CN112898314A

CN112898314A - Preparation and application of deubiquitinase inhibitor

Info

Publication number: CN112898314A
Application number: CN202011232945.8A
Authority: CN
Inventors: 刘丽萍
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2021-06-04

Abstract

The invention aims to provide a compound with single/double inhibitory activity on USP25 and USP28 protease, and provides a compound shown as the following formula I, and racemate, stereoisomer, isotopic marker, nitrogen oxide, solvate, polymorph, metabolite, pharmaceutically acceptable salt or prodrug thereof:

Description

Preparation and application of deubiquitinase inhibitor

Technical Field

The invention relates to the field of medicines, in particular to a preparation method and application of a deubiquitinase inhibitor.

Background

After the autoproteasome inhibitors bortezomib and carfilzomib were approved by the U.S. FDA for the treatment of multiple myeloma in 2003 and 2012, respectively, the ubiquitin-protease system became a hot target for anticancer drugs. The ubiquitin-proteasome pathway (UPP) mediates selective degradation of cellular proteins by ubiquitination, which labels ubiquitin on a target protein, which is recognized and degraded by the 26S proteasome. Ubiquitination is a reversible process, regulated by both ubiquitination and deubiquitination enzymes. Deubiquitination (deubiquitination) is a process of hydrolyzing ubiquitin labeled on a target protein, and deubiquitinase is a key molecule for catalyzing deubiquitination.

Deubiquitinating enzymes (DUBs) are mainly divided into 6 families: ubiquitin C-terminal hydrolases (UCH) family, Ubiquitin Specific Proteases (USP) family, ovarian cancer proteases (OTU) family, mjd (josephin domain) family, JAMM (JAB1/MPN/Mov34 metalloenzyme) family, and monocyte chemoattractant protein-induced protein (MCPIP) family. Ubiquitin (Ub), a small molecule protein widely distributed in the nucleus and cytoplasm of eukaryotic cells, consists of 76 amino acids with a molecular weight of approximately 8.45kDa (Proc. Natl. Acad. Sci.,1985,82(11): 3582-5; J.mol. biol.,2001,314(4): 773-87). The Gly at the C terminal can be connected with the Lys side chain of the target protein, and the main function of the Gly is to label the protein to be hydrolyzed, so that the labeled protein is recognized and degraded by 26s proteasome. Ubiquitination (ubiquitination) modification of proteins refers to a multi-step cascade of protein modification processes including ubiquitin Ub, ubiquitin activating enzyme E1(ubiquitin-activating enzyme), ubiquitin binding enzyme E2(ubiquitin-conjugating enzyme), ubiquitin ligase E3 (ubiquitin ligand), and proteasome (26S) (Cell Death dis.,2018, 9). Firstly, under the action of ATP, ubiquitin Ub connects carboxyl of C terminal Gly with sulfhydryl of ubiquitin activating enzyme E1 to form active thioester bond; secondly, the activated ubiquitin Ub is transferred from the ubiquitin activating enzyme E1 to the active center of the ubiquitin conjugated enzyme E2; thirdly, combining ubiquitin conjugating enzyme E2 with ubiquitin ligase E3 for recognizing substrate S, and connecting carboxyl of Gly in ubiquitin molecules with sulfhydryl of Lys in substrate S to realize transfer of ubiquitin Ub to substrate S; then, other ubiquitin Ub is attached with Gly to Lys side chain in ubiquitin molecule already bound with substrate S, thereby forming polyubiquitin chain. The protein marked by polyubiquitin chains is identified by 26S proteasome, degraded into polypeptide and releases ubiquitin molecules, thereby realizing the selective degradation of the protein.

In 2001 Rebeca Valero et al found a gene encoding ubiquitin specific protease 28(ubiquitin specific protease 28, USP28) on human chromosome 11 (Genome biol.,2001,2 (10)). USP28 protease is a member of Ubiquitin Specific Proteases (USP) family, and is overexpressed in non-small cell lung cancer, breast cancer, colon cancer, bladder cancer and gastric cancer (J.cell. mol.Med.,2015,19(4): 799-805; mol.cancer Res.,2018,16(6): 1000-1012; nat.cell biol., 2007,9(7): 765-771; J.cell. biochem.,2019,120(5): 765-7667-7666), which indicates that USP28 protease may be closely related to tumor development.

Myc protein acts as a transcription factor and is able to activate the expression of genes associated with cell growth and proliferation (Annu. Rev. cell Dev. biol.2000, 16,653; Biochim. Biophys. acta,2015,1849,506). Fbw7 acts as the primary E3 ligase for Myc protein, promoting ubiquitination and degradation of Myc protein (Curr. biol.2004,14,1852; EMBO J.2004,23,2116). Whereas USP28 protease stabilizes Myc protein by modulating Fbw7 mediated degradation (Cell,2006,126(3): 529-542); the deletion of mouse USP28 gene can promote the degradation of Fbw7, the over-expression of USP28 protease can stabilize Fbw7 and its substrate, and promote the oncogenic transformation (Cell Reports,2014,9(3): 1099-1109). Thus, inhibition of USP28 protease can indirectly decrease the stability of Myc protein, thereby inhibiting proliferation of tumor cells.

Histone demethylase LSD1 plays an important role in gene expression regulation (curr. opin. chem. biol.2007,11,561; Epigenomics 2016,8,1103), overexpression phenomenon exists in a large number of malignant tumors, and LSD1 protease is considered to play a very important role in maintenance of Tumor stem cells (hum. pathol.2012,43: 1300; Tumor biol.2013,34: 173; j.ovarian res.2013,6: 75; fertil.steril.2014,101: 740; int.j.gynecol.cancer,2015,25: 1453). In breast cancer cells, USP28 protease can stabilize the LSD1 protein by deubiquitination (Cell Reports,2013,5(1): 224-. Thus, inhibition of USP28 protease can indirectly decrease the stability of LSD1 protein, thereby inhibiting the proliferation of tumor cells.

While mice that lack USP28 show normal longevity, immune function and radiation response by USP 28-related gene knockout (J.Clin.invest.2014,124: 3407; mol.cell.biol.,2014,34(11): 2062-2074). Indicating that the USP28 protease is not essential in mice.

These studies revealed that USP28 protease may be a potentially desirable target for anti-tumor drugs. The USP28 protease inhibitor prevents target protein marked by ubiquitination from being deubiquitinated by inhibiting the deubiquitinating activity of USP28 protease, so that the target protein is excessively degraded, and finally tumor cells are killed. USP28 protease inhibitors, particularly reversible inhibitors thereof, may be a highly desirable class of antineoplastic agents.

Before the gene encoding USP28 was discovered, the gene encoding ubiquitin specific protease 25 (USP 25) was found by Valero et al on human chromosome 21 earlier than 1999. The USP25 protease, a member of the ubiquitin-specific protease family, is involved in various important physiological processes such as myogenesis, endoplasmic reticulum protein degradation, and IL-17-mediated immune system regulation, and has close relation with the development of cancer (Cell mol. Life Sci.,2006,63: 723-675; PLoS ONE,2012,7: e 36542; nat. immunity, 2012,13: 1110-1117; exp. Cell Res.,2010,316: 667-675). Indicating that the USP25 protease may also be closely related to the development of tumors.

Disclosure of Invention

in the formula:

x is CR₅Or N;

y is CR₆Or N;

z is

The dashed bond representation may be a bond or absent;

a is CR₁₀Or N; b is CR₁₁(ii) a c is CR₁₂、NR₁₃O or S; d is CR₁₄Or N;

m is 0, 1,2, 3 or 4;

n is 1,2, 3,4, 5,6, 7 or 8;

p is 0, 1 or 2;

R₁、R₄、R₅and R₁₀-R₁₄Independently of one another, from hydrogen, unsubstituted or substituted (C)₁-C₁₂) Hydrocarbyl, unsubstituted or substituted (C)₁- C₁₂) Hydroxy, unsubstituted or substituted (C)₁-C₁₂) Amino or halogen; said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic hydrocarbon groups or aromatic hydrocarbon groups; the halogen is fluorine, chlorine, bromine or iodine;

R₂and R₃Independently of one another, from hydrogen, unsubstituted or substituted (C)₁-C₁₂) Hydrocarbyl, unsubstituted or substituted (C)₁-C₁₂) Hydroxy or unsubstituted or substituted (C)₁-C₁₂) An amino group; said unsubstituted or substituted (C)₁-C₁₂) An aliphatic or aromatic hydrocarbon group consisting of 1 to 12 carbon atoms and corresponding hydrogen atoms, (C) said substituted₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic hydrocarbon groups or aromatic hydrocarbon groups;

R₇and R₉Independently of one another, from hydrogen, or from hydrogenSubstituted or substituted (C)₁-C₁₂) Hydrocarbyl, unsubstituted or substituted (C)₁-C₁₂) Hydroxy or unsubstituted or substituted (C)₁-C₁₂) An amino group; said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, including nitrogen heterocycle and oxygen heterocycle, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic hydrocarbon groups or aromatic hydrocarbon groups; the R is₇And R₉May be selected from 3-20 membered heterocyclic group containing one, two or more N or 5-20 membered heteroaryl group, which is unsubstituted or optionally substituted with one, two or more nitrogen heterocyclic ring, oxygen heterocyclic ring, further, 3-10 membered heterocyclic group containing only one or two N as hetero atom is preferable;

R₈is hydrogen, unsubstituted or substituted (C)₁-C₁₂) Hydrocarbyl, unsubstituted or substituted (C)₁-C₁₂) Hydroxy, unsubstituted or substituted (C)₁- C₁₂) Amino or halogen; said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic hydrocarbon groups or aromatic hydrocarbon groups; when the dotted bond is a single bond, R₈May be O;

according to an embodiment of the invention, said "free of said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals "in which nitrogen, oxygen, sulfur, phosphorus and halogen may be present in (C)₁-C₁₂) Of aliphatic hydrocarbon radicals(C) in any position of the chain or branch, optionally in a straight or branched chain₁-C₁₂) Preferably may be (C)₁-C₁₀) Aliphatic hydrocarbon group, (C)₁-C₈) Aliphatic hydrocarbon radical or (C)₁-C₆) An aliphatic hydrocarbon group; for example, it may be selected from the following groups: (C)₁-C₆) Aliphatic hydrocarbon group (C)₁-C₆) Aliphatic hydrocarbyloxy, N- (C)₁-C₆) Aliphatic hydrocarbyl amino, N-di- (C)₁-C₃) Aliphatic hydrocarbyl amino group, (C)₁-C₆) Aliphatic hydrocarbyl mercapto, halo (C)₁-C₆) Aliphatic hydrocarbon group, halo (C)₁-C₆) Aliphatic hydrocarbyloxy, (N-mono-or disubstituted) halo (C)₁-C₆) Aliphatic hydrocarbyl amino, halo (C)₁-C₆) Aliphatic hydrocarbyl mercapto group, (C)₁-C₆) Aliphatic hydrocarbyloxy (C)₁-C₆) Aliphatic hydrocarbon group, (C)₁-C₆) Aliphatic hydrocarbyl mercapto group (C)₁-C₆) Aliphatic hydrocarbon group, N- (C)₁-C₆) Aliphatic hydrocarbyl amino group (C)₁-C₆) Aliphatic hydrocarbon group, N-di- (C)₁-C₃) Aliphatic hydrocarbyl amino group (C)₁-C₆) Aliphatic hydrocarbon groups such as methyl, deuterated methyl, ethyl, propyl, isopropyl, cyclopropyl, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N-dimethylaminomethyl, N-dimethylaminoethyl, N-diethylaminoethyl; according to an embodiment of the invention, R₂Can be selected from the group consisting of methyl, deuterated methyl, ethyl, propyl, butyl, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N, N-dimethylaminomethyl, N, N-dimethylaminoethyl, N, N-diethylaminoethyl.

The invention further provides a preparation method of the compound shown in the general formula I, which comprises the following steps:

firstly, respectively preparing an intermediate carboxylic acid A and an intermediate amine B, then carrying out condensation reaction on the intermediate carboxylic acid A and the intermediate amine B to form amide, and then removing a protecting group to obtain a target compound I.

Preparation of intermediate carboxylic acid A

Tert-butyl bromoacetate (chloro) and potassium thioacetate are directly substituted in solvents such as DMF, EtOH and the like to generate 2-acetylthioacetic acid tert-butyl ester; 2-acetyl tert-butyl thioacetate and chlorocyanopyridine/pyrimidine/pyrazine starting material A1-1 using anhydrous DMF as solvent in K₂CO₃、NaOMe、NaOEt、^tReacting at room temperature to 60 ℃ for 1-4 h under the action of alkali such as BuOK and the like to obtain a condensation product A1-2; 3-aminothienopyridine/pyrimidine/pyrazine-2-carboxylic acid tert-butyl ester A-3 is subjected to trifluoroacetic anhydride trifluoroacetylation, sodium hydride hydrogen extraction, methyl iodide or methyl p-toluenesulfonate methylation, and tert-butyl removal by trifluoroacetic acid or hydrochloric acid to obtain an intermediate A1.

Preparation of intermediate amine B

According to an embodiment of the present invention, in addition to the commercially available intermediate amine B, another portion of intermediate B may be prepared by the following steps: under the protection of protecting groups such as Bn, Boc and the like, halogenated aromatic amine and halogenated aromatic ethylamine are subjected to coupling reaction with nitrogen-containing fragments or activated carbon atoms, and then intermediate amine B is obtained through deprotection; or nitro-halogenated aromatic hydrocarbon is substituted or catalytically coupled, and then nitro is reduced to amino to obtain an intermediate amine B. Taking Z as p-bromophenyl and n ═ 2 as an example, the synthetic route is as follows:

halogen (bromine and iodine) substituted aromatic ethylamine B1-1 is protected by benzyl to generate an intermediate B1-2, then the intermediate B1-2 and nitrogen heterocycle are subjected to catalytic coupling to obtain a coupling product intermediate B1-3, and finally, the phenylethylamine intermediate B1 is obtained by hydrogenation deprotection.

Preparation of the target Compound I

According to the embodiment of the invention, the obtained intermediate carboxylic acid A and the intermediate amine B are subjected to condensation reaction to obtain a condensation product, and the trifluoroacetyl group is removed to obtain the target compound I.

Unless otherwise defined, X, Y, Z, R are as defined above₁,R₂,R₃,R₄M and n are defined as formula I.

The present invention also provides a pharmaceutical composition comprising a compound of formula (I), its racemate, stereoisomer, isotopic label, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug as an active ingredient.

According to an embodiment of the present invention, the pharmaceutical composition further comprises a therapeutically effective amount of the compound of formula I or an optical isomer, nitrogen oxide, solvate, pharmaceutically acceptable salt or prodrug thereof and a pharmaceutically acceptable carrier.

The carrier in the pharmaceutical composition is "acceptable" in that it is compatible with (and preferably capable of stabilizing) the active ingredient of the composition and is not deleterious to the subject being treated. One or more solubilizing agents may be used as pharmaceutical excipients for the delivery of the active compound.

The invention further provides the use of a compound of formula (I), racemates, stereoisomers, isotopic labels, nitric oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof, or the use of said pharmaceutical composition in the manufacture of a medicament for the treatment of a disease or disorder associated with the inhibition of USP 28.

The invention further provides the use of a compound of formula (I), racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof or said pharmaceutical composition for the manufacture of a medicament for the treatment of a disease or disorder associated with the inhibition of USP28, USP25 protease.

The invention further provides the use of a compound of formula (I), racemates, stereoisomers, isotopic labels, nitric oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof, or the use of said pharmaceutical composition in the manufacture of a medicament for the treatment of a disease or disorder associated with the inhibition of USP25 and USP 28.

The present invention also provides a method of treating or preventing a disease or disorder associated with modulation of USP28 and/or USP25, which method comprises administering to a patient suffering from at least one of said diseases or disorders a compound of formula (I), racemates, stereoisomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof.

According to embodiments of the invention, the USP25 and/or USP28 related diseases or disorders include cancer, inflammation, autoimmune disease, viral infection and bacterial infection.

According to an embodiment of the invention, the pharmaceutical composition may be in a form suitable for oral administration, such as a tablet, troche, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule, or syrup or elixir. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of: sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a pleasant to the eye and palatable pharmaceutical preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be inert excipients, granulating agents, disintegrating agents, binding agents, and lubricating agents. These tablets may be uncoated or they may be coated by known techniques which mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

According to an embodiment of the invention, the pharmaceutical composition wherein the active ingredient is provided in oral formulation with an inert solid diluent or a soft gelatin capsule wherein the active ingredient is mixed with a water soluble carrier or an oil vehicle; aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending, dispersing or wetting agents. The aqueous suspensions may also contain one or more preservatives, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents; oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, or in a mineral oil. The oil suspension may contain a thickener. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable preparation. These compositions can be preserved by the addition of antioxidants; dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent or one or more preservatives. Suitable dispersing or wetting agents and suspending agents are illustrative of the examples given above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

According to an embodiment of the invention, the pharmaceutical composition may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, or a mineral oil or a mixture thereof. Suitable emulsifiers may be naturally occurring phospholipids, and the emulsions may also contain sweetening, flavoring, preservative and antioxidant agents. Such formulations may also contain a demulcent, a preservative, a colorant and an antioxidant.

According to an embodiment of the invention, the pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles or solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase, and the injection or microemulsion may be injected into the bloodstream of a patient by local mass injection. Alternatively, it may be desirable to administer the solutions and microemulsions in a manner that maintains a constant circulating concentration of the compounds of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is an intravenous pump model Deltec CADD-PLUS. TM.5400.

According to an embodiment of the invention, the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. The 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 parenterally-acceptable, non-toxic diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any blend fixed oil may be used for this purpose. In addition, fatty acids can also be prepared into injections.

According to embodiments of the invention, the compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and therefore will melt in the rectum to release the drug.

As is well known to those skilled in the art, the dosage of a drug administered depends on a variety of factors, including, but not limited to: the activity of the particular compound employed, the age of the patient, the weight of the patient, the health condition of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like; in addition, the optimal treatment regimen, such as the mode of treatment, the daily amount of compound (I) of the formula or the type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

Interpretation of terms:

unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present application, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. The definitions of the groups and the structures of the compounds in such combinations and after the combination are within the scope of the present specification.

Where a range of numerical values is recited in the specification and claims herein, and where the range of numerical values is defined as an "integer," it is understood that the two endpoints of the range are recited and each integer within the range is recited. For example, "an integer of 0 to 6" should be understood to describe each integer of 0, 1,2, 3,4, 5, and 6. "more" means three or more.

The term "halogen" refers to F, Cl, Br and I. In other words, F, Cl, Br, and I may be described as "halogen" in the present specification.

The term "aliphatic hydrocarbon group" includes saturated or unsaturated, straight-chain or branched chain or cyclic hydrocarbon group, the type of the aliphatic hydrocarbon group may be selected from alkyl, alkenyl, alkynyl, etc., the number of carbon atoms of the aliphatic hydrocarbon group is preferably 1 to 12, and may also be 1 to 10, and further preferably ranges from 1 to 6, and specifically may include but is not limited to the following groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 1-ethylethenyl, 1-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 1-hexenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 1-methyl-2-propynyl, 3-butynyl, 1-pentynyl, 1-hexynyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; the aliphatic hydrocarbon group may optionally include one or more other suitable substituents. Examples of such substituents may include hydroxy, halogen, cyano, amino and like groups, for example the aliphatic hydrocarbon group may contain one, two or more halogens, meaning that one, two or more hydrogen atoms of the aliphatic hydrocarbon group may be substituted with an equivalent number of halogens. If the hydrocarbyl group contains more than one carbon, those carbons do not necessarily have to be linked to each other. For example, at least two carbons may be linked via a suitable element or group. That is, the aliphatic hydrocarbon group may optionally contain one, two or more heteroatoms (or be construed as an optional insertion of a heteroatom into the aliphatic hydrocarbon group, optionally a C-C bond and a C-H bond). Suitable heteroatoms will be apparent to those skilled in the art and include, for example, sulfur, nitrogen, oxygen, phosphorus and silicon. The aliphatic hydrocarbyl group containing a heteroatom may be selected fromThe following groups: (C)₁-C₆) Aliphatic hydrocarbyloxy, (C)₁- C₆) Aliphatic hydrocarbyl mercapto, halo (C)₁-C₆) Aliphatic hydrocarbon group, halo (C)₁-C₆) Aliphatic hydrocarbyloxy, halo (C)₁-C₆) Aliphatic hydrocarbyl thio group, (C)₁-C₆) Aliphatic hydrocarbyloxy (C)₁-C₆) Aliphatic hydrocarbon group, (C)₁-C₆) Aliphatic hydrocarbyl mercapto group (C)₁-C₆) Aliphatic hydrocarbon group, N- (C)₁-C₃) Aliphatic hydrocarbyl amino group (C)₁-C₆) Aliphatic hydrocarbon group, N-di- (C)₁-C₃) Aliphatic hydrocarbyl amino group (C)₁-C₆) Aliphatic hydrocarbon groups such as methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N-dimethylaminomethyl, N-dimethylaminoethyl, N-diethylaminoethyl; the "aliphatic hydrocarbon group" moiety contained in the other groups is as explained above.

The term "3-20 membered heterocyclyl" means a saturated monovalent monocyclic or bicyclic hydrocarbon ring comprising 1-5 heteroatoms independently selected from N, O and S, preferably "3-10 membered heterocyclyl". The term "3-10 membered heterocyclyl" means a saturated monovalent monocyclic or bicyclic hydrocarbon ring comprising 1-5, preferably 1-3 heteroatoms selected from N, O and S. The heterocyclic group may be attached to the rest of the molecule through any of the carbon atoms or nitrogen atom (if present). In particular, the heterocyclic group may include, but is not limited to: 4-membered rings such as azetidinyl, oxetanyl; 5-membered rings such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclic group may be benzo-fused. The heterocyclyl group may be bicyclic, for example but not limited to a 5,5 membered ring, such as a hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl ring, or a 5,6 membered bicyclic ring, such as a hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl ring. The nitrogen atom containing ring may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, 2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4] thiadiazinyl, 4, 5-dihydrooxazolyl, or 4H- [1,4] thiazinyl, or it may be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. Unless otherwise indicated, heterocyclyl or heteroaryl includes all possible isomeric forms thereof, e.g. positional isomers thereof. Thus, for some illustrative, non-limiting examples, pyridyl or pyridinylene includes pyridin-2-yl, pyridinylene-2-yl, pyridin-3-yl, pyridinylene-3-yl, pyridin-4-yl, and pyridinylene-4-yl; thienyl or thienylene includes thien-2-yl, thien-3-yl and thien-3-yl.

In any of the methods for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by conventional protecting groups, as described in textbooks or tool books in the art. The protecting group may be removed at a convenient subsequent stage using methods known in the art. One skilled in the art will recognize that other reagents may be used for this deprotection step, depending on the particular protecting group, including but not limited to Pd/C, Pd (OH)₂、PdCl₂、Pd(OAc)₂/Et₃SiH, raney nickel, a suitably selected acid, a suitably selected base, fluoride, and the like.

The target compound may be isolated according to known methods, for example by extraction, filtration, column chromatography, FCC or preparative HPLC.

Depending on their molecular structure, the compounds of the invention may be chiral and may therefore exist in various enantiomeric forms. These compounds may thus be present in racemic or optically active form. The compounds of the invention or intermediates thereof may be separated into enantiomeric compounds by chemical or physical methods well known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are prepared from mixtures by reaction with optically active resolving agents. Examples of suitable resolving agents are optically active acids such as the R and S forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g. N-benzoylproline or N-benzenesulfonylproline) or various optically active camphorsulphonic acids. The chromatographic enantiomeric resolution can also advantageously be carried out with the aid of optically active resolving agents, such as dinitrobenzoylphenylglycine, cellulose triacetate or other carbohydrate derivatives or chirally derivatized methacrylate polymers, which are immobilized on silica gel. Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, for example hexane/isopropanol/acetonitrile.

Those skilled in the art will appreciate that not all nitrogen-containing heterocycles can form N-oxides, as the nitrogen needs to have available lone pairs of electrons for oxidation to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include the oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic and m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as t-butyl hydroperoxide, sodium perborate, and dioxiranes (dioxiranes) such as dimethyldioxirane. These methods for preparing N-oxides have been widely described and reviewed in the literature.

Pharmaceutically acceptable salts may be acid addition salts of the compounds of the invention having sufficient basicity, for example having a nitrogen atom in the chain or ring, for example with the following inorganic acids: for example hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid, or hydrogen sulfates, or acid addition salts with organic acids such as: such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, Mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

In addition, another suitable pharmaceutically acceptable salt of a compound of the invention which is sufficiently acidic is an alkali metal salt (e.g., sodium or potassium salt), an alkaline earth metal salt (e.g., calcium or magnesium salt), an ammonium salt, or a salt with an organic base which affords a physiologically acceptable cation, such as a salt with: sodium ions, potassium ions, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol. By way of example, the pharmaceutically acceptable salts include salts of the group-COOH with: sodium ion, potassium ion, calcium ion, magnesium ion, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol.

In addition, the basic nitrogen-containing groups may be quaternized with the following agents: lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromide, and the like. By way of example, pharmaceutically acceptable salts include hydrochloride, sulfate, nitrate, bisulfate, hydrobromide, acetate, oxalate, citrate, methanesulfonate, formate, or meglumine salts and the like.

Since the compound of the present invention may exist at a plurality of salt-forming sites, the "pharmaceutically acceptable salt" includes not only the salt formed at 1 salt-forming site among the compounds of the present invention but also the salt formed at 2,3 or all of the salt-forming sites among them. For this purpose, the molar ratio of the "pharmaceutically acceptable salt" of the compound of formula (I) to the cation of the acid (anion) or base required for salt formation may vary within wide limits, and may be, for example, 4:1 to 1:4, such as 3:1, 2:1, 1:2, 1:3, etc.

According to the present invention, the pharmaceutically acceptable anion includes anions selected from the group consisting of those generated by ionization of inorganic or organic acids. The "inorganic acid" includes, but is not limited to, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, or nitric acid. The "organic acid" includes, but is not limited to, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, maleic, Fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

Depending on the position and nature of the various substituents, the compounds of the present invention may also contain one or more asymmetric centers. Asymmetric carbon atoms may exist in either the (R) or (S) configuration, with only one asymmetric center yielding a racemic mixture and multiple asymmetric centers yielding a diastereomeric mixture. In some cases, asymmetry may also exist due to hindered rotation about a particular bond, for example, the central bond connects two substituted aromatic rings of a particular compound. Also, the substituents may exist in cis or trans isomeric forms.

The compounds of the invention also include all possible stereoisomers of each, either as a single stereoisomer or as any mixture of said stereoisomers (e.g. the R-or S-isomers, or the E-or Z-isomers) in any proportion. Separation of individual stereoisomers (e.g. individual enantiomers or individual diastereomers) of the compounds of the invention may be achieved by any suitable prior art method (e.g. chromatography, particularly, for example, chiral chromatography).

The term "tautomer" refers to an isomer of a functional group resulting from the rapid movement of an atom in two positions in a molecule. The compounds of the invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; whereas in phenol the enol type predominates. The present invention encompasses all tautomeric forms of the compounds.

In the present invention, reference to compounds also includes isotopically-labeled compounds, which are identical to those shown in formula I, but wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of H, C, N, O, S, F and Cl, such as²H、³H、¹³C、¹¹C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³²P、³⁵S、¹⁸F and³⁶and (4) Cl. Containing isotopes and/or other atoms as described aboveIsotopically-enriched compounds of the present invention, prodrugs thereof, or pharmaceutically acceptable salts of said compounds or said prodrugs are within the scope of the present invention. Certain isotopically-labeled compounds of the present invention, for example, by incorporation of a radioactive isotope (such as³H and¹⁴C) the compounds of (a) are useful in drug and/or substrate tissue distribution assays. Tritium (i.e. tritium³H) And carbon 14 (i.e.¹⁴C) Isotopes are particularly preferred for their ease of preparation and detectability. Again, with heavier isotopes such as deuterium (i.e. deuterium)²H) Substitutions may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in certain circumstances. The compounds of the invention as claimed may be particularly limited to replacement with deuterium or tritium. Furthermore, the absence of hydrogen in the substituents indicating the term deuterium or tritium alone is not meant to exclude deuterium or tritium, but may equally well comprise deuterium or tritium.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present invention sufficient to effect the intended use, including but not limited to the treatment of a disease as defined below. The therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc., can be readily determined by one of ordinary skill in the art. The specific dosage will vary depending on the following factors: the particular compound selected, the dosage regimen to be followed, whether to administer it in combination with other compounds, the timing of administration, the tissue to be administered and the physical delivery system carried.

The term "adjuvant" refers to a pharmaceutically acceptable inert ingredient. Examples of types of excipients include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients enhance the handling characteristics of the pharmaceutical formulation, i.e., make the formulation more amenable to direct compression by increasing flowability and/or cohesiveness. Examples of typical pharmaceutically acceptable carriers suitable for use in the above formulations are: sugars such as lactose, sucrose, mannitol, and sorbitol; starches, such as corn starch, tapioca starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; calcium phosphates such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; stearic acid; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic, cationic and anionic surfactants; a glycol polymer; fatty alcohols; and grain hydrolyzed solids and other nontoxic compatible excipients commonly used in pharmaceutical formulations, such as fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, and the like.

The term "solvate" is those forms of the compounds of the present invention which form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a particular form of solvates in which the coordination is with water. In the present invention, the preferred solvate is a hydrate. Further, pharmaceutically acceptable solvates (hydrates) of the compounds of general formula I according to the invention refer to co-crystals and clathrates of compound I with one or more molecules of water or other solvents in stoichiometric amounts. Solvents that may be used for the solvate include, but are not limited to: water, methanol, ethanol, ethylene glycol and acetic acid.

The term "prodrug", or "prodrug" refers to a compound that is converted in vivo to a compound of the general formula or a particular compound. Such conversion is effected by hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissue. The prodrugs of the present invention may be esters, and esters useful as prodrugs in the present invention are phenyl esters, aliphatic (C1-24) esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For example, a compound of the present invention comprises a hydroxy/carboxy group, i.e., it may be acylated to provide the compound in prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a hydroxyl group on the parent.

The "cancer" of the present invention includes but is not limited to: bladder cancer, breast cancer (e.g., ductal carcinoma), cervical cancer (e.g., squamous cell carcinoma), colorectal cancer (e.g., adenocarcinoma), esophageal cancer (e.g., squamous cell carcinoma), gastric cancer (e.g., adenocarcinoma, medulloblastoma, colon cancer, choriocarcinoma, squamous cell carcinoma), head and neck cancer, hematological cancer (e.g., acute lymphocytic anemia, acute myelogenous leukemia, acute lymphocytic leukemia B-cell, anaplastic large cell lymphoma, B-cell lymphoma, burkitt lymphoma, chronic lymphocytic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic myelogenous leukemia, hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma, T-cell acute lymphocytic leukemia), lung cancer (e.g., bronchoalveolar carcinoma, mesothelioma, mucoepidermoid carcinoma, small cell lung cancer, colon, Non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), lymphoma, nervous system cancer (e.g., glioblastoma, neuroblastoma, glioma), ovarian cancer (e.g., adenocarcinoma), pancreatic cancer (e.g., ductal carcinoma), prostate cancer (e.g., adenocarcinoma), renal cancer (e.g., renal cell carcinoma, renal clear cell carcinoma), sarcoma (e.g., chondrosarcoma, ewing's sarcoma, fibrosarcoma, multisource sarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma), skin cancer (e.g., melanoma, epidermoid carcinoma, squamous cell carcinoma), thyroid cancer (e.g., medullary carcinoma), and uterine cancer, among others.

The term "autoimmune disease" or "autoimmune disorder" as used herein refers to a condition that is immune mediated by attack on self-tissue, but may also involve an immune response to a microorganism. Examples of autoimmune diseases include, but are not limited to: multiple sclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, rheumatoid arthritis, multiple arthritis, local and systemic scleroderma, systemic lupus erythematosus, discoid lupus erythematosus, cutaneous lupus erythematosus (including chilblain lupus erythematosus, lupus nephritis, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, dermatomyositis, polymyositis, idiopathic edema, chronic thyroiditis, Guillain-Barre syndrome, Graves ' disease, myasthenia gravis, sjogren's syndrome, nodular polyarteritis, autoimmune bowel disease, uveitis, autoimmune oophoritis, chronic immune thrombocytopenic purpura, colitis, diabetes, psoriasis, pemphigus vulgaris, proliferative glomerulonephritis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, Chronic arthritis, inflammatory chronic sinusitis, colitis, celiac disease, inflammatory bowel disease, Barlow's esophageal cancer, inflammatory gastritis, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune cardioinflammation, autoimmune encephalitis, and autoimmune-mediated hematologic diseases), and the like.

Detailed Description

The invention is further described with reference to specific examples. The present invention includes, but is not limited to, the following examples.

The experimental methods in the examples described below are all conventional methods unless otherwise specified; the resulting compounds were determined by Varian Mercury-plus 400, 500 or 600MHz NMR spectrometer and Waters Q-TOF-Ultima mass spectrometer¹H NMR and mass spectra; the reagents and biomaterials, if not specifically mentioned, are commercially available.

The following examples and brief description used elsewhere herein:

example 1.

Synthesis of intermediate tert-butyl 2-acetylthioacetate

Potassium thioacetate (239.84g,2.1mol) and anhydrous DMF (500mL) were added to a reaction flask (1L), cooled to 0-5 deg.C, tert-butyl bromoacetate (390.10g,2.0mol) was added dropwise with stirring, and the reaction was continued for 1h at room temperature after the dropwise addition. The reaction mixture was evaporated under reduced pressure at 80 ℃ to remove the solvent, and after cooling, water (500mL) was added to separate an organic layer, and the aqueous layer was extracted with chloroform (150 mL. times.2)Extraction, combination of organic layers, washing with saturated NaCl solution (200 mL. times.3), anhydrous Na₂SO₄Drying, filtering and concentrating under reduced pressure to obtain 2-acetylthioacetic acid tert-butyl ester (380.5 g of red liquid, yield 100%).

R_f＝0.53(PE/EtOAc＝9:1)；¹H NMR(600MHz,CDCl₃)δ3.61(s,2H),2.37(s,3H),1.45(s,9H).

Example 2.

Intermediate A1-2 synthesized from X ═ Y ═ CH, R₁Me is an example.

2-chloro-3-cyano-6-methylpyridine (50.35g,0.330mol), tert-butyl 2-acetylthioacetate (69.06g,0.363mol) and DMF (330mL) were added to a reaction flask (500mL), cooled to 0-5 deg.C, and NaOMe (21.39g,0.396mol) was added in portions and reacted at room temperature for 1 h. The reaction mixture was poured into water (3L) to precipitate a large amount of yellow solid, which was filtered, washed with water to neutrality, and dried to obtain tert-butyl 3-amino-6-methylthioeno [2,3-b ] pyridine-2-carboxylate (yellow solid 75.51g, yield 87%).

R_f＝0.36(PE/Acetone＝17:3)；ESI-MS:265.0[M+H]⁺；¹H NMR(600MHz,CDCl₃)δ7.77(d,J＝8.3Hz,

1H),7.13(d,J＝8.3Hz,1H),5.79(s,2H),2.66(s,3H),1.59(s,9H).

Example 3.

Synthesis of carboxylic acid a1 with X ═ Y ═ CH, R₁Me is an example.

3-amino-6-methylthiophene [2,3-b ] was added to the reaction flask (1L)]Pyridine-2-carboxylic acid tert-butyl ester (75.34g,0.285mol),NaHCO₃(47.89g,0.570mol) and DCM (285mL) were added dropwise with stirring at RT to trifluoroacetic anhydride (71.83g,0.342mol) and the reaction was continued for 0.5h at RT. The reaction mixture was added dropwise with water (200mL), stirred until no gas was generated, the DCM layer was separated, the aqueous phase was extracted with DCM (200 mL. times.2), the organic phases were combined, washed with saturated NaCl solution (200 mL. times.2), anhydrous Na₂SO₄Drying, vacuum concentrating, and recrystallizing with MeOH for several times to obtain 3-trifluoroacetylamino-6-methylthiophene [2,3-b ]]Pyridine-2-carboxylic acid tert-butyl ester (pale yellow solid 93.26g, 91% yield).

R_f＝0.57(PE/EtOAc＝17:3)；ESI-MS:361.0[M+H]⁺；359.1[M-H]^-；¹H NMR(600MHz,CDCl₃)δ 11.14(s,1H),8.46(d,J＝8.6Hz,1H),7.23(d,J＝8.6Hz,1H),2.69(s,3H),1.63(s,9H).

To a reaction flask (500mL) was added 3-trifluoroacetylamino-6-methylthioeno [2,3-b ]]Pyridine-2-carboxylic acid tert-butyl ester (28.83g,80 mmol) and dry DMF (160mL), cooled to 0-5 deg.C and added portionwise 60% NaH (3.36g,84 mmol). After the reaction till no gas is generated, MeI (13.63g,96mmol) is added dropwise at 0-5 ℃, and the temperature is raised to room temperature for reaction for 1 h. HOAc was added dropwise to the reaction solution to adjust pH to 7, water (150mL) was added, chloroform (100 mL. times.3) was added for extraction, the chloroform layers were combined, washed with saturated NaCl solution (100 mL. times.2), and anhydrous Na was added₂SO₄Drying, concentrating under reduced pressure, and recrystallizing with EtOH for multiple times to obtain 3- (N-methyl-N-trifluoroacetyl) amino-6-methylthiophene [2,3-b ]]Pyridine-2-carboxylic acid tert-butyl ester (white solid 24.27g, yield 81%).

R_f＝0.35(PE/EtOAc＝17:3)；ESI-MS:375.1[M+H]⁺；¹H NMR(500MHz,CDCl₃)δ7.83(d,J＝8.3Hz, 1H),7.30(d,J＝8.3Hz,1H),3.36(s,3H),2.72(s,3H),1.57(s,9H).¹³C NMR(125MHz,CDCl₃)δ160.5, 160.1,158.9,157.7(q,J＝36.3Hz),132.7,131.7,130.2,127.3,121.6,116.2(q,J＝288.5Hz),84.5,37.6, 28.2,25.0.

To a reaction flask (250mL) were added tert-butyl 3- (N-methyl-N-trifluoroacetyl) amino-6-methylthieno [2,3-b ] pyridine-2-carboxylate (37.44g,0.10mol), DCM (100mL) and TFA (74.3mL), and reacted at 40 ℃ overnight. The reaction mixture was directly subjected to reduced pressure to remove TFA, and then passed through a short column of silica gel to give 3- (N-methyl-N-trifluoroacetyl) amino-6-methylthioeno [2,3-b ] pyridine-2-carboxylic acid (31.80 g as a yellow solid, yield 100%).

R_f＝0.30(CHCl₃/MeOH＝4:1)；ESI-MS:317.1[M-H]^-；¹H NMR(600MHz,DMSO-d6)δ14.23(br s, 1H),8.32(d,J＝8.3Hz,1H),7.50(d,J＝8.3Hz,1H),3.30(s,3H),2.65(s,3H).

Other intermediate carboxylic acids were synthesized according to the reaction conditions of example 1 using appropriate synthesis precursors.

Example 4.

The preparation of tert-butyl 3- (4- (2-aminoethylphenyl)) -3, 8-diazacyclo [3.2.1] octane-8-carbonate as an organic amine is exemplified.

To a reaction flask (250mL) were added p-bromophenylethylamine B1-1(10.00g,50mmol), KI (0.41g,2.5mmol), and K₂CO₃(16.58g,120mmol) and acetonitrile (100mL), heated to reflux and benzyl chloride (20.89g,165mmol) was added dropwise. After the dropwise addition, the reaction was carried out for 2 hours under reflux. The reaction mixture was filtered to remove inorganic salts, the filtrate was concentrated under reduced pressure to remove acetonitrile by evaporation, and the concentrate was added with chloroform (200mL), washed with saturated NaCl solution (100 mL. times.2), and washed with anhydrous Na₂SO₄Drying, and concentrating under reduced pressure to obtain crude product. The crude product was distilled under reduced pressure to remove excess benzyl chloride and benzyl alcohol as a by-product, to give B1-2 (pale yellow liquid, 18.39g, yield 97%).

To a reaction flask (250mL) was addedB1-2(4.25g,20mmol), azacyclo (9.13g,24mmol), Pd (OAc)₂(449mg, 2mmol)、Xphos(953mg,2mmol)、Cs₂CO₃(13.03g,40mmol) and toluene (80mL), and the N was replaced by evacuation₂The temperature is raised to 100 ℃ and the reaction lasts 18 h. The reaction mixture was filtered to remove insoluble materials, the filtrate was concentrated under reduced pressure, and the concentrate was passed through a silica gel column and eluted with a gradient of PE/EtOAc (19: 1,9: 1) to give B1-3(8.59g, yield 84%).

To a reaction flask (250mL) were added B1-3(8.15g,15.9mmol), Pd/C (0.80g, containing 10% Pd) and MeOH (65mL), and the N was replaced by vacuum₂The reaction was carried out overnight at room temperature. The reaction mixture was filtered to remove insoluble substances, the filtrate was concentrated under reduced pressure to remove methanol, and the concentrate was added with chloroform (200mL), washed with saturated NaCl solution (50 mL. times.3), and washed with anhydrous Na₂SO₄Dried and concentrated under reduced pressure to give B1(5.15g, yield 93%).¹H NMR (400MHz,CDCl₃)δ7.08(d,J＝8.5Hz,2H),6.77(d,J＝8.5Hz,2H),4.33(m,2H),3.37(d,J＝10.0Hz,2H), 2.95(br s,2H),2.92(t,J＝6.8Hz,2H),2.67(t,J＝6.8Hz,2H),1.92(m,4H),1.84(m,2H),1.46(s,9H).

Example 5.

Preparation of Compound I-113(N-4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) phenethyl-6-methyl-3-methylaminothiophene [2,3-b ] pyridine-2-carboxamide) and its hydrochloride

To a reaction flask (10mL) were added 3- (N-methyl-N-trifluoroacetyl) amino-6-methylthioeno [2,3-B ] pyridine-2-carboxylic acid (318mg, 1.0mmol), organic amine B1(398mg,1.2mmol), HOBt (149mg,1.1mmol) and EDCI (230mg,1.2mmol), followed by anhydrous DMF (4mL) and DIEA (524. mu.L, 3.0mmol) and reacted at 60 ℃ for 2 h. The reaction solution was directly concentrated under reduced pressure and subjected to silica gel column chromatography (30g) to give 3- (N-methyl-N-trifluoroacetyl) amino-6-methylthioeno [2,3-b ] pyridine-2-carboxamides.

To a reaction flask (10mL) was added 3- (N-methyl-N-trifluoroacetyl) amino-6-methylthioeno [2,3-b ]]Pyridine-2-carboxamides (1eq), TFA (10eq) and CHCl₃(2mL) and reacted at 60 ℃ for 2 h. Reduced pressure evaporating to drynessSubstance plus K₂CO₃(2-4 eq) and MeOH (2mL) at room temperature for 30 min. Water (10mL) was added to the reaction mixture, followed by CHCl₃(10 mL. times.3), the combined extracts were washed with saturated NaCl solution (10 mL. times.2) and anhydrous Na₂SO₄Drying, and evaporating to dryness under reduced pressure. The dry material was dissolved in MeOH (2mL), 36% HCl solution (1.5-3 eq) was added and stirred at room temperature for 30 min. Filtration with suction and washing with a small amount of MeOH afforded the hydrochloride salt of compound I-113 (yellow solid, yield 36%).

ESI-MS:436.3[M-Cl]⁺.¹H NMR(500MHz,DMSO-d6) δ9.51(d,J＝9.7Hz,1H),9.47(d,

J＝9.7Hz,1H),8.49(d,J＝8.5Hz,1H),7.87(t,J＝4.8Hz,1H),7.30(d,J＝8.5Hz,1H),7.09(d,J＝8.6Hz, 2H),6.84(d,J＝8.6Hz,2H),4.09(s,2H),3.55(d,J＝12.5Hz,2H),3.36(tt,J＝7.8,4.8Hz,2H),3.17(s, 3H),3.08(d,J＝12.5Hz,2H),2.72(t,J＝7.8Hz,2H),2.59(s,3H),1.97(m,2H),1.91(m,2H).¹³C NMR (125MHz,DMSO-d6)δ164.7,158.2,157.6,148.3,146.8,133.5,130.1,129.2,124.4,119.5,114.6,53.7, 50.6,41.0,34.4,32.9,25.4,23.7.

Example compounds I-1-I-194 in table 6 below were synthesized according to the reaction conditions described above for example 9 (compound I-113) using the appropriate synthesis precursors, and the structures are as follows:

table 1: structure of Compound I-1-I-10 and its USP28/25 inhibitory Activity

Table 2: structure of compound I-11-I-105 and its USP28/25 inhibitory activity

Table 3: structure of Compound I-106-I-112 and its USP28/25 inhibitory Activity

Table 4: structure of compound I-113-I-143 and its USP28/25 inhibitory activity

Table 5: structure of compound I-144-I-166 and its USP28/25 inhibitory activity

Table 6: structure of compound I-167-I-190 and its USP28/25 inhibitory activity

Table 7: structure of Compound I-191-I-194 and its USP28/25 inhibitory Activity

IC represented by symbol correspondence in Table 8₅₀The ranges are as follows:

example 6.

USP28 activity was determined by the ubiquitin-rhodamine 110 method.

Purified USP28 and the Ubiquitin-Rhodamine 110 substrate (Ubiquitin-Rhodamine 110) used to determine the activity of DUBs were both from R&D Systems. Test compounds were first prepared as 10mM stock solutions in DMSO, then buffered [ 20mM Tris-HCl (pH 8.0), 2mM CaCl₂3mM BME, 0.01% Prionix, 0.01% Triton X-100]Diluted to the required concentration (with DMSO content. ltoreq.0.5%), mixed with USP28 (final concentration 4nM) in 96-well plates and incubated for 30min at room temperature, then substrate (ubiquitin-rhodamine 110) is added to 125 nM. The final volume of the whole reaction was 20. mu.L. The detection of the released fluorescence (excitation wavelength 485nm, emission wavelength 535nm) on the microplate reader was started immediately after the addition of the substrate. The inhibition of USP25 by the test compound was calculated according to the following formula:

inhibition [% 1- [ (test compound + fluorescence of substrate-fluorescence of test compound (no substrate)) ]

Calculating the IC according to the inhibition rate of the test compound on the USP28 under different concentrations₅₀The values, results are shown in tables 1-7.

Example 7.

USP25 activity was determined by the ubiquitin-rhodamine 110 method.

Purified USP25 and the Ubiquitin-Rhodamine 110 substrate (Ubiquitin-Rhodamine 110) used to determine the activity of DUBs were both from R&D Systems. Test compounds were first prepared as 10mM stock solutions in DMSO, then buffered [ 20mM Tris-HCl (pH 8.0), 2mM CaCl₂3mM BME, 0.01% Prionix, 0.01% Triton X-100]Diluting to desired concentration (wherein DMSO content is less than or equal to 0.5%), mixing with USP25 (final concentration 15nM) in 96-well plate, incubating at room temperature for 30min, and adding substrate (ubiquitin-rhodamine 110) to 125%nM. The final volume of the whole reaction was 20. mu.L. The detection of the released fluorescence (excitation wavelength 485nm, emission wavelength 535nm) on the microplate reader was started immediately after the addition of the substrate, and the inhibition of the test compound against USP25 was calculated according to the following formula:

Calculating the IC according to the inhibition rate of the test compound on the USP25 under different concentrations₅₀The values, results are shown in tables 1-7.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A compound of formula I, and racemates, stereoisomers, isotopic labels, nitric oxides, solvates, polymorphs, metabolites, pharmaceutically acceptable salts or prodrugs thereof:

in the formula:

x is CR₅Or N;

y is CR₆Or N;

z is

The dashed bond representation may be a bond or absent;

m is 0, 1,2, 3 or 4;

n is 1,2, 3,4, 5,6, 7 or 8;

p is 0, 1 or 2;

R₁、R₄、R₅and R₁₀-R₁₄Independently of one another, from hydrogen, unsubstituted or substituted (C)₁-C₁₂) Hydrocarbyl, unsubstituted or substituted (C)₁-C₁₂) Hydroxy, unsubstituted or substituted (C)₁-C₁₂) Amino or halogen; said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic hydrocarbon groups or aromatic hydrocarbon groups; the halogen is fluorine, chlorine, bromine or iodine;

R₇and R₉Independently of one another, from hydrogen, unsubstituted or substituted (C)₁-C₁₂) Hydrocarbyl, unsubstituted or substituted (C)₁-C₁₂) Hydroxy or unsubstituted or substituted (C)₁-C₁₂) An amino group; said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, including nitrogen heterocycle and oxygen heterocycle, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic hydrocarbon groups or aromatic hydrocarbon groups; the R is₇And R₉May be selected from 3-20 membered heterocyclic group containing one, two or more N or 5-20 membered heteroaryl group, which is unsubstituted or optionally substituted with one, two or more nitrogen heterocyclic ring, oxygen heterocyclic ring, further, 3-10 membered heterocyclic group containing only one or two N as hetero atom is preferable;

R₈is hydrogen, unsubstituted or substituted (C)₁-C₁₂) Hydrocarbyl, unsubstituted or substituted (C)₁-C₁₂) Hydroxy, unsubstituted or substituted (C)₁-C₁₂) Amino or halogen; said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic hydrocarbon groups or aromatic hydrocarbon groups; when the dotted bond is a single bond, R₈May be O;

preferably, said "is free of said unsubstituted or substituted (C)₁-C₁₂) Is aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of 1-12 carbon atoms and corresponding hydrogen atoms, and the unsubstituted or substituted (C)₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms₁-C₁₂) Aliphatic or aromatic hydrocarbon radicals "in which nitrogen, oxygen, sulfur, phosphorus and halogen may be present in (C)₁-C₁₂) (C) is a linear or branched aliphatic hydrocarbon group, and may be in any position of the linear or branched aliphatic hydrocarbon group₁-C₁₂) Preferably may be (C)₁-C₁₀) Aliphatic hydrocarbon group, (C)₁-C₈) Aliphatic hydrocarbon radical or (C)₁-C₆) An aliphatic hydrocarbon group; for example, it may be selected from the following groups: (C)₁-C₆) Aliphatic hydrocarbon group (C)₁-C₆) Aliphatic hydrocarbyloxy, N- (C)₁-C₆) Aliphatic hydrocarbyl amino, N-di- (C)₁-C₃) Aliphatic hydrocarbyl amino group, (C)₁-C₆) Aliphatic hydrocarbyl mercapto, halo (C)₁-C₆) Aliphatic hydrocarbon group, halo (C)₁-C₆) Aliphatic hydrocarbyloxy, (N-mono-or disubstituted) halo (C)₁-C₆) Aliphatic hydrocarbyl amino, halo (C)₁-C₆) Aliphatic hydrocarbyl mercapto group, (C)₁-C₆) Aliphatic hydrocarbyloxy (C)₁-C₆) Aliphatic hydrocarbon group, (C)₁-C₆) Aliphatic hydrocarbyl mercapto group (C)₁-C₆) Aliphatic hydrocarbon group, N- (C)₁-C₆) Aliphatic hydrocarbyl amino group (C)₁-C₆) Aliphatic hydrocarbon group, N-di- (C)₁-C₃) Aliphatic hydrocarbyl amino group (C)₁-C₆) Aliphatic hydrocarbon groups such as methyl, deuterated methyl, ethyl, propyl, isopropyl, cyclopropyl, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N-dimethylaminomethyl, N-dimethylaminoethyl, N-diethylaminoethyl; preferably, R₂Can be selected from the group consisting of methyl, deuterated methyl, ethyl, propyl, butyl, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N, N-dimethylaminomethyl, N, N-dimethylaminoethyl, N, N-diethylaminoethyl.

2. The compound of formula I according to claim 1, and racemates, stereoisomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof, characterized in that: z is

The R is₇And R₉May be selected from unsubstituted or optionally substituted by one, two or more groups, containing one, two orMore N3-20-membered heterocyclic group or 5-20-membered heteroaryl group, and further, preferably 3-10-membered heterocyclic group containing only one or two N as hetero atoms.

3. The compound of formula I according to claim 1 or 2, and racemates, stereoisomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof, characterized in that: the structure of formula I is further selected from the following structures of formula II or formula III:

in the formulae II and III, R₁、R₂、R₃、R₄、R₆、R₉X, Y and Z are as defined for formula I.

4. A compound of formula I according to any one of claims 1-3, and racemates, stereoisomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof.

5. A process for the preparation of a compound of formula I according to any one of claims 1 to 4, and racemates, stereoisomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, pharmaceutically acceptable salts or prodrugs thereof, comprising the steps of:

carrying out condensation reaction on the intermediate carboxylic acid A and the intermediate amine B to obtain a condensation product, and removing trifluoroacetyl group to obtain a target compound I;

unless otherwise defined, X, Y, Z, R are as defined above₁,R₂,R₃,R₄,m,nThe definition is the same as formula I.

6. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 4 and racemates, stereoisomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof.

7. Use of a compound of formula I according to any one of claims 1 to 4, and racemates, stereoisomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs thereof, or a pharmaceutical composition according to claim 6, for the manufacture of a medicament for the treatment of a disease or disorder associated with the inhibition of USP28 and/or USP 25.

8. The use according to claim 7, wherein the USP28 and/or USP25 associated disease or disorder comprises cancer, inflammation, autoimmune disease, viral infection and bacterial infection.