TW201738221A - New himbacine analogue and uses thereof in medicines - Google Patents

Abstract

The present invention relates to the fields of organic chemistry and pharmacology. Specifically, the present invention relates to a new himbacine analogue, the compound having a structure represented by formula (I), relates to a stereoisomer, a tautomer, a prodrug, or a pharmaceutically acceptable salt, or a solvate of the compound represented by formula (I) and a pharmaceutical composition comprising the same, and relates to uses thereof.

Description

New Xibazin analogues, pharmaceutical compositions thereof and their use in medicine

The present invention relates to the field of organic chemistry and pharmacy, in particular, the present invention relates to a novel bisulphonin analog having a structure of the formula (Ι) and stereoisomers, tautomers of a compound of the formula (Ι) A construct, prodrug or pharmaceutically acceptable salt or solvate and pharmaceutical composition containing the same, and uses thereof.

Cardiovascular and cerebrovascular events include acute coronary syndrome (ACS), myocardial infarction, cerebral thrombosis, etc., with high morbidity and high mortality. The World Health Organization also pointed out that ischemic cardiovascular and cerebrovascular events are the leading cause of death. At present, more than 3 million people die of cardiovascular and cerebrovascular diseases every year in China, accounting for 40.3% of all deaths. The pathological cause of such diseases is due to the accumulation of thrombus after platelet activation, which leads to ischemia of the body tissues. Therefore, antithrombotic therapy is the main way to prevent cardiovascular and cerebrovascular events.

Platelet adhesion is the activation process of platelets, including the release of a variety of autocrine and paracrine factors, including adenosine diphosphate (ADP), thrombin, adrenaline, thromboxane A2, which not only platelets The initial reaction acts as a signal amplification and maintenance, and causes the platelets in the blood circulation to gradually form a thrombus. The initiation of multiple clotting factors on the surface of platelets promotes the conversion of prothrombin to thrombin, which promotes platelet aggregation and promotes the conversion of fibrinogen to fibrin, which is the main cause of clotting and hemostasis. The response of platelets to thrombin is mediated by platelet surface G protein-coupled receptors, the thrombin receptor. Further studies have found that thrombin receptors can be initiated by proteolytic hydrolysis, so thrombin receptors are also known as Protease Activated Receptors (PARs). There are four PARs subtypes that are widely distributed in tissues, PAR-1, PAR-3 and PAR-4 are activated by thrombin, and PAR-2 is activated by trypsin or tryptase. Human platelets express only two receptors, PAR-1 and PAR-4, of which PAR-1 plays a major role in thrombin-mediated platelet initiation. Murine platelets express both PAR-1 and PAR-3 receptors.

Oral antithrombotic drugs that are currently on the market act by inhibiting the platelet activation pathway. The cyclooxygenase inhibitor aspirin inhibits the production of thromboxane A2. Thiophene pyridines (such as clopidogrel) can irreversibly bind to the ADP receptor P2Y12 on the surface of platelets, thereby inhibiting platelet activation of ADP. The platelet activation pathway induced by ADP and thromboxane A2 is a critical step for conventional pathological thrombosis and hemostasis. Therefore, the combination of cyclooxygenase inhibitors and thienopyridines inevitably leads to antithrombotic effects. The risk of bleeding complications increases. Therefore, although antiplatelet therapy has achieved good results in clinical practice, the risk of bleeding is still worthy of attention.

The PAR-1 mediated platelet activation pathway is primarily responsible for pathological thrombosis. PAR-1 receptor inhibitors block thrombin-mediated platelet initiation without affecting thrombin-mediated fibrinogen cleavage, while PAR-1 receptor inhibitors also do not affect platelet adhesion, initiation or aggregation pathways Related factors such as collagen, vWF, ADP and procoagulant (see Coughlin SR.; J Thromb Haemost., 2005, 3: 1800-1814), so PAR-1 receptor inhibitors have both antithrombotic effects and possible It does not increase the risk of bleeding and is an ideal antiplatelet drug. In addition, thrombin receptor antagonists can be combined with aspirin, clopidogrel, etc. to increase antithrombotic effects. Thrombin receptor antagonists are also expected to be developed as new drugs against arteriosclerosis and cancer.

Since the discovery of thrombin receptors, many pharmaceutical companies have been working on new drug development studies targeting thrombin receptors. A series of patent applications for thrombin receptor antagonists have been disclosed, such as WO03089428, which discloses a class of bisulphonin derivatives; and WO2002085855 discloses a 2-iminopyrrolidine derivative. In the phase III clinical trial, the thrombin receptor antagonist SCH530348 developed by Merck showed that SCH530348 significantly reduced the incidence of cardiovascular diseases such as myocardial infarction or cerebral thrombosis, but also known that bleeding adverse reactions, especially stroke, In patients with transient cerebral ischemia and cerebral hemorrhage, the risk of intracranial hemorrhage increases, which greatly reduces the current population of thrombin receptor antagonists. The development of new thrombin receptor antagonists with fewer bleeding side effects and better efficacy remains a major challenge.

The present invention is designed to have a compound represented by the formula (I), and the compound of the present invention has a large structural difference from the compound specifically disclosed in the prior art, and exhibits an excellent effect and effect.

In order to overcome the deficiencies of the prior art, it is an object of the present invention to provide novel xibacine analogs having the formula (Ι) structure, as well as their stereoisomers, tautomers, or pharmaceutically acceptable Accepted salts or solvates, or metabolites and metabolic precursors or prodrugs,

A compound having the formula (Ι) or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof:

among them:

R ^{1 is} selected from heterocyclic, aryl or heteroaryl, wherein said heterocyclic, aryl or heteroaryl may be further optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano and nitro , amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)OR ³ , -OC(O)R ³ , -C Substituted by a substituent of (O)R ³ , —NHC(O)R ³ , —C(O)NR ³ R ⁴ wherein the alkoxy group, alkyl group, cycloalkyl group, heterocyclic group, aryl group is substituted Or a heteroaryl group may be further optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, hetero Substituted by a substituent of an aryl group;

R ^{2 is} selected from -C(O)OR ³ , -CN, -C(O)NR ³ R ⁴ , heterocyclyl, aryl or heteroaryl, wherein said heterocyclyl, aryl or heteroaryl Further optionally, one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl, hetero Substituted with a substituent of aryl, -C(O)OR ³ , -OC(O)R ³ , -C(O)R ³ , -NHC(O)R ³ , -C(O)NR ³ R ⁴ ; The alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group described therein may be further optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, Substituted with a substituent of -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl or heteroaryl;

X is selected from CH or N;

n is selected from 0, 1, 2 or 3, and when n = 0, X = N, R ^{2 is} selected from heteroaryl, wherein said heteroaryl group may be further optionally selected from one or more selected from halogen , hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)OR ³ , -OC Substituted by a substituent of (O) R ³ , -C(O)R ³ , -NHC(O)R ³ , -C(O)NR ³ R ⁴ ; wherein the alkoxy group, alkyl group, naphthenic group Or a heterocyclic group, an aryl group or a heteroaryl group may be further optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, Substituted by a substituent of a cycloalkyl, aryl or heteroaryl group;

R ³ and R ⁴ are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the heterocyclic group, aryl group or heteroaryl group may be further optionally Substituted by one or more substituents selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, alkoxy, alkyl or cycloalkyl.

Preferably, a compound of the formula (II) or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof:

among them:

R ^{1 is} selected from a heterocyclic group, an aryl group or a heteroaryl group, and the heterocyclic group, aryl group or heteroaryl group is optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -C(O)OR ³ , -OC(O)R ³ , -C(O)R ^{3, -NHC (O) R 3} , -C (O) NR 3 R 4 substituents of; the alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl any of Substituted by one or more substituents selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, heteroaryl;

In the structure of the formula (II-1), R ^{2 is} selected from -C(O)OR ³ , -CN, -C(O)NR ³ R ⁴ , heterocyclic group, aryl or heteroaryl group, said heterocyclic ring The aryl, aryl or heteroaryl group is optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl , aryl, heteroaryl, -C(O)OR ³ , -OC(O)R ³ , -C(O)R ³ , -NHC(O)R ³ , -C(O)NR ³ R ⁴ Substituted with a substituent; the alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine Substituted with a substituent of -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl or heteroaryl;

In the structure of formula (II-2), R ^{2 is} selected from heteroaryl; the heteroaryl is optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)OR ³ , -OC(O)R ³ , -C(O)R ³ , -NHC( Substituted with a substituent of R ³ , —C(O)NR ³ R ⁴ ; the alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group optionally being one or more Substituted by a substituent selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, heteroaryl;

R ³ and R ⁴ are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, and the heterocyclic group, aryl group or heteroaryl group is optionally one or more Substituted by a substituent selected from a halogen, a hydroxyl group, a cyano group, a nitro group, an amine group, an alkoxy group, an alkyl group or a cycloalkyl group.

Most preferably, typical compounds of the invention include, but are not limited to:

The present invention relates to a compound of any one of the formula (I), or a stereoisomer, tautomer or prodrug thereof, in a pharmaceutically acceptable salt or solvate, wherein the pharmaceutically acceptable salt is the compound and the inorganic Conventional non-toxic salts formed by acid/organic acids or inorganic bases/organic bases, which may be mono-, di-, tri- or poly-salts, as determined by the salt-forming functional groups contained in the compounds. Wherein if the compound of the formula (I) contains a basic functional group, it can be combined with sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, tartaric acid, fumaric acid, maleic acid, citric acid, acetic acid, formic acid, methanesulfonic acid, p-toluene Sulfonic acid, oxalic acid or succinic acid form acid addition salts which can be synthesized according to conventional chemical methods from the reaction of a compound of the invention comprising a basic functional group with a corresponding inorganic acid/organic acid; if the compound of formula (I) contains an acidic functional group Forming a stable alkali metal salt, an alkaline earth metal salt or an optionally substituted ammonium salt with an alkaline agent such as a hydroxide, a carbonate, a hydrogencarbonate, an alkoxide and an ammonia or an organic a base such as trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, tromethamine or other basic amino acids such as lysine, ornithine or arginine; these salts may be conventional Chemical methods are synthesized from the reaction of a compound of the invention comprising an acidic functional group with a corresponding inorganic base/organic base.

The invention further relates to pharmaceutically acceptable solvates of the compounds, including conventional solvates, such as solvates formed by the presence of a solvent in the preparation of the compounds of the invention, solvates formed from the presence of water or ethanol. It can be used as a non-limiting example.

Another aspect of the invention relates to an isomer of any one of the compounds of formula (I), wherein one or more carbon-carbon double bonds are present in the compound of formula (I), and cis-trans isomers may be present Unless otherwise specified, the present invention encompasses all possible cis-trans isomers or mixtures of different ratios of isomers; while one or more chiral centers are present in the compounds of formula (I), unless otherwise stated. The present invention encompasses all racemic, racemic mixtures, enantiomers, diastereomers and mixtures of diastereomers which are theoretically possible. The chemical structural formulas and chemical names referred to in the present invention include all isomers which are theoretically possible in the compounds.

If the compound of formula (I) is present as a mixture of diastereomers or enantiomers or as a mixture thereof in a selected synthesis, the compound of formula (I) can be isolated as follows Optically pure stereoisomers: chromatographic separation on a carrier material, or if the racemic compound is capable of forming a salt, it can also be diastereomeric with an optically active base or optically active acid as an auxiliary. The body salt is subjected to fractional crystallization. Examples of suitable chiral stationary phases for diastereomeric thin layer chromatography or column chromatography are modified tannin supports (becoming a Pirkle phase) and high molecular weight hydrocarbons such as triethylsulfonyl cellulose. In order to isolate a racemic group containing an acidic group in the compound of the formula (I), and an optically active base such as (-)-nicotine, (+)- and (-)-phenylethylamine, quinine base , L-lysine, L-arginine and D-arginine form diastereomeric salts with different solubility, the less soluble components are separated in solid form, pure enantiomers The diastereomer salt obtained by the above method is obtained. The racemate containing a basic group such as an amino group in the compound of the formula (I) can be used as described above with an optically active acid such as (+)-camphor-10-sulfonic acid, D- and L-tartaric acid, D- and L-lactic acid, D- and L-mandelic acid are converted to the pure enantiomers.

Another aspect of the invention relates to an isotopic substitution of a compound of formula (I), or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, wherein any one of the compounds may be present At least one hydrogen atom is replaced by a deuterium atom or at least one carbon or fluorine atom is replaced by a corresponding isotope.

Another aspect of the invention relates to a pharmaceutical composition comprising a compound of any one of formula (I), or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable drug Carrier used.

Another aspect of the invention relates to any one of the compounds of formula (I), or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition for the preparation of blood coagulation Use in drugs for enzyme receptor antagonists.

Another aspect of the invention relates to any one of the compounds of formula (I), or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition for the preparation of blood coagulation Use in a medicament for an enzyme receptor antagonist, wherein the thrombin receptor antagonist is a PAR1 receptor antagonist.

Another aspect of the invention relates to any one of the compounds of formula (I), or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition for the preparation of platelets Use in drugs for agglutination inhibitors.

Another aspect of the invention relates to any one of the compounds of formula (I), or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition for the preparation of a therapeutic And/or use in a medicament for preventing a thrombin receptor-related disease, wherein the thrombin receptor-related disease is selected from the group consisting of arterial and venous thrombosis, acute coronary syndrome, restenosis, stable angina, heart rate Disorders, myocardial infarction, hypertension, heart failure, stroke, inflammatory disease, pulmonary embolism and other lung diseases, gastrointestinal diseases, rheumatism, asthma, chronic liver fibrosis, tumors and skin diseases.

Another aspect of the invention relates to any one of the compounds of formula (I), or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition and another A product of a cardiovascular drug, which is used as a combination product at the same time or separately for the treatment of cardiovascular diseases, wherein the other type of cardiovascular drug is selected from the group consisting of aspirin, clopidogrel, ticlopidine, abciximab, Antiplatelet aggregation drugs such as tirofiban or eptifibatide.

Detailed description of the invention

Terms used in the specification and patent claims have the following meanings unless stated to the contrary.

"Halogen" means fluoro, chloro, bromo, iodo.

"Alkyl" means a saturated aliphatic hydrocarbon group comprising straight and branched chain groups of 1 to 20 carbon atoms. Preferred are alkyl groups having 1 to 12 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, N-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3- Methyl butyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl Base, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2, 4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3- Dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2 -ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl 3-ethylpentyl, n-decyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3 , 3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferably, it is a lower alkyl group having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl Base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Base, 2,3-dimethylbutyl and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from the group consisting of halogen, hydroxy, cyanide. Base, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, heteroaryl.

"Cycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably a cycloalkyl ring comprising 3 to 10 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptene Alkenyl, cyclooctyl and the like. Polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups.

"Spirocycloalkyl" means a polycyclic group of 5 to 20 members which shares a carbon atom (referred to as a spiro atom) between the monocyclic rings. These may contain one or more double bonds, but none of the rings are fully conjugated. π electronic system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan. The spirocycloalkyl group is classified into a monospirocycloalkyl group, a bispirocycloalkyl group or a polyspirocycloalkyl group, preferably a monospirocycloalkyl group and a bispirocycloalkyl group, depending on the number of common spiro atoms between the ring and the ring. . More preferably 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan monospirocycloalkyl.

"Bridge cycloalkyl" means 5 to 20 members, any two rings sharing two carbon-free all-carbon polycyclic groups, which may contain one or more double bonds, but none of the rings have a total The π-electron system of the yoke. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl group, preferably a bicyclic ring, a tricyclic ring or a tetracyclic ring, more preferably a bicyclic ring or a tricyclic ring.

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring wherein the ring to which the parent structure is attached is a cycloalkyl group, non-limiting examples include indanyl, tetrahydrogen Naphthyl, benzocycloheptyl and the like. The cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, and NR ³ R ^4, alkoxy, alkyl, cycloalkyl, aryl, heteroaryl.

"Heterocyclyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising from 3 to 20 ring atoms wherein one or more of the ring atoms are selected from N, O or S(O) _m ( Wherein m is an integer from 0 to 2) and the remaining ring atoms are C. It preferably comprises from 3 to 12 ring atoms, wherein from 1 to 4 are heteroatoms, more preferably the cycloalkyl ring contains from 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, acridinyl, pyridazinyl, morpholinyl, thiomorpholinyl, oxazinyl, and the like; polycyclic heterocyclic groups include spiro, thick Heterocyclic groups of the ring and bridged ring.

"Spiroheterocyclyl" means a polycyclic heterocyclic group of 5 to 20 members in which one atom (referred to as a spiro atom) is shared between the monocyclic rings, wherein one or more ring atoms are selected from N, O or S(O) _m (where m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are C. These may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan. The spiroheterocyclyl group is classified into a monospiroheterocyclyl group, a dispiroheterocyclic group or a polyspiroheterocyclic group, preferably a monospiroheterocyclic group and a dispiroheterocyclic group, depending on the number of common spiro atoms between the ring and the ring. . More preferably 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan monospirocycloalkyl.

"Fused heterocyclyl" refers to 5 to 20 members, each ring in the system sharing an adjacent pair of atomic polycyclic heterocyclic groups with other rings in the system, and one or more rings may contain one or more a bond, but none of the rings have a fully conjugated π-electron system in which one or more ring atoms are selected from heteroatoms of N, O or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are C. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan. It may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocycloalkyl group according to the number of constituent rings, preferably a bicyclic or tricyclic ring, more preferably a 5 member/5 member or a 5 member/6 member bicyclic fused heterocyclic ring. base.

"Bridge heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 14 members, any two rings sharing two atoms which are not directly bonded, these may contain one or more double bonds, but none of the rings have a complete conjugation A π-electron system in which one or more ring atoms are selected from heteroatoms of N, O or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are C. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic group, preferably a bicyclic ring, a tricyclic ring or a tetracyclic ring, more preferably a bicyclic ring or a tricyclic ring.

The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heterocyclic group; the heterocyclic group may be optionally substituted or unsubstituted When substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, ring Alkyl, aryl, heteroaryl.

"Aryl" means a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (ie, a ring that shares a pair of adjacent carbon atoms), a polycyclic ring having a conjugated π-electron system (ie, having adjacent pairs) The ring group of a carbon atom is preferably 6 to 10 members such as a phenyl group and a naphthyl group. The aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is an aryl ring; the aryl group may be substituted or unsubstituted, when When substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, Aryl, heteroaryl.

"Heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms include oxygen, sulfur and nitrogen. It is preferably 6 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazole. Base. The heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heteroaryl ring; the heteroaryl may be optionally substituted or unsubstituted When substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, Cycloalkyl, aryl, heteroaryl.

"Alkoxy" means -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like. The alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, heteroaryl.

"Optional" or "optionally" means that the subsequently described event or environment may, but need not, occur, including where the event or environment occurs or does not occur. For example, "heterocyclic group optionally substituted by an alkyl group" means that an alkyl group may be, but not necessarily, present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group. .

"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

The invention further encompasses pharmaceutical compositions comprising a compound of the invention, or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof.

The pharmaceutical composition of the present invention may be any pharmaceutical form that can be taken: such as tablets, sugar-coated tablets, film-coated tablets, enteric coated tablets, capsules, hard capsules, soft capsules, oral liquids, mouths. Containing agents, granules, granules, pills, powders, ointments, elixirs, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops or patches.

The pharmaceutical composition of the present invention is preferably in the form of a unit dose of a pharmaceutical preparation.

In the pharmaceutical composition of the present invention, a unit dose of the medicament may contain 0.1 to 1000 mg of the pharmaceutically active substance of the present invention, and the balance is a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be from 0.01 to 99.99% by weight based on the total weight of the formulation.

The composition of the present invention is used in a dosage according to the condition of the patient, such as 1-3 times a day. One 1-10 tablets, etc.

Preferably, the composition of the present invention is an oral preparation or an injection.

Wherein the oral preparation is selected from the group consisting of a capsule, a tablet, a dropping pill, a granule, a concentrated pill, an oral liquid, and a mixture.

Wherein the injection is selected from the group consisting of an injection solution, a lyophilized powder injection, and an aqueous injection.

The pharmaceutical composition of the present invention, which is orally administered, may contain a usual excipient such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent or a wetting agent. If necessary, the tablets can be coated.

Suitable fillers include cellulose, mannitol, lactose or other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone or a starch derivative, preferably sodium starch glycolate. A suitable lubricant is magnesium stearate. A suitable wetting agent is sodium lauryl sulfate.

The pharmaceutical composition of the present invention can be prepared into a solid oral composition by a usual method such as mixing, filling, tableting or the like. Repeated mixing allows the active material to be distributed throughout the composition using a large amount of filler.

The oral liquid preparation may be in the form of an aqueous or oily suspension, solution, emulsion, syrup or elixir, or it may be a dry product which may be formulated with water or other suitable carrier before use. Such liquid preparations may contain conventional additives such as suspending agents such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats. Emulsifiers such as lecithin, sorbitan monooleate or gum arabic; non-aqueous vehicles (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters such as glycerides, propylene glycol or ethanol; preservatives For example, p-hydroxybenzyl ester, propyl paraben or sorbic acid, and if desired, may contain conventional flavoring or coloring agents.

For injection, the liquid unit dosage form prepared contains the active substance of the invention and a sterile vehicle. This compound can be suspended or dissolved depending on the carrier and concentration. The solution is usually prepared by dissolving the active substance in a carrier, sterilizing it by filtration before filling it into a suitable vial or ampoule, and then sealing. Excipients such as a local anesthetic, preservative and buffer may also be dissolved in such a carrier. To increase its stability, the composition can be frozen after filling the vial and the water removed under vacuum.

The pharmaceutical composition of the present invention may optionally be added to a suitable pharmaceutically acceptable carrier when prepared as a medicament, the pharmaceutically acceptable carrier being selected from one or more of the following: mannitol, sorbitol, sodium metabisulfite, Sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid , acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, anthraquinone derivatives, fiber And its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin, earth temperature 80, agar, calcium carbonate, calcium hydrogencarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, Phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, and the like.

The pharmaceutical dosage form of the present invention is not completely limited thereto, and it can be prepared into more dosage forms such as dropping pills, sustained release preparations and the like which can be administered in any form.

The invention is further illustrated by the following specific examples, in which the synthesis of the representative compounds and the related structural identification data are given in the following examples, which are intended to illustrate the invention and not to limit the invention, according to the essence of the invention. Simple modifications made by the invention are within the scope of the claimed invention.

Example 1:

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridyl]-vinyl]dodecyl hydrogen -1-methyl-3-oxonaphtho[2,3-c]furan-6-yl]aminoacetic acid (Ι-1)

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridinyl]ethenyl]dodecahydro-1-methyl-3 -oxonaphtho[2,3-c]furan-6-yl]amino acetic acid(Ι-1)

first step:

Compound 1a (30 g, 0.275 mol) and pyridine (32 g) were added to 300 mL of DCM, and the temperature was lowered to 0 ° C under nitrogen atmosphere; trifluoromethanesulfonic acid anhydride (93 g, 0.33 mol) was added dropwise, and the reaction system was allowed to rise to room temperature; The reaction system was poured into 400 mL of water, extracted with 150 mL of dichloromethane, and the organic phase was washed once with 2 N of dilute hydrochloric acid (100 mL), washed once with saturated brine, and dried and evaporated to give a brown liquid compound 1b (62 g, yield 93 %).

The second step:

Compound 1b (62g, 0.257mol), 1c (43.2g, 0.308mol), potassium carbonate (106g, 0.77mol), Pd ( PPh 3) 4 were added to 400 mL of toluene was purged with nitrogen three times, the temperature of the system Raise to 90 ° C, reflux reaction for 6 h; after the system is cooled to room temperature, pour into 400 mL of water, the system is filtered through diatomaceous earth, the filtrate is washed once with water, once with saturated brine, dried and concentrated to give 145 g of brown liquid; Purification by column chromatography (PE: EA = 20:1).

third step:

Under a nitrogen atmosphere, n-butyl lithium was added dropwise to a solution of diisopropylamine THF (280 mL) at -40 °C, and reacted at -30 °C for 1.5 h; 1d (48 g, 0.256 mol) was dissolved in 500 mL of THF, nitrogen. Under protection, the temperature was lowered to -60 ° C, the above LDA was added dropwise to the system, and the reaction was kept at -60 ° C for 1 h; 1e (53 g, 0.308 mol) was added dropwise to the system, and the reaction was carried out at -50 ° C for 1.5 h; Pour 1 L of saturated ammonium chloride, and the aqueous phase was extracted with ethyl acetate (400 mL×2). , reddish brown liquid compound 1f (38 g, yield 79.7%).

the fourth step:

Under N ₂ protection, 0 ^o C, THF of LHMDS was added dropwise to a solution of 1f (600 mg, 1.8 mmol) in THF (6 mL), added, and kept for 30 min; Ti(i-OPr) ₄ THF ( 1 mL) was added dropwise to the system for 10 min; 1 g (see Bioorganic & Medicinal Chemistry Letters, 20, 2010, 6676 for synthesis) (200 mg, 0.61 mmol) in THF (1.5 mL) was added dropwise to the system, then The reaction was returned to room temperature for 3 h; 20 mL of an aqueous solution of saturated sodium potassium tartrate was added to the system, extracted with ethyl acetate, and dried and dried. After purification by column chromatography, a pale white solid was obtained for 1 h (130 mg, yield 22%).

the fifth step:

1 h (0.8 g, 1.6 mmol) was dissolved in concentrated hydrochloric acid at room temperature, and the reaction was carried out at 120 ° C for 3 hours; the compound 1i was obtained by spin-drying and directly transferred to the next step.

The sixth step:

The reaction was carried out with N ₂ at room temperature, and the mixture was stirred for 15 min at room temperature. The aqueous solution of glyoxylic acid (65 mg, 0.43 mmol) was added to the system, and the reaction was carried out for 30 min at room temperature; NaBH ₃ CN (28 mg, 0.43 mmol) was added to the system, and reacted at room temperature for 1 h; a small amount of water-methanol was added to the system for quenching, and the mixture was concentrated by spin-drying, and then purified by thin layer chromatography (MeCN: H ₂ O = 5: 1) The product was concentrated to give a pale yellow solid compound I-1 (50 mg, yield 24%).

1H NMR (DMSO, 400 MHz): d 8.65 (1H, s), 7.95 (1H, d, J = 6.0 Hz), 7.48 (1H, d, J = 8.0 Hz), 7.40 (2H, m), 7.33 ( 1H, d, J = 9.6 Hz), 7.05 (1H, s), 6.55 (2H, d, J = 3.6 Hz), 4.74 (1H, m), 3.46 (2H, m), 3.21 (1H, m), 2.65 (1H, m), 2.35 (2H, t, J = 1.6 Hz), 2.03 (2H, m), 1.85 (2H, m), 1.30-1.18 (12H, m), 0.80 (2H, m), 0.77 (2H, m);

ESI-MS: 479.2 [M+H]+

Example 2:

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridyl]-vinyl] dodehydrogen -1-methyl-3-oxonaphtho[2,3-c]furan-6-yl]aminoacetonitrile (Ι-2)

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridinyl]ethenyl]dodecahydro-1-methyl-3 -oxonaphtho[2,3-c]furan-6-yl]amino acetonitrile(Ι-2)

1i (0.39g, 0.92mmol), bromoacetonitrile (0.14g, 1.21mmol), DIPEA (0.36g, 2.78mmol) were added to acetonitrile under nitrogen protection for 5h at 40 ° C; 100 mL of NaHCO ₃ solution, extracted with ethyl acetate, EtOAc (EtOAc) ).

1H NMR (DMSO, 400 MHz): d 8.78 (1H, s), 7.82 (1H, d, J = 6.4 Hz), 7.47 (1H, m), 7.37 (1H, m), 7.28 (4H, m), 7.10 (1H, m), 6.61-6.53 (2H, m), 4.75 (1H, m), 3.64 (2H, m), 2.73 (2H, m), 2.39 (2H, m), 2.01-1.91 (4H, m), 1.61-0.89 (12H, m);

ESI-MS: 460.2 [M+H]+

Example 3: N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridyl]-vinyl ] dodehydro-1-methyl-3-oxonaphtho[2,3-c]furan-6-yl]-N-(4-methylpyrimidinyl-2)-ammonium (Ι-3)

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridinyl]ethenyl]dodecahydro-1-methyl-3 -oxonaphtho[2,3-c]furan-6-yl]- N-(4-methylpyrimidin-2-yl) amino(Ι-3)

The 1i (150mg, 0.36mmol), 3a (92mg, 0.71mmol), DIPEA (1mL) in 5mL DMSO was added to, under N _2, the reaction was heated to 150 ^o C 10H; reaction was cooled to room temperature, water was added 20mL The solid was precipitated, filtered, and the filter cake was washed with water to give white crystals of y-3 (40 mg, yield 21.9%).

1H NMR (DMSO, 400 MHz): d 8.70 (1H, s), 8.04 (1H, d, J = 4.0 Hz), 7.74 (1H, d, J = 11.2 Hz), 7.37 (1H, m), 7.34 ( 1H, m), 7.28 (1H, m), 7.02 (1H, m), 6.55 (2H, m), 6.31 (1H, d, J = 4.8 Hz), 4.83 (1H, d, J = 7.6 Hz), 4.68 (1H, m), 3.82 (1H, m), 2.67 (1H, m), 2.53 (1H, m), 2.31 (2H, m), 2.22 (3H, s), 2.02 (2H, m), 1.88 (2H, m), 1.37 (3H, s), 1.24-0.80 (8H, m);

ESI-MS: 513.35 [M+H]+

Example 4: N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridyl]-vinyl ] dodehydro-1-methyl-3-oxonaphtho[2,3-c]furan-6-yl]-N-(benzoxazolyl-2)-ammonium (Ι-4)

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2-pyridinyl]ethenyl]dodecahydro-1-methyl-3 -oxonaphtho[2,3-c]furan-6-yl]- N-(benzo[d]oxazol-2-yl) amino(Ι-4)

At room temperature 1i (110mg, 0.26mmol), 4a (80mg, 0.52mmol), potassium carbonate (69mg, 0.52mmol) in 10mL DMF was added to, under N _2, the reaction was heated to 100 ^o C 10H; reaction system After cooling to room temperature, 20 mL of water and ethyl acetate (20 mL × 2) were added, and the organic phase was combined, dried and concentrated, and purified by high-pressure preparative chromatography to afford white solid Ι-4 (29 mg, yield: 20.7%).

1H NMR (DMSO, 400 MHz): d 8.82 (1H, s), 7.87 (1H, d, J = 2.0 Hz), 7.48 (1H, s), 7.47 (2H, m), 7.46-7.06 (6H, m ), 6.63 (2H, m), 5.43 (1H, d, J = 12.6 Hz), 4.80 (1H, d, J = 12.6 Hz), 3.85 (1H, m), 2.76 (1H, m), 2.44 (2H , d, J = 8.0 Hz), 2.28 (2H, m), 2.02 (3H, m), 1.48 (3H, s), 1.36-1.01 (7H, m);

ESI-MS: 538.60 [M+H]+

Example 5: N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-(3'-fluoro-1,1'-biphenyl)-4- Vinyl] ethyl dodecyl-1-methyl-3-oxonaphtho[2,3-c]furan-6-yl]carbamate (Ι-5)

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[3'-fluoro-(1,1'-biphenyl)-4-yl]ethenyl]dodecahydro -1-methyl-3-oxonaphtho[2,3-c]furan-6-yl] carbamic acid ethyl ester (Ι-5)

first step:

At 0 ^o C, the NaH (139mg, 2.7mmol) was added into 5a (1.78g, 2.7mmol) in 10 mL THF solution, the reaction was continued 1 h; dropwise 1g (450mg, 1.38mmol) in THF (10 mL) solution, the reaction warmed to room temperature 2 h; the system with aqueous saturated NH ₄ Cl quenched, extracted with ethyl acetate, the organic phase was dried and concentrated to give compound 5b was used directly in the next step.

The second step:

5c (0.48g, 3.4mmol), Pd(PPh ₃ ) ₄ (140mg, 0.7mmol) was added to 5b (0.66g, 1.3mmol) of toluene-EtOH-Na ₂ CO ₃ aqueous solution (10mL - 5mL) -5mL) in a mixture, N ₂ protection warmed to 85 ^o C overnight; 50 mL of water was added to the system, extracted with ethyl acetate, the organic phases were combined, dried and concentrated by silica gel column chromatography to give a white solid Ι-5 ( 100 mg, yield 15.6%).

1H NMR (DMSO, 400 MHz): d 8.65 (1H, s), 7.54 (2H, d, J = 8.0 Hz), 7.40 (4H, m), 7.27 (1H, d, J = 10.8 Hz), 7.04 ( 1H, s), 6.47 (1H, d, J = 16.0 Hz), 5.95 (1H, m), 4.72 (1H, d, J = 4.0 Hz), 4.55 (1H, m), 4.10 (2H, m), 3.56 (1H, m), 2.70 (1H, m), 2.37 (2H, m), 2.04 (2H, m), 1.90 (2H, m), 1.44 (3H, s), 1.27 (6H, m), 0.97 (2H, m), 0.91 (2H, m);

ESI-MS: 492.20 [M+H]+

Example 6: N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-4-(2-pyridyl)-styryl]dodecyl-1-methyl Ethyl 3-oxonaphtho[2,3-c]furan-6-yl]carbamate (Ι-6)

N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[4-(pyridin-2-yl)-phenyl]ethenyl]dodecahydro-1-methyl-3 -oxonaphtho[2,3-c]furan-6-yl] carbamic acid ethyl ester (Ι-6)

At 0 ^o C, Ar protection, the n -BuLi was added dropwise 6a (590mg, 1.9mmol) in THF (6 mL) solution, and reacted for 30 min at 0 ^o C; 1g was added dropwise to the system (250mg, 0.77 mmol) in THF (6 mL) added and the reaction 1 h; saturated NH ₄ Cl solution to quench the reaction, extracted with ethyl acetate, the organic phase was concentrated to dryness purified by silica gel column chromatography (PE by: white solid 2): EA = 1 I-6 (140 mg, yield 38.2%).

1H NMR (DMSO, 400 MHz): d 8.69 (1H, s), 7.98 (2H, d, J = 6.8 Hz), 7.75 (2H, d, J = 8.6 Hz), 7.44 (2H, d, J = 6.8 Hz), 7.25 (1H, d, J = 7.6 Hz), 6.48 (1H, d, J = 7.6 Hz), 5.97 (1H, m), 4.73 (1H, m), 4.5 (1H, m), 4.10 ( 2H, d, J = 6.0 Hz), 3.50 (1H, m), 2.69 (1H, m), 2.39 (2H, m), 2.04 (2H, m), 1.93 (2H, m), 1.43 (3H, s ), 1.23 (6H, m), 1.07 (2H, m), 0.92 (2H, m);

ESI-MS: 475.25 [M+H]+

Biological Test Example 1: Calcium ion transport inhibition assay

The model described below indicates that the compound of the present invention is a PAR-1 receptor inhibitor. In various cell types, activation of the PAR-1 receptor by a selective PAR-1 agonist triggers an intracellular signaling pathway that causes the endoplasmic reticulum to release calcium ions. In the KNRK cell line expressing human PAR1, calcium release from the receptor initiated by SFLLR was measured by fluorescence technique using a calcium ion selective probe. The emission intensity of the fluorescence is directly proportional to the activity and concentration of the PAR-1 antagonist. This method can determine the effect of the compounds of the invention on PAR-1 mediated calcium ion transport.

First, the experimental materials:

Reagents: HBSS buffer, HEPES, probenecid, BSA, Calcium 4 dye was purchased from Invitrogen. Reagents such as TFLLR-NH ₂ and SCH-79797 are supplied by SEREP, France.

Cell line: KNRK cell line stably expressing human PAR1.

Fluorescence microscope: CellLux (PerkinElmer).

Compounds I-1 to I-6: supplied by the Tianshili Pharmaceutical Group Co., Ltd. Research Institute.

Buffer and stock solution:

Assay buffer: 1×HBSS buffer, formulated as a buffer containing 20 mM HEPES, 2.5 mM probenecid, 0.1% BSA, pH = 7.4 (probenecid and BSA need to be freshly prepared);

Loading buffer: 1 × HBSS buffer, formulated with 20 mM HEPES, 2.5 mM probenecid, 0.1% BSA, pH = 7.4 (probenecid and BSA need to be freshly prepared), 2 μM calcium 4 dye buffer;

Compound stock solution (5 x CPD): The compound was first dissolved in 100% DMSO to give a final concentration of 10 mM. During the experiment, the above stock solution was prepared into a 5-fold concentration solution with an assay buffer;

6 × TFLLR-NH ₂ : HaTRAP was diluted to a final concentration of 30 uM with an assay buffer.

Final reaction volume: 20 uL loading buffer, 5 uL 5 x CPD, 5 uL 6 x TFLLR-NH ₂ (haTRAP, final concentration 5 uM).

Second, the experimental steps:

1. Pre-treat the sterile 384-well plate with 1 time Matrigel and place it at 37 ° C for 15-30 min;

2. Add 2×10 ⁴ KNRK cells stably expressing human PAR1 to each well of the above-mentioned treated sterile 384-well plate, and culture in a cell culture incubator for 24 hours;

3. Remove the cell culture medium from the 384-well plate and add 20 uL of loading buffer containing Calcium 4 dye;

4. Incubate in the incubator for 60 min in the dark;

5, then add 5uL 5 times compound stock solution (1% final concentration of DMSO) to the culture well, continue to culture for 15 min, then add 5 uL 6 times TFLLR-NH ₂ and record the fluorescence intensity under a fluorescence microscope for 100 seconds. .

6. Inhibitor activity in the experimental results is expressed as the percentage of the fluorescence intensity of the inhibitor at different concentrations as a percentage of the blank control haTRAP excitation fluorescence intensity. The inhibition rate of the test compound for PAR-1 was calculated by the following formula: IR = ( F _NC - F _TC ) / F _NC %

F _NC : Fluorescence intensity of negative control wells

F _TC : test the fluorescence intensity of the compound pores

At a concentration of 10 μM, the calcium signal antagonism was >60%, and the derivative of the present invention was identified as a PAR-1 receptor antagonist.

7, half the test compound inhibitory concentration IC ₅₀ can be calculated by the inhibition rate at different concentrations.

Third, the experimental results:

The inhibitory rate of the PAR-1 receptor at different concentrations of the compounds of the invention is as follows:

Half of the compounds of the invention inhibit the concentration IC ₅₀ was measured as follows:

Conclusion: The test compound of the present invention has a significant inhibitory effect on PAR-1 mediated calcium transport. The IC _{50 of} calcium signal antagonism is in the range of 0.18-2.1 μM, and the activity is better or similar to that of the marketed drug SCH530348. The compound was identified as a PAR-1 receptor antagonist.

Biological Test Example 2: Pharmacokinetic Testing of Compounds of the Invention

The drug concentrations in the plasma and brain tissues of the compounds of Examples I-2, I-3 and I-6 of the present invention were investigated at different times, and the pharmacokinetic behavior of the compounds of the present invention in rats was investigated, and their pharmacokinetic characteristics were evaluated.

Experimental animal

Eight healthy adult SD rats, half male and half female, were divided into 4 groups on average. Purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

2. Dosage

This study is a single dose administration experiment. Eight SD rats were divided into four groups, with an average of 4 groups, 2 in each group, which were SCH530348 group, I-2 group, I-3 group and I-6 group. Fasted for 12 h before administration. The dose administered was 5 mg/kg.

3. Drug preparation

Weigh the appropriate amount of the drug, add 0.5% sodium carboxymethylcellulose to grind until the sample is evenly suspended, the sample concentration is 2.0mg / ml, prepared at the time of use.

4. Blood collection plan

After the rats were administered, 0.5 mL of blood was taken from the eyelids at 0 min, 5 min, 15 min, 25 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and the plasma was taken by centrifugation at 4500 rpm for 10 minutes. Store in a -20 ° C refrigerator.

5. Sample preparation

Take 50uL of plasma sample, add 20uL of 1ug/ml diazepam internal standard solution, add 10uL of methanol, 400uL of ethyl acetate, vortex for 3min, centrifuge at 17000r/min for 10min, take supernatant of 300uL, and blow dry with nitrogen. After that, it was reconstituted with 100 uL of methanol:water = 1:1, and vortexed for 1 min, and then directly injected for LC-MS analysis.

The pharmacokinetic parameters of the compounds of the invention are as follows:

Conclusion: The half-life and blood concentration of the compounds of the present invention in the body I-2 and I-6 are better than those of the marketed drug SCH530348, and the ratio of the drug concentration of the I-2 and I-6 compounds in the brain tissue/plasma is far. It is much smaller than SCH530348, suggesting that the compounds I-2 and I-6 of the present invention have weaker ability to cross the blood-brain barrier, and the risk of cerebral hemorrhage may be greatly reduced, and has a better clinical application prospect.

no

no

no

Claims (15)

A compound having the formula (Ι) or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof: Wherein: R ^{1 is} selected from heterocyclic, aryl or heteroaryl; R ^{2 is} selected from -C(O)OR ³ , -CN, -C(O)NR ³ R ⁴ , heterocyclyl, aryl or hetero Aryl; X is selected from CH or N; n is selected from 0, 1, 2 or 3, and when n = 0, X = N, R ^{2 is} selected from heteroaryl; R ³ and R ⁴ are each independently selected from hydrogen Atom, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl. The compound of claim 1, or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, wherein R ^{1 is} selected from heterocyclic, aryl or hetero Aryl, said heterocyclyl, aryl or heteroaryl optionally being selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl , cycloalkyl, heterocyclic, aryl, heteroaryl, -C(O)OR ³ , -OC(O)R ³ , -C(O)R ³ , -NHC(O)R ³ , -C Substituting (O) a substituent of NR ³ R ⁴ ; the alkoxy group, alkyl group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group optionally being selected from one or more selected from the group consisting of halogen, hydroxyl, Substituted by a substituent of a cyano group, a nitro group, an amine group, a -NR ³ R ⁴ , an alkoxy group, an alkyl group, a cycloalkyl group, an aryl group or a heteroaryl group; R ^{2 is} selected from -C(O)OR ³ , -CN, -C(O)NR ³ R ⁴ ,heterocyclyl, aryl or heteroaryl, optionally substituted by one or more selected from the group consisting of halogen, hydroxy, Cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)OR ³ , -OC(O) R ^{3, -C (O) R 3} , -NHC (O) R 3, -C (O) NR 3 R 4 substituents of; the alkoxy, alkyl, cycloalkyl, heterocyclyl Or an aryl or heteroaryl group optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, hetero Substituted by a substituent of aryl; X is selected from CH or N; n is selected from 0, 1 or 2, and when n = 0, X = N, R ^{2 is} selected from heteroaryl, said heteroaryl Optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, - Substituted by a substituent of C(O)OR ³ , -OC(O)R ³ , -C(O)R ³ , -NHC(O)R ³ , -C(O)NR ³ R ⁴ ; The oxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy Substituted by a substituent of a group, an alkyl group, a cycloalkyl group, an aryl group or a heteroaryl group; R ³ and R ⁴ are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group. , the heterocyclic group, Aryl or heteroaryl group optionally substituted with one or more substituents selected from halo, hydroxy, cyano, nitro, amino, alkoxy, alkyl or cycloalkyl substituents. The compound of claim 1 or 2, or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, wherein the compound is selected from the group consisting of: Wherein: R ^{1 is} selected from a heterocyclic group, an aryl group or a heteroaryl group, and the heterocyclic group, aryl group or heteroaryl group is optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, and amine. , -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)OR ³ , -OC(O)R ³ , -C(O Substituting a substituent of R ³ , —NHC(O)R ³ , —C(O)NR ³ R ⁴ ; said alkoxy group, alkyl group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group The base is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, heteroaryl In the structure of formula (II-1), R ^{2 is} selected from -C(O)OR ³ , -CN, -C(O)NR ³ R ⁴ , heterocyclyl, aryl or heteroaryl, said The heterocyclic group, aryl or heteroaryl group is optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, hetero Cyclo, aryl, heteroaryl, -C(O)OR ³ , -OC(O)R ³ , -C(O)R ³ , -NHC(O)R ³ , -C(O)NR ³ R ⁴ substituents; said alkoxy, alkyl, cycloalkyl, Cycloalkyl, aryl or heteroaryl group optionally substituted with one or more substituents selected from halo, hydroxy, cyano, nitro, amino, -NR ³ R ^4, alkoxy, alkyl, cycloalkyl, aryl Substituted by a substituent of a heteroaryl group; in the structure of the formula (II-2), R ^{2 is} selected from a heteroaryl group; and the heteroaryl group is optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, and nitrate. Base, amine group, -NR ³ R ⁴ , alkoxy group, alkyl group, cycloalkyl group, heterocyclic group, aryl group, heteroaryl group, -C(O)OR ³ , -OC(O)R ³ ,- Substituted by a substituent of C(O)R ³ , -NHC(O)R ³ , -C(O)NR ³ R ⁴ ; the alkoxy group, alkyl group, cycloalkyl group, heterocyclic group, aryl group Or a heteroaryl group optionally selected from one or more selected from the group consisting of halogen, hydroxy, cyano, nitro, amine, -NR ³ R ⁴ , alkoxy, alkyl, cycloalkyl, aryl, heteroaryl Substituted by a substituent; R ³ and R ⁴ are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, and the heterocyclic group, aryl group or heteroaryl group is optionally selected. Substituted by one or more substituents selected from halogen, hydroxy, cyano, nitro, amine, alkoxy, alkyl or cycloalkyl. The compound of claim 1 or 2, or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, wherein the compound is selected from the group consisting of: The compound of claim 1 or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, wherein the isomer of the compound comprises all theoretically possible Cis trans isomers, cis and trans isomer mixtures, racemates, racemic mixtures, enantiomers, diastereomers and mixtures of diastereomers. The compound of claim 1, or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, wherein at least one hydrogen atom may be present in the compound by a ruthenium atom. Substitution or at least one carbon or fluorine atom is replaced by the corresponding isotope. The compound of claim 1, or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, wherein the pharmaceutically acceptable salt of the compound is the compound Conventional non-toxic salts formed with inorganic/organic acids or inorganic bases/organic bases. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof. And a pharmaceutically acceptable carrier. A compound according to any one of claims 1 to 7 or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or as claimed in the patent application Use of the pharmaceutical composition according to item 8 for the preparation of a medicament for a thrombin receptor antagonist. The use of claim 9, wherein the thrombin receptor antagonist is a PAR1 receptor antagonist. A compound according to any one of claims 1 to 7 or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or as claimed in the patent application Use of the pharmaceutical composition of item 8 in the manufacture of a medicament for a platelet aggregation inhibitor. A compound according to any one of claims 1 to 7 or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, or as claimed in the patent application Use of the pharmaceutical composition according to item 8 for the preparation of a medicament for treating and/or preventing a thrombin receptor-related disease. The use according to claim 12, wherein the thrombin receptor-related disease is selected from the group consisting of arterial and venous thrombosis, acute coronary syndrome, restenosis, stable angina pectoris, cardiac rhythm disorder, myocardial infarction, High blood pressure, heart failure, stroke, inflammatory disease, pulmonary embolism and other lung diseases, gastrointestinal diseases, rheumatism, asthma, chronic liver fibrosis, tumors and skin diseases. A compound according to any one of claims 1 to 7 or a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt or solvate thereof, and another cardiovascular The use of a drug as a combined product, at the same time, or separately for the preparation of a medicament for the treatment of cardiovascular diseases. The use according to claim 14, wherein the other type of cardiovascular drug is selected from the group consisting of aspirin, clopidogrel, ticlopidine, abciximab, tirofiban or eptifibatide. Antiplatelet aggregation drug.