同时将吸有50％FeCl3溶液的小片滤纸(1cm×1.5cm)敷于右侧股动脉上，其下置小片塑料薄膜(2.5×2.0cm)，用于保护周围组织。10min后去掉滤纸条，用温生理盐水冲洗血管及局部组织，将被包裹的血管剪下，将血管壁粘附的血栓剥离到锡箔纸上称湿重，室温过夜称干重。
2.4实验流程：实验流程如图9，将犬前肢和两侧股动脉分离后，稳定10min，开始给药。给药的同时用50％FeCl3滤纸包裹股动脉建立AT模型，包裹10min后去除；建立AVST模型，40min后取出。连续观察 给药后120min，试验全部结束。
3.检测指标
3.1血小板聚集率：从股静脉取血2.7ml，迅速注入含有0.3ml 3.8％枸橼酸钠的离心管中，充分混匀。810rpm(100g)离心8min，取PRP，3500rpm(1863g)离心8min，取PPP做血小板聚集。
分别于给药前(0min)，给药后5min，15min采血测定血小板聚集率，计算血小板聚集抑制率。计算公式如下：
Figure PCTCN2015086173-appb-000020
3.2凝血功能测定：分别于给药前0min，给药5min，15min采血用血小板凝血因子分析仪测定凝血功能(PT、TT、APTT)。血样3510rpm离心8min得到贫血小板血浆(platelet poor plasma,PPP)。按照试剂盒操作要求进行试剂重建及保存，每个血样于测试杯中，再分别加入PT、TT、APTT试剂，立即开始测试，测试结束记录结果。
3.3 ACT的测定：给药前0min，给药5min，15min取全血，加白陶土，37℃水浴锅内，记录全血凝固时间。
3.4血栓重量：实验结束后，剪下形成血栓的血管段，称其湿重，室温下过夜测得血栓干重。计算血栓形成抑制率。
Figure PCTCN2015086173-appb-000021
4实验结果
4.1多肽2静脉单次给药对FeCl3诱导的家兔股动脉血栓形成的影响，结果见图10和表15。。不同浓度的多肽2静脉单次给药，可减轻FeCl3诱导的家兔股动脉血栓干重，抑制FeCl3诱导的家兔股动脉血栓形成。与生理盐水组相比，多肽2小剂量3.0mg/kg组统计学有显著差异(P<0.05),多肽2中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有极显著差异(P<0.01)，多肽2中剂量组抗动脉血栓效果优于比伐卢定组。
表15多肽2静脉单次给药对FeCl3诱导的家兔股动脉血栓形成的影响
Figure PCTCN2015086173-appb-000022
组别 剂量(mg/kg) n 血栓干重(mg) 血栓抑制率(％)
生理盐水组 — 8 2.9±1.9 —
比伐卢定 6.0 8 1.5±0.9* 48.20
依诺肝素 200U 7 1.5±0.4* 48.26
多肽2小剂量 3.0 6 1.4±0.5* 52.99
多肽2中剂量 6.0 8 1.2±0.7** 56.90
多肽2大剂量 12.0 8 1.1±0.9** 60.82
与生理盐水组相比，*P<0.05，**P<0.01。
4.2多肽2静脉单次给药对家兔股动静脉旁路血栓形成的影响
如图11和表16结果所示，不同浓度的多肽2静脉单次给药，可以减轻家兔股动静脉旁路血栓干重，抑制家兔股动静脉旁路血栓形成。与生理盐水组相比，多肽2中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有极显著差异(P<0.01)，多肽2中剂量组抗动静脉旁路血栓效果明显优于比伐卢定和依诺肝素组。
表16多肽2静脉单次给药15min对家兔股动静脉旁路血栓形成的影响
Figure PCTCN2015086173-appb-000023
组别 剂量(mg/kg) n 血栓干重(mg) 血栓抑制率(％)
生理盐水组 - 8 23.6±7.0 —
比伐卢定 6.0 8 20.8±6.3 11.86
依诺肝素 200U 7 17.3±6.5 26.88
多肽2小剂量 3.0 6 20.2±4.3 14.48
多肽2中剂量 6.0 8 13.5±5.3** 42.80
多肽2大剂量 12.0 8 10.6±6.8** 55.24
与生理盐水组相比，*P<0.05，**P<0.01。
4.3多肽2静脉单次给药对家兔血小板聚集功能的影响
结果如图12和表17所示，多肽2各剂量组静脉单次给药可抑制家兔血小板聚集。停药后随着时间的 延长，对血小板聚集的抑制作用逐渐减弱。
多肽2各剂量组静脉单次给药5min，可不同程度地抑制家兔血小板聚集。与生理盐水组相比，多肽2中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有显著差异(P<0.05)，多肽2中剂量组抗血小板作用明显优于比伐卢定和依诺肝素组。给药15min，对家兔血小板聚集的抑制作用逐渐减弱。与生理盐水组相比，多肽2大剂量12.0mg/kg有显著差异(P<0.05)。
表17多肽2静脉单次给药对家兔血小板聚集功能的影响(血小板聚集抑制率，％，
Figure PCTCN2015086173-appb-000024
)
组别 剂量(mg/kg) n 给药5min 给药15min
生理盐水 - 8 0.6±5.8 3.2±6.0
比伐卢定 6.0 8 5.2±5.8 4.6±6.0
依诺肝素 200 7 9.5±6.2 16.1±6.5
多肽2小剂量 3.0 6 17.2±6.7 6.3±7.0
多肽2中剂量 6.0 8 47.5±5.8*#￥& 15.4±6.0
多肽2大剂量 12.0 7 72.5±6.2*#￥&@ 46.0±6.5*#￥&@
与生理盐水组相比，*P<0.05；与比伐卢定组相比，#P<0.05；与依诺肝素组相比，￥P<0.05；与多肽2小剂量组相比，&P<0.05；与多肽2中剂量组相比，@P<0.05
4.4多肽2静脉单次给药对家兔凝血功能的影响
结果表明，多肽2给药后，可以延长家兔APTT、PT、TT和ACT。停药后随着时间的延长，对上述凝血指标的的延长作用逐渐减弱。
4.4.1对家兔APTT的影响
结果如图13和表18和19结果所示。多肽2各剂量组可不同程度地延长家兔APTT，随着时间的延长，15min对APTT的延长作用逐渐减弱。给药5min，与生理盐水组相比，多肽2中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有显著差异(P<0.05)；给药15min时，与生理盐水组相比，多肽2中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有显著差异(P<0.05)。
表18多肽2静脉单次给药对家兔APTT的影响(s，x±Se)
组别 剂量(mg/kg) n 给药前 给药5min 给药15min
生理盐水 - 8 23.5±1.0 23.9±3.6 21.8±2.7
比伐卢定 6.0 8 21.6±1.0 50.4±3.6* 30.9±2.7
依诺肝素 200 7 22.5±1.1 48.7±3.9* 40.9±2.9*
多肽2小剂量 3.0 6 20.6±1.2 24.4±4.2#￥ 20.0±3.1#
多肽2中剂量 6.0 8 24.1±1.0 50.5±3.6*& 34.9±2.7*&
多肽2大剂量 12.0 8 23.3±1.0 54.4±3.6*& 37.0±2.7*&
与生理盐水组相比，*P<0.05；与比伐卢定组相比，#P<0.05；与伊诺肝素组相比，￥P<0.05；与多肽2小剂量组相比，&P<0.05。
表19多肽2静脉单次给药对家兔APTT的影响(延长倍数，
Figure PCTCN2015086173-appb-000025
)
Figure PCTCN2015086173-appb-000026
4.4.2对家兔PT的影响
结果如图14、表20和表21所示。多肽2各剂量组可不同程度地延长家兔PT，随着时间的延长，15min对PT的延长作用逐渐减弱。
给药5min，与生理盐水组相比，多肽2中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有显著差异(P<0.05)；给药15min时，与生理盐水组相比，多肽2中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有显著差异(P<0.05)。
表20多肽2静脉单次给药对家兔PT的影响(S，x±Se)
组别 剂量(mg/kg) n 给药前 给药后5min 给药15min
生理盐水 - 8 6.2±0.2 6.0±1.1 5.8±0.7
比伐卢定 6.0 8 6.4±0.2 18.8±1.1* 10.2±0.7*
依诺肝素 200 7 5.8±0.2 6.7±1.1# 6.7±0.8#
多肽2小剂量 3.0 6 6.0±0.3 8.9±1.2# 7.0±0.8
多肽2中剂量 6.0 8 6.3±0.2 12.9±1.1*#￥ 8.9±0.7*
多肽2大剂量 12.0 8 6.5±0.2 19.2±1.1*￥& 12.5±0.7*￥&@
表21多肽2静脉单次给药对家兔PT的影响(延长倍数，
Figure PCTCN2015086173-appb-000027
)
Figure PCTCN2015086173-appb-000028
4.4.3对家兔TT的影响
结果如表22所示，多肽2各剂量组给药5min、15min，可以延长家兔TT超过检测限180s。
表22多肽2静脉单次给药对家兔TT的影响(S，
Figure PCTCN2015086173-appb-000029
)
组别 剂量(mg/kg) n 给药前 给药5min 给药15min
生理盐水 - 8 21.2±0.8 20.3±0.8 19.1±0.7
比伐卢定 6.0 8 22.4±5.2 >180s >180s
依诺肝素 200 7 20.7±4.2 >180s >180s
多肽2小剂量 3.0 6 21.0±2.6 >180s >180s
多肽2中剂量 6.0 8 22.3±1.9 >180s >180s
多肽2大剂量 12.0 8 21.8±6.4 >180s >180s
4.4.4多肽2对家兔ACT的影响
结果如图15、表23和表24所示，多肽2各剂量组可不同程度地延长家兔ACT，随着时间的延长，15min对ACT的延长作用逐渐减弱。
给药5min，与生理盐水组相比，多肽2大剂量12.0mg/kg组统计学有显著差异(P<0.05)。
给药15min时，与生理盐水组相比，多肽2小剂量3.0mg/kg、中剂量6.0mg/kg、大剂量12.0mg/kg组统计学有显著差异(P<0.05)。
表23多肽2静脉单次给药对家兔ACT的影响(s，
Figure PCTCN2015086173-appb-000030
)
组别 剂量(mg/kg) n 给药前 给药5min 给药15min
生理盐水 - 8 240.0±24.0 226.9±35.3 208.1±24.1
比伐卢定 6.0 8 305.6±24.0 443.8±35.3* 375.0±24.1*
依诺肝素 200 7 285.0±25.6 915.0±37.7*# 610.7±25.7*#
多肽2小剂量 3.0 6 357.5±27.7 352.5±40.7￥ 337.5±27.8*￥
多肽2中剂量 6.0 8 232.5±24.0 356.3±35.3￥ 324.4±24.1*￥
多肽2大剂量 12.0 8 315.0±24.0 505.6±35.3*￥&@ 418.1±24.1*￥
与生理盐水组相比，*P<0.05；与比伐卢定组相比，#P<0.05；
与依诺肝素组相比，￥P<0.05；
与多肽2小剂量组相比，&P<0.05；与多肽2中剂量组相比，@P<0.05。
表24多肽2静脉单次给药对家兔ACT的影响(延长倍数，
Figure PCTCN2015086173-appb-000031
)
Figure PCTCN2015086173-appb-000032
多肽2静脉单次给药，可抑制FeCl3诱导的家兔股动脉血栓形成并剂量依赖性地抑制家兔股动静脉旁路血栓形成，其中剂量及大剂量组抗血栓效果优于比伐卢定组；多肽2可剂量依赖性抑制家兔血小板聚集，影响家兔凝血功能，延长APTT、PT、TT和ACT。
测试例8
本测试例用于说明多肽2对进展性缺血脑卒中的保护作用。
1、实验动物：健康、成年SD大鼠60只，雄性，体质量250g～300g，购自西安交通大学医学院动物中心。动物生产许可证号：SCXK(陕)2012-003。
2、受试药品：多肽2，肽含量为97.21％；对照药：依诺肝素注射液，赛诺菲(北京)制药有限公司生产，批号：4SH69。
3、实验方法：
3.1溶液的配置：多肽2：用前配成2mg/ml的母液。
3.2分组及给药：采用颈静脉插管给药的方式于造模15min开始给药，首剂加维持60min，给药组多肽2剂量为0.25mg/kg+0.75mg/kg/h，模型组给生理盐水。
3.3、实验操作：线栓法(颈总动脉插入法)：颈部取正中切口切开皮肤，分离出颈总、颈外、颈内动脉，在近心端结扎颈总动脉，远心端结扎颈外动脉。用小型血管夹暂时性夹闭颈内动脉远心端，在颈总、颈外、颈内动脉分叉处预留结扎线，用眼科剪在颈总动脉近颈内动脉处剪一小口，使MCAO栓线有一定程度的弯曲，沿颈总动脉插入颈内动脉，插线时栓线向操作者方向弯曲，向前缓慢推进约18mm(自动脉分叉处计)，有阻力感时停止进线，在分叉处结扎预留线以固定栓线，造模15min后劲静脉首剂加维持给要60min，给药结束后颈部伤口常规缝合。
4、检测指标
4.1神经行为学评分：于术后4h、8h、24h对两组大鼠进行神经行为学评分，以Longa五级四分法为标准：0分:无神经缺损症；1分:提尾时对侧前肢内收，不能全伸直；2分:向对侧旋转；3分:行走时向对侧倾倒；4分:不能行走或昏迷。1-4分为有效模型。
4.2 TTC染色观察梗死病变及梗死体积术后24h后处死动物，取脑，进行TTC染色用绘图软件计算梗死体积。
4.3脑指数、脑含水量：各组取大鼠在手术造模后24h将大鼠断头迅速剥离出全脑称湿重,计算脑指数，脑指数＝脑湿重/体重*100％。
5、试验结果
表25和表26试验结果显示，多肽2剂量0.5+1.25和1.0+2.5(mg/kg+mg/kg/h))使术后24小时脑梗死体积从(23.41±10.08)％分别降至(11.12±6.56)％和(8.01±6.66)％，神经行为学损伤也有改善，说明对实验性脑梗塞有保护作用。
表25多肽2对神经行为学的影响(x±s)
组别 剂量(mg/kg+mg/kg/h) n 4h 8h 24h
假手术组 — 5 0 0 0
模型组 — 19 2.32±0.75 2.37±0.83 2.50±0.99
依诺肝素组 90U/kg+120U/kg/h 8 0.63±0.74** 0.88±0.64** 1.38±0.74*
多肽2一组 0.5+1.25 7 1.00±0.58** 1.00±0.58** 1.00±0.58**
多肽2二组 1.0+2.5 7 0.86±1.07** 0.86±0.90** 1.14±0.69**
与模型组比，*P＜0.05，**P＜0.01
表26多肽2对脑梗死体积和脑指数的影响(x±s)
组别 剂量(mg/kg+mg/kg/h) n 脑梗死体积比(％) 脑指数(％)
假手术组 — 5 0±0 0.6175±0.0511**
模型组 — 19 23.41±10.08 0.7167±0.0555
依诺肝素组 90U/kg+120U/kg/h 8 10.22±5.38** 0.6819±0.0356
多肽2一组 0.5+1.25 7 11.12±6.56** 0.6648±0.0374*
多肽2二组 1.0+2.5 7 8.01±6.66** 0.6830±0.0322
与模型组比，*P＜0.05，**P＜0.01
测试例9
本测试例用于说明多肽2对凝血酶诱导的小鼠肺栓塞的保护作用。
1、实验动物：昆明种小鼠，雄性，6～8周龄，20～25g，由西安交通大学实验动物中心提供，动物生产许可证号SCXK(陕)2012-003。
2、实验试剂
2.1凝血酶sigma公司，批号T4648，来源于牛血清，规格1000U/瓶。
2.2依诺肝素注射液0.4ml:4000AxalU，赛诺菲(北京)制药有限公司，分装批准文号：国药准字J20090094。
2.3多肽2委托吉尔生化(上海)有限公司，批号P131029-ML360794，肽含量为97.21％。
3、实验仪器
BC-2800Vet型迈瑞全自动血球分析仪，深圳迈瑞生物医疗电子股份有限公司。
4、实验方法
小鼠随机分为7组，每组10只。第一组生理盐水组，第二组模型对照组；第三组阳性对照依诺肝素0.5mg/kg，第四组阳性对照组比伐卢定8mg/kg，；第五组多肽2小剂量组2.5mg/kg，第六组多肽2中剂量组5.0mg/kg，第七组多肽2大剂量组10.0mg/kg。除生理盐水组、模型对照组尾静脉注射200ul生理盐水外，其余各组分别按剂量给药。2min后生理盐水组尾静脉注射100ul生理盐水，其余各组尾静脉注射100ul凝血酶1500u/kg(80-90％致死剂量)。给药后观察小鼠反应，15min内死亡情况。15min内未死亡小鼠进行处理。
(1)记录死亡率：记录15min内急性肺栓塞小鼠的死亡情况，记录存活时间，计算死亡率；
(2)测定肺系数：死亡后立即解剖切除肺组织之外支气管和脂肪组织，用蒸馏水洗净肺组织，在用滤纸吸干肺表面的水分后称肺质量。取出肺脏称重，计算肺系数。肺系数＝肺质量/体质量×100％；
(3)血小板数目测定：15min内未死亡小鼠，眼球取血，EDTA抗凝后，测量血小板数目，立即解剖取出肺脏称重测定肺系数。
5、实验结果：LPS模型组小鼠死亡12/13只，死亡率为85％，与生理盐水组相比两组率的差别有非常显著意义(P<0.01)。依诺肝素0.5mg/kg组、比伐卢定8mg/kg和多肽2大剂量10.0mg/kg组死亡1/10只，死亡率为10％，分别与模型组相比，两组死亡率的差别有非常显著意义(P<0.01)。多肽2中剂量5.0mg/kg组死亡3/10只，死亡率为30％，分别与模型组相比，两组死亡率的差别有非常显著意义(P<0.01)；多肽2两个组与依诺肝素0.5mg/kg、比伐卢定8mg/kg分别相比，两组死亡率的差别无显著意义(P>0.05)。
多肽2小剂量2.5mg/kg组死亡6/10只，死亡率为60％，与模型组相比，两组死亡率的差别无显著意义(P>0.05)；与依诺肝素250U/kg、比伐卢定8mg/kg相比，两组死亡率的差别有显著意义(P<0.01)；与多肽2中剂量5.0mg/kg、多肽2大剂量10.0mg/kg相比，两组死亡率的差别有显著意义(P<0.05)。模型组小鼠肺系数显著增大，与生理盐水组相比统计学差异有非常显著意义(P<0.01)；比伐卢定8mg/kg组小鼠肺系数减小，与模型组相比统计学差异有显著意义(P<0.05)；多肽2大剂量10.0mg/kg组小鼠肺系数减小，与模型组相比统计学差异有非常显著意义(P<0.01)。结果见表27，图16，多肽2对凝血酶诱导的小鼠肺栓塞有保护作用。
表27多肽2对凝血酶诱导的小鼠肺栓塞的影响作用
组别 剂量(mg/kg) n 死亡率(％) 肺系数(g/100g) 血小板数目(×109)
空白对照组 — 10 0** 0.82±0.12** 1069.4±407.01
模型组 — 13 85 1.22±0.26 65.45±5.1
依诺肝素组 250IU/kg 10 10**## 1.02±0.33 459.83±49.66
比伐卢定组 8.0 10 10**## 0.99±0.07* 411.89±175.52
多肽2大剂量组 10.0 10 10**# 0.83±0.14** 529.57±104.28
多肽2中剂量组 5.0 10 30**# 1.08±0.27 207.83±33.09
多肽2小剂量组 2.5 10 60 0.94±0.16* 165.33±20.2
与模型组相比，*P<0.05，**P<0.01；与多肽2小剂量组相比，#P<0.05，##P<0.01
上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明所提供的凝血酶和血小板GP Ⅱ b/Ⅲa受体多靶点拮抗化合物具有直接、可逆、特异抗凝血酶功能，还具有抑制GP Ⅱ b/Ⅲa受体的作用，可以在较小的剂量下达到抗凝抗栓效果。同时降低出血风险、避免联合用药带来的剂量匹配、出血、作用协调等问题。
Figure PCTCN2015086173-appb-000033

Claims (22)

  1. 一种抗凝血和拮抗血小板GPⅡb/Ⅲa受体的多靶点化合物，所述化合物具有如式(1)所示的结构：
    A-L-B-L’-C  式(1)
    其中，其中A和B为与凝血酶结合位点，C为与血小板GPⅡb/Ⅲa受体结合位点，L为第一连接基团，L’为第二连接基团。
  2. 根据权利要求1所述的化合物，其特征在于，L’的结构如式(2)所示：
    ((Gly)n1-(Ser)n2)n3  式(2)
    其中n1为1、2、3或4；n2为0或1；n3为0、1、2或3。
  3. 根据权利要求1所述的化合物，其特征在于，L’的结构如式(3)所示：
    (Glu-Ala-Ala-Ala-Lys)n1  式(3)
    其中n1为0、1、2或3。
  4. 根据权利要求1所述的化合物，其特征在于，L’的结构如式(4)所示：
    (Arg-Val-Leu-Ala-Glu-Ala)n1  式(4)
    其中n1为0、1、2或3。
  5. 根据权利要求1-4中任意一项所述的化合物，其特征在于，A的结构如式(5)所示：
    A1-A2-A3-A4  式(5)
    其中A1为D-Phe；A2为Pro或Pip；A3为Arg、Lys、Orn或Har；A4为Pro、D-Pro或Ser。
  6. 根据权利要求5所述的化合物，其特征在于，B的结构如式(6)所示：
    B1-B2-B3-B4-B5  式(6)
    其中B1为任意两个酸性氨基酸组成的二肽；
    B2为Val、Leu、Ile、Nle或Phe；
    B3为Hyp，Ser，Pro或一种N-甲基氨基酸；
    B4为任意两个酸性氨基酸组成的二肽；
    B5为选自Tyr、Trp、Phe、Leu、Nle、Ile、Val、Cha和Pro中的一种的氨基酸，或者B5为含有Tyr、Trp、Phe、Leu、Nle、Ile、Val、Cha和Pro中的至少一种氨基酸的二肽。
  7. 根据权利要求1-4和6中任意一项所述的化合物，其特征在于，C的结构如式(7)所示：
    Cys-Har-C1-Asp-Trp-Pro-C2  式(7)
    其中C1为Gly或Ser，C2为Cys或Cys的-OH被-NH2取代后获得的结构，式(7)中的两个巯基之间形成二硫键。
  8. 根据权利要求7所述的化合物，其特征在于，L的结构如式(8)所示：
    L1-L2-L3-L4-Gly-Asp-L5  式(8)
    其中L1为Gly、Ala、Val或Gly-Gly；L2为Gly或Cys；L3为Gly、Gly-Gly、Gly-Gly-Gly或右旋氨基酸；L4为Asn或Gln；L5选自Phe、Tyr、Phe的苯环被取代后获得的衍生物和Tyr的苯环被取代后获得的衍生物中的一种。
  9. 根据权利要求8所述的化合物，其特征在于，A-L-B选自SEQ ID No.1至SEQ ID No.3所示多肽序列中的一种。
  10. 根据权利要求1-4、6、8和9中任意一项所述的化合物，其特征在于，所述化合物具有如式(9)所 示的结构：
    X-A-L-B-L’-C-Y  式(9)
    其中，X选自氢、一个或两个C1-C6的烷基、一个或两个C2-C10的酰基、苄氧羰基或叔丁氧羰基中的一种；Y选自OH、C1-C6的烷氧基、氨基、一个或两个C1-C4的烷基取代的氨基中的一种。
  11. 根据权利要求10所述的化合物，其特征在于，所述化合物包括SEQ ID No.4至SEQ ID No.7所示的多肽结构中的一种多肽序列。
  12. 根据权利要求11所述的化合物，其特征在于，所述化合物选自SEQ ID No.4至SEQ ID No.7所示的多肽结构中的一种。
  13. 权利要求1-12中任意一项所述的化合物的盐。
  14. 根据权利要求13所述的化合物的盐，其特征在于，所述盐为所述化合物的醋酸盐或所述化合物的三氟乙酸盐。
  15. 权利要求1-12中任意一项所述的化合物的制备方法，其特征在于，该方法包括以下步骤：
    (1)采用固相合成法按照多肽序列从羧基端开始依次接入保护型氨基酸或片段，得到侧链全保护氨基酸多肽-Wang树脂；
    (2)用酸解试剂对侧链全保护氨基酸多肽-Wang树脂进行酸解得到线性多肽粗品；
    (3)对线性多肽粗品进行环化形成二硫键，然后采用高压制备液相进行纯化，得到多肽序列。
  16. 一种药物组合物，其特征在于，所述药物组合物的活性成分包括权利要求1-12中任意一项所述的化合物或者权利要求13和14中任意一项所述的盐。
  17. 根据权利要求16所述的药物组合物，其特征在于，所述药物组合物的剂型为注射剂、片剂、胶囊剂、丸剂、散剂、颗粒剂、悬浮剂或乳剂。
  18. 权利要求1-12所述的化合物或权利要求13-14所述的盐在制备预防和治疗外周动脉血栓、动静脉旁路血栓形成的药物中的应用。
  19. 权利要求1-12所述的化合物或权利要求13-14所述的盐在制备用于治疗急性冠脉综合征、经皮冠状动脉介入治疗或PCI放置冠脉内支架治疗中血栓形成的药物中的应用。
  20. 权利要求1-12所述的化合物或权利要求13-14所述的盐在制备预防和治疗进展性缺血性脑卒中形成的药物中的应用。
  21. 权利要求1-12所述的化合物或权利要求13-14所述的盐在制备预防和治疗急性肺栓塞的药物中的应用。
  22. 权利要求1-12所述的化合物或权利要求13-14所述的盐在制备预防和治疗器官组织移植中血栓形成的药物中的应用。
PCT/CN2015/086173 2015-08-05 2015-08-05 有抗凝血和抗血小板活性的多靶点化合物及制法和用途 WO2017020282A1 (zh)

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JP2018526284A JP6530564B2 (ja) 2015-08-05 2015-08-05 抗凝固・抗血小板活性を有するマルチターゲット化合物及びその製法並びに用途
CN202110662996.2A CN113201049B (zh) 2015-08-05 2015-08-05 有抗凝血和抗血小板活性的多靶点化合物及制法和用途
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EP15900056.1A EP3333177B1 (en) 2015-08-05 2015-08-05 Multi-target compound with anticoagulation and antiplatelet activity, preparation method therefor, and use thereof
CN202110662995.8A CN113773369B (zh) 2015-08-05 2015-08-05 有抗凝血和抗血小板活性的多靶点化合物及制法和用途
ES15900056T ES2886576T3 (es) 2015-08-05 2015-08-05 Compuesto de múltiples dianas con actividad anticoagulante y antiplaquetaria, método de preparación para el mismo y uso del mismo
CN202110661682.0A CN113201048B (zh) 2015-08-05 2015-08-05 有抗凝血和抗血小板活性的多靶点化合物及制法和用途
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CN113773369B (zh) 2022-11-08
CN113201049B (zh) 2022-10-04
US20180305409A1 (en) 2018-10-25
JP2018525442A (ja) 2018-09-06
US11643439B2 (en) 2023-05-09
JP6530564B2 (ja) 2019-06-12
ES2886576T3 (es) 2021-12-20
EP3333177B1 (en) 2021-07-14
CN113201048A (zh) 2021-08-03
EP3333177A4 (en) 2018-12-26
CN108137653B (zh) 2021-07-13
CN108137653A (zh) 2018-06-08
CN113201049A (zh) 2021-08-03
EP3333177A1 (en) 2018-06-13
CN113201048B (zh) 2022-10-04

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