CN103467481B

CN103467481B - Dihydropyridine compounds, a combination thereof thing, preparation method and purposes

Info

Publication number: CN103467481B
Application number: CN201210187107.2A
Authority: CN
Inventors: 程建军; 秦继红; 叶斌
Original assignee: SHANGHAI HUILUN TECHNOLOGY Co Ltd
Current assignee: Shanghai Huilun Jiangsu Pharmaceutical Co ltd; Shanghai Huilun Pharmaceutical Co ltd
Priority date: 2012-06-07
Filing date: 2012-06-07
Publication date: 2016-08-31
Anticipated expiration: 2032-06-07
Also published as: CN103467481A

Abstract

Dihydropyridine compounds disclosed by the invention, for having the compound of below general formula (I):Wherein, R₁Selected from hydrogen, halogen, alkyl, replacement alkyl, alkyl oxy, substituted alkyl oxy, alkyl amine group, substituted alkyl amine group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic radical, substituted heterocyclic radical, ester group, amide groups；R₂Selected from hydrogen, cyano group, alkyl, replacement alkyl, thiazolinyl, substituted alkenyl, acyl group；Or, R₂With R₃Formed and ring；R₃Selected from alkyl, replace alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl；Or, R₃With R₂Or R₄Formed and ring；R₄Selected from hydrogen, alkyl, replacement alkyl；Or, R⁴With R³Or R⁵Formed and ring；R₅Selected from alkyl, replace alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl；Or, R₅With R₄Formed and ring.The invention also discloses the application in terms for the treatment of cancer drug of the compounds process for production thereof of this logical formula (I), the compositions of the compound containing this logical formula (I) and this pharmaceutical composition.

Description

Dihydropyridine compound, composition, preparation method and application thereof

Technical Field

The invention relates to dihydropyridine compounds, a preparation method thereof, a pharmaceutical composition containing the same as an active ingredient, and application thereof as a medicament for treating diseases related to tyrosine kinase c-Met, in particular c-Met-related cancers.

Background

Cancer is the first killer threatening the life and health of human beings all over the world. Although medical advances have made many new approaches to cancer treatment in humans, cancer is still currently considered to be an unsolved medical problem. The causes of cancer are many, and in recent years, the nature of tumors has been elucidated due to the development of molecular oncology, molecular pharmacology and other disciplines, and it has been recognized that the nature of cellular carcinogenesis is cell immortalization due to disorder of cell signaling pathways.

Protein tyrosine kinases (PTKs, tyrosine kinases for short) are the most common growth factor receptors and are closely related to the development and progression of tumors, as the most important members involved in cell signaling.

Tyrosine kinase activity is too high, leading to activation of its downstream signaling pathways, which leads to cell transformation, proliferation, resistance to apoptosis, promotion of cell survival, and ultimately, tumor formation. Therefore, in recent years, the trend of development of antitumor drugs has shifted from traditional cytotoxic drugs to drugs aiming at intracellular abnormal signal transduction, and related drugs are sequentially applied to clinic. Compared with the traditional cytotoxic antitumor drugs, the molecular targeted drug has strong curative effect and small toxic and side effects, and gradually becomes a hot spot in the research and development of the current antitumor drugs.

Tyrosine kinases are classified into receptor-type tyrosine kinases and non-receptor-type tyrosine kinases.

Examples of receptor-type tyrosine kinases include Epidermal Growth Factor Receptor (EGFR) family, Vascular Endothelial Growth Factor Receptor (VEGFR) family, platelet-derived growth factor receptor (PDGFR) family, Fibroblast Growth Factor Receptor (FGFR) family, and the like.

Non-receptor tyrosine kinases such as the Src kinase family, Jak, FAK, etc., each kinase family comprising multiple subtypes.

The hepatocyte growth factor receptor c-Met is one of the receptor type tyrosine kinases (Park et al, Proc. Natl. Acad. Sci. USA 84:6379-83, 1987; Bottaro et al, Science 2S 1:802-4,1991) and consists of a highly glycosylated outer alpha and beta subunit together with an extracellular domain, a transmembrane fragment and a cytoplasmic tyrosine kinase domain. The endogenous ligand is Hepatocyte Growth Factor (HGF) (Nature,327:239-242 (1987); J.cell biol.,111:2097-2108(1990)), and the ligand binding induces dimerization of c-Met, generates an autophosphorylated activated receptor, promotes downstream signal transduction, and mediates multiple responses in tumor cells, including proliferation of epithelial cells and endothelial cells, stimulation of epithelial cell motility, cell survival and morphological change, and promotion of invasion, etc. In addition, HGF regulates angiogenesis, which is important for tumor growth and spread. The overexpression of c-Met and its ligands in a variety of tumors, including thyroid, ovarian, pancreatic, etc., also suggests a role in the development of these tumors. At present, the advantages of c-Met as a target of action of antitumor drugs have been gradually elucidated (Nature Reviews Cancer,2012,12, 89-103).

In primary tumors and secondary tumor metastases where c-Met receptor activation plays a critical role, biological agents targeting HGF or c-Met (ribozymes, antibodies, and antisense RNA) can inhibit tumor production, and selective small molecule inhibitors targeting c-Met are also predicted to have therapeutic potential. Patents such as WO2009091374, WO2009149836, WO2011003604, WO2011042367, WO2011042368, CN200910247948.6, CN201010175273.1 and the like all contain selective c-Met small molecule inhibitors, and preparation methods and applications thereof.

Dihydropyridine compounds as tyrosine kinase inhibitors, particularly c-Met inhibitors, have never been reported.

Disclosure of Invention

One of the technical problems to be solved by the present invention is to provide a dihydropyridine compound.

The second technical problem to be solved by the present invention is to provide a method for preparing the dihydropyridine compound.

The invention also provides a pharmaceutical composition containing the dihydropyridine compound.

The fourth technical problem to be solved by the invention is to provide the application of the dihydropyridine compound.

The dihydropyridines of the first aspect of the invention are compounds having the following general formula (I):

wherein,

R₁selected from hydrogen, halogen, alkyl and substituted alkylA group, alkyloxy, substituted alkyloxy, alkylamino, substituted alkylamino, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, ester group, amide group;

R₂selected from hydrogen, cyano, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl; or, R₂And R₃Forming a fused ring;

R₃selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; or, R₃And R₂Or R₄Forming a fused ring;

R₄selected from hydrogen, alkyl, substituted alkyl; or, R⁴And R³Or R⁵Forming a fused ring;

R₅selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; or, R₅And R₄Forming a union ring.

More specifically, the compound of the general formula (I) of the present invention is preferably one selected from the following compounds (I-1) to (I-46):

the compound of the general formula (I) is any one of enantiomer, diastereoisomer and conformational isomer or a mixture of any two or three of enantiomer, diastereoisomer and conformational isomer.

The compound of the general formula (I) is a pharmaceutically acceptable derivative.

The compounds of general formula (I) according to the invention may be present in the form of pharmaceutically acceptable salts.

The pharmaceutically acceptable salt is hydrochloride, sulfate, phosphate, acetate, trifluoroacetate, methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, tartrate, maleate, fumarate, succinate or malate of the compound shown in the general formula (I).

The compound of the general formula (I) as the second aspect of the present invention can be prepared from the reaction formula of the following scheme 1 through the corresponding intermediate of the structural formula (II),

route 1:

wherein R is₁，R₂，R₃，R₄The definition is the same as above; PG is an amine protecting group. PG is preferably t-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyl, 3, 4-dimethoxybenzyl.

In particular, R In Intermediate (II)₁In the case of H, the preparation is shown in scheme 2: nitrifying 5-methylthiophene-2-formic acid with fuming nitric acid to obtain an intermediate of a structural formula 2, esterifying the intermediate of the structural formula 2 into an intermediate of a structural formula 3, reducing nitro of the intermediate of the structural formula 3 into amino by adopting reduced iron powder to obtain an intermediate of a structural formula 4, and cyclizing the intermediate of the structural formula 4 into an intermediate of a structural formula 5 in the presence of acetic anhydride and nitrite; removing acetyl of the intermediate of the structural formula 5 to obtain an intermediate of a structural formula 6, reducing ester groups in the intermediate of the structural formula 6 into alcohol groups to obtain an intermediate of a structural formula 7, and oxidizing the intermediate of the structural formula 7 to obtain an intermediate of a structural formula (II);

route 2:

in Intermediate (II) R₁When H is not present, the preparation is as shown in scheme 3: nitrifying 5-methylthiophene-2-formic acid with fuming nitric acid to obtain an intermediate of a structural formula 2, esterifying the intermediate of the structural formula 2 into an intermediate of a structural formula 3, reducing nitro of the intermediate of the structural formula 3 into amino by adopting reduced iron powder to obtain an intermediate of a structural formula 4, and cyclizing the intermediate of the structural formula 4 into an intermediate of a structural formula 5 in the presence of acetic anhydride and nitrite; removing acetyl of the intermediate of the structural formula 5 to obtain an intermediate of a structural formula 6, and iodinating the intermediate 6 of the structural formula 6 to obtain an intermediate of a structural formula 8; protecting pyrazole amino of the intermediate of the structural formula 8 with an amino protecting group to obtain an intermediate of a structural formula 9; then, coupling by using iodine of the intermediate of the structural formula 9 to obtain an intermediate of a structural formula 10; reducing the ester group of the intermediate of the structural formula 10 into an alcohol group to obtain an intermediate of the structural formula 11, and oxidizing the alcohol group in the intermediate of the structural formula 11 into an aldehyde group to obtain an intermediate of the structural formula (II);

route 3:

the coupling is selected from Suzuki coupling and Buchwald coupling.

When the desired compound is a compound of formula (Ia) and R' ═ R₃＝R₅Its preparation is shown in scheme 4:

route 4:

when the desired compound to be prepared is a compound of the formula (Ib), and R₃And R₅In different cases, the preparation method is shown in scheme 5:

route 5:

when the desired compound is a compound of the formula (Ic), R₃And R₅Is different and R₄When H is excluded, the preparation is as shown in scheme 6:

route 6:

when the compound to be produced is a compound represented by the general formula (Id), it can be produced as shown in scheme 7:

route 7:

wherein X is CH₂Or O.

When the compound to be produced is a compound represented by the general formula (Ie), the production process is shown in scheme 8:

route 8:

when the compound to be produced is a compound represented by the general formula (If), the production process is as shown in scheme 9:

route 9:

wherein X is CH₂Or O.

The dihydropyridine-containing pharmaceutical composition as the third aspect of the invention, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable excipient.

A pharmaceutical composition as a third aspect of the invention, wherein said pharmaceutical composition comprises a therapeutically effective amount of a pharmaceutically acceptable derivative of a compound of formula (I) and a pharmaceutically acceptable excipient.

A pharmaceutical composition as a third aspect of the invention, wherein said pharmaceutical composition comprises a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of general formula (I) and a pharmaceutically acceptable excipient.

The pharmaceutical composition is prepared into tablets, capsules, aqueous suspensions, oily suspensions, dispersible powders, granules, pastilles, emulsions, syrups, creams, ointments, suppositories or injections.

As the fourth aspect of the invention, the application of the compound shown in the general formula (I) in preparing a preparation for regulating the catalytic activity of protein kinase is provided.

As the fourth aspect of the invention, the application of the pharmaceutically acceptable derivatives of the compounds in the general formula (I) in preparing preparations for regulating the catalytic activity of protein kinases is provided.

As the fourth aspect of the invention, the use of the pharmaceutically acceptable salt of the compound shown as the general formula (I) in the preparation of a preparation for regulating the catalytic activity of protein kinase.

As a fourth aspect of the invention, there is provided a use of the pharmaceutical composition for the manufacture of a medicament for the treatment of a disease associated with a protein kinase.

The protein kinase is c-Met receptor tyrosine kinase.

The protein kinase related disease is cancer.

The cancer is thyroid cancer, colorectal cancer, gastric cancer, renal cancer, liver cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, bladder cancer, head and neck cancer, pancreatic cancer, gallbladder cancer, osteosarcoma, rhabdomyosarcoma, MFH/fibrosarcoma, glioblastoma/astrocytoma, melanoma, or mesothelioma.

The dihydropyridine compounds of general formula (I) according to the present invention are also useful for the study of biological or pharmacological phenomena, the study of signal transduction pathways involving tyrosine kinases, and the comparative evaluation of novel tyrosine kinase inhibitors.

Detailed Description

The present invention provides compounds of general formula (I) as defined above, methods of preparing such compounds, pharmaceutical compositions using such compounds and methods of using such compounds.

Listed below are definitions of various terms used to describe the compounds of the present invention. These definitions apply to the terms used throughout the specification (unless otherwise limited in specific instances), whether used individually or as part of a larger group.

Unless otherwise defined, the term "alkyl" (used alone or as part of another group) as used herein refers to a monovalent group derived from an alkane that contains from 1 to 12 carbon atoms. Preferred alkyl groups have 1 to 6 carbon atoms. Alkyl is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4-dimethylpentyl, octyl, 2, 4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl and the like.

The substituents of the "substituted alkyl" are selected from the following groups: alkyl, halogen (e.g., fluorine, chlorine, bromine, iodine), alkoxy, amino/amino, haloalkyl (e.g., trichloromethyl, trifluoromethyl), aryl, aryloxy, alkylthio, hydroxy, cyano, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, urea, or mercapto.

The term "aryl" as used herein (alone or as part of another group) refers to monocyclic or polycyclic aromatic rings, e.g., phenyl, substituted phenyl, and the like, as well as fused groups such as naphthyl, phenanthryl, and the like. Thus, an aryl group comprises at least one ring having at least 6 atoms, up to five such rings (of which up to 22 atoms are included), and adjacent carbon atoms or suitable heteroatoms have alternating (conjugated) double bonds between them. Preferred aryl groups contain 6 to 14 carbon atoms in the ring.

"substituted aryl" may be optionally substituted with one or more groups including, but not limited to, halo (such as fluoro, chloro, bromo), alkyl (such as methyl, ethyl, propyl), substituted alkyl (such as trifluoromethyl), cycloalkyl, alkoxy (such as methoxy or ethoxy), hydroxy, carboxy, carbamoyl (-C (═ O) NR' R), alkoxycarbonyl (-CO)₂R), amino/amino, nitro, cyano, alkenyloxy, aryl, heteroaryl, sulfonyl (-SO)₂R), wherein R, R 'and R' are the alkyl groups.

The term "heteroaryl" as used herein (used alone or as part of another group) refers to substituted and unsubstituted aromatic 5 or 6 membered monocyclic groups, 9 or 10 membered bicyclic groups, and 11 to 14 membered tricyclic groups, which have at least one heteroatom (O, S or N) in at least one ring. The fused rings forming the bicyclic and tricyclic groups described above may contain only carbon atoms and may be saturated or partially saturated, provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom. The nitrogen and sulfur atoms may be oxidized, and the nitrogen atom may be quaternized. Bicyclic or tricyclic heteroaryl groups must include at least one ring that is fully aromatic, but the other fused ring or rings may be aromatic or non-aromatic. Heteroaryl groups may be attached at any available nitrogen or carbon atom of any ring.

A "substituted heteroaryl" ring system may contain zero, one, two or three substituents selected from: halogen, alkyl, substituted alkyl, alkenyl, bulky, aryl, nitro, cyano, hydroxy, alkoxy, alkylthio, -CO₂H、-C(＝O)H、-CO₂-alkyl, -C (═ O) alkyl, phenyl, benzyl, phenylethyl, phenyloxy, phenylthio, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, heteroaryl, -NR 'R ", -C (═ O) NR' R", -CO₂NR'R"、-C(＝O)NR'R"、-NR'CO₂R"、-NR'C(＝O)R"、-SO₂NR ' R ' and-NR ' SO₂R ", wherein R 'and R" are each independently selected from hydrogen, alkyl, substituted alkyl, and cycloalkyl, or R' and R "together form a heterocycloalkyl or heteroaryl ring.

Examples of monocyclic heteroaryl groups include pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl and the like.

Examples of bicyclic heteroaryls include indolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, chromonyl, coumarinyl, benzofuranyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridyl, and the like.

Examples of tricyclic heteroaryl groups include carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and the like.

The term "heterocycle" (used alone or as part of another group) as used herein refers to a cycloalkyl (non-aromatic) group in which one carbon atom in the ring is replaced by a heteroatom selected from O, S or N and up to 3 additional carbon atoms may be replaced by the heteroatom. The term "heterocyclyl", as used herein (alone or as part of another group), refers to a stable, saturated or partially unsaturated monocyclic ring system containing 5 to 7 ring atoms (carbon atoms and other atoms selected from nitrogen, sulfur and/or oxygen). The heterocyclic ring may be a 5,6 or 7 membered monocyclic ring and contain one, two or three heteroatoms selected from nitrogen, oxygen and/or sulphur. The heterocyclic ring may be optionally substituted, meaning that the heterocyclic ring may be substituted at one or more substitutable ring positions with one or more groups independently selected from: alkyl, heterocycloalkyl, heteroaryl, alkoxy, nitro, monoalkylamino, dialkylamino, cyano, halogen, haloalkyl, alkanoyl, amino/aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkylamido, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy and aryl, said aryl being optionally substituted with halogen, alkyl and alkoxy. Examples of such heterocycloalkyl groups include, but are not limited to: piperidine, morpholine, homomorpholine, piperazine, thiomorpholine, pyrrolidine and azetidine.

The term "alkoxy" as used herein (alone OR as part of another group) refers to an alkyl group, preferably having 1 to 6 carbon atoms, such as — OR, where R is the alkyl group, attached through an oxygen atom.

The term "amino" (used alone or as part of another group) as used herein refers to-NH₂. The "amino" group may be optionally substituted with one or two substituents (-NR 'R "), where R' and R" may be the same or different, such as alkyl, aryl, arylalkyl, alkenyl, alkynyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, alkyl, heterocycloalkylalkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, antelopylalkyl, alkoxyalkyl, alkylthio, carbonyl, or carboxyl. These substituents may be further substituted with a carboxylic acid or any of the alkyl or aryl substituents listed herein. In some embodiments, amino is substituted with carboxy or carbonyl, forming an N-acyl or N-carbamoyl derivative group.

The term "haloalkyl" as used herein (alone or in combination with a halogen)With or as part of another group) refers to a halogen atom attached through an alkyl group, such as-CF₃。

The term "acyl" (used alone or as part of another group) as used herein refers to an alkyl group or — C (═ O) R, where R is the alkyl group, attached through a carbonyl group.

The term "alkoxycarbonyl" (used alone OR as part of another group) as used herein refers to-C (═ O) OR, where R is the alkyl group.

The term "arylalkyl" or "aralkyl" (used alone or as part of another group) as used herein refers to an aromatic ring (e.g., benzyl) attached through an alkyl group as described above.

The term "aminoalkyl" (used alone or as part of another group) as used herein refers to an amino group (-NR 'R') which is attached through an alkyl group.

The term "arylalkylamino" (used alone or as part of another group) as used herein refers to an aryl group attached through an alkyl group, which is attached through an amino group.

The term "heteroatom" refers to O, S or N, independently selected. It should be noted that any heteroatom that does not satisfy valency is considered to have a hydrogen atom attached, thereby satisfying the valency.

The term "halogen" refers to an independently selected fluorine, chlorine, bromine or iodine.

The term "cycloalkyl" (used alone or as part of another group) as used herein refers to a fully or partially saturated hydrocarbon ring of 3 to 9 carbon atoms, preferably 3 to 7 carbon atoms. Furthermore, cycloalkyl groups may be substituted. "substituted cycloalkyl" refers to a ring having one, two, or three substituents selected from: halogen, alkyl, substituted alkyl (wherein the substituents are as defined above for the alkyl substituents), alkenyl, alkynyl, nitro, cyano, oxo (═ O), hydroxy, alkoxy, alkylthio, -CO₂H、-C(＝O)H、-CO₂-alkyl, -C (═ O) alkyl, keto, ═ N-OH, ═ N-O-alkyl, aryl, heteroaryl, five or six membered ketal (i.e. 1, 3-dioxane or 1, 3-dioxane), -NR 'R ", -C (═ O) NR' R", -CO₂NR'R”、-C(＝O)NR'R"、-NR'CO₂R"、-NR'C(＝O)R"、-SO₂NR ' R ' and-NR ' SO₂R ", wherein each of R 'and R" is independently selected from hydrogen, alkyl, substituted alkyl and cycloalkyl, or R' and R ", together form a heterocycloalkyl or heteroaryl ring.

The term "anti-cancer agent" includes any known agent useful for treating cancer, including: (1) cytotoxic drugs: nitrogen mustards, such as melphalan, cyclophosphamide; platinum coordination complexes such as cisplatin, carboplatin, and oxaliplatin; (2) antimetabolite antineoplastic agents: 5-fluorouracil, capecitabine, methotrexate, calcium folinate, raltitrexed, purine antagonists (e.g., 6-thioguanine and 6-mercaptopurine); (3) hormones: 17 alpha-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostandrosterone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, clorenyl estrol, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, toremifene; (4) tyrosine kinase inhibitors: EGFR inhibitors including Gefitinib (Gefitinib), Erlotinib (Erlotinib), Cetuximab (Cetuximab), Herceptin (Herceptin), and the like; VEGF inhibitors such as anti-VEGF antibodies (Avastin) and small molecule inhibitors such as Sunitinib, Vandetanib, Cediranib; Bcr-Abl inhibitors such as Imatinib (Imatinib), Dasatinib (Dasatinib); src inhibitors, MEK kinase inhibitors, MAPK kinase inhibitors, PI3K kinase inhibitors, c-Met inhibitors, ALK inhibitors, and the like; (5) drugs acting on tubulin such as vinblastine drugs, paclitaxel drugs, epothilone drugs such as Ixabepilone (Ixabepilone), and the like; (6) topoisomerase I inhibitors such as topotecan, irinotecan; (7) histone Deacetylase (HDAC) inhibitors such as Vorinostat; (8) proteasome inhibitors such as Bortezomib (Bortezomib); (9) other classes of anticancer drugs such as aurora kinase (aurora kinase) inhibitors, biological response modifiers, growth inhibitors, glutamine antagonists, anti-angiogenic and anti-vascular drugs, matrix metalloproteinase inhibitors, and the like.

"mammal" includes humans and domestic animals such as cats, dogs, pigs, cattle, sheep, goats, horses, rabbits, and the like. Preferably, for the purposes of the present invention, the mammal is a human.

"optional" or "optionally" means that the subsequently described environmental event may or may not be present, and that the description includes instances where the event or environment occurs and instances where it does not. For example, "optionally substituted aryl" means that the aryl group may or may not be substituted and that the description includes both substituted aryl and unsubstituted aryl groups.

By "pharmaceutically acceptable derivative" is meant any non-toxic salt, ester salt, amide salt, or other derivative that, when administered to a recipient, is capable of providing, directly or indirectly, a compound of the present invention or an inhibitory active metabolite or residue thereof.

"pharmaceutically acceptable excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, dispersant, diluent, preservative, suspending agent, stabilizer, dye/colorant, flavoring agent, surfactant, wetting agent, isotonic agent, solvent, or emulsifier that has been approved by the national food and drug administration as being useful for human or livestock.

"pharmaceutically acceptable salts" include acid addition salts and base addition salts.

"pharmaceutically acceptable acid addition salts" refers to salts which retain the biological effects and properties of the free base, do not have biological or other undesirable consequences, and are formed with inorganic acids such as, but not limited to, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, and organic acids such as, but not limited to, the following: formic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, p-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, mucic acid, naphthalene-2-sulfonic acid, Naphthalene-1, 5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, fumaric acid, succinic acid, tartaric acid, thiocyanic acid, undecylenic acid, and the like.

"pharmaceutically acceptable base addition salts" refers to salts that retain the biological effects and properties of the free acid and are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, the salts of: primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, methylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, isopropylamine, diethanolamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benzphetamine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperidine, piperazine, N-ethylpiperidine, polyamine resins, and the like. Preferred organic bases are isopropylamine, diethylamine, ethanolamine, triethylamine, dicyclohexylamine, choline and caffeine.

"pharmaceutical composition" refers to a formulation of a compound of the present invention with a generally accepted vehicle for delivering biologically active compounds to a mammal, such as a human. Such media include all pharmaceutically acceptable carriers, diluents or excipients therefor.

A "therapeutically effective amount" refers to an amount of a compound of the present invention which, when administered to a mammal (preferably a human), is sufficient to effect treatment of a disease or condition associated with the mammal (preferably a human) as defined below. The amount of a compound of the invention that constitutes a "therapeutically effective amount" will depend, for example, on the activity of the particular compound employed; the metabolic stability and length of action of the compound; the age, weight, general health, sex, and diet of the patient; mode and time of administration; the rate of excretion; combined medication; the severity of the particular condition or disorder; and the individual undergoing treatment, but it can be routinely determined by one of ordinary skill in the art based on his own knowledge and this disclosure.

"treating" or "treatment" as used herein encompasses the treatment of a disease or disorder associated with a mammal, preferably a human, having the disease or disorder associated therewith and includes:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal has a disease but has not yet been diagnosed as having it;

(ii) inhibiting the disease or disorder, i.e., arresting its development;

(iii) ameliorating the disease or condition, i.e., causing regression of the disease or condition;

(iv) stabilizing the disease or condition.

As used herein, the terms "disease" and "condition" may be used interchangeably or may be different, as a particular disease or condition may not have a known predisposition (and thus the cause has not been studied), and therefore has not been considered a disease but merely as an abnormal condition or syndrome, wherein the clinician has more or less identified a particular syndrome. The compounds of the invention and their structures shown herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, geometric, or conformational) forms, which may be defined as (R) -/(S) -or (D) -/(L) -or (R, R) -/(R, S) -/(S, S) -, according to the absolute stereochemical definition for an amino acid. The present invention is meant to include all such possible isomers, as well as their racemic, enantiomerically enriched, and optionally pure forms. Optically active (+) and (-), (R) -and (S) -and (R, R) -/(R, S) -/(S, S) -or (D) -and (L) -isomers can be prepared using chiral synthesis, chiral resolution, or can be resolved using conventional techniques such as, but not limited to, High Performance Liquid Chromatography (HPLC) using a chiral column. When the compounds described herein contain an alkenyl double bond or other geometrically asymmetric center, the compounds include both E and Z geometric isomers unless otherwise specified. Likewise, all tautomeric forms are also included.

"stereoisomers" refers to compounds made up of the same atoms bonded with the same chemical bonds but having different three-dimensional structures, which are not interchangeable. The present invention encompasses various stereoisomers and mixtures thereof and includes "enantiomers" which refer to two stereoisomers whose molecules are nonsuperimposable mirror images of each other, and "diastereomers"; diastereoisomers refer to stereoisomers in which the molecules have two or more chiral centers and are in a non-mirror relationship between the molecules.

"tautomer" refers to a proton that moves from one atom of a molecule from an original position to another position on the same molecule. The invention includes tautomers of any of the compounds.

In addition, unless otherwise indicated, the compounds of the present invention also include compounds that differ in structure only in the presence of one or more isotopically enriched atoms. For example, having the structure of the invention except that "deuterium" or "tritium" is used in place of hydrogen, or¹⁸F-fluorine labeling: (¹⁸Isotope of F) instead of fluorine, or with¹¹C-，¹³C-, or¹⁴C-enriched carbon (C¹¹C-，¹³C-, or¹⁴C-carbon labeling;¹¹C-，¹³c-, or¹⁴C-isotopes) instead of carbon atoms are within the scope of the invention. Such compounds are useful as analytical tools or probes in, for example, biological assays, or as tracers for in vivo diagnostic imaging of disease, or as tracers for pharmacodynamic, pharmacokinetic or receptor studies.

The invention also provides the following methods: proliferative diseases, such as cancer, are treated via modulation of c-Met kinase by administering to a patient in need of such treatment (simultaneously or sequentially) a therapeutically effective amount of a compound of general formula (I) as defined above in combination with at least one other anti-cancer agent. In a preferred embodiment, the proliferative disease is cancer.

In particular, the compounds of general formula (I) are useful in the treatment of a variety of cancers, most particularly those that rely on c-Met activation. c-Met activation can be modulated by gene amplification, mutation(s), and/or HGF stimulation, wherein HGF is provided by tumor (autocrine) or host (paracrine) tissues. In general, the compounds of the invention may be used to treat the following cancers:

(A) solid tumors including gastric cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer), colon cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, esophageal cancer, gallbladder cancer, bladder cancer, pancreatic cancer, thyroid cancer, prostate cancer, and skin cancer (including squamous cell carcinoma);

(B) hematopoietic tumors of lymphoid lineage, including Acute Lymphoblastic Leukemia (ALL), acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma;

(C) hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia;

(D) tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma;

(E) tumors of the central and peripheral nervous system, including astrocytomas, neuroblastomas, gliomas, and schwannomas;

(F) other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, thyroid follicular cancer, and kaposi's sarcoma.

The compounds of formula (I) may also be used in the treatment of any disease process characterized by abnormal proliferation of cells, such as benign prostate hyperplasia, neurofibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, inflammatory bowel disease, transplant rejection, endotoxic shock and fungal infections.

The compounds of formula (I) modulate the level of RNA and DNA synthesis in cells. Thus, these agents may be used to treat viral infections (including but not limited to HIV, human papilloma virus, herpes virus, poxviruses, EB virus, sindbis virus and adenovirus).

The compounds of formula (I) are useful in the chemoprevention of cancer. Chemoprevention is defined as inhibiting the development of aggressive cancer or inhibiting tumor recurrence by blocking the initial mutagenic event or by blocking the progression of pre-malignant cells that have suffered damage.

The compounds of general formula (I) are useful for inhibiting tumor angiogenesis and metastasis.

The compounds of the present invention may also be used in combination (either together or sequentially) with known anticancer agents (including, but not limited to, those mentioned above under "anticancer agents") or anticancer therapies such as radiation therapy.

Certain compounds of formula (I) can generally be prepared according to the following schemes 1 to 9. Tautomers and solvates (e.g., hydrates, ethanolates) of the compounds of formula (I) are also within the scope of the invention. Methods for the preparation of solvates are generally known in the art. Thus, the compounds of the present invention may be in free form or in the form of a hydrate. In the processes described below, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g.tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, p-methoxybenzyl and the like. Suitable protecting groups for amino groups include t-butoxycarbonyl, benzyloxycarbonyl, acetyl, benzoyl, trifluoroacetyl, p-methoxybenzyl and the like. Suitable protecting groups for carboxylic acids include alkyl, aryl or arylalkyl esters. Suitable protecting groups for the NH function of a heteroaryl group such as, for example, an indole or indazole ring include t-butyloxycarbonyl, benzyloxycarbonyl, acetyl, benzoyl, 2-trimethylsilanyl-ethoxymethyl, p-methoxybenzyl and the like.

Protecting Groups may be added or removed according to methods known to those skilled in the art (Greene, t.w., Protective Groups in organic Synthesis, 1999, 3 rd edition, Wiley) and standard techniques described herein. The protecting group may also be a polymer resin such as Wang resin, Rink resin or 2-chlorotrityl chloride resin.

Also, while these protected derivatives of the compounds of the present invention may not be pharmacologically active themselves, they may be administered to a mammal and then metabolized in vivo to form the compounds of the present invention which are pharmacologically active. Such derivatives are therefore described as "prodrugs". All prodrugs of the compounds of the present invention are included within the scope of the present invention.

The dihydropyridines of the general formula (I) according to the invention can be prepared by the following process.

A process for the preparation of compounds of general formula (I) by reaction of the corresponding intermediates of formula (II) according to scheme 1,

route 1:

wherein R is₁，R₂，R₃，R₄The definition is the same as above; PG is an amine protecting group. PG is preferably t-butoxycarbonyl or p-methoxybenzyl.

route 2:

in Intermediate (II) R₁When H is not present, the preparation is as shown in scheme 3: nitrifying 5-methylthiophene-2-formic acid with fuming nitric acid to obtain an intermediate of a structural formula 2, esterifying the intermediate of the structural formula 2 into an intermediate of a structural formula 3, reducing nitro of the intermediate of the structural formula 3 into amino by adopting reduced iron powder to obtain an intermediate of a structural formula 4, and cyclizing the intermediate of the structural formula 4 into an intermediate of a structural formula 5 in the presence of acetic anhydride and nitrite; removing acetyl of the intermediate of the structural formula 5 to obtain an intermediate of a structural formula 6, and iodinating the intermediate 6 of the structural formula 6 to obtain an intermediate of a structural formula 8; protecting pyrazole amino of the intermediate of the structural formula 8 with an amino protecting group to obtain an intermediate of a structural formula 9; then using the structural formulaCoupling the iodine of the intermediate of 9 to obtain an intermediate of a structural formula 10; reducing the ester group of the intermediate of the structural formula 10 into an alcohol group to obtain an intermediate of the structural formula 11, and oxidizing the alcohol group in the intermediate of the structural formula 11 into an aldehyde group to obtain an intermediate of the structural formula (II);

route 3:

the coupling is selected from Suzuki coupling and Buchwald coupling.

route 4:

when the desired compound to be prepared is a compound of the formula (Ib), and R₃When different from R5, the preparation method is shown in scheme 5:

route 5:

route 6:

route 7:

wherein X is CH₂Or O.

route 8:

route 9:

wherein X is CH₂Or O.

Wherein the following are common abbreviations:

DMF: n, N-dimethylformamide;

DMSO, DMSO: dimethyl sulfoxide;

CDCl₃: deuterated chloroform;

¹h NMR: nuclear magnetic resonance hydrogen spectroscopy;

MS mass spectrum

ESI-MS: electrospray ionization mass spectrometry;

s: a single peak;

d: double peaks;

t: a triplet;

dd: double peak;

br: broad peak;

m: multiple peaks;

DEG C: c, centigrade degree;

mol: molar ratio;

TLC: thin layer chromatography.

Other compounds of the invention not specifically disclosed in the above schemes can be prepared by similar methods using appropriate starting materials by those skilled in the art.

All compounds of the invention prepared as above in free base or acid form can be converted into their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid. Salts of the compounds prepared above may be converted to their free base or acid forms by standard techniques.

The compounds of the present invention include all crystalline forms, amorphous forms, anhydrates, hydrates, solvates, and salts thereof. Furthermore, all compounds of the invention comprising an ester group and an amide group can be converted into the corresponding acids by methods known to the person skilled in the art or by the methods described herein. Likewise, compounds of the invention comprising a carboxylic acid group can be converted into the corresponding esters and amides by methods known to those skilled in the art. Other substitutions and substitutions on the molecule may also be made by methods known to those skilled in the art (e.g., hydrogenation, alkylation, reaction with acid chlorides, etc.).

To prepare the cyclodextrin inclusion complexes of the invention, the compounds of general formula (I) as defined in the summary of the invention above may be dissolved in a pharmacologically acceptable solvent such as, but not limited to, an alcohol (preferably ethanol), a ketone (e.g. acetone)) Or an ether (e.g. diethyl ether) and mixed at 20 to 80 ℃ with an aqueous solution of α -, β -or γ -cyclodextrin, preferably β -cyclodextrin, or the acid of a compound of formula (I) as defined in the summary of the invention above, in the form of an aqueous solution of its salt (e.g. sodium or potassium salt), can be blended with cyclodextrin and then with an equivalent amount of acid (e.g. HCl or H)₂SO₄) To provide the corresponding cyclodextrin inclusion compound.

At this point or after cooling, the corresponding cyclodextrin inclusion compound crystals can crystallize out. Or when the compound of formula (I) is oily and crystalline, it can be converted to the corresponding cyclodextrin inclusion compound by adding an aqueous solution of cyclodextrin with stirring at room temperature for a long period of time (e.g., 1 hour to 14 days). The inclusion compound can then be isolated as a solid or as crystals by filtration and drying.

Cyclodextrins for use in the present invention are commercially available (e.g., from Aldrich Chemical Co.), or can be prepared by one skilled in the art using known methods. See, for example, Croft, A.P. et al, "Synthesis of chemical modified Cyclodextrins", Tetrahedron 1983,39,9, 1417-. Suitable cyclodextrins include the various types of inclusion complexes prepared with compounds of formula (I) above.

By selecting appropriate amounts of cyclodextrin and water, a reproducible inclusion compound of the active substance content can be obtained according to the stoichiometric composition. The inclusion compound may be used in a dry, water-absorbing form or in a form which contains water but is less water-absorbing. Typical molar ratios of cyclodextrin to compound of formula (I) are 2:1 (Cyclodextrin: Compound).

The pharmaceutical composition comprising the compound of formula (I) as an active ingredient may be in a form suitable for oral administration, for example, as tablets, capsules, aqueous suspensions, oily suspensions, dispersible powders or granules, syrups and the like. Orally-administrable compositions may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients or carriers suitable for the manufacture of tablets. These excipients or carriers may be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as microcrystalline cellulose, sodium carboxymethylcellulose, corn starch or alginic acid; binding agents, for example starch, gelatin, polyvinylpyrrolidone or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, water-soluble taste masking substances (such as hydroxypropyl-methylcellulose or hydroxypropyl-cellulose) or time delay substances (such as ethyl cellulose, cellulose acetate butyrate) may be used.

The capsule includes hard gelatin capsule and soft gelatin capsule. Hard gelatin capsules are prepared by mixing the active ingredient with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin; soft gelatin capsules are prepared by mixing the active ingredient with a water-soluble carrier, such as polyethylene glycol, or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials and excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone and acacia; dispersing or wetting agents may be a naturally occurring phosphatide (e.g. lecithin) or a condensation product of an alkylene oxide with a fatty acid (e.g. polyoxyethylene stearate) or a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g. heptadecaethylene-oxycetanol) or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (such as polyoxyethylene sorbitol monooleate) or a condensation product of ethylene oxide with a partial ester derived from a mixture of a fatty acid and a hexitol (e.g. polyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives (for example ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of antioxidants such as butylated hydroxyanisole or alpha-tocopherol.

Dispersible powders and granules comprise the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are those already mentioned above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. Dispersible powders and granules can be prepared by the addition of water to prepare an aqueous suspension.

Syrups may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. These formulations may also contain a demulcent, a preservative, a flavoring agent, a coloring agent and an antioxidant.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifiers may be naturally occurring phosphatides (e.g. soy bean lecithin), esters or partial esters derived from mixtures of fatty acids and hexitols (e.g. sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide (e.g. polyoxyethylene sorbitan monooleate). The emulsions may also contain sweetening agents, flavouring agents, preservatives and antioxidants.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Among the acceptable carriers and solvents that may be employed are water, Ringer's solution, isotonic sodium chloride solution and dextrose solution.

The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is first dissolved in a mixture of soybean oil and lecithin. Then, the resulting oil solution was introduced into a mixture of water and glycerin and treated, thereby forming a microemulsion.

Injectable solutions or microemulsions may be introduced into the bloodstream of a patient by local bolus injection or the solution or microemulsion may be administered in a manner so as to maintain a constant circulating concentration of the compound of the invention. To maintain such a constant concentration, a continuous intravenous administration device such as an infusion pump may be used.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension for intramuscular or subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent, for example, a solution in 1, 3-butanediol. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (such as oleic acid) may be used in the preparation of injectables.

The compounds of formula (I) may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. These materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of different molecular weights and fatty acid esters of polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions, etc., comprising the compounds of formula (I) may be prepared and used.

The compounds of the present invention may be administered in intranasal form by topical use of suitable intranasal vehicles and delivery devices, or by transdermal routes using transdermal skin patches well known to those skilled in the art. The compounds of the present invention may also be administered in the form of suppositories using bases such as: cocoa butter, glycerogelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of different molecular weights, and fatty acid acetates of polyethylene glycols.

When the compounds of the present invention are administered to a human subject, the daily dosage will generally be determined by the prescribing physician, and will generally vary with the age, weight, sex, and response of the patient, as well as the severity of the patient's symptoms. Generally, an effective daily dose for a 70kg patient is about 0.001mg/kg to 100mg/kg, preferably 0.01mg/kg to 50mg/kg, more preferably 1mg/kg to 25 mg/kg.

If formulated as a fixed dose, these combination products are treated with the compounds of the present invention within the dosage ranges described above and other pharmaceutically active agents within their approved dosage ranges. When the combined preparation is not suitable, the compound of formula (I) may also be administered sequentially with known anticancer or cytotoxic agents. The present invention is not limited by the order of administration; the compounds of formula (I) may be administered before or after administration of known anticancer drug(s) or cytotoxic drug(s).

The compounds of the invention are inhibitors of c-Met mediated diseases or c-Met mediated disorders. The terms "c-Met mediated disease" and "c-Met mediated disorder" refer to any disease state or other deleterious disorder in which c-Met is known to have a role. The terms "c-Met mediated disease" and "c-Met mediated disorder" also refer to those diseases or disorders that are alleviated by treatment with a c-Met inhibitor. Such diseases and disorders include, but are not limited to, cancer and other proliferative disorders.

Thus, the compounds are useful for treating, for example, the following diseases or conditions in mammals, especially humans: stomach, lung, esophagus, pancreas, kidney, colon, thyroid, brain, breast, prostate, and other solid tumor cancers; atherosclerosis; modulating angiogenesis; thrombosis and pulmonary fibrosis.

The compounds of the present invention are also useful in the study of biological or pharmacological phenomena, the study of signaling pathways involving tyrosine kinases, and the comparative evaluation of novel tyrosine kinase inhibitors.

The compounds referred to herein include, but are not limited to, the structural types given in scheme 1 and scheme 9 above, and can be obtained by applying similar methods using appropriate starting materials by those skilled in the art.

Examples

The specific synthetic preparations (for preparing the compounds of the invention) and biological examples (assays to demonstrate the utility of the compounds of the invention) provided below are intended to aid in the practice of the invention and should not be construed as limiting the scope of the invention.

Synthetic preparation example 1

Preparation of 2, 6-dimethyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-1)

Step 1 the reaction formula is as follows:

the method comprises the following steps: acetic anhydride (14.36g,140.7mmol) was added to a three-necked flask, the temperature was reduced to-50 ℃, fuming nitric acid (5.67g,90.0mmol) was added dropwise, 5-methylthiophene 2-carboxylic acid (4.0g,28.13mmol) was slowly added in portions after the addition was completed, and stirring was carried out at-20 ℃ for 2 hours after the addition was completed. The reaction solution was poured into ice water, allowed to stand, filtered, and dried to obtain 5-methyl-4-nitro-thiophene-2-carboxylic acid (4.0g, 76%) as a yellow solid.

Step 2 the reaction formula is as follows:

the method comprises the following steps: 5-methyl-4-nitro-thiophene-2-carboxylic acid (4.0g,21.37mmol) was dissolved in methanol (40mL), concentrated sulfuric acid (5mL) was added, and the mixture was stirred under reflux for 20 hours. Cooled to room temperature, the methanol was evaporated under reduced pressure and the residue was dissolved in ethyl acetate (200mL), washed with water and concentrated to give 5-methyl-4-nitro-thiophene-2-carboxylic acid methyl ester as a brown oil (2.5g, 62%).

Step 3 the reaction formula is as follows:

the method comprises the following steps: 5-methyl-4-nitro-thiophene-2-carboxylic acid methyl ester (3.34g,16.6mmol) was dissolved in a mixed solvent of methanol (25mL) and water (10mL), and ammonium chloride (4.39g, 82mmol) and reduced iron powder (4.2g, 75mmol) were added, followed by stirring under reflux for 6 hours. Cooling to room temperature, filtering under reduced pressure, concentrating the filtrate, dissolving the residue in ethyl acetate, washing with water, washing with saturated saline, drying, and concentrating to dryness to obtain crude 4-amino-5-methyl-thiophene-2-carboxylic acid methyl ester (2.1g, 74%). ESI-MS: M/z 172(M + H).

Step 4 the reaction formula is as follows:

the method comprises the following steps: methyl 4-amino-5-methyl-thiophene-2-carboxylate (0.4g,2.16mmol) was dissolved in toluene (20mL), KOAc (0.424g,4.23mmol), 18-crown-6 (57.07mg) and acetic anhydride (0.727g,7.13mmol) were added, the mixture was heated to 95 ℃ with stirring, isoamylnitrite (0.607g,5.18mmol) was added, the mixture was heated to 100 ℃ with stirring overnight. After cooling to room temperature, ethyl acetate EtOAc (100mL) was added, and the system was washed with water, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to give methyl 1-acetyl-1H-thieno [3,2-c ] pyrazole-5-carboxylate (0.34g, 70%). ESI-MS: M/z 225(M + H).

Step 5 the reaction formula is as follows:

the method comprises the following steps: 1-acetyl-1H-thieno [3,2-c ] pyrazole-5-carboxylic acid methyl ester (10.0g,42mmol) and anhydrous potassium carbonate (23.2g,168mmol) were added together to anhydrous methanol (375mL), stirred at room temperature for 20 minutes, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to give 1H-thieno [3,2-c ] pyrazole-5-carboxylic acid methyl ester (8.0g, 97%) as a brown solid.

Step 6 the reaction formula is as follows:

the method comprises the following steps: methyl 1H-thieno [3,2-c ] pyrazole-5-carboxylate (8.0g,33.6mmol) was dissolved in anhydrous tetrahydrofuran (160mL) under nitrogen. The temperature was reduced to-20 ℃ and lithium aluminium hydride (3.8g,100.7mmol) was added in portions and stirred for 3 hours. Water (3.8mL) was carefully added dropwise, allowed to stand for a while, and filtered. The filtrate was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give 1H-thieno [3,2-c ] pyrazole-5-methanol (3.54g, 69%) as a white solid.

Step 7 the reaction formula is as follows:

the method comprises the following steps: 1H-thieno [3,2-c ] pyrazole-5-methanol (1.0g,6.5mmol) was dissolved in methylene chloride (115mL), Dess-Martin oxidant (3.3g,7.8mmol) was added, and the mixture was stirred at room temperature for 3 hours. After the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to give 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (0.90g, 91%) as a white solid.

Step 8 the reaction formula is as follows:

the method comprises the following steps: 1H-thieno [3,2-c]Pyrazole-5-carbaldehyde (700mg,4.6mmol) and 3-aminocrotonic acid nitrile (831mg,10.1mmol) were dissolved in glacial acetic acid (15mL), and the mixture was heated to 90 ℃ and stirred for 1 hour. Distilling off acetic acid under reduced pressure, and purifying the residue by column chromatography to obtain the target product 2, 6-dimethyl-4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (820mg, 66%) as an off-white solid. MS:282(M + H);¹H NMR(300MHz,DMSO-d₆)13.02(s,1H),9.68(s,1H),7.75(d,1H),7.04(s,1H),4.79-4.80(d,1H),2.03(s,6H)。

synthetic preparation example 2

Preparation of 2-ethyl-6-methyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-2)

Step 1 the reaction formula is as follows:

the method comprises the following steps: under nitrogen protection, anhydrous tetrahydrofuran (100mL) was cooled to-70 ℃ and n-butyllithium (2.5M n-hexane solution) was added12.8mL, 32mmol) and stirred for a while anhydrous acetonitrile (1.15g,28mmol) was added dropwise. After stirring for 3 minutes, ethyl propionate (2.04g,20mmol) was added dropwise while keeping the temperature of the reaction system below-66 ℃. After the addition, the system was heated to-45 ℃ and stirred for 2 hours, and then 1N hydrochloric acid (64mL) was added dropwise to quench. The reaction was concentrated, the residue was extracted with ether, the extracts were combined, dried over anhydrous sodium sulfate, and concentrated to give a residual oil of 3-oxovaleronitrile (1.802g, 93% crude yield) which was used directly in the next reaction.¹H NMR(300MHz,CDCl₃)：3.48(s,2H),2.62(q,2H),1.11(t,3H)。

Step 2 the reaction formula is as follows:

the method comprises the following steps: 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (200mg,1.314mmol) (see synthetic preparation example 1 for preparation procedure) and 3-oxopentanenitrile (191mg,1.972mmol) were dissolved together in dichloromethane (20mL), piperidine (11mg,0.131mmol), glacial acetic acid (12mg,0.197mmol) were added, and the reaction was refluxed under nitrogen overnight. The reaction solution was cooled to room temperature, and anhydrous ethanol (20mL) was added to dilute the reaction solution, followed by stirring for ten minutes and filtration, the filtrate was concentrated to dryness, ethyl acetate (5mL) and petroleum ether (10mL) were added to the residue, followed by stirring for five minutes and filtration, and the solid was dried to give 2- ((1H-thieno [3,2-c ] pyrazol-5-yl) methanoyl) -3-oxovaleronitrile (190mg, 62.5%). ESI-MS: 232[ M + H ].

Step 3 the reaction formula is as follows:

the method comprises the following steps: 2- ((1H-thieno [3, 2-c)]Pyrazol-5-yl) methylene) -3-oxovaleronitrile (140mg,0.61mmol) and 3-aminocrotonitrile (109mg,1.33mmol) were dissolved together in glacial acetic acid (10mL), and the mixture was heated to 100 ℃ and stirred for 1 hour. Cooling to room temperature, concentrating the reaction solution under reduced pressure, and purifying the residue by column chromatography (dichloromethane: methanol: 80:1)Reacting to obtain a target compound 2-ethyl-6-methyl-4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (120mg, 67%). ESI-MS: 296[ M + H ]]；¹H NMR(300MHz,DMSO-d₆)13.00(br,1H),9.62(br,1H),7.75(s,1H),7.03(s,1H),4.79(s,1H),2.27-2.36(q,2H),2.04(s,3H),1.14(t,3H，J＝7.2Hz)。

Synthetic preparation example 3

Preparation of 2-isobutyl-6-methyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-3)

Step 1 the reaction formula is as follows:

the method comprises the following steps: under the protection of nitrogen, anhydrous tetrahydrofuran (100mL) is cooled to-70 ℃, n-butyllithium (2.5M n-hexane solution, 12.8mL, 32mmol) is added, then anhydrous acetonitrile (1.15g,28mmol) is added dropwise, after stirring is completed for 3 minutes, ethyl isovalerate (2.6g,20mmol) is added dropwise, and the temperature of a reaction system is kept to be not more than-66 ℃ in the process of dropwise addition. After the addition, the temperature is raised to minus 45 ℃ and the mixture is stirred for 2 hours. The reaction was quenched dropwise with 1N hydrochloric acid (40mL), concentrated under reduced pressure, the residue was extracted with diethyl ether, the extracts were combined, dried, concentrated, and the residue was used as an oil for the next reaction, 5-methyl-3-oxohexanenitrile (2.68g, 100% crude yield).¹H NMR(300MHz,CDCl₃)0.93(d,6H),2.16(m,1H),2.49(t,2H),3.43(s,2H)。

Step 2 the reaction formula is as follows:

the method comprises the following steps: 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (200mg,1.31mmol) (see synthetic preparation 1 for the preparation procedure), 5-methyl-3-oxohexanenitrile (329mg,2.63mmol) was dissolved in dichloromethane (20mL), glacial acetic acid (12mg,0.20mmol) and piperidine (11mg,0.13mmol) were added, and the mixture was stirred under nitrogen and refluxed overnight. The reaction solution was cooled, and the reaction solution was diluted with anhydrous ethanol (20mL), stirred for ten minutes and filtered, the filtrate was concentrated to dryness, and the residue was subjected to column chromatography (dichloromethane: methanol 300:1) to give 2- ((1H-thieno [3,2-c ] pyrazol-5-yl) methanoyl) -5-methyl-3-oxohexanenitrile (294mg, 86%). MS (ESI +)260[ M + H ].

Step 3 the reaction formula is as follows:

the method comprises the following steps: 2- ((1H-thieno [3, 2-c)]Pyrazol-5-yl) methylene) -5-methyl-3-oxohexanenitrile (100mg,0.605mmol), 3-aminocrotonitrile (70mg,0.848mmol) were dissolved together in glacial acetic acid (8mL), heated to 100 ℃, stirred for 1 hour, cooled to room temperature, concentrated to dryness under reduced pressure, and the residue was purified on a silica gel preparation plate (dichloromethane: methanol 20:1) to obtain 2-isobutyl-6-methyl-4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (73mg, 59%). MS (ESI +)324[ M + H]；¹H NMR(300MHz,DMSO-d₆)9.56(br,1H),7.83(s,1H),7.04(s,1H),4.83(s,1H),2.12-2.27(m,2H),2.04(s,3H),1.91-2.00(m,1H),0.92(dd,6H,J＝9.9,6.0Hz)。

Synthetic preparation example 4

Preparation of 2-methyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -6- (3,3, 3-trifluoropropyl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-4)

Step 1 the reaction formula is as follows:

the method comprises the following steps: anhydrous tetrahydrofuran (100mL) was cooled to-74 ℃ under nitrogen, n-butyllithium (2.5M in n-hexane, 16mL, 40mmol) was added, and anhydrous acetonitrile (1.437g,35mmol) was added dropwise. After stirring for 3 minutes, ethyl 4,4, 4-trifluorobutyrate (4.253g,25mmol) was added dropwise while keeping the temperature of the reaction system at-69 ℃. After the addition, the temperature is raised to minus 45 ℃ and the mixture is stirred for 2 hours. The reaction was quenched dropwise with 1N hydrochloric acid (50mL), concentrated under reduced pressure, the residue was extracted with ether, the extracts were combined, dried, concentrated, and the residue (3.908g, 94% crude yield) was used directly in the next reaction¹H NMR(300MHz,CDCl₃)2.43-2.55(m,2H),2.89(t,2H,J＝7.5Hz),3.55(s,2H)。

Step 2 the reaction formula is as follows:

the method comprises the following steps: 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (304mg,2.0mmol) (see synthetic preparation 1 for the preparation procedure), 6,6, 6-trifluoro-3-oxohexanenitrile (660mg,4.0mmol) was dissolved in dichloromethane (20mL), glacial acetic acid (12mg,0.20mmol) and piperidine (11mg,0.13mmol) were added, and the mixture was stirred under reflux under nitrogen overnight. The reaction solution was cooled, and anhydrous ethanol (20mL) was added to dilute the reaction solution, followed by stirring for ten minutes, filtration, concentration of the filtrate to dryness, and column chromatography of the residue (dichloromethane: methanol ═ 200:1) to give 2- ((1H-thieno [3,2-c ] pyrazol-5-yl) methanoyl) -6,6, 6-trifluoro-3-oxohexanenitrile (340mg, 57%). MS (ESI +)300[ M + H ].

Step 3 the reaction formula is as follows:

the method comprises the following steps: 2- ((1H-thieno [3, 2-c)]Pyrazol-5-yl) methylene) -6,6, 6-trisDissolving fluoro-3-oxohexanenitrile (200mg,0.67mmol) and 3-aminocrotonitrile (70mg,0.848mmol) together in glacial acetic acid (8mL), heating to 100 deg.C, stirring for 1 hour, cooling to room temperature, concentrating under reduced pressure to dryness, and purifying the residue on silica gel preparation plate (dichloromethane: methanol ═ 20:1) to obtain 2-methyl-4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -6- (3,3, 3-trifluoropropyl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (63mg, 26%). MS (ESI +)364[ M + H]；¹H NMR(300MHz,DMSO-d₆)9.56(br,1H),7.83(s,1H),7.04(s,1H),4.83(s,1H),2.04(s,3H),1.97(m,2H),1.83(m,2H)。

Synthetic preparation example 5

Preparation of 2- (2-methoxyethyl) -6-methyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-5)

Step 1 the reaction formula is as follows:

the method comprises the following steps: n-BuLi (31mL,77mmol) was added to-78 deg.C THF (350mL) followed by the dropwise addition of acetonitrile (2.9g, 70mmol) and, after the addition, stirring at low temperature for 1 h. Methyl 2-methoxypropionate (5.9g, 50mmol) was then added dropwise, the temperature was raised to-45 ℃ and stirred for 2h, quenched with 2N hydrochloric acid (160mL) at low temperature, the reaction was slowly raised to room temperature, extracted with diethyl ether, the organic phases combined and concentrated to give 5-methoxy-3-oxopentanenitrile (3.5g, 55%).

Step 2 the reaction formula is as follows:

the method comprises the following steps: 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (350mg,2.3mmol) (see synthetic preparation 1 for the preparation procedure) was dissolved in dichloromethane (5mL), and then 5-methoxy-3-oxovaleronitrile (292mg,2.3mmol), piperidine (11mg,0.13mmol), acetic acid (99mg,1.643mmol) and 4A molecular sieves were added, and after completion of addition, the reaction was heated to reflux and stirred overnight. The reaction was cooled, concentrated and the residue was used directly in the next step.

Step 3 the reaction formula is as follows:

the method comprises the following steps: the residue obtained in the above step (600mg,2.3mmol) was dissolved in glacial acetic acid (6mL), 3-aminocrotonitrile (190mg,2.3mmol) was added, and the mixture was heated to 100 ℃ and stirred for 1 h. After cooling, the reaction was concentrated to dryness and the residue was purified by preparative TLC to give 25mg, 3% of product. MS: [ M +1]＝326；¹H NMR(300MHz,CDCl₃)7.80(s,1H),7.02(s,1H),6.40(br,1H),4.67(s,1H),3.99-3.90(m,2H),3.41(s,3H),2.93-2.88(m,2H)，2.01(s,3H)。

Synthetic preparation example 6

Preparation of 2- (4-fluorophenyl) -6-methyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-6)

Step 1 the reaction formula is as follows:

the method comprises the following steps: 1H-thieno [3,2-c]Pyrazole-5-carbaldehyde (150mg,0.99mmol) (see synthetic preparation example 1 for the preparative procedure) and 4-fluorobenzoylacetonitrile (177mg, 1.08mmol) were co-dissolved in dichloromethane (15mL), acetic acid (9mg,0.15mmol) and piperidine (8mg, 0.10mmol) were added, and the mixture was refluxed under nitrogen atmosphereThe reaction was carried out overnight. The reaction mixture was cooled, and dichloromethane (30mL) was added to dilute the reaction mixture, followed by filtration, concentration of the filtrate to dryness, and column chromatography of the residue (dichloromethane: methanol: 100:1) to give 2- (4-fluorobenzoyl) -3- (1H-thieno [3, 2-c)]Pyrazol-5-yl) acrylonitrile (259mg, 88%). MS (ESI +) 298[ M +1]]⁺。

Step 2 the reaction formula is as follows:

the method comprises the following steps: reacting 2- (4-fluorobenzoyl) -3- (1H-thieno [3, 2-c)]Pyrazol-5-yl) acrylonitrile (259mg,0.87mmol) and aminocrotonitrile (157mg,1.917mmol) were dissolved in acetic acid (15mL), and the mixture was heated to 100 ℃ and stirred for 1 hour. After cooling, the reaction was concentrated to dryness and purified by preparative TLC (dichloromethane: methanol ═ 20:1) to give 2- (4-fluorophenyl) -6-methyl-4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (62mg, 20%). HPLC showed 97% purity; LC-MS (ESI +)362[ M +1]]⁺；¹H NMR(300MHz,CDCl₃)7.76(br,1H),7.54-7.59(m,2H),7.19(t,2H,J＝8.7Hz),7.04(s,1H),6.36(s,1H),2.24(s,3H)。

Synthetic preparation example 7

Preparation of 2- (4-methoxyphenyl) -6-methyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-7)

Step 1 the reaction formula is as follows:

the method comprises the following steps: methyl p-methoxybenzoate (2.0g,12mmol) was dissolved in dry toluene (50mL), cooled to 0 deg.C and sodium hydride (1.2g, 30mmol) was added. After stirring for 10 min acetonitrile (1.23g, 30mmol) was added and the temperature was slowly raised to 110 ℃ and stirred overnight. After cooling to room temperature, filtration was carried out, and the residue was washed with toluene, collected and dried to give 1-cyano-2- (4-methoxyphenyl) -2-oxopropanenitrile sodium salt (2.2g, 93%).

Step 2 the reaction formula is as follows:

the method comprises the following steps: 1-cyano-2- (4-methoxyphenyl) -2-oxopropanenitrile sodium salt (500mg,3.29mmol), 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (980mg,4.93mmol) (see synthetic preparation 1 for the preparation procedure) were added to dichloromethane (50mL), glacial acetic acid (490mg,4.93mmol) and piperidine (280mg,3.29mmol) were added, and the mixture was stirred under reflux under nitrogen overnight. The reaction was cooled, and dichloromethane (30mL) was added to dilute the reaction, filtered, and the filtrate was washed with water, dried and concentrated to dryness to give 2- (4-methoxybenzoyl) -3- (1H-thieno [3,2-c ] pyrazol-5-yl) acrylonitrile (1.0g), which was used as crude in the next reaction. LC-MS (ESI +)310[ M + H ].

Step 3 the reaction formula is as follows:

the method comprises the following steps: 2- (4-methoxybenzoyl) -3- (1H-thiophene [3, 2-c)]Pyrazole-5-yl) acrylonitrile (300mg, 0.92mmol) and aminocrotonic acid nitrile (152mg, 1.84mmol) were dissolved together in glacial acetic acid (20mL), N₂Heated to 100 ℃ under protection and stirred for 1 hour. Cooled to room temperature, the reaction was concentrated to dryness and purified on silica gel plates (DCM: MeOH ═ 10:1) to give 2- (4-methoxyphenyl) -6-methyl-4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (35mg, 10%). MS (ESI +)374[ M + H]。¹H NMR(300MHz,CDCl₃)9.85(s,1H),7.84(s,1H),7.50-7.47(m,2H),7.10-7.05(m,3H),4.92(s,1H),3.85(s,3H),2.10(s,3H)。

Synthetic preparation example 8

Preparation of 2-methyl-6- (pyridin-4-yl) -4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-8)

Step 1 the reaction formula is as follows:

the method comprises the following steps: methyl isonicotinate (1.0g,7.30mmol) was dissolved in toluene (30mL), cooled to 0 deg.C, and stirred for 10 min with the addition of sodium hydride (60%, 0.58g, 14.6mmol) carefully. Acetonitrile (1.50g, 36.5mmol) was added to the above solution, and the mixture was slowly warmed to 80 ℃ and stirred for 4 hours. Cooling to room temperature, filtering, washing the filter residue with toluene, collecting the filter residue, and drying to obtain 1-cyano-2-oxo-2- (pyridin-4-yl) ethane sodium salt (1.2g, 98%)

Step 2 the reaction formula is as follows:

the method comprises the following steps: 1-cyano-2-oxo-2- (pyridin-4-yl) ethane sodium salt (300mg,2.96mmol), 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (300mg,1.97mmol) (see synthetic preparation 1 for the preparation procedure) were added together to dichloromethane (20mL), acetic acid (178mg,2.96mmol) and piperidine (82mg,1.97mmol) were added, and stirred under reflux under nitrogen overnight. The reaction mixture was cooled to room temperature, and dichloromethane (30mL) was added to dilute the reaction mixture, followed by filtration, concentration of the filtrate to dryness, and column chromatography of the residue (DCM: MeOH ═ 100:1) was performed to give 2-isonicotinoyl-3- (1H-thieno [3,2-c ] pyrazol-5-yl) acrylonitrile (600 mg). (DCM: MeOH ═ 20:1), Rf ═ 0.5 LC-MS (ESI +)281[ M + H ].

Step 3 the reaction formula is as follows:

the method comprises the following steps: 2-Isonicotinoyl-3- (1H-thieno [3, 2-c)]Pyrazol-5-yl) (550mg, 1.96mmol) and aminocrotonitrile (400mg, 4.91mmol) were dissolved together in acetic acid (20mL), and the mixture was heated to 100 ℃ and stirred for 1 hour. Cooled to room temperature, the reaction was concentrated to dryness and purified on silica gel prep. plate (DCM: MeOH ═ 10:1) to give 2-methyl-6- (pyridin-4-yl) -4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (80mg, 129%). MS (ESI +)362[ M + H ]]；¹H NMR(300MHz,CDCl₃)13.15(br,1H),10.07(s,1H),8.76-8.75(m,2H),7.84(br,1H),7.56(m,2H),7.15(s,1H),5.01(s,1H,),2.10(s,3H)。

Synthetic preparation example 9

Preparation of 6-methyl-8- (1H-thieno [3,2-c ] pyrazol-5-yl) -2,3,4, 8-tetrahydro-1H-quinolizine-7, 9-dicarbonitrile of formula (I-9)

Step 1 the reaction formula is as follows:

the method comprises the following steps: to a solution of 2-piperidone (4.96g,50mmol) in dichloromethane (200mL) was added MeOTf (methyl trifluoromethanesulfonate) (10.2g,62mmol), and after the addition, the reaction mixture was reacted at room temperature for 18 hours. To the reaction solution were added solid sodium carbonate (20g) and water (20mL), stirred for 10 minutes, filtered, and the filtrate was dried over anhydrous sodium sulfate, concentrated to leave a crude 6-methoxy-2, 3,4, 5-tetrahydropyridine (4.52g, 81%) as a residue, which was used in the next reaction without purification. LC-MS (ESI +): 114[ M +1]]⁺。

Step 2 the reaction formula is as follows:

the method comprises the following steps: the crude 6-methoxy-2, 3,4, 5-tetrahydropyridine obtained in step 1 (4.52g,40mmol) was dissolved in anhydrous tetrahydrofuran (150mL), tert-butyl cyanoacetate (5.98g,42.3mmol) was added, and the mixture was heated to 70 ℃ under reflux overnight. The reaction mixture was concentrated, and the residue was subjected to column chromatography (petroleum ether: ethyl acetate 10:1) to give tert-butyl 2-cyano-2- (piperidin-2-enyl) acetate (3.2g, 36%). LC-MS (ESI +) 223[ M +1]]⁺。

Step 3 the reaction formula is as follows:

the method comprises the following steps: tert-butyl 2-cyano-2- (piperidin-2-enyl) acetate (100mg,0.45mmol) was added to 6M hydrochloric acid (16mL), heated to 100 ℃ and stirred for 15 minutes, concentrated to dryness, and the residue (piperidin-2-enyl) acetonitrile (55mg, 100% crude yield) was used directly in the next reaction.

Step 4 the reaction formula is as follows:

the method comprises the following steps: (piperidin-2-enyl) acetonitrile (55mg crude) obtained in the above step was dissolved in glacial acetic acid (16mL), and 2- ((1H-thieno [3, 2-c) prepared in step 1 of Synthesis preparation 2 was added]Pyrazol-5-yl) methylene) -3-oxobutanenitrile (65mg,0.3mmol) was heated to 100 ℃ and stirred for 1 hour. The reaction mixture was concentrated to dryness, and the residue was subjected to column chromatography (dichloromethane: methanol ═ 50:1) to give 6-methyl-8- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -2,3,4, 8-tetrahydro-1H-quinolizine-7, 9-dicarbonitrile (63mg, 66%). HPLC: 98.4 percent; LC-MS (ESI +) 322[ M +1]]⁺；¹H NMR(300MHz,CDCl₃)7.74(s,1H),6.96(s,1H),4.60(s,1H),3.53-3.60(m,2H),2.75(t,2H,J＝4.7Hz),2.27(s,3H),1.79-1.93(m,4H)。

Following the procedure of synthetic preparation 9, compounds I-10 to I-12 in the following table can be prepared, respectively, by replacing 2- ((1H-thieno [3,2-c ] pyrazol-5-yl) methylene) -3-oxobutanenitrile therein with the corresponding intermediate, respectively:

synthetic preparation example 13:

preparation of 6-methyl-8- (1H-thieno [3,2-c ] pyrazol-5-yl) -1,3,4, 8-tetrahydropyrido [2,1-c ] [1,4] oxazine-7, 9-dicarbonitrile of structural formula (I-13)

Step 1 the reaction formula is as follows:

the method comprises the following steps: 3-Morpholinone (303mg,3mmol) was dissolved in dichloromethane (20mL), MeOTF (methyl triflate) (610mg,3.72mmol) was added, and the mixture was stirred at room temperature for 18 hours. Adding Na₂CO₃Powder (5g) and water (1mL) were stirred for half an hour and filtered, the filtrate was dried over anhydrous sodium sulfate and concentrated to give crude 5-methoxy-3, 6-dihydro-2H-1, 4-oxazine (261mg, 75%) which was used in the next step without purification.

Step 2 the reaction formula is as follows:

the method comprises the following steps: the crude 5-methoxy-3, 6-dihydro-2H-1, 4-oxazine (261mg, 75%) obtained in the above step was co-dissolved with tert-butyl cyanoacetate (461mg,2.81mmo) in anhydrous tetrahydrofuran (10mL), and the mixture was stirred under reflux overnight. Cooled to room temperature, concentrated and the residue chromatographed (petroleum ether: ethyl acetate 10:1) to give tert-butyl 2-cyano-2- (morphol-3-enyl) acetate (106mg, 21%) as a white solid.

Step 3 the reaction formula is as follows:

the method comprises the following steps: tert-butyl 2-cyano-2- (morpholin-3-enyl) acetate (189mg,0.842mmol) was added to 6M hydrochloric acid (6mL) and heated to 100 deg.C with stirring for 15 minutes. Cooled to room temperature, concentrated under reduced pressure to give 2- (morphin-3-enyl) acetonitrile (103mg, 99% crude yield) as a pale yellow solid, which was used directly in the next step without purification.

Step 4 the reaction formula is as follows:

the method comprises the following steps: the crude 2- (morpholino-3-enyl) acetonitrile (103mg, 0.83mmol) obtained in the above step was dissolved in glacial acetic acid (6mL), and 2- ((1H-thieno [3, 2-c) was added]Pyrazol-5-yl) methylene) -3-oxobutanenitrile (preparative synthetic preparation 2, step 1, 102mg,0.468mmol) was heated to 100 ℃ and stirred for 15 minutes. Cooled to room temperature, the solvent was evaporated under reduced pressure, and the residue was purified by preparative TLC (dichloromethane: methanol ═ 15: 1) to give a pale yellow solid (29mg, 19%). HPLC purity: 95 percent;¹H NMR(300MHz,CDCl₃)7.75(s,1H),6.98(s,1H),4.53-4.75(m,3H),3.89-4.09(m,2H),3.47-3.67(m,2H),2.30(s,3H)。

following the procedure of synthetic preparation 13, compounds I-14 to I-16, respectively, were prepared by substituting the corresponding intermediate for 2- ((1H-thieno [3,2-c ] pyrazol-5-yl) methylene) -3-oxobutanenitrile therein:

synthetic preparation example 17:

preparation of 1,2, 6-trimethyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-17)

Step 1 the reaction formula is as follows:

the method comprises the following steps: methyl 1H-thieno [3,2-c ] pyrazole-5-carboxylate (8.5g, 46.7mmol) (see synthetic preparation example 1 for the preparation procedure), potassium carbonate (7.75g, 56mmol) were added to DMF (150mL), p-methoxybenzyl chloride (7.3g, 46.7mmol) was slowly added dropwise to the reaction solution, and stirring was carried out at ordinary temperature for 1 hour. DMF was evaporated under reduced pressure and the residue was taken up in ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give methyl 1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylate (13.8g, 98%).

Step 2 the reaction formula is as follows:

the method comprises the following steps: lithium aluminum hydride (1.36g, 35.76mmol) was suspended in tetrahydrofuran (100mL), cooled to 0 deg.C, and a solution of methyl 1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylate (7.2g, 23.8mmol) in tetrahydrofuran (100mL) was added dropwise, followed by stirring at room temperature for 2 hours. While chilled in ice water, quench slowly and dropwise with water (1.4mL) and filter. The residue was washed with a mixed solvent of dichloromethane and methanol (3:1), filtered, the filtrate was concentrated, and column chromatography was performed to give (1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) methanol (5.94g, 91%).

Step 3 the reaction formula is as follows:

the method comprises the following steps: (1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) methanol (5.94g, 21.7mmol) was dissolved in dichloromethane (150mL), and dess-martin oxidant (13.8g, 32.5mmol) was added to react at room temperature for 2 hours under nitrogen protection. The solvent was concentrated to dryness and the residue was chromatographed to give 1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (3.76g, 64%).

Step 4 the reaction formula is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (500mg, 1.84mmol), aminocrotonic acid (332mg, 4.04mmol) were dissolved in glacial acetic acid (50mL), heated to 100 ℃ and stirred for 1 hour, the acetic acid was distilled off under reduced pressure, and the residue was subjected to column chromatography directly to give 4- (1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (590mg, 80%)

Step 5 the reaction formula is as follows:

the method comprises the following steps: 4- (1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (590mg, 1.47mmol) was dissolved in DMF (30mL), cooled to 0 ℃ and cesium carbonate (719mg, 2.2mmol) was added to the reaction solution. After stirring for 15 minutes, iodomethane (209mg, 1.47mmol) was added, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane, washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to give 4- (1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) -1,2, 6-trimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (611mg, 100%).

Step 6 the reaction formula is as follows:

the method comprises the following steps: compound 5(611mg, 1.47mmol) was dissolved in trifluoroacetic acid (20mL), heated to 90 ℃ and stirred overnight. Concentrating trifluoroacetic acid, drying, and performing column chromatography to obtain target product 1,2, 6-trimethyl-4- (1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (43mg, 10%). MS (ESI +): 296[ M +1]]；¹H NMR(300MHz,DMSO-d₆)7.81(s,1H),7.02(s,1H),4.72(s,1H),3.19(s,3H),2.20(s,6H)。

Synthetic preparation example 18:

preparation of 1-ethyl-2, 6-dimethyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-18)

This compound was prepared by following the procedure of preparation example 17, substituting methyl iodide therein with ethyl bromide。MS(ESI+)310[M+1]；¹H NMR(300MHz,DMSO-d₆)7.81(br,1H),7.03(s,1H),4.74(s,1H),3.66(q,2H),2.25(s,6H),1.14(t,3H)。

Synthetic preparation example 19

Preparation of 1-benzyl-2, 6-dimethyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-19)

This compound was prepared by following the procedure of preparation 17, substituting benzyl bromide for methyl iodide therein. MS (ESI +): 372[ M +1]]；¹H NMR(300MHz,DMSO-d₆)13.21(br,1H)，7.87(s,1H),7.42(m,3H),7.33(m，1H)，7.19(m，2H)，7.09(s，1H)，4.97(s,2H),4.89(s，1H)，2.16(s,6H)。

Synthetic preparation example 20

Preparation of 3-oxo-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -3,4,6,7,8, 9-hexahydro-1H-furo [3,4-c ] quinolizine-5-carbonitrile of the formula (I-20) by the following specific reaction scheme:

the method comprises the following steps: 1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (304mg,2.0mmol) (see synthetic preparation example 1 for preparation procedure) and furan-2, 4(3H,5H) -dione (200mg,2.0mmol) were added to n-pentanol (5mL), and stirred at reflux for 1 hour. Cool to room temperature, add freshly prepared 2- (piperidin-2-enyl) acetonitrile (367mg, 3.0mmol), glacial acetic acid (4mL), and stir at 100 ℃ for 1.5 hours. Cooled to room temperature, concentrated under reduced pressure and the residue purified by preparative TLC (dichloromethane: methanol 10:1) to give the title product as a pale yellow solid (59mg, 9%). MS (ESI +):339[ M +1 ].

Synthetic preparation example 21

Preparation of 3-oxo-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -1,3,4,6,8, 9-hexahydrofuro [3',4':5,6] pyrido [2,1-c ] [1,4] oxazine-5-carbonitrile of formula (I-21) the specific reaction scheme is as follows:

the method comprises the following steps: 1H-thieno [3,2-c]Pyrazole-5-carbaldehyde (223mg,1.469mmol) (see synthetic preparation example 1 for the preparation procedure) and furan-2, 4(3H,5H) -dione (147mg,1.469mmol) were added to n-pentanol (5mL), and stirred at reflux for 1 hour. Cool to room temperature, add freshly prepared 2- (morphin-3-enyl) acetonitrile (273mg, 2.2mmol), glacial acetic acid (4mL), and stir at 100 ℃ for 1.5 hours. Cooled to room temperature, concentrated under reduced pressure and the residue was purified by preparative TLC (dichloromethane: methanol 10:1) to give the title product as a pale yellow solid (60mg, 12%). LC-MS (ESI +):341[ M +1]]⁺；¹H NMR(300MHz,CDCl₃)13.04(s,1H),7.76(d,1H),7.07(s,1H),5.09-5.10(d,2H),4.94(s,1H),4.52-4.65(m,2H),3.93-3.99(m,2H),3.50-3.57(m,2H)。

Synthetic preparation example 22

Preparation of 3, 6-dimethyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -4, 7-dihydro-isoxazolo [5,4-b ] pyridine-5-carbonitrile of formula (I-22) the specific reaction scheme is as follows:

the method comprises the following steps: 2- ((1H-thieno [3, 2-c)]Pyrazol-5-yl) methylene) -3-oxobutanenitrile (175mg,0.81mmol) (see synthetic preparation 2, step 1) was dissolved in glacial acetic acid (10mL), 3-methylisoxazol-5-amine (79mg,0.81mmol) was added, and the mixture was heated to 100 ℃ and stirred for 1 hour. Cooled to room temperature, the reaction was concentrated to dryness, and the residue was purified by preparative TLC (dichloromethane: methanol ═ 20:1) to give the title compound (30mg, 13%). HPLC showed 95.2% purity; LC-MS (ESI +) 298[ M +1]]⁺；¹H NMR(300MHz,DMSO-d₆)10.96(br,1H),7.79(s,1H),7.10(s,1H),5.27(s,1H),2.12(s,3H),1.87(s,3H)。

Synthetic preparation example 23:

preparation of 6- (4-fluorophenyl) -3-methyl-4- (1H-thieno [3,2-c ] pyrazol-5-yl) -4, 7-dihydroisoxazolo [5,4-b ] pyridine-5-carbonitrile of formula (I-23) the specific reaction scheme is as follows:

the method comprises the following steps: reacting 2- (4-fluorobenzoyl) -3- (1H-thieno [3, 2-c)]Pyrazol-5-yl) acrylonitrile (480mg,1.614mmol) (see synthetic preparation 6 for the preparation procedure) was dissolved in glacial acetic acid (30mL), 3-methylisoxazol-5-amine (190mg,1.937mmol) was added, and the mixture was heated to 100 ℃ and stirred for 1 hour. Cool to room temperature, concentrate the reaction to dryness, and purify the residue on a silica gel prep pad (DCM: MeOH ═ 20:1) to give compound 3(20mg, 3.3%). MS (ESI +)378[ M +1]]⁺。¹H NMR(300MHz,DMSO-d₆)13.02(br,1H),11.23(br,1H),7.65(t,2H,J＝4.4Hz),7.62(s,1H),7.37(t,2H,J＝7.2Hz),7.18(s,1H),5.44(s,1H₉),1.94(s,3H)。

Synthetic preparation example 24

Preparing 2, 6-dimethyl-4- (3-methyl-1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-24),

the reaction route of the step 1 is as follows:

the method comprises the following steps: a sodium cake (5.13g, 223mmol) was added to methanol (30mL) and reacted until the sodium cake completely disappeared to give a sodium methoxide solution. To a solution of methyl 1-acetyl-1H-thieno [3,2-c ] pyrazole-5-carboxylate (10.0g,44.6mmol) (see synthetic preparation example 1 for the preparation procedure) in methanol (100mL) was added the sodium methoxide solution prepared above, and the resulting mixture was stirred at room temperature for 30 minutes. A solution of iodine (28.3g,112mmol) in DMF (30mL) was added dropwise to the reaction mixture. After the addition was complete, the reaction was heated to 60 ℃ and stirred overnight. The reaction was cooled to room temperature, concentrated to dryness, the residue was extracted with ethyl acetate and water, the ethyl acetate phase was washed with water, washed with saturated brine, dried, concentrated to a certain volume, and filtered to give methyl 3-iodo-1H-thieno [3,2-c ] -5-carboxylate (10.3g, 73%). LC-MS (ESI +): 309[ M + H ].

The reaction route of the step 2 is as follows:

the method comprises the following steps: 3-iodo-1H-thieno [3,2-c]-methyl 5-carboxylate (5.0g, 16.23mmol), DMAP (397mg, 3.25mmol) and Et₃N (1.81g,17.85mmol) was dissolved in dichloromethane (100mL) and Boc was added dropwise₂O (4.25g,19.47mmol), reacting at room temperature for 30 min after dropwise addition, concentrating the reaction solution to dryness, and purifying the residue by silica gel column chromatography to obtain 1-tert-butoxycarbonyl-3-iodo-1H-thieno [3,2-c ]]Pyrazole-5-carboxylic acid methyl ester (5.9g, 89%). LC-MS (ESI +): 409[ M + H]⁺。

The reaction route of the step 3 is as follows:

the method comprises the following steps: reacting 1-tert-butyloxycarbonyl-3-iodo-1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (5.5g,13.47mmol) was dissolved in 1, 4-dioxane (100mL) and Pd (dppf) Cl was added₂(986mg,1.347mmol), dimethylzinc (1.15M in toluene, 17.6mL, 20.2mmol) was added dropwise, and after completion of addition, nitrogen gas was replaced three times, and the mixture was heated to 100 ℃ and stirred for 1 hour. Cooling the mixture to the room temperature,adding dichloromethane (300mL) to dilute the reaction solution, dropwise adding 1N hydrochloric acid to neutralize the reaction solution until the pH value is about 7, washing the organic phase with water, washing with saturated salt water, drying, concentrating, and performing column chromatography on the residue (dichloromethane: methanol: 125:1) to obtain 3-methyl-1H-thieno [3, 2-c: (3-methyl-1H-thieno [3,2-c ])]Pyrazole-5-carboxylic acid methyl ester (1.96g, 74%). LC-MS (ESI +): 197[ M + H]⁺。

The reaction route of the step 4 is as follows:

the method comprises the following steps: 3-methyl-1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (1.46g,7.45mmol) was dissolved in DMF (30mL), potassium carbonate (2.06g,14.9mmol) was added, PMBCl (1.4g,8.94mmol) was added dropwise, and after the addition was completed, stirring was carried out at room temperature for 4 hours. The reaction mixture was concentrated to dryness, the residue was dissolved in methylene chloride (100mL), washed with water, washed with saturated brine, dried, concentrated, and the residue was subjected to column chromatography (PE: EA: 10:1) to give 1- (4-methoxybenzyl) -3-methyl-1H-thieno [3,2-c ]]Pyrazole-5-carboxylic acid methyl ester (0.96g, 41%). LC-MS (ESI +): 317[ M + H ]]⁺。

Step 5 the reaction scheme is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -3-methyl-1H-thieno [3, 2-c)]Pyrazole-5-carboxylic acid methyl ester (429mg,1.36mmol) was dissolved in THF (20mL) and LiBH was added₄(118mg,5.43mmol), stirring at room temperature for 3 hours, adding dichloromethane (30mL) to dilute the reaction solution, adding 1N hydrochloric acid dropwise to adjust the pH to about 7, washing the organic phase with water, washing with saturated brine, drying, and performing column chromatography on the residue (PE: EA: 10:1) to obtain 1- (4-methoxybenzyl) -3-methyl-1H-thieno [3,2-c ]]Pyrazole-5-methanol (390mg, crude yield 100%). LC-MS (ESI +): 289[ M + H ]]⁺。

The reaction route of the step 6 is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -3-methyl-1H-thieno [3,2-c]Pyrazole-5-methanol (390mg,1.354mmol) is dissolved in dichloromethane (25mL), Dess-Martin oxidant (1.15g,2.708mmol) is added to react for 3 hours at room temperature under the protection of nitrogen, TLC (PE: EA ═ 2:1) shows that the reaction is finished, the reaction solution is concentrated, and the residue is subjected to column chromatography (PE: EA ═ 30:1) to obtain 1- (4-methoxybenzyl) -3-methyl-1H-thieno [3,2-c ]]Pyrazole-5-carbaldehyde (279mg, 71%). LC-MS (ESI +): 287[ M + H]⁺。

Step 7 the reaction scheme is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -3-methyl-1H-thieno [3, 2-c)]Pyrazole-5-carbaldehyde (275mg, 0.96mmol) and aminocrotonitrile (174mg, 2.12mmol) were dissolved in glacial acetic acid (10mL), and heated to 100 ℃ for reaction for 1 hour. The reaction mixture was concentrated, and the residue was 4- (1- (4-methoxybenzyl) -3-methyl-1H-thieno [3, 2-c)]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (399mg, crude yield 100%) was used directly in the next reaction. LC-MS (ESI +): 416[ M + H ]]⁺。

The reaction route of the step 8 is as follows:

the method comprises the following steps: 4- (1- (4-methoxybenzyl) -3-methyl-1H-thieno [3, 2-c) obtained in the above step]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile crude product (399mg,0.96mmol) is dissolved in trifluoroacetic acid (10mL), heated to 85 ℃ and refluxed for 24 hours, and the reaction is complete. The reaction mixture was concentrated to dryness, and methylene chloride (20mL) was added to the residue using saturated carbonWashing with sodium hydrogen carbonate solution, washing with water and saturated salt solution, drying, concentrating, purifying the residue by silica gel preparation plate after column chromatography to obtain the target product 2, 6-dimethyl-4- (3-methyl-1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (120mg, 42%). HPLC purity: 98.3 percent; MS (ESI +): 296[ M + H ]]；¹H NMR(300MHz,DMSO-d₆)12.6(br,1H),9.68(br,1H),6.97(s,1H),4.77(s,1H),2.31(s,3H),2.06(s,6H)。

Compounds I-25 to I-28 can be prepared using the intermediate 1- (4-methoxybenzyl) -3-methyl-1H-thieno [3,2-c ] pyrazole-5-carbaldehyde prepared in preparation 24, respectively, following the procedure described above for the preparation of compounds I-9, I-13, I-6, I-22.

Synthetic preparation example 29

Preparation of 4- (3-Ethyl-1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-29)

The reaction route of the step 1 is as follows:

the method comprises the following steps: 1-tert-Butoxycarbonyl-3-iodo-1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (8.2g,20mmol) (see synthetic preparation 24) was dissolved in 1, 4-dioxane (200mL) and Pd (dppf) Cl was added₂(1.46g,2mmol), diethyl ether was added dropwiseZinc (1M toluene solution, 30mL, 30mmol), nitrogen replacement three times after addition, heating to 100 ℃ and stirring for 1 hour. Adding dichloromethane (200mL) to dilute the reaction solution, dropwise adding 1N hydrochloric acid to neutralize the reaction solution to about pH 7, washing the organic phase with water, washing with saturated salt water, drying, concentrating, and performing column chromatography on the residue (dichloromethane: methanol: 400:1) to obtain 3-ethyl-1H-thieno [3,2-c ]]Crude pyrazole-5-carboxylic acid methyl ester (4.46g, 106% crude yield). LC-MS (ESI +) 211[ M +1]]⁺。

The reaction route of the step 2 is as follows:

the method comprises the following steps: 3-ethyl-1H-thieno [3, 2-c)]Crude pyrazole-5-carboxylic acid methyl ester (9.73g,46.28mmol) was dissolved in anhydrous tetrahydrofuran (250mL), cooled to-15 deg.C and LiAlH was added₄(5.27g,138.83mmol), stirring for 2 hours under nitrogen protection, and adding LiAlH₄(1.32g,34.72mmol), warmed to room temperature and stirred for 1 hour. Adding water (10mL) dropwise carefully, stirring for 10 minutes, adding methanol (100mL) and dichloromethane (100mL), stirring for ten minutes, filtering, adding methanol (50mL) and dichloromethane (50mL) to the filter residue, stirring for ten minutes, filtering, combining the filtrates, washing with water, washing with saturated common salt water, drying, concentrating, and performing column chromatography on the residue (dichloromethane: methanol ═ 100:1) to obtain 3-ethyl-1H-thieno [3,2-c ] thiophene]Pyrazole-5-methanol (3.85g, 46%). LC-MS (ESI +):183[ M +1]]⁺。

The reaction route of the step 3 is as follows:

the method comprises the following steps: 3-ethyl-1H-thieno [3, 2-c)]Pyrazole-5-methanol (1.82g, 10mmol) is dissolved in dichloromethane (200mL), Dess-Martin oxidant (6.36g, 15mmol) is added, the reaction is carried out at room temperature for 2 hours under the protection of nitrogen, the reaction solution is concentrated, and the residue is subjected to column chromatography (dichloromethane: methanol 300:1) to obtain 3-ethyl-1H-thieno [3,2-c]Pyrazole-5-carbaldehyde (1.75g, 97%). LC-MS (ESI +): 181[ M +1]]⁺。

The reaction route of the step 4 is as follows:

the method comprises the following steps: 3-ethyl-1H-thieno [3, 2-c)]Pyrazole-5-carbaldehyde (400mg,2.2mmol) and aminocrotonitrile (397mg,4.84mmol) were dissolved in glacial acetic acid (20mL) and heated to 100 ℃ for 1 hour. The reaction solution was concentrated, and the residue was subjected to column chromatography (dichloromethane: methanol ═ 80:1) to give the objective compound 4- (3-ethyl-1H-thieno [3, 2-c)]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (136mg, 20%). HPLC showed 98.2% purity; LC-MS (ESI +): 310[ M +1]]⁺；¹H NMR(300MHz,CDCl₃)8.99(br,1H),6.66(s,1H),4.36(s,1H),2.55-2.65(m,2H),1.87(s,6H),1.11(t,3H,J＝7.5Hz)。

Compounds I-30 to I-33 can be prepared, respectively, by using the intermediate 3-ethyl-1H-thieno [3,2-c ] pyrazole-5-carbaldehyde prepared in synthetic preparation 29, following the procedure described above for the preparation of compounds I-9, I-13, I-6, I-22.

Synthetic preparation example 34

Preparation of 4- (3-methoxy-1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-34)

The reaction route of the step 1 is as follows:

the method comprises the following steps: methyl 3-iodo-1H-thieno [3,2-c ] pyrazole-5-carboxylate (10.0g, 32.4mmol) (see synthetic preparation example 1 for the preparation procedure) was dissolved in DMF (100ml), potassium carbonate (9.0g, 64.9mmol) was added under ice water cooling, p-methoxybenzyl chloride (6.9g, 38.9mmol) was added dropwise, and the mixture was stirred at room temperature for 2 hours. Water was added to the reaction system, followed by extraction with ethyl acetate, and the combined ester layers were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (EA/PE ═ 1/8), whereby methyl 3-iodo-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylate (13.9g, yield 100%) was obtained as a nearly white solid.

The reaction route of the step 2 is as follows:

the method comprises the following steps: 3-iodine-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylic acid methyl ester (0.86g, 2mmol), sodium methoxide (0.44g, 8mmol), 3,4,7, 8-tetramethylphenanthroline (0.24g, 1mmol), and cuprous iodide (0.38g, 2mmol) were mixed in a single-neck bottle, and anhydrous methanol (50mL) was added and stirred at 80 ℃ for 16 hours under nitrogen protection. Cooling to room temperature, adding water into the system, acidifying with 1N hydrochloric acid, extracting with dichloromethane, combining the extract, drying with anhydrous sodium sulfate, and concentrating to obtain crude 3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylic acid (1.1g) and dark green solid. LC-MS showed a content of the target product (M +1 ═ 319) of about 30%.

The reaction route of the step 3 is as follows:

the method comprises the following steps: under the protection of nitrogen, the 3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] obtained in the previous step]The crude pyrazole-5-carboxylic acid (1.1g) was dissolved in THF (100mL) and the temperature was reducedAdding LiAlH to the mixture at the temperature of-10 DEG C₄(500mg) and stirred for 2 hours. Carefully dropwise adding water to quench the reaction, standing and filtering. Washing the filter residue with dichloromethane, extracting the filtrate with dichloromethane, mixing dichloromethane phases, drying with anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain (3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol (210mg, 35% yield in two steps), a pale yellow-green product. LC-MS: M +1 ═ 305.

The reaction route of the step 4 is as follows:

the method comprises the following steps: under the protection of nitrogen, (3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol (140mg, 0.46mmol) was dissolved in dry dichloromethane (20mL), cooled to-10 ℃, Dess-Martin reagent (293mg, 0.69mmol) was added, and stirring was carried out for 2 hours. The mixture was warmed to room temperature and stirred for 2 hours. Dropwise adding saturated sodium bicarbonate water solution, separating dichloromethane phase, extracting water phase with dichloromethane, mixing dichloromethane, drying, concentrating, and purifying by column chromatography (ethyl acetate/petroleum ether ═ 1/4) to obtain 3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3,2-c ═ p]Pyrazole-5-carbaldehyde (115mg, yield 83%) as a yellow solid.¹H NMR(300MHz,CDCl₃):9.80(s,1H),7.23(d,2H,J＝8.4Hz),7.02(s,1H),6.88(d,2H,J＝8.4Hz),5.24(s,2H),4.04(s,3H),3.81(s,3H)。

Step 5 the reaction scheme is as follows:

the method comprises the following steps: 3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (237mg, 0.78mmol) was dissolved in glacial acetic acid (7mL), aminocrotonitrile (142mg, 1.73mmol) was added, and the mixture was stirred at 90 ℃ for 1 hour. Acetic acid was evaporated under reduced pressure and the residue was crude 4- (3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile as a brown oil. LC-MS:432[ M +1 ].

The reaction route of the step 6 is as follows:

the method comprises the following steps: trifluoroacetic acid (10mL) was added to the brown oil and the mixture was stirred under reflux for 16 hours. Trifluoroacetic acid is distilled off under reduced pressure, the residue is cooled by ice water, saturated aqueous sodium bicarbonate solution is basified, and dichloromethane is extracted. The combined extracts were dried over anhydrous sodium sulfate, concentrated and purified by preparative TLC (dichloromethane/methanol ═ 20:1) to give 4- (3-methoxy-1H-thieno [3, 2-c)]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile, 90mg of a pale yellow solid, yield 37%. MS (312, M + H);¹H NMR(DMSO-d₆300MHz) 12.01(s,1H),9.69(s,1H),6.94(s,1H),4.78(s,1H),3.89(s,3H),2.03(s, 6H); HPLC, purity 96.8%.

The following compounds can be prepared respectively by using 3-methoxy-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-formaldehyde as a raw material and adopting a preparation method similar to the preparation method of the compounds I-9, I-13 and I-6:

synthetic preparation example 38

Preparation of 4- (3-amino-1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-38)

The reaction route of the step 1 is as follows:

the method comprises the following steps: 1-tert-butyl, 5-methyl-3-iodo-1H-thieno [3,2-c]Pyrazole-1, 5-dicarboxylic acid ester (0.82g, 2mmol), Pd₂(dba)₃(183mg,0.2mmol), Xantphos (347mg,0.6mmol) and cesium carbonate (980mg,3mmol) were dissolved in 1, 4-dioxane (20mL), benzophenone imine (540mg,3mmol) was added under nitrogen, and the mixture was heated to 100 ℃ and stirred for 6 hours. Cooling to room temperature, evaporating the solvent under reduced pressure, adding water and ethyl acetate to the residue, separating the organic phase, drying over anhydrous sodium sulfate, and concentrating. Purifying the residue by column chromatography to obtain light yellow solid 1-tert-butyl, 5-methyl-3- ((diphenylmethylene) amino) -1H-thieno [3,2-c]Pyrazole-1, 5-dicarboxylate (0.68g, 74%).

The reaction route of the step 2 is as follows:

the method comprises the following steps: 1-tert-butyl, 5-methyl-3- ((diphenylmethanoyl) amino) -1H-thieno [3,2-c ] pyrazole-1, 5-dicarboxylate (0.68g, 1.47mmol) was dissolved in methanol (10mL), hydroxylamine hydrochloride (0.112g, 1.62mmol) was added, and the mixture was stirred at room temperature for 6 hours. The solvent was evaporated under reduced pressure, and the residue was dissolved in methylene chloride, washed with a saturated aqueous solution of sodium hydrogencarbonate, dried over anhydrous sodium sulfate, and subjected to column chromatography to give 1-tert-butyl, 5-methyl-3-amino-1H-thieno [3,2-c ] pyrazole-1, 5-dicarboxylate (0.35g, 80%).

The reaction route of the step 3 is as follows:

the method comprises the following steps: 5-methyl-3-amino-1H-thieno [3,2-c]Pyrazole-1, 5-dicarboxylic acid ester (0.35g, 1.18mmol) was dissolved in acetonitrile (20mL), and 4-dimethylaminopyridine (159mg, 1.30)mmol) and di-tert-butyl dicarbonate (Boc)₂O) (284mg, 1.30mmol), and stirred at reflux for 2 hours. Cooling to room temperature, evaporating under reduced pressure to remove solvent, and purifying the residue by column chromatography to obtain white solid 1-tert-butyl, 5-methyl 3- ((tert-butyloxycarbonyl) amino) -1H-thieno [3,2-c]Pyrazole-1, 5-dicarboxylic acid ester (0.32g, 68%).

The reaction route of the step 4 is as follows:

the method comprises the following steps: 1-tert-butyl, 5-methyl 3- ((tert-butoxycarbonyl) amino) -1H-thieno [3,2-c ] pyrazole-1, 5-dicarboxylate (0.32g, 0.805mmol) was dissolved in anhydrous tetrahydrofuran (10mL), and lithium borohydride (70mg, 3.2mmol) was added under nitrogen and stirred at room temperature for 5 hours. After the reaction was completed, the temperature was lowered with ice water, and diluted hydrochloric acid (1N, 0.5mL) was added dropwise and stirred for 0.5 hour after the addition. The solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate, washed with water, dried, concentrated and subjected to column chromatography. Tert-butyl 3- ((tert-butoxycarbonyl) amino) -5- (hydroxymethyl) -1H-thieno [3,2-c ] pyrazole-1-carboxylate (0.22g, 74%) was obtained.

Step 5 the reaction scheme is as follows:

the method comprises the following steps: ((tert-Butoxycarbonyl) amino) -5- (hydroxymethyl) -1H-thieno [3,2-c ] pyrazole-1-carboxylic acid tert-butyl ester (0.22g, 0.6mmol) was dissolved in dichloromethane (10mL), Dess-Martin oxidant (509mg, 1.2mmol) was added, and the mixture was stirred at room temperature for 2 hours under nitrogen. The reaction solution was concentrated and purified by column chromatography to give tert-butyl 3- ((tert-butoxycarbonyl) amino) -5-formyl-1H-thieno [3,2-c ] pyrazole-1-carboxylate (0.17g, 77%).

The reaction route of the step 6 is as follows:

the method comprises the following steps: tert-butyl 3- ((tert-butoxycarbonyl) amino) -5-carboxaldehyde-1H-thieno [3,2-c ] pyrazole-1-carbamate (124mg, 0.336mmol) and 3-aminocrotonitrile (61mg, 0.738mmol) were added together to glacial acetic acid (5mL) and stirred at 95 ℃ for 15 minutes. Acetic acid was removed under reduced pressure and the residue was purified by preparative TLC to give tert-butyl (5- (3, 5-dicyano-2, 6-dimethyl-1, 4-dihydropyridin-4-yl) -1H-thieno [3,2-c ] pyrazol-3-yl) carbamate (49mg, 36%).

Step 7 the reaction scheme is as follows:

the method comprises the following steps: (5- (3, 5-dicyano-2, 6-dimethyl-1, 4-dihydropyridin-4-yl) -1H-thieno [3, 2-c)]Pyrazol-3-yl) carbamic acid tert-butyl ester (67mg,0.169mmol) was dissolved in dichloromethane (5mL), and trifluoroacetic acid (1mL) was added thereto, followed by stirring at room temperature for 5 hours. The reaction solution was diluted with ethyl acetate (10mL), solid sodium carbonate (3g) was added, stirred for 0.5 hour, filtered, the filtrate was concentrated, and preparative TLC purification (dichloromethane: methanol 10:1) was performed to give 4- (3-amino-1H-thieno [3, 2-c)]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (35mg, 70%). ESI-MS:297(M + H);¹H NMR(300MHz,DMSO-d₆)11.46(s,1H),9.69(s,1H),6.78(s,1H),5.08(s,2H),4.69(s,1H),2.03(s,6H)。

synthetic preparation example 39

Preparation of 2, 6-dimethyl-4- (3- (pyridin-3-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-39)

The reaction route of the step 1 is as follows:

the method comprises the following steps: 1-tert-Butoxycarbonyl-3-iodo-1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (200mg,0.49mmol) (preparation procedure see synthetic preparation 24), pyridine-3-boronic acid (90mg,0.74mmol), Pd (dppf) Cl₂-CH₂Cl₂(42mg,0.049mmol) and Cs₂CO₃(639mg,1.96mmol) was added to a mixed solvent of 1, 4-dioxane (10mL) and water (4mL), replaced with nitrogen three times, heated to 120 ℃ and stirred for 30 minutes. Cooling to room temperature, concentrating the reaction solution, and performing column chromatography on the residue (dichloromethane: methanol: 75:1) to obtain 3- (pyridine-3-yl) -1H-thieno [3, 2-c)]Pyrazole-5-carboxylic acid methyl ester (65mg, 51%). LC-MS (ESI +)260[ M +1]]⁺。

The reaction route of the step 2 is as follows:

the method comprises the following steps: 3- (pyridin-3-yl) -1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (487mg,1.878mmol) was dissolved in tetrahydrofuran (20mL), cooled to-20 ℃, added with lithium aluminum hydride (214mg,5.635mmol) under nitrogen protection, and after addition, heated to room temperature for reaction for 3 hours. Cooling the reaction solution with ice water, adding water (0.4mL) dropwise to quench the reaction, filtering, washing the filter residue with methanol for three times, concentrating the filtrate, and performing column chromatography on the residue (dichloromethane: methanol ═ 20:1) to obtain (3- (pyridin-3-yl) -1H-thieno [3,2-c ], (3-pyridin-3-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol (276mg, 64%). LC-MS (ESI +): 232[ M + H ]]⁺。

The reaction route of the step 3 is as follows:

the method comprises the following steps: reacting (3- (pyridine-3-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol (270mg,1.167mmol) in waterAdding Dess-Martin oxidant (990mg,2.335mmol) into dichloromethane (30mL), reacting at room temperature for 3 hr under nitrogen protection, concentrating the reaction solution, and performing column chromatography to obtain 3- (pyridin-3-yl) -1H-thieno [3,2-c ] residue]Pyrazole-5-carbaldehyde (88mg, 33%). LC-MS (ESI +): 230[ M + H [ ]]⁺。

The reaction route of the step 4 is as follows:

the method comprises the following steps: 3- (pyridin-3-yl) -1H-thieno [3,2-c]Pyrazole-5-carbaldehyde (88mg,0.384mmol) and aminocrotonitrile (69mg,0.884mmol) were dissolved in glacial acetic acid (5mL) and heated to 95 ℃ for 2 hours. Cooling to room temperature, concentrating the reaction solution, and performing column chromatography on the residue to obtain 2, 6-dimethyl-4- (3- (pyridine-3-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (52mg, 38%). HPLC showed 97% purity;¹H NMR(300MHz,DMSO)13.43(s,1H),9.77(s,1H),8.99(s,1H),8.54-8.56(m,1H),8.14(t,1H,J＝3.8Hz),7.49-7.53(m,1H),7.18(s,1H),4.91(s,1H),2.06(s,6H)；LC-MS(ESI+)：359[M+H]⁺。

synthetic preparation example 40

Preparation of 2, 6-dimethyl-4- (3- (pyridin-4-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-40)

The method comprises the following steps: the title compound was prepared by the method of EXAMPLE 39 substituting pyridine-4-boronic acid for pyridine-3-boronic acid in step 1. LC-MS (ESI +): 359[ M + H]⁺；¹H NMR(300MHz,DMSO)13.58(s,1H),9.76(s,1H),8.65(d,2H,J＝4.8Hz),7.72(d,2H,J＝5.4Hz),7.20(s,1H),4.92(s,1H),2.06(s,6H)。

Synthesis preparation example 41

Preparation of 2, 6-dimethyl-4- (3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-41)

The reaction route of the step 1 is as follows:

the step is 1-tert-butyloxycarbonyl-3-iodo-1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (2.5g,6.12mmol) (preparation see synthetic preparation 24), 1-methyl-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazole (3.82g,18.37mmol), Pd (dppf) Cl₂(896mg,1.22mmol) and Cs₂CO₃(3.99g,12.24mmol) was added to a mixed solvent of 1, 4-dioxane (125mL) and water (50mL), and the mixture was replaced with nitrogen three times, heated to 100 ℃ and reacted for 1 hour. Cooling to room temperature, concentrating the reaction mixture, dissolving the residue in dichloromethane (100mL) and water (30mL), separating the organic phase, washing with water, washing with saturated brine, drying, concentrating, and subjecting the residue to column chromatography (dichloromethane/methanol ═ 200/1) to give 3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3,2-c ] compound]Pyrazole-5-carboxylic acid methyl ester (888mg, 55.3%). LC-MS (ESI +): 263[ M +1]]⁺。

The reaction route of the step 2 is as follows:

the method comprises the following steps: 3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (888mg, 3.386mmol) was dissolved in DMF (25mL) and K was added₂CO₃(936mg, 6.772mmol), PMBCl (636mg, 4.063mmol) was added dropwise and, after the addition was complete, stirring was carried out for 4 hours. Concentrating the reaction solutionConcentrating, dissolving the residue in dichloromethane (50mL) and water (20mL), washing the organic phase with water, washing with saturated saline, drying, concentrating, and performing column chromatography (PE: EA ═ 10:1) to obtain the compound 1- (4-methoxybenzyl) -3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3, 2-c)]Pyrazole-5-carboxylic acid methyl ester (722mg, 55.8%). LC-MS (ESI +):383[ M +1]]⁺.

The reaction route of the step 3 is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (722mg, 1.89mmol) was dissolved in anhydrous THF (20mL) and LiBH was added₄(165mg, mmol) and stirred for 3 hours. Adding dichloromethane (30mL) to dilute the reaction solution, dropwise adding 1N hydrochloric acid to adjust the pH to about 7, washing the organic phase with water, washing with saturated salt water, drying, and performing column chromatography (PE: EA: 6:1) to obtain a compound (1- (4-methoxybenzyl) -3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol (403mg, 60%). LC-MS (ESI +) 355M +1]⁺。

The reaction route of the step 4 is as follows:

the method comprises the following steps: (1- (4-methoxybenzyl) -3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3, 2-c) obtained in the above step]Pyrazol-5-yl) methanol (403mg, 1.138mmol) is dissolved in dichloromethane (20mL), Dess-Martin reagent (996mg, 2.276mmol) is added to react for 3 hours at room temperature under the protection of nitrogen, TLC (PE: EA ═ 2:1) shows that the reaction is finished, the reaction liquid is concentrated, and the residue is subjected to column chromatography (dichloromethane) to obtain the compound 1- (4-methoxybenzyl) -3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3, 2-c)]Pyrazole-5-carbaldehyde (391mg, 97%). LC-MS (ESI +) 353M +1]⁺。

Step 5 the reaction scheme is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3, 2-c)]Pyrazole-5-carbaldehyde (391mg, 1.11mmol) and aminocrotonitrile (200mg, 2.44mmol) were dissolved in glacial acetic acid (10mL), heated to 100 ℃ and stirred for 1 hour. The reaction was concentrated and the residue was 4- (1- (4-methoxybenzyl) -3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (533mg, 99% crude yield) was used in the next reaction without purification. LC-MS (ESI +) 482[ M +1]]⁺。

The reaction route of the step 6 is as follows:

the method comprises the following steps: the residue (533mg, 1.107mmol) from the above step was dissolved in trifluoroacetic acid (10mL), heated to 85 ℃ and stirred for 24 hours. Cooling to room temperature, concentrating the reaction mixture to dryness, adding dichloromethane (30mL) to the residue, washing with saturated sodium bicarbonate solution, washing with water and saturated sodium chloride solution, drying, concentrating, and performing column chromatography on the residue (dichloromethane: methanol ═ 50:1) to obtain the target compound 2, 6-dimethyl-4- (3- (1-methyl-1H-pyrazol-4-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (100mg, 25%). LC-MS (ESI +)362[ M +1]]⁺；¹H NMR(300MHz,CD₃OD)7.90(s,1H),7.82(s,1H),6.97(s,1H),4.72(s,1H),3.95(s,3H),2.12(s,6H)。

Synthesis preparation example 42

Preparation of 2, 6-dimethyl-4- (3- (thien-2-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-42)

The reaction route of the step 1 is as follows:

the method comprises the following steps: reacting 1-tert-butyloxycarbonyl-3-iodo-1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (2.0g, 4.9mmol), thiophene-2-boronic acid (940mg, 7.35mmol), Pd (dppf) Cl₂(717mg, 0.98mmol) and Cs₂CO₃(4.79g, 14.7mmol) was added to 1, 4-dioxane (50mL) and water (20mL), replaced with nitrogen three times, heated to 100 ℃ and stirred for 1 hour. Cooling to room temperature, concentrating the reaction mixture, dissolving the residue in dichloromethane (100mL), washing with water, washing with saturated brine, drying, concentrating, and subjecting the residue to column chromatography (dichloromethane: methanol: 200:1) to obtain 3- (thien-2-yl) -1H-thieno [3,2-c ]]Pyrazole-5-carboxylic acid methyl ester (761mg, 59%). LC-MS (ESI +) 265[ M +1]]⁺。

The reaction route of the step 2 is as follows:

the method comprises the following steps: reacting 3- (thien-2-yl) -1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (468mg, 1.77mmol) in DMF (15mL) and K was added₂CO₃(489mg,3.54mmol), PMBCl (333mg, 2.12mmol) was added dropwise, and after the addition was completed, the mixture was stirred at room temperature for 4 hours. Concentrating the reaction solution under reduced pressure, dissolving the residue in dichloromethane, washing with water, washing with saturated saline, drying, concentrating, and performing column chromatography (PE: EA: 10:1) to obtain 1- (4-methoxybenzyl) -3- (thiophen-2-yl) -1H-thieno [3,2-c ]]Pyrazole-5-carboxylic acid methyl ester (680mg, 100%). LC-MS (ESI +) 385M +1]⁺。

The reaction route of the step 3 is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -3- (thiophen-2-yl) -1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (680mg, 1.77mmol) was dissolved in anhydrous tetrahydrofuran (20mL), and LiBH was added₄(154mg, 1.08mmol), and stirred at room temperature for 3 hours. Adding dichloromethane (30mL) to dilute the reaction solution, adding 1N hydrochloric acid dropwise to adjust pH to about 7, washing the organic phase with water, washing with saturated salt water, drying, and performing column chromatography (PE: EA: 10:1) to obtain (1- (4-methoxybenzyl) -3- (thiophene-2-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol (208mg, yield 33%). LC-MS (ESI +) 357[ M +1]]⁺。

The reaction route of the step 4 is as follows:

the method comprises the following steps: reacting (1- (4-methoxybenzyl) -3- (thiophen-2-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol (208mg, 0.49mmol) was dissolved in dichloromethane (10mL), Dess-Martin reagent (312mg, 0.74mmol) was added, and the mixture was stirred at room temperature for 3 hours under nitrogen atmosphere. Concentrating the reaction solution, and performing column chromatography (eluting with dichloromethane) to obtain (1- (4-methoxybenzyl) -3- (thiophene-2-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) formaldehyde (173mg, 84%). LC-MS (ESI +) 355M +1]⁺。

Step 5 the reaction scheme is as follows:

the method comprises the following steps: (1- (4-methoxybenzyl) -3- (thiophen-2-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) formaldehyde (173mg, 0.49mmol) and aminocrotonitrile (88mg, 1.08mmol) were dissolved in glacial acetic acid (5mL), and the mixture was heated to 100 ℃ and stirred for 1 hour. Cooling to room temperature, concentrating the reaction solution under reduced pressure, and performing column chromatography on the residue (PE: EA ═ 6)1) obtaining 4- (1- (4-methoxybenzyl) -3- (thiophene-2-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (225mg, 95%). LC-MS (ESI +):484[ M +1]]⁺。

The reaction route of the step 6 is as follows:

the method comprises the following steps: 4- (1- (4-methoxybenzyl) -3- (thiophen-2-yl) -1H-thieno [3,2-c]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (215mg, 0.445mmol) was dissolved in trifluoroacetic acid (7.5mL), heated to 70 ℃, and stirred for 8 hours. The reaction mixture was concentrated to dryness, and the residue was dissolved in dichloromethane (30mL), washed with saturated sodium bicarbonate solution, washed with saturated brine, dried, concentrated, and subjected to column chromatography (dichloromethane: methanol ═ 50:1) to give 2, 6-dimethyl-4- (3- (thien-2-yl) -1H-thieno [3,2-c ] -2]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile (32mg, 20%). HPLC showed 95.54% purity; LC-MS (ESI +)364[ M +1]]⁺；¹H NMR(300MHz,CDCl₃)13.17(br,1H),9.74(br,1H),7.53(d,1H,J＝4.5),7.31(s,1H),7.13-7.18(m,2H),4.89(s,1H),2.06(s,6H)。

Synthetic preparation example 43

Preparation of 2, 6-dimethyl-4- (3-morpholino-1H-thieno [3,2-c ] pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile of formula (I-43)

The reaction route of the step 1 is as follows:

the method comprises the following steps: methyl 3-iodo-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylate (0.86g, 2mmol) (see synthetic preparation 34 for preparative procedures), morpholine (0.87g, 10mmol), potassium carbonate (1.38g, 10mmol), L-proline (68mg, 0.5mmol), cuprous iodide (0.38g, 2mmol) were mixed in a single-neck flask, anhydrous DMSO (5mL) was added, and stirred at 80 ℃ for 20 hours under nitrogen. Cooling to room temperature, adding water into the system, extracting with ethyl acetate, combining the extracts, drying with anhydrous sodium sulfate, concentrating, and separating by column chromatography to obtain 1- (4-methoxybenzyl) -3-morpholine-1H-thieno [3,2-c ] pyrazole-5-methyl formate as a yellow-green solid of 0.20g with a yield of 23%.

The reaction route of the step 2 is as follows:

the method comprises the following steps: under the protection of nitrogen, 1- (4-methoxybenzyl) -3-morpholine-1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (0.19g, 0.49mmol) is dissolved in anhydrous tetrahydrofuran (20mL), cooled to-20 deg.C, LiAlH is added₄(34mg, 0.89mmol) and stirred for 2 hours. Carefully dropwise adding water to quench the reaction, extracting dichloromethane, drying the dichloromethane phase with anhydrous sodium sulfate, and concentrating to obtain (1- (4-methoxybenzyl) -3-morpholine-1H-thieno [3,2-c ]]Pyrazol-5-yl) methanol 0.19g of crude product, crude yield 100%.

The reaction route of the step 3 is as follows:

the method comprises the following steps: (1- (4-methoxybenzyl) -3-morpholino-1H-thieno [3,2-c ] pyrazol-5-yl) methanol (0.19g, 0.49mmol) was dissolved in dry dichloromethane (10mL) under nitrogen protection, cooled to-5 ℃, Dess-Martin reagent (285mg, 0.67mmol) was added, and stirring was carried out for 2 hours. Saturated aqueous sodium bicarbonate solution was added dropwise to separate a dichloromethane phase, dried, concentrated, and purified by column chromatography (EA/PE ═ 1/4) to give 1- (4-methoxybenzyl) -3-morpholino-1H-thieno [3,2-c ] pyrazole-5-carbaldehyde as a yellow solid (110 mg, yield 63%).

The reaction route of the step 4 is as follows:

the method comprises the following steps: 1- (4-methoxybenzyl) -3-morpholino-1H-thieno [3,2-c ] pyrazole-5-carbaldehyde (357mg, 1.0mmol) was dissolved in glacial acetic acid (5mL), aminocrotonitrile (180mg, 2.2mmol) was added, and the mixture was heated to 90 ℃ and stirred for 1 hour. Acetic acid is removed by evaporation under reduced pressure, and the residual brown oily substance is a crude product of 4- (1- (4-methoxybenzyl) -3-morpholine-1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile, and the crude product is not purified and is directly used for the next step.

Step 5 the reaction scheme is as follows:

the method comprises the following steps: 4- (1- (4-methoxybenzyl) -3-morpholine-1H-thieno [3, 2-c) obtained in the above step]Trifluoroacetic acid (10mL) was added to the crude pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile, and the mixture was stirred at 80 ℃ overnight. Cooling to room temperature, evaporating the solvent under reduced pressure, cooling the residue with ice water, dropwise adding saturated aqueous sodium bicarbonate solution for alkalization, extracting with dichloromethane, extracting with ethyl acetate, combining the extracts, drying with anhydrous sodium sulfate, concentrating, and performing column chromatography (dichloromethane/methanol, 50/1-20/1) to obtain 2, 6-dimethyl-4- (3-morpholine-1H-thieno [3,2-c ])]Pyrazol-5-yl) -1, 4-dihydropyridine-3, 5-dicarbonitrile, 180mg of a yellow solid, yield 50%. MS of 367[ M + H]；¹H NMR(DMSO-d₆,300MHz)11.97(s,1H),9.67(s,1H),6.93(s,1H),4.78(s,1H),3.71(m,4H),3.16(m,4H),2.03(s,6H)。

Synthetic preparation example 44

Preparation of 6-methyl-8- (3-morpholino-1H-thieno [3,2-c ] pyrazol-5-yl) -2,3,4, 8-tetrahydro-1H-quinolizine-7, 9-dicarbonitrile of formula (I-44)

To synthesize 1- (4-methoxybenzyl) -3-morphinanyl-1H-thieno [3,2-c ] in preparation 43]The target compound can be prepared by adopting pyrazole-5-formaldehyde as a raw material and following the similar method. MS 407[ M + H ]]；¹H NMR(DMSO-d₆,300MHz)12.01(br,1H),6.93(s,1H),4.74(s,1H),3.72(m,4H),3.57(m,2H),3.18(m,4H),2.62(m,2H),1.75-1.63(m,4H)。

Synthetic preparation example 45

Preparation of 6-methyl-8- (3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) -1,3,4, 8-tetrahydropyrido [2,1-c ] [1,4] oxazine-7, 9-dicarbonitrile of structural formula (I-45)

The reaction route of the step 1 is as follows:

the method comprises the following steps: methyl 3-iodo-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylate (1.29g, 3mmol) (see synthetic preparation 34 for the preparation steps), N-methylpiperazine (0.905g, 9mmol), potassium carbonate (2.08g, 15mmol), L-proline (104mg, 0.9mmol), cuprous iodide (0.29g, 1.5mmol) were mixed in a single-neck flask, added DMSO (5mL) under nitrogen and stirred at 80 ℃ overnight. Cooling to room temperature, adding water into the system, extracting with ethyl acetate, combining the extract, drying with anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain 1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazole-5-methyl formate as a yellow-green solid 0.42g with the yield of 35%.

The reaction route of the step 2 is as follows:

the method comprises the following steps: under the protection of nitrogen, 1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c]Pyrazole-5-carboxylic acid methyl ester (250mg, 0.624mmol) is dissolved in anhydrous tetrahydrofuran (20mL), cooled to-20 deg.C, LiAlH is added₄And stirred for 2 hours. Carefully dropwise adding water to quench the reaction, extracting with dichloromethane, drying the dichloromethane phase with anhydrous sodium sulfate, and concentrating to obtain (1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol crude product 200mg, crude yield 74%.

The reaction route of the step 3 is as follows:

the method comprises the following steps: under nitrogen, (1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) methanol (200mg, 0.537mmol) was dissolved in dry dichloromethane (10mL), cooled to-5 ℃, Dess-Martin reagent (312mg, 0.736mmol) was added, and stirred for 2 hours. Saturated aqueous sodium bicarbonate solution was added dropwise, and the organic phase was separated, dried, concentrated, and purified by column chromatography (dichloromethane/methanol ═ 50/1) to give (1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) carbaldehyde as a yellow solid (144 mg) in 72% yield.

The reaction route of the step 4 is as follows:

the method comprises the following steps: to a solution of (1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) methanol (250mg, 0.675mmol) in methylene chloride (15mL) were added cyanoacetone sodium salt (78mg, 0.742mmol), piperidine (6mg,0.07mmol), acetic acid (51mg, 0.843mmol) and 4A molecular sieves (0.2g), and after completion, the mixture was heated to reflux and stirred overnight. The reaction was cooled, filtered, and the solid was washed with saturated sodium bicarbonate solution and water, dissolved in ethanol, insoluble material was filtered, the filtrate was concentrated to dryness, and the residue was subjected to column chromatography to give (E) -2- ((1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) methylene) -3-oxobutyronitrile (275mg, 94%).

Step 5 the reaction scheme is as follows:

the method comprises the following steps: 2- ((1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) methylene) -3-oxybutyronitrile (500mg, 1.15mmol) and 2- (morphol-3-enyl) acetonitrile (210mg,1.70mmol) were dissolved in glacial acetic acid (10mL) under nitrogen protection and stirred at 90 ℃ for 1 hour. Acetic acid was evaporated under reduced pressure and the residual brown oil was crude 8- (1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3,2-c ] pyrazol-5-yl) -6-methyl-1, 3,4, 8-tetrahydropyrido [2,1-c ] [1,4] oxazine-7, 9-dicarbonitrile, which was directly subjected to the next reaction without purification.

The reaction route of the step 6 is as follows:

the method comprises the following steps: the 8- (1- (4-methoxybenzyl) -3- (4-methylpiperazin-1-yl) -1H-thieno [3, 2-c) obtained in the above step]Pyrazol-5-yl) -6-methyl-1, 3,4, 8-tetrahydropyrido [2,1-c][1,4]Trifluoroacetic acid (10mL) was added to the crude oxazine-7, 9-dicarbonitrile and the mixture was stirred under reflux overnight. Cooling to room temperature, evaporating under reduced pressure to remove solvent, cooling the residue with ice water, adding saturated aqueous solution of sodium bicarbonate for alkalization, extracting with dichloromethane, and extracting with ethyl acetateThe extraction solutions are combined, dried by anhydrous sodium sulfate, concentrated and subjected to preparative TLC to obtain the target compound, 83mg of yellow solid and 17% yield in two steps. MS 422[ M + H]；¹H NMR(CDCl₃,300MHz)6.79(s,1H),4.57(m,3H),4.00(d,2H),3.52(d,2H),3.37(t,4H),2.57(t,4H),2.44(s,3H),2.27(s,3H)。

Synthesis preparation example 46:

preparation of 4- (3- (2-hydroxyethoxy) -1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile of structural formula (I-46)

The reaction route of the step 1 is as follows:

the method comprises the following steps: methyl 3-iodo-1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylate (1.29g, 3mmol) (see synthetic preparation 34 for the preparation steps), 3,4,7, 8-tetramethyl-1, 10-phenanthroline (472mg, 2mmol), cesium carbonate (4.89g, 15mmol), ethylene glycol mono tert-butyl ether (106mg, 0.9mmol), cuprous iodide (0.29g, 1.5mmol) were mixed in a single-neck bottle, toluene (5mL) was added, and stirring was performed overnight at 100 ℃ under nitrogen protection. After cooling to room temperature, water was added to the system, extraction was performed with ethyl acetate, and the extracts were combined, dried over anhydrous sodium sulfate, and concentrated to give a crude product of 3- (2-tert-butoxyethoxy) -1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazole-5-carboxylic acid as a yellow-green solid (3.4 g).

The reaction route of the step 2 is as follows:

the method comprises the following steps: 3- (2-tert-butoxyethoxyethoxy) obtained in the above step1- (4-methoxybenzyl) -1H-thieno [3,2-c ] yl]Dissolving the crude pyrazole-5-formic acid in anhydrous tetrahydrofuran (60mL), cooling to-20 ℃, and adding LiAlH₄(1.05g, 27.66mmol) and stirred for 2 hours. Carefully dropwise adding water to quench the reaction, extracting with dichloromethane, drying the dichloromethane phase with anhydrous sodium sulfate, concentrating, and performing silica gel column chromatography to obtain (3- (2-tert-butoxyethoxy) -1- (4-methoxybenzyl) -1H-thieno [3, 2-c)]Pyrazol-5-yl) methanol, 120mg of yellow solid, 10% yield in two steps.

The reaction route of the step 3 is as follows:

the method comprises the following steps: (3- (2-tert-butoxyethoxy) -1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) methanol (120mg, 0.307mmol) was dissolved in dry dichloromethane (10mL) under nitrogen protection, cooled to-5 ℃, Dess-Martin reagent (179mg, 0.42mmol) was added, and stirred for 2 hours. Saturated aqueous sodium bicarbonate solution was added dropwise to separate a dichloromethane phase, and the dichloromethane phase was dried, concentrated, and purified by column chromatography (dichloromethane/methanol ═ 50/1) to give 3- (2-t-butoxyethoxy) -1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) carbaldehyde as a yellow solid (100 mg) with a yield of 83%.

The reaction route of the step 4 is as follows:

the method comprises the following steps: under nitrogen, 3- (2-tert-butoxyethoxy) -1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) carbaldehyde (100mg, 0.257mmol) was dissolved in glacial acetic acid (5mL), aminocrotonitrile (47mg, 0.566mmol) was added, and the mixture was heated to 90 ℃ and stirred for 1.5 hours. Acetic acid was evaporated under reduced pressure and the residual oil was crude 4- (3- (2-tert-butoxyethoxy) -1- (4-methoxybenzyl) -1H-thieno [3,2-c ] pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile which was directly subjected to the next step without purification.

Step 5 the reaction scheme is as follows:

the method comprises the following steps: 4- (3- (2-tert-butoxyethoxy) -1- (4-methoxybenzyl) -1H-thieno [3, 2-c) obtained in the above step]Trifluoroacetic acid (10mL) was added to the crude pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile, and the mixture was stirred under reflux overnight. Cooling to room temperature, evaporating solvent under reduced pressure, cooling the residue with ice water, adding saturated aqueous sodium bicarbonate solution for alkalization, extracting with dichloromethane, extracting with ethyl acetate, mixing the extractive solutions, drying with anhydrous sodium sulfate, concentrating, and separating by TLC to obtain white solid 4- (3- (2-hydroxyethoxy) -1H-thieno [3,2-c ]]Pyrazol-5-yl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarbonitrile (28mg, 32% yield over two steps). MS:342[ M + H ]]；¹H NMR(CDCl₃300MHz)：12.07(s,1H),9.69(s,1H),6.93(s,1H),4.77(s,1H),4.18(t,2H),3.68(t,2H),2.03(s,6H)。

Biological test example 1: in vitro inhibition of c-Met enzyme by compounds

The inhibition of the c-Met enzyme by compounds in vitro was determined using the method of Mobility Shift Assay.

Experimental methods

1. 1.25 Xkinase buffer solution and stop solution are prepared

1.1 containing no MnCl₂62.5mM HEPES kinase buffer, pH 7.5;

0.001875％Brij-35；

12.5mM MgCl₂；

2.5mM DTT；

1.2 containing MnCl₂1.25 times the kinase buffer.

62.5mM HEPES,pH 7.5；

0.001875％Brij-35；

12.5mM MgCl₂；

12.5mM MnCl₂；

2.5mM DTT；

1.3 stop solution

100mM HEPES,pH 7.5；

0.015％Brij-35；

0.2％Coating Reagent#3；

50mM EDTA。

2. Preparation of Compound solution

2.1 dilution of the Compound

20. mu.L of 50mM compound and 80. mu.L of 100% DMSO were added to an EP tube to prepare 100. mu.L of 10mM compound. Another EP tube was filled with 30. mu.L of 10mM compound and 70. mu.L of 100% DMSO to prepare 100. mu.L of 3mM compound.

In the second well of the 96-well plate, 95. mu.L of 100% DMSO and 5. mu.L of 3mM compound were added, and the other wells 60. mu.L of 100% DMSO were added. mu.L of compound from well 2 was added to well 3, and 3-fold dilutions were made sequentially down for a total of 10 concentrations. The compound concentration ranged from 150uM to 7.6 nM.

2.2 transfer of 5 times the amount of Compound to the reaction plate

10. mu.L of each of the above 96-well plates was transferred to another 96-well plate, and 90. mu.L of ultrapure water was added thereto. Thus, the second to eleventh wells were compounds dissolved in 10% DMSO, and the first and twelfth wells were 10% DMSO. From the above 96-well plate, 5. mu.L of the solution was transferred to a 384-well reaction plate. Thus, 5 μ L of 5-fold compound dissolved in 10% DMSO and 5 μ L of 10% DMSO were present in 384-well plates. mu.L of 250mM EDTA was added to the negative control wells.

3. Kinase reaction

3.1 preparation of 2.5 times enzyme solution

The kinase was added to 1.25 fold kinase buffer to form a 2.5 fold enzyme solution.

3.2 preparation of 2.5 times the substrate solution

FAM-labeled polypeptide and ATP were added to 1.25 fold kinase buffer to form a 2.5 fold substrate solution.

3.3 addition of enzyme solution to 384-well plates

There were 5 μ L of 5-fold compounds dissolved in 10% DMSO in 384-well plates.

mu.L of a 2.5-fold enzyme solution was added to the 384-well reaction plate.

Incubate for 10 minutes at room temperature.

3.4 addition of substrate solution to 384-well plates

mu.L of a 2.5-fold substrate solution was added to the 384-well reaction plate.

3.5 kinase reaction and termination

Incubate at 28 ℃ for 1 hour. The reaction was stopped by adding 25. mu.L of stop solution.

Reading data by a Caliper

Conversion data were read on the Caliper.

5. Inhibition rate calculation

Conversion data was copied from the Caliper. The conversion was converted to inhibition data. Wherein max refers to the conversion rate of a DMSO control, and min refers to the conversion rate of an enzyme-free control.

Inhibition rate (max-conversion)/(max-min) × 100

Biological test example 1: results of the experiment

Remarking: "+" represents 500nM<IC₅₀<5 uM; "+ +" represents 50nM<IC₅₀<500 nM; "+ + + +" represents IC₅₀<50nM。

Claims

1. Dihydropyridine compounds, characterized by having the following general formula (I):

wherein,

R₁selected from the group consisting of hydrogen, methyl, ethyl, methoxy, amino, hydroxyethoxy, pyridyl, thienyl, morpholinyl, and mixtures thereof,

R₂Is selected from cyano;

R₃is selected from C₁-C₆Alkyl, methoxyethyl, phenyl which may be substituted by fluorine, methoxy, pyridine; or, R₂And R₃And the carbon atoms to which they are attached form a ring

R₄Selected from hydrogen, methyl, ethyl, benzyl; or, R₄And R₃And form a piperidine ring and a morpholine ring;

R₅selected from methyl or p-fluorophenyl; or, R₅And R₄And form morpholine ring and piperidine ring.

2. The dihydropyridines according to claim 1, wherein the compound of the general formula (I) is one selected from the group consisting of the following compounds (I-1) to (I-46):

3. dihydropyridines according to claim 1, characterized in that the compounds of the general formula (I) are present in the form of pharmaceutically acceptable salts.

4. A dihydropyridine compound as claimed in claim 3 wherein the pharmaceutically acceptable salt is the hydrochloride, sulphate, phosphate, acetate, trifluoroacetate, methanesulphonate, trifluoromethanesulphonate, p-toluenesulphonate, tartrate, maleate, fumarate, succinate or malate salt of the compound of formula (I).

5. A process for the preparation of dihydropyridines according to claim 1, wherein the compounds of the general formula (I) are prepared from the corresponding intermediates of the formula (II) by reaction of the following scheme 1,

route 1:

wherein R is₁，R₂，R₃，R₄，R₅As defined in claim 1; PG is an amine protecting group.

6. The process according to claim 5, wherein PG is t-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyl, 3, 4-dimethoxybenzyl.

7. The process of claim 5, wherein R is present In Intermediate (II)₁In the case of H, the preparation is shown in scheme 2: nitrifying 5-methylthiophene-2-formic acid with fuming nitric acid to obtain an intermediate of a structural formula 2, esterifying the intermediate of the structural formula 2 into an intermediate of a structural formula 3, reducing nitro of the intermediate of the structural formula 3 into amino by adopting reduced iron powder to obtain an intermediate of a structural formula 4, and cyclizing the intermediate of the structural formula 4 into an intermediate of a structural formula 5 in the presence of acetic anhydride and nitrite; removing acetyl of the intermediate of the structural formula 5 to obtain an intermediate of a structural formula 6, reducing ester groups in the intermediate of the structural formula 6 into alcohol groups to obtain an intermediate of a structural formula 7, and oxidizing the intermediate of the structural formula 7 to obtain an intermediate of a structural formula (II);

route 2:

8. the process of claim 5, wherein R is present In Intermediate (II)₁When H is not present, the preparation is as shown in scheme 3: nitrifying 5-methylthiophene-2-formic acid with fuming nitric acid to obtain an intermediate of a structural formula 2, esterifying the intermediate of the structural formula 2 into an intermediate of a structural formula 3, reducing nitro of the intermediate of the structural formula 3 into amino by adopting reduced iron powder to obtain an intermediate of a structural formula 4, and cyclizing the intermediate of the structural formula 4 into an intermediate of a structural formula 5 in the presence of acetic anhydride and nitrite; removing acetyl of the intermediate of the structural formula 5 to obtain an intermediate of a structural formula 6, and iodinating the intermediate 6 of the structural formula 6 to obtain an intermediate of a structural formula 8; protecting pyrazole amino of the intermediate of the structural formula 8 with an amino protecting group to obtain an intermediate of a structural formula 9; then, coupling by using iodine of the intermediate of the structural formula 9 to obtain an intermediate of a structural formula 10; reducing the ester group of the intermediate of the structural formula 10 into an alcohol group to obtain an intermediate of the structural formula 11, and oxidizing the alcohol group in the intermediate of the structural formula 11 into an aldehyde group to obtain an intermediate of the structural formula (II);

route 3:

9. the method of claim 8, wherein the coupling is selected from the group consisting of a Suzuki coupling, a Buchwald coupling.

10. The process according to claim 5, wherein when the compound to be prepared is a compound of formula (Ia) and R' ═ R₃＝R₅Its preparation is shown in scheme 4:

route 4:

11. the process according to claim 5, wherein when the compound to be prepared is a compound of formula (Ib) and R is₃And R₅In different cases, the preparation method is shown in scheme 5:

route 5:

12. the process according to claim 5, wherein when the compound to be produced is a compound of the formula (Ic), R is₃And R₅Is different and R₄When H is excluded, the preparation is as shown in scheme 6:

route 6:

13. the process of claim 5, wherein when the compound to be prepared is of the formula (Id), the process is as shown in scheme 7:

route 7:

wherein X is CH₂Or O.

14. The process according to claim 5, wherein when the compound to be prepared is a compound of the formula (Ie), the process is as shown in scheme 8:

route 8:

15. the process according to claim 5, wherein when the compound to be prepared is a compound of the formula (If), the process is as shown in scheme 9:

route 9:

wherein X is CH₂Or O.

16. A pharmaceutical composition comprising a dihydropyridine compound of claim 1 wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable excipient.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition is formulated as a tablet, capsule, aqueous suspension, oily suspension, dispersible powder, granule, lozenge, emulsion, syrup, cream, ointment, suppository, or injection.

18. A pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of general formula (I) according to claim 1 and a pharmaceutically acceptable excipient.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is formulated as a tablet, capsule, aqueous suspension, oily suspension, dispersible powder, granule, lozenge, emulsion, syrup, cream, ointment, suppository, or injection.

20. Use of a dihydropyridine compound of claim 1 in the preparation of a preparation for modulating the catalytic activity of a protein kinase; the protein kinase is c-Met receptor tyrosine kinase.

21. Use of a pharmaceutically acceptable salt of a dihydropyridine compound of claim 1 in the preparation of a preparation for modulating the catalytic activity of a protein kinase; the protein kinase is c-Met receptor tyrosine kinase.

22. Use of a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment of a protein kinase related disease; the protein kinase is c-Met receptor tyrosine kinase.

23. The use according to claim 22, wherein the protein kinase related disease is cancer.

24. The use of claim 23, wherein the cancer is thyroid cancer, colorectal cancer, gastric cancer, renal cancer, liver cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, bladder cancer, head and neck cancer, pancreatic cancer, gall bladder cancer, osteosarcoma, rhabdomyosarcoma, malignant fibrous histiocytoma, fibrosarcoma, glioblastoma, astrocytoma, melanoma, or mesothelioma.

25. Use of a pharmaceutical composition according to claim 18 for the manufacture of a medicament for the treatment of a disease associated with a protein kinase; the protein kinase is c-Met receptor tyrosine kinase.

26. The use according to claim 25, wherein the protein kinase related disease is cancer.

27. The use of claim 26, wherein the cancer is thyroid cancer, colorectal cancer, gastric cancer, renal cancer, liver cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, bladder cancer, head and neck cancer, pancreatic cancer, gall bladder cancer, osteosarcoma, rhabdomyosarcoma, malignant fibrous histiocytoma, fibrosarcoma, glioblastoma, astrocytoma, melanoma, or mesothelioma.