WO2017107262A1 - 基于萜类衍生物的par-1抑制剂及其制备方法和在治疗血栓性疾病中的用途 - Google Patents

基于萜类衍生物的par-1抑制剂及其制备方法和在治疗血栓性疾病中的用途 Download PDF

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WO2017107262A1
WO2017107262A1 PCT/CN2016/070851 CN2016070851W WO2017107262A1 WO 2017107262 A1 WO2017107262 A1 WO 2017107262A1 CN 2016070851 W CN2016070851 W CN 2016070851W WO 2017107262 A1 WO2017107262 A1 WO 2017107262A1
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compound
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pharmaceutically acceptable
acceptable salt
preparation
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娄红祥
孙斌
刘军
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山东大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
    • C07D213/46—Oxygen atoms
    • C07D213/48—Aldehydo radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/55—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/56—Amides

Definitions

  • the invention belongs to the technical field of blood anticoagulation, in particular to a novel PAR-1 inhibitor based on quinone derivatives, a preparation method thereof and a pharmaceutical composition comprising the same and a salt thereof, and the PAR-1 inhibitor And the use of their salts in the manufacture of a medicament for the prevention and/or treatment of a thrombotic disease.
  • anti-platelet drugs for clinical use in the prevention and treatment of thrombotic diseases are mainly divided into three categories.
  • the first category is drugs that affect platelet metabolism enzymes, such as cyclooxygenase inhibitors, thromboxane A2 (TXA2) inhibitors, and phosphoric acid.
  • the second type is adenosine diphosphate (ADP) inhibitors, such as ticlopidine, clopidogrel, and ticagrelor, which inhibit the platelet P2Y 12 receptor. Platelet aggregation; the third type is platelet GP II b /III a receptor antagonists, such as abciximab and eptifibatide, which competitively block fibrinogen and platelet surface GP II b /III a Body binding inhibits platelet aggregation.
  • ADP adenosine diphosphate
  • TXA2 or ADP inhibitors block pathology.
  • the process of thrombosis also affects the normal hemostatic function of the human body, which in turn increases the probability and risk of bleeding in patients.
  • Protease-activated receptor-1 is a member of the G-protein coupled receptor family. Humans have four receptor subtypes of PAR-1, PAR-2, PAR-3 and PAR-4. And only PAR-1 and PAR-4 are distributed on the surface of platelets. PAR-1 plays a more important role in thrombin-mediated platelet activation. Thrombin activates PAR-1 at lower concentrations and further activates platelets, resulting in rapid aggregation of platelets. Inhibition of the PAR-1 receptor blocks the process of thrombin-mediated platelet aggregation and pathological thrombus expansion, but does not affect the normal protective hemostasis of the human body involved in TXA2 and ADP. Therefore, PAR-1 is an ideal antiplatelet drug target.
  • Vorapaxar Sulfate is a PAR-1 inhibitor pioneered by Merck, and was approved by the FDA in May 2014 for use in patients suffering from heart attacks or in blocked leg arteries. Patients and can further reduce the risk of heart attack and stroke. Since Wolapasha blocks platelet aggregation by inhibiting PAR-1, Therefore, the drug does not affect the normal hemostasis process and reduces the risk of accidental bleeding. Although Wolapasha has good anticoagulant activity, it has a complicated structure (having 7 chiral centers), a long synthetic route (the linear synthesis step is 16 steps), and a disadvantage of high production cost.
  • Natural products are important sources for the development of new drugs due to their diverse variety, complex structure, clear chirality and fixed conformation.
  • Diterpenoids andrographolide and sesquiterpenoids Hydroquinone dialdehydes are common important active natural products with the following structural formula:
  • the object of the present invention is to provide an anti-platelet anti-thrombotic disease drug, which has the advantages of clear target, novel, low bleeding risk, high safety, outstanding activity effect, low preparation cost and the like. It can be a promising drug for the treatment of thrombotic diseases.
  • R 1 and R 2 each represent: a hydrogen atom, a halogen atom, a hydroxyl group, a (C 1 -C 4 )alkyl group, a (C 1 -C 4 )alkoxy group or (C 1 - C 4 ) hydroxyalkyl;
  • R 1 and R 2 together form a spiro or heterospiro ring having 3-7 atoms;
  • R 3 and R 4 each represent a hydrogen atom, a hydroxyl group, a (C 1 -C 4 )alkyl group, a (C 1 -C 4 )hydroxyalkyl group or a (C 1 -C 4 )alkoxy group;
  • R 3 represents -C(O)R 6 , -C(O)OR 6 , -CO(O)R 6 , -COSOR 6 , -C(O)NR 6 R 7 ; or R 4 represents -O(O CR 8 , -OSOR 8 , -OSO 2 R 8 , -NHC(O)OR 8 , -NHC(O)R 8 , -NHCONHR 8 , -NHC(O)NR 8 R 9 , -NHSO 2 R 8 ;
  • R 4 represents an oxygen atom and a carbon atom to which it is bonded forms a double bond, ie a ketone carbonyl group
  • R 5 represents a halogen atom, a trifluoromethoxy group or a trimethyl group
  • R 6 , R 7 , R 8 and R 9 are independently selected from a hydrogen atom, a (C 1 -C 6 )alkyl group, an ester group, a carboxylic acid group, a phenyl group and a benzyl group.
  • R 1 and R 2 each represent a hydrogen atom or a (C 1 -C 4 )hydroxyalkyl group, or R 1 and R 2 together form a heterospiro ring having 3-7 atoms;
  • R 3 is (C 1 -C 4 ) hydroxyalkyl, methoxymethyl, formic acid, methyl formate, aldehyde or formamide;
  • R 4 is hydroxy, methoxy or ketocarbonyl;
  • R 5 is a halogen atom or three Fluoromethyl.
  • the R 1 and R 2 together form a heterospiro ring as a three-membered oxygen ring.
  • the pharmaceutically acceptable salt of the above compound is preferably a salt of the above compound with a mineral acid such as hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid; or a salt of an organic acid such as methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid.
  • a mineral acid such as hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid
  • an organic acid such as methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid.
  • (C 1 -C 4) alkoxy denotes a group -OR, wherein R is a (C 1 -C 4) alkyl.
  • (C 1 -C 4 )hydroxyalkyl denotes a (C 1 -C 4 )alkyl group substituted by a hydroxy group.
  • spirocyclic means that R 1 and R 2 are bonded by a covalent bond and together with a spiro atom on a six-membered carbocyclic ring form a cyclic structure.
  • heterospiro means a spiro ring composed of R 1 , R 2 and a spiro atom, and contains one or more hetero atoms such as an oxygen atom, a nitrogen atom or a sulfur atom.
  • the invention further provides methods of preparing compounds of formula (I) which are obtainable as detailed below.
  • the method for producing the above compound comprises reacting a compound represented by the formula (II) with a compound represented by the formula (III) to obtain a compound represented by the formula (I).
  • R 1 , R 2 , R 3 , R 4 and R 5 have the definitions as defined above. Further, R 3 and R 4 may also represent a co-formed 4 to 8 membered carbocyclic or heterocyclic ring.
  • the compound represented by the above formula (II) is prepared by using andrographolide as a starting material, and dehydrating rearrangement reaction with aluminum oxide to obtain a compound represented by the formula (IV), and the compound of the formula (IV)
  • the compound is epoxidized with m-CPBA, acetalized with 2,2-dimethoxypropane, and finally reacted with ozone to obtain a compound of the formula (II).
  • R 1 , R 2 , R 3 and R 4 have the definitions as defined above. Further, R 3 and R 4 may also represent a co-formed 4 to 8 membered carbocyclic or heterocyclic ring.
  • the present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing and/or treating a thrombotic disease.
  • thrombotic diseases include thrombosis, atherosclerosis, restenosis, hypertension, angina pectoris, heart failure, emergency infarction, glomerulonephritis, or peripheral vascular disease.
  • the present invention also provides a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • the pharmaceutical composition comprises one or more pharmaceutically or food acceptable excipients.
  • the excipients used may be solid or liquid.
  • Solid form preparations include powders, tablets, dispersion granules, capsules, pills, and suppositories. Powders and tablets may contain from about 5% to about 95% of the active ingredient. Suitable solid adjuvants can be magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration.
  • the liquid form preparations include solutions, suspensions and emulsions, and examples thereof are aqueous solutions for parenteral injection or water-propylene glycol, or oral solutions in which sweeteners and contrast agents are added. In addition, it can also be used as a small water needle for injection, a freeze-dried powder for injection, a large infusion or a small infusion.
  • the pharmaceutical composition is a solid oral preparation, a liquid oral preparation or an injection.
  • the pharmaceutical composition is a tablet, a dispersible tablet, an enteric coated tablet, a chewable tablet, an orally disintegrating tablet, a capsule, a sugar coating agent, a granule, a dry powder, an oral solution, a small water injection needle for injection, and an injection. Freeze dry powder, large infusion or small infusion.
  • the compounds of formula (I) and their pharmaceutically acceptable salts prepared according to the present invention are useful in the preparation of prophylactic and/or therapeutic thrombosis, atherosclerosis, restenosis, hypertension, An agent for angina pectoris, heart failure, emergency infarction, glomerulonephritis, or peripheral vascular disease.
  • another subject of the present invention is a method of treating a mammal suffering from thrombosis, atherosclerosis, restenosis, hypertension, angina pectoris, heart failure, emergency infarction, glomerulitis or peripheral vascular disease.
  • the method comprises administering a therapeutically effective amount of a compound of formula (I) as described above.
  • terapéuticaally effective amount refers to the amount of therapeutic agent required to treat, ameliorate, a targeted disease or condition or to exhibit a detectable therapeutic effect.
  • the compounds of the invention are effective over a relatively wide dosage range.
  • the dose at which the compound of the formula (I) of the present invention is actually administered can be determined by a doctor according to the relevant circumstances. These conditions include: the subject's physical condition, route of administration, age, weight, individual response to the drug, severity of symptoms, and the like.
  • the compound of formula (I) above, or a pharmaceutically acceptable salt thereof, may also be used in combination with at least one other cardiovascular disease agent during the course of treatment.
  • Other cardiovascular disease drugs covered have an atomic composition or structure that is different from the compound of formula (I).
  • Other cardiovascular disease drugs that can be used in combination with the novel compounds of the invention include those having antithrombotic, antiplatelet aggregation, antiatherosclerosis, anti-restenosis and/or anticoagulant activity, which are useful in therapy and Thrombosis-related diseases including thrombosis, atherosclerosis, restenosis, hypertension, angina pectoris, arrhythmia, heart failure Exhaustion, myocardial infarction, glomerulonephritis, thrombotic stroke, thromboembolic stroke, peripheral vascular disease, other cardiovascular diseases, and other diseases in which thrombin and its receptors have pathological effects.
  • the compound of the invention has the advantages of clear target, novel structure, low bleeding risk, high safety, outstanding activity effect, low preparation cost, and the like, and has a good application prospect in preparing a medicament for treating thrombotic diseases.
  • the starting material for preparing the compound of the present invention is andrographolide or hydrazine dialdehyde.
  • andrographolide is a common active natural product, which is cheap and easy to obtain.
  • Using andrographolide as a raw material through the structural modification and modification of 6 to 7 steps under the premise of ensuring the conformation of its key chiral center, a variety of compounds with novel structures can be prepared and obtained rapidly and on a large scale. Good economy.
  • the andrographolide 500 mg was weighed and placed in a round bottom flask, and pyridine was added thereto to dissolve, and aluminum oxide (200 mg) was added to the solution, followed by heating and stirring. After completion of the reaction, the system was cooled to room temperature, and the alumina was removed by filtration, and the solvent was evaporated under reduced pressure.
  • the compound 3 (400 mg) was placed in a three-necked flask, dichloromethane and pyridine were added, and O 3 was introduced under low temperature. After the reaction was completed, the system was diluted with dichloromethane, washed with water, dried over magnesium sulfate and filtered. The solvent was evaporated under reduced pressure and purified by column chromatography to afford compound 4 as colorless oil.
  • the compound 5 or 6 (0.5 mmol) was placed in a flask, dissolved in tetrahydrofuran, and a solution of n-butyllithium in hexane (2.5 M/L) was added dropwise under low temperature. After the addition, the system was allowed to warm to room temperature and stirring was continued. After adding 0.4 mmol of a solution of the compound 4 in tetrahydrofuran, the mixture was stirred until the end of the reaction. The reaction was quenched with saturated aqueous ammonium chloride, and ethyl acetate was evaporated. A white solid compound 7 or 8 was obtained.
  • the pure compound 7 or 8 (500 mg) was weighed and placed in a round bottom flask, dissolved in methanol, added to Amberlyst-15 (100 mg), stirred, and after completion of the reaction, the solvent was filtered off, and the white product was purified by column chromatography. I-1 or I-2.
  • the compound I-1 (0.5 mmol) was dissolved in dichloromethane, and TEMPO, K 2 CO 3 -NaHCO 3 buffer, TBAI (0.05 mmol) and NCS were added under low temperature, and stirred at room temperature until the end of the reaction. ester, with distilled water, dried over anhydrous Na 2 SO 4, filtered, and the solvent was evaporated under reduced pressure, purified by column chromatography to give white solid I-7.
  • Example 6 Determination of biological activity (PAR-1 inhibitory activity) of a compound
  • the HEK293-G ⁇ 15-PAR1 cell line (HD Biosciences stable cell line) was quickly taken out from the liquid nitrogen tank and shaken continuously in a 37 ° C water bath until it was completely thawed.
  • the cell suspension was quickly added to pre-warmed medium (90% DMEM + 10% FBS + 1 X Pen / Strep), placed in a centrifuge, centrifuged at 1000 rpm for 10 minutes. The centrifuge tube was taken out, the supernatant was discarded, fresh pre-warmed medium was added to the centrifuge tube, the cells were resuspended, the cell suspension was added to the culture dish, and cultured at 37 ° C, 5% CO 2 .
  • pre-warmed medium 90% DMEM + 10% FBS + 1 X Pen / Strep
  • the cells When the cells are over 80 to 90% of the culture dish, gently wash the cells with 0.05% trypsin-EDTA, remove some of the digestive juice, incubate the cells for 2-3 minutes, stop the digestion with the new medium, and gently blow the cells with the pipette tip.
  • the cells are resuspended and, in general, passaged from 1:4 to 1:8 every 2 to 3 days.
  • 1 ⁇ Matrigel (Brand: BD, Cat#: 356230) was added to a clean 384-well cell plate, incubated at 37 ° C for 30 minutes, and then centrifuged at 500 rpm for 30 seconds to remove the coating solution.
  • the cell pellet was collected by digestion, resuspended to 3 ⁇ 10 5 cells/mL with medium, and 50 ⁇ L per well was added to the coated cell plate, followed by incubation at 37 ° C, 5% CO 2 overnight.
  • the 30 mM DMSO stock solution was first diluted to 10 mM with DMSO, and then diluted 4 times from 10 mM for a total of 10 concentrations.
  • Ten DMSO concentration gradients of the compounds were added to the assay buffer at a ratio of 1:20 to prepare a working solution of the compound (5 times the final reaction concentration). After that, the working solution of the compound was transferred to a 384-well compound plate according to the following layout for use.
  • Positive control diluted 40 mM DMSO stock solution of reference compound SCH79797 to 2 mM;
  • Negative control 5% DMSO in assay buffer
  • the 10 mM DMSO stock solution of the agonist haTRAP was diluted to 18 ⁇ M (6 times the final reaction concentration of 3 ⁇ M) with the assay buffer, and then transferred to a 384-well compound plate at least 25 ⁇ l/well for use.
  • the 384-well compound plate (FLIPR) was transferred to a 5 ⁇ l/well agonist to the cell plate according to the FLIPR setup procedure while reading the fluorescence signal for each well in the cell plate.
  • the inhibition rate (%) of the compound in each well on the cell plate was calculated from the fluorescence signal values of the positive control and the negative control on each cell plate.
  • the positive control contained a high concentration of the reference compound (400 ⁇ M of SCH79797) as a 100% inhibition control; the negative control contained no compound, only DMSO (1% DMSO) as the compound solvent, which was a 0% inhibition control.
  • the calculated inhibition rate and the corresponding compound concentration were introduced into the relevant software for mapping, and the IC 50 value of the compound was calculated according to the 4-PL dose effect formula.
  • the IC 50 result of the reference compound is also one of the criteria for testing the quality of each experiment.

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Abstract

一种新的基于萜类衍生物的PAR-1抑制剂、它们的制备方法以及包括它们和它们盐的药物组合物，以及所述PAR-1抑制剂和它们的盐在制备用于预防和/或治疗血栓性疾病的药物中的用途；该化合物具有靶点明确、结构新颖，出血风险小，安全性高，活性效果突出，制备成本低廉等优点，在制备治疗血栓性疾病的药物中具有很好的应用前景。

Description

基于萜类衍生物的PAR-1抑制剂及其制备方法和在治疗血栓性疾病中的用途 技术领域
本发明属于血液抗凝固技术领域，具体涉及一种新的基于萜类衍生物的PAR-1抑制剂、它们的制备方法以及包括它们和它们盐的药物组合物，以及所述PAR-1抑制剂和它们的盐在制备用于预防和/或治疗血栓性疾病的药物中的用途。
背景技术
在我国随着社会老龄化进程的加快，心血管病发病人数快速增长，该类疾病病程长、费用贵、致死致残率高。其中，由于血管栓塞引起的心血管疾病的发病率逐年上升，目前已经成为我国重大的公共卫生问题。
研究表明，血小板过度活化在血栓形成和血栓性疾病的发生和发展过程中起到重要作用。因此，抑制血小板的过度活化，减少血小板的黏附、聚集和释放是防治血栓性疾病的重要手段。目前，临床上用于防治血栓性疾病的抗血小板药物主要分为三大类，第一类是影响血小板代谢酶的药物，如环氧合酶抑制剂、血栓素A2(TXA2)抑制剂和磷酸二酯酶(PDE)抑制剂等；第二类是二磷酸腺苷(ADP)抑制剂，如噻氯匹定、氯吡格雷和替卡格雷等，通过抑制血小板的P2Y12受体，从而抑制血小板的聚集；第三类是血小板GP IIb/IIIa受体拮抗剂，如阿昔单抗和依替巴肽等，通过竞争性阻断纤维蛋白原与血小板表面GP IIb/IIIa受体结合，抑制血小板聚集。上述的传统抗血小板药物大多是通过抑制TXA2或ADP对抗血栓的形成，但由于TXA2和ADP均参与正常的止血过程，并在该过程中起到重要作用，因此TXA2或ADP抑制剂在阻滞病理性血栓形成过程的同时也影响了人体正常的止血功能，进而增加了患者发生出血的概率和风险。
血小板蛋白酶激活受体(Protease-activated receptor-1,PAR)是G蛋白偶联受体家族的一员，人类有PAR-1、PAR-2、PAR-3和PAR-4四种受体亚型，而分布于血小板表面的只有PAR-1和PAR-4。在凝血酶介导的血小板激活过程中，PAR-1起着更重要的作用。凝血酶在较低的浓度下便可激活PAR-1，并进一步激活血小板，导致血小板的快速聚集。抑制PAR-1受体能够阻断由凝血酶介导的血小板聚集和病理性血栓扩大的过程，但同时不会影响由TXA2和ADP参与的人体正常保护性止血过程。因此，PAR-1是一个理想的抗血小板药物靶点。
硫酸沃拉帕沙(Vorapaxar Sulfate)是由默沙东公司首创的PAR-1抑制剂，并已于2014年5月获得FDA批准上市，该药用于遭受心脏病发作的患者或腿部动脉有堵塞的患者，并可进一步降低心脏病发作和中风的风险。由于沃拉帕沙是通过抑制PAR-1阻断血小板的聚集， 因此该药物不会影响正常的止血过程，减少病人意外出血的风险。尽管沃拉帕沙具有良好的抗凝血活性，但是其存在结构复杂(具有7个手性中心)，合成路线较长(其线性合成步骤为16步)和制备成本高昂的缺点。
天然产物由于其具有种类多样、结构复杂、手性明确、构象固定等特点，是研发新药的重要来源。以廉价易得的天然产物作为起始原料或药物前体，对其进行结构改造和修饰，可以迅速构建并大规模制备药物，同时还可有效降低生产成本。二萜类化合物穿心莲内酯和倍半萜类化合物水蓼二醛是常见的重要活性天然产物，其结构式如下：
Figure PCTCN2016070851-appb-000001
其结构中手性中心的构象与沃拉帕沙结构中对应的关键手性中心构象完全一致。因此，以二萜类化合物和倍半萜类化合物为前体，在确保其关键手心中心构象保持不变的前提下，对其进行结构修饰与改造，是快速发现新的PAR-1抑制剂类药物的重要途径。
发明内容
针对上述现有技术的不足，本发明的目的是提供一种抗血小板抗血栓疾病的药物，该药物具有靶点明确、新颖，出血风险小，安全性高，活性效果突出，制备成本低廉等优势，可以作为治疗血栓性疾病的很有前景的药物。
为实现上述目的，本发明采用下述技术方案：
一种式(I)所示的化合物或其药学上可接受的盐，
Figure PCTCN2016070851-appb-000002
其中，
Figure PCTCN2016070851-appb-000003
代表是单键或双键；R1和R2都分别代表：氢原子、卤素原子、羟基、(C1-C4)烷基、(C1-C4)烷氧基或(C1-C4)羟烷基；
或R1和R2共同形成一个双键；
或R1和R2共同形成一个具有3-7个原子的螺环或杂螺环；
R3和R4都分别代表：氢原子、羟基、(C1-C4)烷基、(C1-C4)羟烷基或(C1-C4)烷氧基；
或R3代表-C(O)R6、-C(O)OR6、-CO(O)R6、-COSOR6、-C(O)NR6R7；或R4代表-O(O)CR8、-OSOR8、-OSO2R8、-NHC(O)OR8、-NHC(O)R8、-NHCONHR8、-NHC(O)NR8R9、-NHSO2R8；
或R4代表氧原子，并与其相连的碳原子形成双键，即酮羰基；
R5代表卤素原子、三氟甲氧基或三氟甲基；
R6、R7、R8和R9独立选自氢原子、(C1-C6)烷基、酯基、羧酸基、苯基及苄基。
优选的，所述R1和R2都分别代表氢原子或(C1-C4)羟烷基，或R1和R2共同形成一个具有3-7个原子的杂螺环；R3为(C1-C4)羟烷基、甲氧甲基、甲酸基、甲酸甲酯基、醛基或甲酰胺基；R4为羟基、甲氧基或酮羰基；R5为卤素原子或三氟甲基。
进一步优选的，所述R1和R2共同形成杂螺环为三元氧环。
上述化合物的实例如下：
Figure PCTCN2016070851-appb-000004
上述化合物药学上可接受的盐优选为上述化合物与无机酸，例如盐酸、硫酸、硝酸或氢溴酸形成的盐；或有机酸，例如甲磺酸、甲苯磺酸或三氟乙酸形成的盐。
除非另外指出，术语“(C1-C4)烷氧基”表示基团-O-R，其中R为(C1-C4)烷基。
术语“(C1-C4)羟烷基”表示被羟基取代的(C1-C4)烷基基团。
术语“螺环”表示R1和R2间通过共价键相连，并且与六元碳环上的螺原子共同形成一个环状结构。
术语“杂螺环”表示由R1、R2及螺原子构成的螺环中，包含有一个或多个杂原子，例如：氧原子、氮原子或硫原子等。
本发明另外提供了制备式(I)化合物的方法，它们可以如下面详述地得到。
上述化合物的制备方法包括使式(II)所示的化合物与式(III)所示的化合物反应得到式(I)所示的化合物。
Figure PCTCN2016070851-appb-000005
其中，R1、R2、R3、R4和R5具有如前所述的定义。此外，R3和R4还可代表共同形成的4至8元碳环或杂环。
上述式(II)所示的化合物的制备方法为：以穿心莲内酯为起始原料，与三氧化二铝发生脱水重排反应制得式(IV)所示的化合物，将式(IV)的化合物通过与m-CPBA发生环氧化反应，再与2,2-二甲氧基丙烷发生缩醛化反应，最后与臭氧发生反应，制得式(II)所示的化合物。
Figure PCTCN2016070851-appb-000006
其中，R1、R2、R3和R4具有如前所述的定义。此外，R3和R4还可代表共同形成的4至8元碳环或杂环。
本发明还提供上述化合物或其药学上可接受的盐在制备预防和/或治疗血栓性疾病的药物中的用途。例如在制备PAR-1抑制剂中的用途。这些血栓性疾病包括血栓形成、动脉粥样硬化、再狭窄症、高血压、心绞痛、心脏衰竭、紧急梗死、肾小球炎或周边血管疾病等。
本发明还提供一种药物组合物，该药物组合物包含上述的化合物或其药学上可接受的盐。
优选的，所述药物组合物还包含一种或多种药学上可接受的载体、赋形剂和/或稀释剂。
优选的，所述药物组合物包含一种或多种药学上或食品学上可接受的辅料。所用的辅料可为固态或液态。固态形式的制剂包括粉剂、片剂、分散颗粒、胶囊、药丸及栓剂。粉剂及片剂可包含约5％至约95％的活性成分。适当的固体辅料可以是碳酸镁、硬脂酸镁、滑石粉、糖或者乳糖。片剂、粉剂、药丸及胶囊为适于口服用的固态剂型。液态形式的制剂包括溶液、悬浮液及乳液，其实施例为非经肠注射用水溶液或水-丙二醇溶液，或添加甜味剂及造影剂的口服溶液。此外，还可制成注射用小水针、注射用冻干粉针、大输液或小输液。
优选的，所述药物组合物为固体口服制剂、液体口服制剂或注射剂。
进一步优选的，所述药物组合物为片剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小水针、注射用冻干粉针、大输液或小输液。
我们已经发现，根据本发明制备的式(I)所示的化合物和它们药学上可接受的盐是可以用于制备预防和/或治疗血栓形成、动脉粥样硬化、再狭窄症、高血压、心绞痛、心脏衰竭、紧急梗死、肾小球炎或周边血管疾病的药剂。
因此，本发明的另一个主题是，一种治疗遭受血栓形成、动脉粥样硬化、再狭窄症、高血压、心绞痛、心脏衰竭、紧急梗死、肾小球炎或周边血管疾病的哺乳动物的方法，所述方法包括施用治疗有效量的如上所述的式(I)的化合物。
本文使用的术语“治疗有效量”表示，治疗、改善靶向的疾病或病症或者表现出可检测的治疗效果所需的治疗剂的量。
本发明的化合物在相当宽的剂量范围内是有效的。实际服用本发明式(I)所示的化合物的剂量可由医生根据有关的情况来决定。这些情况包括：被治疗者的身体状态、给药途径、年龄、体重、对药物的个体反应，症状的严重程度等。
在治疗过程中，上述式(I)化合物或其药学上可接受的盐可还以与至少一种其它心血管疾病药物合用。所涵盖的其它心血管疾病药物的原子组成或结构均异于式(I)的化合物。可与本发明新颖化合物组合使用的其它心血管疾病药物包括具有抗血栓形成、抗血小板聚集、抗动脉粥样硬化、抗再狭窄症及/或抗凝血活性的药物，这些药物可用于治疗与血栓形成相关的疾病，其包括血栓形成、动脉粥样硬化、再狭窄症、高血压、心绞痛、心律不整、心脏衰 竭、心肌梗死、肾小球炎、血栓形成性中风、血栓栓塞性中风、周边血管疾病、其他心血管疾病以及凝血酶及其受体在其中其病理作用的其他疾病。
本发明的有益效果：
(1)本发明的化合物具有靶点明确、结构新颖，出血风险小，安全性高，活性效果突出，制备成本低廉等优点，在制备治疗血栓性疾病的药物中具有很好的应用前景。
(2)制备本发明的化合物的起始原料为穿心莲内酯或水蓼二醛。其中，穿心莲内酯为常见的活性天然产物，廉价、易得。以穿心莲内酯为原料，在确保其关键手性中心构象不变的前提下，通过6至7步的结构修饰与改造，可快速、大规模制备并获得多种具有全新结构的化合物，同时具有良好的经济性。
具体实施方式
结合实施例对本发明作进一步的说明，应该说明的是，下述说明仅是为了解释本发明，并不对其内容进行限定。
实施例1：化合物I-1和I-2的制备
(1)步骤1：化合物1的制备
称取穿心莲内酯(500mg)置于圆底烧瓶中，加入吡啶加以溶解，向溶液中加入三氧化二铝(200mg)，加热搅拌。反应结束后，体系降至室温，过滤除去氧化铝，减压蒸去溶剂，柱层析纯化(DCM/MeOH)得白色固体化合物1。
MS m/z 333(M+1)；1H NMR(400MHz,DMSO)δ7.65(s,1H),6.74(dd,J＝15.8,10.1Hz,1H),6.12(d,J＝15.8Hz,1H),5.05(d,J＝4.9Hz,1H),4.89(s,2H),4.73(s,1H),4.42(s,1H),4.14(dd,J＝7.4,2.7Hz,1H),3.84(dd,J＝10.9,2.6Hz,1H),3.31–3.14(m,2H),2.36(d,J＝10.5Hz,2H),1.98(dd,J＝14.1,7.5Hz,1H),1.72(d,J＝13.0Hz,1H),1.65–1.50(m,2H),1.46–1.25(m,2H),1.23–1.11(m,2H),1.09(s,3H),0.76(s,3H).
Figure PCTCN2016070851-appb-000007
(2)步骤2：化合物2的制备
取纯品化合物1(0.3mmol)和K2CO3(0.5mmol)置于圆底烧瓶中，加入二氯甲烷，体系降 温至0℃，缓慢加入m-CPBA，加毕，体系升至室温。反应结束后，加入纯水和二氯甲烷萃取，食盐水洗涤，硫酸镁干燥，过滤，减压蒸去溶剂，柱层析纯化得白色固体化合物2。
MS m/z 349(M+1)；1H NMR(400MHz,Acetone)δ7.48(s,1H),6.49(dd,J＝15.5,9.9Hz,1H),6.15(d,J＝15.6Hz,1H),4.84(s,2H),4.53(s,1H),4.15(d,J＝10.8Hz,1H),3.69(s,1H),3.38(dd,J＝34.6,15.0Hz,3H),2.85(s,1H),2.72(d,J＝3.5Hz,1H),2.51(d,J＝4.5Hz,1H),2.20(d,J＝9.8Hz,1H),1.93–1.52(m,6H),1.43(dd,J＝26.2,13.5Hz,2H),1.23(s,4H),0.99(s,3H).
Figure PCTCN2016070851-appb-000008
(3)步骤3：化合物3的制备
称取化合物2(6.91g)置于圆底烧瓶中，加入二氯甲烷溶解，相继加入2，2-二甲氧基丙烷(25mL)和对甲苯磺酸吡啶嗡(PPTS，800mg),反应结束后，加水，水层用二氯甲烷萃取，合并有机层，硫酸镁干燥，过滤，减压蒸去溶剂，柱层析纯化得白色固体化合物3。
MS m/z 389(M+1)；1H NMR(400MHz,Acetone)δ7.49(s,1H),6.54(dd,J＝15.6,9.9Hz,1H),6.17(d,J＝15.7Hz,1H),4.85(d,J＝1.4Hz,2H),4.06(d,J＝11.6Hz,1H),3.45(dd,J＝9.6,4.3Hz,1H),3.23(d,J＝11.6Hz,1H),2.73(dd,J＝4.7,1.6Hz,1H),2.53(d,J＝4.7Hz,1H),2.26(d,J＝9.9Hz,1H),2.09–1.96(m,2H),1.92–1.76(m,2H),1.71(s,1H),1.57(dd,J＝12.9,4.0Hz,1H),1.53–1.41(m,2H),1.40–1.34(m,4H),1.28(s,4H),1.27–1.16(m,8H).
Figure PCTCN2016070851-appb-000009
(4)步骤4：化合物4的制备
取化合物3(400mg)置于三囗烧瓶中，加入二氯甲烷和吡啶，低温条件下，通入O3，反应结束后，体系中加入二氯甲烷稀释，用水洗，硫酸镁干燥，过滤，减压蒸去溶剂，柱层析快速纯化，得无色油状化合物4，纯化后投下一步反应。
Figure PCTCN2016070851-appb-000010
(5)步骤5：化合物7或8的制备
取化合物5或6(0.5mmol)置于烧瓶中，加入四氢呋喃溶解，低温条件下滴加正丁基锂的己烷溶液(2.5M/L)，加毕，体系升至室温并继续搅拌，后加入化合物4的四氢呋喃溶液0.4mmol，搅拌至反应结束，滴加饱和氯化铵溶液淬灭反应，加入乙酸乙酯，有机层水洗，硫酸镁干燥，过滤，减压蒸去溶剂，柱层析纯化得白色固体化合物7或8。
化合物7:MS m/z 478(M+1)；1H NMR(400MHz,CDCl3),δ:8.75(d,J＝2Hz,1H),7.79(d,J＝8Hz,1H),7.44(m,1H),7.35(d,8Hz,1H),7.24(m,2H),7.08(m,1H),6.54(d,J＝3.2Hz,2H),4.11(d,J＝11.6Hz,1H),3.51(dd,J＝6Hz,4Hz,1H),3.30(d,J＝11.6Hz,1H),2.92(d,J＝4Hz,1H),2.61(d,J＝4.4Hz,1H),2.41(t,J＝4.4Hz,1H),1.96(m,2H),1.84(m,1H),1.76(m,1H),1.62(m,1H),1.52(m,2H),1.44(s,3H),1.38(s,3H),1.29(s,3H),1.25(s,3H),1.21(m,3H).
化合物8:MS m/z 527(M+1)；1H NMR(400MHz,CDCl3)δ8.69(s,1H),7.70(dd,J＝26.0,8.2Hz,3H),7.54(dd,J＝18.4,7.6Hz,2H),7.18(d,J＝8.5Hz,1H),6.47(s,2H),4.02(d,J＝11.5Hz,1H),3.43(dd,J＝9.8,3.8Hz,1H),3.22(d,J＝11.7Hz,1H),2.84(d,J＝3.4Hz,1H),2.53(d,J＝4.0Hz,1H),2.34(d,J＝5.6Hz,1H),2.05–1.84(m,2H),1.78(d,J＝13.9Hz,1H),1.68(dd,J＝13.4,5.2Hz,1H),1.58(dd,J＝13.5,4.9Hz,1H),1.45(t,J＝11.8Hz,2H),1.37(s,3H),1.30(d,J＝8.1Hz,4H),1.20(d,J＝15.1Hz,8H).
Figure PCTCN2016070851-appb-000011
(6)步骤6：化合物I-1或I-2的制备
称取纯品化合物7或8(500mg)置于圆底烧瓶中，加入甲醇溶解，加入Amberlyst-15(100mg)，搅拌，反应结束后，过滤、蒸去溶剂，柱层析纯化的白色产物化合物I-1或I-2。
I-1：MS m/z 438(M+1)；1H NMR(400MHz,CDCl3),δ:8.74(s,1H),7.82(d,J＝8Hz,1H),7.46(q,J＝7.6Hz,1H),7.35(d,J＝7.6Hz,1H),7.25(2H),7.11(t,J＝8.4,1H),6.52(m,1H),6.52(m,1H),4.27(d,J＝10.8Hz,1H),3.53(d,J＝9.2Hz,1H),3.40(m,1H),3.21(m,2H),2.92(d,3.6Hz,1H),2.61(d,4Hz,1H),2.38(d,7.6Hz,1H),1.96(m,2H),1.81(m,1H),1.74(m,1H),1.62(m,1H),1.52(m,2H),1.30(s,3H),1.19(m,2H),1.04(s,3H).
I-2：MS m/z 488(M+1)；1H NMR(400MHz,CDCl3)δ8.76(d,J＝1.9Hz,1H),7.87–7.78(m,2H),7.75(d,J＝7.6Hz,1H),7.63(dt,J＝15.4,7.7Hz,2H),7.30–7.26(m,1H),6.50(dt,J＝15.3,12.2Hz,2H),5.31(s,0H),4.27(d,J＝10.7Hz,1H),3.59–3.47(m,1H),3.45–3.27(m,3H),2.92(d,J＝3.6Hz,1H),2.62(d,J＝4.3Hz,1H),2.39(d,J＝9.1Hz,1H),1.95(d,J＝11.2Hz,2H),1.88–1.79(m,1H),1.78–1.69(m,1H),1.69–1.60(m,1H),1.53(dd,J＝18.2,7.1Hz,2H),1.32(s,3H),1.27–1.23(m,1H),1.21(s,1H),1.05(s,3H).
Figure PCTCN2016070851-appb-000012
实施例2：化合物I-3和I-4的制备
将二茂二氯化钛85mg和锌粉40mg至于四氢呋喃溶液中，将化合物I-1或I-2(60mg)与1,4-环己二烯(80μL)的四氢呋喃溶液滴加至反应体系中。反应结束后，加入磷酸二氢钠饱和水溶液，搅拌至反应结束，随后过滤，加乙酸乙酯，水洗三遍，硫酸钠干燥，过滤，蒸除溶剂，硅胶柱层析分离纯化得到化合物I-3或I-4。
I-3:MS m/z 442(M+1)；1H NMR(400MHz,CDCl3),δ:8.68(s,1H),7.84(dd,J＝6Hz,2Hz,1H),7.45(dd,J＝8Hz,6Hz,1H),7.34(d,J＝7.6Hz,1H),7.29(1H),7.25(1H),7.08(t,J＝8.4Hz,1H),4.10(d,J＝11.2Hz,1H),3.74(m,3H),3.58(d,J＝12Hz,1H),3.48(dd,J＝6.4Hz,4Hz,1H),3.14(m,2H),2.59(m,1H),2.27(m,1H),1.84(m,3H),1.65(m,2H),1.49(m,2H),1.18(s,3H),0.92(s,3H),0.79(m,2H).
I-4：MS m/z 492(M+1)；1H NMR(400MHz,CDCl3),δ:8.72(s,1H),7.88(d,J＝8Hz,1H),7.79(s,1H),7.75(d,J＝7.6Hz,1H),7.67(d,J＝7.6Hz,1H),7.62(d,J＝7.6Hz,1H),7.33(d,J＝8Hz,1H),4.10(m,2H),3.78(d,J＝12Hz,1H),3.59(d,J＝12Hz,1H),3.51(dd,J＝10.8Hz,4.8Hz,1H),3.15(d,J＝12.4Hz,2H),2.92(br,2H),2.64(t,J＝12.8Hz,1H),2.28(m,1H),2.04(s,1H),1.91(m,2H),1.66(m,3H),1.53(m,3H),1.25(s,3H),1.19(s,3H).
Figure PCTCN2016070851-appb-000013
实施例3：化合物I-5和I-6的制备
化合物I-1(0.1mmol)溶于THF(2mL)中，加入NaH(0.2mmol)，搅拌，滴入CH3I(0.25mmol)，随后搅拌至反应结束。加入乙酸乙酯和水萃取，水洗涤，无水Na2SO4干燥，过滤，减压蒸去溶剂，柱层析纯化得I-5和I-6。
I-5：MS m/z 452(M+1)；1H NMR(400MHz,CDCl3),δ:8.75(d,J＝1.6Hz,1H),7.82(d,J＝7.2Hz,1H),7.44(m,1H),7.35(d,J＝8Hz,1H),7.24(m,2H),7.09(td,J＝8.4Hz,2Hz,1H),6.54(m,2H),3.93(d,J＝9.2Hz,1H),3.87(d,J＝7.2Hz,1H),3.33(s,3H),3.28(d,J＝9.2Hz,1H),2.94(d,J＝3.6Hz,1H),2.60(d,J＝4.4Hz,1H),2.39(d,J＝9.2Hz,1H),1.95(s,1H),1.92(t,J＝2Hz,1H),1.78(m,1H),1.70(m,1H),1.65(m,2H),1.56(dd,J＝12.8Hz,4Hz,1H),1.51(m,1H),1.27(s,3H),1.21(m,2H),1.07(s,3H).
I-6：MS m/z 452(M+1)；1H NMR(400MHz,CDCl3),δ:8.75(s,1H),7.83(d,J＝7.2Hz,1H),7.44(dd,J＝14.4Hz,8Hz,1H),7.35(d,J＝7.6Hz,1H),7.27(m,2H),7.11(m,1H),6.54(m,2H),4.17(d,J＝8.8Hz,1H),3.37(s,3H),3.26(m,2H),3.00(dd,J＝12Hz,4.4Hz,1H),2.92(d,J＝3.6Hz,1H),2.61(d,J＝4.4Hz,1H),2.37(d,J＝8.4Hz,1H),1.94(m,2H),1.70(m,2H),1.60(m,2H),1.52(m,1H),1.25(s,3H),1.21(m,2H),1.03(s,3H).
Figure PCTCN2016070851-appb-000014
实施例4：化合物I-7、I-8和I-9的制备
(1)化合物I-7的制备
取化合物I-1(0.5mmol)溶于二氯甲烷中，低温条件下，加入TEMPO、K2CO3-NaHCO3缓冲液、TBAI(0.05mmol)及NCS，室温搅拌至反应结束，加入乙酸乙酯，用蒸馏水，无水Na2SO4干燥，过滤，减压蒸去溶剂，柱层析纯化，得白色固体I-7。
I-7：MS m/z 436(M+1)；1H NMR(400MHz,CDCl3),δ:10.01(s,1H),8.85(d,J＝2Hz,1H),8.07(dd,J＝8Hz,2.4Hz,1H),7.62(m,4H),7.24(td,J＝8Hz,1.6Hz,1H),6.48(m,2H),5.16(br,1H),2.80(d,J＝4.4Hz,1H),2.54(d,J＝4.8Hz,1H),2.44(d,J＝8.8Hz,1H),1.97(m,4H),1.51(d,J＝13.2Hz,1H),1.37(m,4H),1.12(s,3H),0.86(s,3H).
Figure PCTCN2016070851-appb-000015
(2)化合物I-8的制备
将化合物I-7(0.4mmol)溶于异戊烷、叔丁醇和THF的混合溶剂中，冰浴条件下滴加NaClO2-NaH2PO4缓冲液，室温搅拌至反应结束。加乙酸乙酯，用蒸馏水洗涤，柱层析纯化，得白色固体I-8。
I-8：MS m/z 452(M+1)；1H NMR(400MHz,CDCl3),δ:12.41(br,1H),8.84(d,J＝2Hz,1H),8.08(dd,J＝8Hz,2Hz,1H),7.59(m,4H),7.23(td,J＝8.4Hz,1.6Hz,1H),6.48(m,2H),4.40(br,1H),3.12(dd,J＝12Hz,4Hz,1H),2.81(d,J＝4Hz,1H),2.57(d,J＝4.4Hz,1H),2.36(d,J＝7.6Hz,1H),2.07(m,1H),1.94(m,1H),1.84(m,1H),1.56(m,1H),1.46(m,1H),1.39(m,1H),1.31(s, 3H),1.26(m,2H),0.94(s,3H).
Figure PCTCN2016070851-appb-000016
(3)化合物I-9的制备
取化合物I-8(0.5mmol)溶解于DMF中，随后加入K2CO3(1.25mmol)，并慢慢滴加CH3I(2.5mmol)，反应体系搅拌至反应完全。加入乙酸乙酯，用蒸馏水洗涤，无水Na2SO4干燥，柱层析纯化，得化合物I-9。
I-9：MS m/z 466(M+1)；1H NMR(400MHz,CDCl3),δ:8.75(s,1H),7.81(d,J＝4Hz,1H),7.43(q,J＝4.8,1H),7.34(d,J＝5.2Hz,1H),7.24(2H),7.08(t,J＝5.2Hz,1H),6.53(br,2H),3.71(s,3H),3.25(d,J＝8Hz,1H),3.16(td,J＝8Hz,2.4Hz,1H),2.93(s,1H),2.63(d,J＝2.8Hz,1H),2.39(d,J＝5.2Hz,1H),2.10(m,1H),2.04(m,1H),1.95(m,2H),1.81(m,1H),1.71(d,J＝9.2Hz,1H),1.52(d,J＝7.2Hz,1H),1.46(s,3H),1.25(m,2H),0.92(s,3H).
Figure PCTCN2016070851-appb-000017
实施例5：化合物I-10的制备
将化合物I-1(0.05mmol)溶于无水DCM，向反应体系中加入PCC(0.15mmol)，搅拌至反应结束，过滤，滤液中加入乙酸乙酯，蒸馏水洗涤，Na2SO4干燥，减压蒸除溶剂，得白色固体。随后，加入DCM溶解，依次加入TEMPO、K2CO3-NaHCO3缓冲液、TBAI(0.005mmol) 及NCS，搅拌至反应结束。加入乙酸乙酯稀释，用蒸馏水洗涤，Na2SO4干燥，过滤减压蒸去溶剂，柱层析纯化得化合物I-10。
I-10：MS m/z 450(M+1)；1H NMR(400MHz,CDCl3),δ:8.84(d,J＝2Hz,1H),8.08(m,1H),7.61(m,4H),7.25(m,1H),6.50(m,2H),2.94(dd,J＝14.8Hz,6Hz,1H),2.87(d,J＝4Hz,1H),2.62(d,J＝4.4Hz,1H),2.22(m,2H),1.90(m,2H),1.75(m,1H),1.61(d,J＝10.8Hz,1H),1.49(m,2H),1.41(m,2H),1.27(s,3H),1.22(s,3H).
Figure PCTCN2016070851-appb-000018
实施例6：化合物的生物活性(PAR-1抑制活性)测定
1.细胞培养
1.1细胞复苏
将HEK293-Gα15-PAR1细胞株(HD Biosciences稳转细胞株)从液氮罐内迅速取出，37℃水浴中不停摇晃，直到全部融化。迅速将细胞悬液加入预热的培养基(90％DMEM+10％FBS+1X Pen/Strep)中，放入离心机，1000转/分钟，离心10分钟。将离心管取出，弃去上清液，向离心管内加入新鲜预热的培养基，重悬细胞，将细胞悬液加入培养皿，37℃，5％CO2培养。1.2传代
当细胞长满至培养皿80～90％，用0.05％胰酶-EDTA轻轻洗细胞，去除部分消化液后孵育细胞2～3min，用新的培养基终止消化，枪头轻轻吹打细胞并将细胞重悬，通常情况下，每2～3天按1：4至1：8传代。
2.钙离子内流实验
2.1细胞板包被
实验前一天，在干净的384孔细胞板中加入1×Matrigel(Brand：BD，Cat#：356230)，在37℃孵育30分钟，然后500转/分倒置离心30秒去除包被液。
2.2铺板
消化收集细胞沉淀，用培养基重悬至3×105细胞/mL，每孔50μL加入包被好的细胞板，后37℃,5％CO2孵育过夜。
2.3缓冲液配制
实验当天，配制新鲜的实验缓冲液和0.5×Calcium 4(Brand：Molecular Devices，Cat#：R8141)上样缓冲液。
2.4化合物的配制
先将30mM的DMSO储存液用DMSO稀释至10mM，后从10mM开始4倍倍比稀释，共10个浓度。将化合物的10个DMSO浓度梯度按1：20的比例加入到实验缓冲液中，制备成化合物的工作液(终反应浓度的5倍)。后将化合物的工作液按照下图布局转至384孔化合物板中待用。
阳性对照：将参照化合物SCH79797的40mM DMSO储存液稀释至2mM；
阴性对照：实验缓冲液配制的5％DMSO
2.5 PAR-1激动剂haTRAP的配制
用实验缓冲液将激动剂haTRAP的10mM DMSO储存液稀释至18μM(终反应浓度3μM的6倍)，后至少以25μl/孔转入384孔化合物板中待用。
2.6染料孵育
取出孵育过夜的细胞板，300转/分倒置离心30秒去除细胞培养基，每孔加入20μL新鲜配制的0.5×Calcium 4上样缓冲液，后37℃,5％CO2孵育1小时。
2.7加入化合物
按照布局从化合物板中转移5μL/孔的化合物工作液至细胞板中，然后再次置于37℃,5％CO2孵育15分钟。
2.8加激动剂读取荧光信号
按FLIPR设置程序从384孔化合物板(FLIPR)转5μl/孔激动剂到细胞板，同时读取细胞板中每孔的荧光信号。
3.数据分析
根据每块细胞板上的阳性对照和阴性对照的荧光信号值计算该细胞板上每孔中化合物的抑制率(％)。阳性对照含有高浓度的参照化合物(400μM的SCH79797)，为100％抑制对照；阴性对照不含任何化合物，只有作为化合物溶剂的DMSO(1％DMSO)，为0％抑制对照。将计算所得抑制率和相对应的化合物浓度导入相关软件作图，并按照4-PL剂量效应公式计算出该化合物的IC50值。参照化合物的IC50结果亦是检验每次实验质量的标准之一。
部分化合物的活性筛选结果如表1所示。
表1 部分化合物剂量效应结果
  SCH79797 I-1 I-2 I-3 I-4 I-5 I-6 I-7 I-8 I-9 I-10
Slope 0.95 2.87 4.62 2.09 5.00 2.59 1.53 1.02 1.97 0.97 1.23
IC50(μM) 8.71 7.76 7.95 8.91 8.76 7.07 10.96 3.52 31.64 0.66 19.71
活性筛选结果表明：化合物I-1至I-10均具有显著的体外抗PAR-1活性，其IC50值在0.66-19μM之间。其中，化合物I-7和I-9的体外PAR-1抑制活性较阳性对照药SCH79797有明显的提高，其IC50值分别为3.52μM和0.66μM。

Claims (14)

  1. 一种式(I)所示的化合物或其药学上可接受的盐，
    Figure PCTCN2016070851-appb-100001
    其中，
    Figure PCTCN2016070851-appb-100002
    代表是单键或双键；R1和R2都分别代表：氢原子、卤素原子、羟基、(C1-C4)烷基、(C1-C4)烷氧基或(C1-C4)羟烷基；
    或R1和R2共同形成一个双键；
    或R1和R2共同形成一个具有3-7个原子的螺环或杂螺环；
    R3和R4都分别代表：氢原子、羟基、(C1-C4)烷基、(C1-C4)羟烷基或(C1-C4)烷氧基；
    或R3代表-C(O)R6、-C(O)OR6、-CO(O)R6、-COSOR6或-C(O)NR6R7；
    或R4代表-O(O)CR8、-OSOR8、-OSO2R8、-NHC(O)OR8、-NHC(O)R8、-NHCONHR8、-NHC(O)NR8R9或-NHSO2R8；
    或R4代表氧原子，并与其相连的碳原子形成双键，即酮羰基；
    R5代表卤素原子、三氟甲氧基或三氟甲基；
    R6、R7、R8和R9独立选自氢原子、(C1-C6)烷基、酯基、羧酸基、苯基及苄基。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐，其特征在于，所述R1和R2都分别代表氢原子或(C1-C4)羟烷基，或R1和R2共同形成一个具有3-7个原子的杂螺环；R3为(C1-C4)羟烷基、甲氧甲基、甲酸基、甲酸甲酯基、醛基或甲酰胺基；R4为羟基、甲氧基或酮羰基；R5为卤素原子或三氟甲基。
  3. 根据权利要求2所述的化合物或其药学上可接受的盐，其特征在于，所述R1和R2共同形成的杂螺环为三元氧环。
  4. 根据权利要求1至3任一项所述的化合物或其药学上可接受的盐，其特征在于，其是选自下列结构的化合物：
    Figure PCTCN2016070851-appb-100003
  5. 根据权利要求1所述的化合物或其药学上可接受的盐，其特征在于，所述化合物药学上可接受的盐为所述化合物与无机酸或有机酸形成的盐。
  6. 权利要求1-5中的任一项所述的化合物或其药学上可接受的盐在制备PAR-1抑制剂中的用途。
  7. 权利要求1-5中的任一项所述的化合物或其药学上可接受的盐在制备预防和/或治疗血栓性疾病的药物中的用途。
  8. 根据权利要求7所述的用途，其特征在于，所述血栓性疾病包括血栓形成、动脉粥样硬化、再狭窄症、高血压、心绞痛、心脏衰竭、紧急梗死、肾小球炎或周边血管疾病。
  9. 一种权利要求1-5中的任一项所述的化合物或其药学上可接受的盐的制备方法，其特征在于，所述化合物的制备方法包括使式(II)所示的化合物与式(III)所示的化合物反应得到式(I)所示的化合物；
    Figure PCTCN2016070851-appb-100004
    其中，R1、R2、R3、R4和R5如在权利要求1中所定义；或R3和R4代表共同形成的4至8元碳环或杂环。
  10. 根据权利要求9所述的制备方法，其特征在于，式(II)所示的化合物的制备方法为：以穿心莲内酯为起始原料，与三氧化二铝发生脱水重排反应制得式(IV)所示的化合物，将式(IV)的化合物通过与m-CPBA发生环氧化反应，再与2,2-二甲氧基丙烷发生缩醛化反应，最后与臭氧发生反应，制得式(II)所示的化合物；
    Figure PCTCN2016070851-appb-100005
    其中，R1、R2、R3和R4如在权利要求1中所定义；或R3和R4代表共同形成的4至8元碳环或杂环。
  11. 一种药物组合物，其包含权利要求1-5中任一项所述的的化合物或其药学上可接受的盐。
  12. 根据权利要求11所述的药物组合物，其特征在于，所述药物组合物还包括一种或多种药学上可接受的载体、赋形剂和/或稀释剂。
  13. 根据权利要求11或12所述的药物组合物，其特征在于，所述药物组合物为固体口服制剂、液体口服制剂或注射剂。
  14. 根据权利要求13所述的药物组合物，其特征在于，所述药物组合物为片剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小水针、注射用冻干粉针、大输液或小输液。
PCT/CN2016/070851 2015-12-22 2016-01-14 基于萜类衍生物的par-1抑制剂及其制备方法和在治疗血栓性疾病中的用途 WO2017107262A1 (zh)

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CN110627710B (zh) * 2019-09-29 2020-06-05 山东大学 一种新型par-1抑制剂及其制备方法和在预防和/或治疗血栓性疾病中的用途
CN110804012B (zh) * 2019-10-30 2021-06-08 山东大学 一种还原缩硫醛或缩硫酮脱硫的方法
CN112759548B (zh) * 2020-12-31 2021-11-02 山东大学 一种par-1抑制剂及其制备方法
CN115043820B (zh) * 2022-05-27 2024-03-22 山东大学 Par-1抑制剂及其类似物的制备方法和在血栓性疾病防治中的应用

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