A class of ubiquitination-specific protease inhibitors and preparation method and application thereof

Abstract

The present invention relates to a compound represented by formula I and its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug, as well as a pharmaceutical composition comprising the same, a preparation method thereof, and medical use thereof. The compound has the activity of inhibiting USP28 and / or USP25, and the structure of formula I is as follows:

Description

[0001] This application is a divisional application of the invention patent application with the application date of May 9, 2019, application number 201910385956.0, and invention name “A class of ubiquitination-specific protease inhibitors and preparation methods and applications thereof”. Technical Field

[0002] The present invention relates to the field of medicine, and in particular to a new class of ubiquitination-specific protease inhibitors and a preparation method and use thereof. Background Art

[0003] The normal function of cells depends on intracellular protein homeostasis, and maintaining this homeostasis depends on the dynamic balance of protein synthesis and degradation. Cells mainly remove proteins that are no longer needed, such as damaged or completed proteins, through the proteasome degradation pathway. Proteins degraded through the proteasome pathway are usually marked by polyubiquitin chains linked to lysine at position 48. The polyubiquitin tag of proteins is the result of a series of enzymes, mainly including E1, E2, and E3. E1 activates ubiquitin by forming a high-energy thioester bond between its own cysteine ​​residue and the C-terminal carboxyl group of ubiquitin composed of 76 amino acids; the activated ubiquitin is transferred to the cysteine ​​residue of E2 (there are about 50 E2 conjugating enzymes in mammals); then, under the action of E3 ligase (there are about 500 E3 in mammalian cells), E2 conjugating enzyme transfers ubiquitin to a lysine residue of a target protein. In essence, the E3 ligase simply brings together the E2 coupling enzyme and the substrate, allowing ubiquitin to be transferred from E2 to the target protein (Annu. Rev. Biochem. 2009, 78, 477 & 2018, 87, 697; J. Am. Soc. Nephrol. 2006, 17, 1807). In a series of cellular activities, ubiquitin-mediated protein degradation through proteasomes is an essential regulatory means, such as cell cycle, apoptosis (Front Cell. Dev. Biol. 2018, 6, 11; Cell Death Differ. 1999, 6, 303; J. Cell. Mol. Med. 2002, 6, 25), DNA damage checkpoint control (DNA Repair 2010, 9, 1229; Biochim. Biophys. Acta 2014, 1843, 150; Cell Death Differ. 2010, 17, 78; ISRN Mol. Biol. 2012, 146748), etc.

[0004] Compared with ubiquitination, cells also have the ability to deubiquitinate in order to more accurately regulate protein homeostasis. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs). DUBs are a class of specific proteolytic enzymes (Physiol. Rev. 2013, 93, 1289; Oncogene 2012, 31, 2373; Biochem. J. 2015, 465, 1; BMC Biochem. 2008, 9 Suppl 1, S3; Protein Sci. 2014, 23, 344). In mammals, there are about 100 known DUBs, which are divided into several families, including the ubiquitin-specific protease (USP) family, the ubiquitin C-terminal hydrolase (UCH) family, the ovarian tumor protease (OTU) family, and the Machado-Josephin domain (MJD) family.

[0005] We know that the dysregulation of the proteasome-mediated protein degradation system is closely related to a variety of human diseases, including some diseases of the tumor, immune and nervous systems (Front Mol. Neurosci. 2014, 7, 70; Cardiovasc. Res. 2010, 85, 251; Essays Biochem. 2005, 41, 187; Front Biosci. 2014, 19, 886; Cancer Biol. Ther. 2002, 1, 337; IUBMB Life 2015, 67, 544; Acta Neuropathol. 2009, 118, 329; Int. J. Biochem. Cell. Biol. 2018, 101, 80; J. Clin. Oncol. 2013, 31, 1231; Cancer Metastasis Rev.2017,36,635; Circ.Res.2013,112,1046; Drug Resist.Updat.2015,23,1; Biochim.Biophys.Acta 2014,1843,13; Cancer Metastasis Rev.2017,36,683; CancerSci.2009,100,24).

[0006] USP28 is a ubiquitin-specific protease that plays an important role in maintaining the levels of proteins such as c-MYC (Nat. Cell. Biol. 2007, 9, 765), LSD1 (Cell Rep. 2013, 5, 224), HIF1alpha (Blood 2012, 119, 1292), Notch1 (J. Clin. Invest. 2014, 124, 3407), 53BP1 (Mol. Cell. Biol. 2014, 34, 2062) and CLASPIN (Cell 2006, 126, 529), preventing them from being degraded while they are still functioning. Almost all of these substrates, especially c-MYC, play an important role in tumorigenesis and development. There is also evidence that USP28 is overexpressed in tumors and patients with high expression levels have a poor prognosis (Tumor Biol. 2014, 35, 4017; BBA-Mol. Basis. Dis. 2019, 1865, 599; Biochem. Pharmacol. 2018, 150, 280; Oncotarget, 2017, 8, 39627; Transl. Oncol. 2017, 10, 80; J. Cell. Mol. Med. 2015, 19, 799). This makes USP28 an attractive target for tumor therapy.

[0007] As a transcription factor, c-MYC activates the expression of genes related to cell growth and proliferation (Biochim.Biophys.Acta,2015,1849,506;Annu.Rev.Cell Dev.Biol.2000,16,653;TrendsBiochem.Sci.1997,22,177;Adv.Cancer Res.1996,70,95;Lung Cancer 2001,34Suppl 2,S43). Almost all growth regulatory signaling pathways ultimately require c-MYC to function, making c-MYC one of the most promising targets in tumor therapy (Cancer Lett.2003,197,125;Expert Opin.Ther.Targets2003,7,623;Cell 2004,117,153-156;Semin.Cancer Biol.2006,16,318). However, research experience over the past few decades has shown that it is almost impossible to find small molecule compounds that directly regulate c-MYC activity (Biochim. Biophys. Acta 2015, 1849, 525). As a second choice, people are currently trying to find ways to indirectly inhibit c-MYC function. One method is to take advantage of the unstable nature of c-MYC protein. FBW7 is the main E3 ligase of c-MYC, promoting its ubiquitination and degradation (Curr. Biol. 2004, 14, 1852; EMBO J. 2004, 23, 2116), while USP28 plays the opposite role in this process (Nat. Cell Biol. 2007, 9, 765). Therefore, inhibiting USP28 can potentially reduce the stability of c-MYC and thus slow down or prevent the proliferation of tumor cells.

[0008] LSD1 is a histone demethylase that plays an important role in epigenetic regulation of gene expression (Curr. Opin. Chem. Biol. 2007, 11, 561; Epigenomics 2016, 8, 1103). LSD1 has been found to be overexpressed in a large number of malignant tumors, and LSD1 is believed to play a very important role in the maintenance of tumor stem cells (Hum. Pathol. 2012, 43, 1300; Fertil. Steril. 2014, 101, 740; Int. J. Cancer, 2011, 128, 574; J. Ovarian Res. 2013, 6, 75; Int. J. Gynecol. Cancer, 2015, 25, 1453; PLoS One, 2015, 10, e0118002; Tumor Biol. 2013, 34, 173; World J. Gastroenterol. 2012, 18, 6651). In breast cancer cells, LSD1 deficiency causes the loss of stem cell populations and reduces the proliferation potential of cells (Cell Rep. 2013, 5, 224). In addition, LSD1 has also been identified as a key regulator in tumor immunity (Cell 2018, 174, 549). Therefore, inhibiting USP28 can destabilize LSD1 and c-MYC, two extremely important oncoproteins, and reduce their intracellular levels, thereby preventing tumor cell proliferation.

[0009] The embryonic development of mice with USP28 knockout was normal and they grew normally after birth. The adult mice showed no obvious unhealthy conditions and their fertility was not weakened, indicating that USP28 is not essential in mice (J. Clin. Invest. 2014, 124, 3407; Mol. Cell. Biol. 2014, 34, 2062). However, USP28-deficient mice showed resistance to colon cancer induced by APC mutation (J. Clin. Invest. 2014. 124, 3407), suggesting that USP28 is a valuable therapeutic target at least in colon cancer.

[0010] Recent studies have made people realize the important role of cell senescence in individual aging (Nat. Rev. Mol. Cell. Biol. 2007, 8, 729; Exp. Gerontol. 2001, 36, 1619; Nat. Med. 2015, 21, 1424; J. Physiol. Anthropol. 2007, 26, 365; Mol. Biol. Cell, 2015, 26, 4524; Curr. Opin. Cell. Biol. 1991, 3, 230; Adv. Exp. Med. Biol. 2017, 1002, 189; Mech. Ageing Dev. 2008, 129, 460; J. Cell. Biochem. 2007, 101, 1355; Nat. Rev. Nephrol. 2017, 13, 77; Nature 2014.509,439). More importantly, clearing senescent cells can improve the health of elderly animals (Nature 2011,479,232; J. Clin. Invest. 2018,128,1217; Nat. Med. 2017.23,775-781; Clin. Pharmacol. Ther. 2013,93,105). The senescence-associated secretory phenotype (SASP) refers to the phenomenon that senescent cells can secrete a large number of cytokines, many of which can induce inflammatory responses (J. Clin. Invest. 2013,123,966). Existing studies have shown that USP28 is required for the process of cell senescence (Genes Dev. 2017,31,1933), so inhibiting USP28 may have a beneficial effect on health in the elderly.

[0011] USP25 is a very close homologous gene to USP28, and similar to USP28-deficient mice, mice lacking USP25 do not show any unhealthy traits (Nat. Immunol. 2012, 13, 1110). However, these two deubiquitinases are localized in different regions of the cell (USP28 in the nucleus and USP25 in the cytoplasm), and their substrate spectra are also different. Tankyrase is one of the substrates of USP25 (Cell Rep. 2017. 31, 1024). It is a poly-ADP-ribosyltransferase that participates in a variety of biological processes, such as the Wnt signaling pathway, telomere length maintenance, and vesicle trafficking. Blocking the function of USP25 can lead to the attenuation of Wnt signaling (Genes Dev. 2017, 31, 1024). Considering the known role of Wnt signaling in cancer, it can be predicted that inhibiting USP25 will also have a beneficial effect on tumor treatment. Based on the homology between USP28 and USP25, it is foreseeable that any small molecules targeting USP28 will also target USP25, but this may in turn increase their value in treating tumors.

[0012] It is reported that USP25 can negatively regulate IL17-mediated immune response by deubiquitinating TRAF5 and TRAF6 (Nat. Immunol. 2012, 13, 1110). Further studies have shown that USP25 can also deubiquitinate TRAF3 protein to regulate TLR4-dependent innate immune response (PLoS One 2013, 8, e80976). Therefore, inhibiting USP25 may be beneficial to the body's immune response against tumors and infections. Summary of the invention

[0013] In order to improve the problems existing in the prior art and provide a novel structure having USP28 / USP25 inhibitory activity, the present invention provides a compound represented by the following formula I and its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug:

[0014]

[0015] in:

[0016] X is C-R5 or N;

[0017] m is 0, 1, 2, 3, 4, 5 or 6;

[0018] n is 1 or 2;

[0019] Y is any of the following:

[0020] Z is nitrogen (NH), oxygen (O), sulfur (S) or methylene (CH2); the dotted bond indicates that it can be a bond or does not exist;

[0021] p and q are the same or different and are independently selected from 0, 1, 2 or 3;

[0022] R1, R2, R5 are the same or different and are independently selected from hydrogen, halogen, hydroxyl, amino and optionally unsubstituted or substituted (C1-C 12 ) aliphatic hydrocarbon group, the unsubstituted (C1-C 12 ) aliphatic hydrocarbon group is an aliphatic hydrocarbon group composed of 1 to 12 carbon atoms and corresponding hydrogen atoms, and the substituted (C1-C 12 ) aliphatic hydrocarbon groups are (C1-C 12 ) aliphatic hydrocarbon group;

[0023] R3 is unsubstituted or substituted (C1-C 12 ) aliphatic hydrocarbon group, the unsubstituted (C1-C 12 ) Aliphatic hydrocarbon groups are aliphatic hydrocarbon groups consisting of 1 to 12 carbon atoms and corresponding hydrogen atoms. 12 ) aliphatic hydrocarbon groups are (C1-C 12 ) aliphatic hydrocarbon group;

[0024] Each R4, R6 and R8 are the same or different and are independently selected from hydrogen, halogen, hydroxyl, amino and optionally unsubstituted or substituted (C1-C 12 ) aliphatic hydrocarbon group, the unsubstituted (C1-C 12 ) Aliphatic hydrocarbon groups are aliphatic hydrocarbon groups consisting of 1 to 12 carbon atoms and corresponding hydrogen atoms. 12 ) aliphatic hydrocarbon groups are (C1-C 12 ) aliphatic hydrocarbon group;

[0025] R7 is selected from hydrogen, halogen, hydroxy, amino and optionally unsubstituted or substituted (C1-C 12 ) aliphatic hydrocarbon group, the unsubstituted (C1-C 12 ) Aliphatic hydrocarbon groups are aliphatic hydrocarbon groups consisting of 1 to 12 carbon atoms and corresponding hydrogen atoms. 12 ) aliphatic hydrocarbon groups are (C1-C 12 ) aliphatic hydrocarbon, provided that, when R6 is H, R7 is not H;

[0026] or R7 is selected from 3-20 membered heterocyclyl or 5-20 membered heteroaryl which is unsubstituted or optionally substituted by one, two or more R9;

[0027] R9 is selected from hydrogen, halogen, hydroxy, amino and optionally unsubstituted or substituted (C1-C 12 ) aliphatic hydrocarbon group, the unsubstituted (C1-C 12 ) Aliphatic hydrocarbon groups are aliphatic hydrocarbon groups consisting of 1 to 12 carbon atoms and corresponding hydrogen atoms. 12 ) aliphatic hydrocarbon groups are (C1-C 12 ) aliphatic hydrocarbon group.

[0028] According to an embodiment of the present invention, the “unsubstituted (C1-C 12 ) Aliphatic hydrocarbon groups are aliphatic hydrocarbon groups consisting of 1 to 12 carbon atoms and corresponding hydrogen atoms. 12 ) aliphatic hydrocarbon groups are (C1-C 12 ) aliphatic hydrocarbon group, wherein halogen, such as oxygen, sulfur, nitrogen, phosphorus may be present in (C1-C 12 ) on the straight or branched chain of the aliphatic hydrocarbon group, or at any position of the straight or branched chain, the (C1-C 12 The aliphatic hydrocarbon group may preferably be (C1-C 10) aliphatic (C1-C8) aliphatic (C1-C6) aliphatic; for example, it can be selected from the following groups: (C1-C6) aliphatic, (C1-C6) aliphatic oxy, N-(C1-C6) aliphatic amine, N,N-di-(C1-C3) aliphatic amine, (C1-C6) aliphatic thiol, halogenated (C1-C6) aliphatic, halogenated (C1-C6) aliphatic Oxygen, (mono- or di-N-substituted) halogenated (C1-C6) aliphatic amine, halogenated (C1-C6) aliphatic thiol, (C1-C6) aliphatic oxy (C1-C6) aliphatic, (C1-C6) aliphatic thiol (C1-C6) aliphatic, N-(C1-C6) aliphatic amino (C1-C6) aliphatic, N,N-di-(C1-C3) aliphatic amino ( C1-C6) aliphatic hydrocarbon group, for example, can be methyl, ethyl, propyl, isopropyl, cyclopropyl, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N,N-dimethylaminomethyl, N,N-dimethylaminoethyl, N,N-diethylaminoethyl; According to an embodiment of the present invention, R3 can be selected from 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;

[0029] According to an embodiment of the present invention, Y can be selected from According to an embodiment of the present invention, the R7 can be selected from a 3-20 membered heterocyclic group or a 5-20 membered heteroaryl group which is unsubstituted or optionally substituted by one, two or more R9 and contains one, two or more N, and further, is preferably a 3-10 membered heterocyclic group which contains only one or two N as heteroatoms.

[0030] According to an embodiment of the present invention, R7 may be selected from the following groups which are unsubstituted or optionally substituted by one, two or more R9:

[0031]

[0032] According to an embodiment of the present invention, the structure of formula I is further selected from the following structure of formula II:

[0033]

[0034] In Formula II, R1, R2, R3, R4, X and Y are as defined in Formula I.

[0035] According to an embodiment of the present invention, the following compounds (I-1 to I-85) or their tautomers, optical isomers, nitrogen oxides, solvates, pharmaceutically acceptable salts or prodrugs are preferred:

[0036]

[0037]

[0038]

[0039]

[0040] The present invention further provides a method for preparing the compound of formula I, comprising the following steps:

[0041] The intermediate carboxylic acid A and the intermediate amine B are reacted with a peptide coupling reagent under alkaline conditions to form an amide, and then the protecting groups trifluoroacetyl (Tfac) and tert-butyloxycarbonyl (Boc) are removed to obtain the target compound I:

[0042]

[0043] (wherein, R'4 and Y' are respectively R4 and Y of Formula I, or R4 and Y whose active groups such as hydroxyl and amino groups are protected by Boc)

[0044] Reagents and reaction conditions: a) Amide coupling reaction: the coupling reagent is selected from EDCI-HOBt, BOP, HATU, the base is selected from DEA, TEA, EDCI or DMAP; the solvent is selected from DCM or DMF; b) De-Tfac reaction: the base is selected from K2CO3 or NaOMe, and the solvent is selected from MeOH; c) De-Boc reaction: dilute hydrochloric acid-methanol.

[0045] According to an embodiment of the present invention, the intermediate A can be prepared by the following steps (the raw materials can be obtained from commercial sources):

[0046]

[0047] Reagents and reaction conditions: a) DMF; b) NaOMe / DMF; c) (Tfac)2O / NaHCO3 / CHCl3; d) NaH / DMF, R3-I; e) TFA / DCM.

[0048] According to an embodiment of the present invention, part of the intermediate BI (such as B-Ia or B-Ib) can be obtained by commercial routes or classical synthesis methods:

[0049]

[0050] R'4, R'6, R'7 and R'8 are respectively R4, R6, R7 and R8 of the general formula I or R4, R6, R7 and R8 whose hydroxyl group and amino group are protected by Boc (when R4, R6, R7 and R8 contain active groups such as hydroxyl group or amino group)

[0051] According to an embodiment of the present invention, another part of intermediate B-II can be prepared by the following steps:

[0052]

[0053] R'4 and R'6 are respectively R4 and R6 of the general formula I or R4 and R6 whose hydroxyl or amino groups are protected by Boc (when R4 and R6 contain active groups such as hydroxyl or amino groups)

[0054] Reagents and reaction conditions: a) BnCl, KI, K2CO3 / MeCN; b) H-R7-Boc, Pd(OAc)2, X-phos, Cs(CO3)2, toluene; c) HCO2NH4, Pd(OH)2 / C, MeOH.

[0055] Unless otherwise defined, X, R1, R2, R3, R4, R6, R7, R8, m, n, p, and q are defined as in Formula I.

[0056] According to the embodiments of the present invention, the formula II covered by the general formula I can be synthesized by the above-mentioned general preparation method, and the corresponding raw material structure used can be clearly identified according to the structure of formula II.

[0057] The present invention also provides a pharmaceutical composition comprising a compound of formula (I), its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug as an active ingredient.

[0058] According to an embodiment of the present invention, the pharmaceutical composition further comprises a therapeutically effective amount of the compound of formula I or its tautomer, optical isomer, nitrogen oxide, solvate, pharmaceutically acceptable salt or prodrug and a pharmaceutically acceptable carrier.

[0059] The carrier in the pharmaceutical composition is "acceptable" in that it is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject being treated. One or more solubilizing agents may be used as pharmaceutical excipients for delivery of the active compound.

[0060] The present invention further provides the use of the compound of formula (I), its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug, or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder associated with the inhibition of USP28.

[0061] The present invention further provides the use of the compound of formula (I), its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug, or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder associated with the inhibition of USP25.

[0062] The present invention further provides the use of the compound of formula (I), its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug, or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder associated with the inhibition of USP25 and USP28.

[0063] The present invention also provides a method for treating or preventing a disease or disorder associated with the regulation of USP28 and / or USP25, the method comprising administering to a patient suffering from at least one of the diseases or disorders a compound of formula (I), a racemate, stereoisomer, tautomer, isotopically labeled, nitrogen oxide, solvate, polymorph, metabolite, ester, pharmaceutically acceptable salt or prodrug thereof.

[0064] According to an embodiment of the present invention, the diseases or disorders associated with USP25 and / or USP28 include cancer, inflammation, autoimmune diseases, viral infections and bacterial infections.

[0065] According to an embodiment of the present invention, the pharmaceutical composition can be in a form suitable for oral administration, such as tablets, lozenges, pastilles, water or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions can be prepared according to any known method for preparing pharmaceutical compositions in the art, and such compositions can contain one or more ingredients selected from the following: sweeteners, flavoring agents, coloring agents and preservatives to provide pleasing and palatable pharmaceutical preparations. Tablets contain active ingredients and non-toxic pharmaceutically acceptable excipients suitable for preparing tablets for mixing. These excipients can be inert excipients, granulating agents, disintegrants, binders, and lubricants. These tablets can be uncoated or can be coated with known techniques that provide sustained-release effects over a long period of time by masking the taste of the drug or delaying disintegration and absorption in the gastrointestinal tract.

[0066] According to an embodiment of the present invention, in the pharmaceutical composition, the active ingredient is mixed with an inert solid diluent or a soft gelatin capsule in which the active ingredient is mixed with a water-soluble carrier or an oily solvent to provide an oral preparation; the aqueous suspension contains the active substance and an excipient suitable for preparing an aqueous suspension for mixing. Such excipients are suspending agents, dispersants or wetting agents. The aqueous suspension may also contain one or more preservatives, one or more coloring agents, one or more flavoring agents and one or more sweeteners; the oil suspension may be prepared by suspending the active ingredient in a vegetable oil or a mineral oil. The oil suspension may contain a thickener. The above-mentioned sweeteners and flavoring agents may be added to provide a palatable preparation. These compositions may be preserved by adding an antioxidant; dispersible powders and granules suitable for preparing water suspensions may be provided with active ingredients and dispersants or wetting agents, suspending agents or one or more preservatives for mixing by adding water. Suitable dispersants or wetting agents and suspending agents may illustrate the above examples. Other excipients such as sweeteners, flavoring agents and coloring agents may also be added. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0067] According to an embodiment of the present invention, the pharmaceutical composition can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil, or a mineral oil or a mixture thereof. A suitable emulsifier can be a naturally occurring phospholipid, and the emulsion can also contain a sweetener, a flavoring agent, a preservative and an antioxidant. Such preparations can also contain a demulcent, a preservative, a coloring agent and an antioxidant.

[0068] According to an embodiment of the present invention, the pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or solvents that may be used include 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 the oil phase. The injection or microemulsion may be injected into the patient's bloodstream by local mass injection. Alternatively, it is preferred that the solution and microemulsion be administered in a manner that maintains a constant circulating concentration of the compound of the invention. To maintain this constant concentration, a continuous intravenous drug delivery device may be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.

[0069] According to an embodiment of the present invention, the pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration. Can be prepared by known techniques with the above-mentioned suitable dispersants or wetting agents and suspending agents. Aseptic injection preparations can also be sterile injection solutions or suspensions prepared in parenteral acceptable nontoxic diluents or solvents. In addition, sterile fixed oils can be used as solvents or suspension media conveniently. For this purpose, any blended fixed oils can be used. In addition, fatty acids can also prepare injections.

[0070] According to an embodiment of the present invention, the compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and which will dissolve in the rectum to release the drug.

[0071] As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to the following factors: the activity of the specific compound used, the age of the patient, the weight of the patient, the health status of the patient, the behavior of the patient, the diet of the patient, the administration time, the administration method, the excretion rate, the combination of drugs, etc.; in addition, the best treatment method such as the treatment mode, the daily dosage of the compound of formula (I) or the type of pharmaceutically acceptable salt can be verified according to traditional treatment plans. Beneficial effects of the present invention:

[0072] The present invention provides a class of ubiquitination-specific protease inhibitors with novel structures. Experimental verification shows that the compounds of the present invention have good inhibitory activity against USP28 and / or USP25, which is more than 15 times higher than the activity of the inhibitors in the prior art.

[0073] Terminology explanation:

[0074] Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be arbitrarily combined and combined with each other. The group definitions and compound structures after such combination and combination shall fall within the scope recorded in the specification of this application.

[0075] When the numerical range described in the specification and claims of this application is defined as an "integer", it should be understood that the two endpoints of the range and each integer in the range are recorded. For example, "an integer from 0 to 6" should be understood as recording each integer of 0, 1, 2, 3, 4, 5 and 6. "More" means three or more.

[0076] 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.

[0077] The term "aliphatic hydrocarbon group" includes saturated or unsaturated, linear or branched chain or cyclic hydrocarbon groups. The type of the aliphatic hydrocarbon group can be selected from alkyl, alkenyl, alkynyl, etc. The number of carbon atoms of the aliphatic hydrocarbon group is preferably 1 to 12, and can also be 1 to 10, and a further preferred range is 1 to 6. Specifically, it 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, vinyl, 1-propenyl, 2-propenyl, 1- Methylvinyl, 1-butenyl, 1-ethylvinyl, 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 the above-mentioned substituents may include groups such as hydroxyl, halogen, cyano and amino, for example, the aliphatic hydrocarbon group may contain one, two or more halogens, which means that one, two or more hydrogen atoms of the aliphatic hydrocarbon group may be replaced by an equal number of halogens. If the hydrocarbon group contains more than one carbon, then those carbons do not necessarily have to be connected to each other. For example, at least two of the carbons may be connected via a suitable element or group. That is, the aliphatic group may optionally contain one, two or more heteroatoms (or be interpreted as optionally inserting heteroatoms into the aliphatic group, optionally C—C bonds and C—H bonds). Suitable heteroatoms are obvious to those skilled in the art and include, for example, sulfur, nitrogen, oxygen, phosphorus and silicon. The aliphatic group containing heteroatoms may be selected from the following groups: (C1-C6) aliphatic oxy, (C1-C6) aliphatic thiol, halogenated (C1-C6) aliphatic, halogenated (C1-C6) aliphatic oxy, halogenated (C1-C6) aliphatic thiol, (C1-C6) aliphatic oxy (C1-C6) aliphatic, (C1-C6) aliphatic thiol (C1-C6) aliphatic, N-(C1-C3) aliphatic amine (C1-C6) aliphatic Hydrocarbon group, N,N-di-(C1-C3) aliphatic hydrocarbon amino group (C1-C6) aliphatic hydrocarbon group, for example, it can be 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 "aliphatic hydrocarbon group" part contained in other groups is the same as the above explanation.

[0078] The term "3-20 membered heterocyclyl" means a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5 heteroatoms independently selected from N, O and S, preferably a "3-10 membered heterocyclyl". The term "3-10 membered heterocyclyl" means a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5, preferably 1-3 heteroatoms selected from N, O and S. The heterocyclyl may be connected to the rest of the molecule through any one of the carbon atoms or the nitrogen atom (if present). In particular, the heterocyclyl may include, but is not limited to: a 4-membered ring, such as azetidinyl, oxetanyl; a 5-membered ring, 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 heterocyclic group may be bicyclic, for example, but not limited to, a 5,5-membered ring, such as a hexahydrocyclopenta[c]pyrrole-2(1H)-yl ring, or a 5,6-membered bicyclic ring, such as a hexahydropyrrolo[1,2-a]pyrazine-2(1H)-yl ring. The ring containing the nitrogen atom 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. According to the present invention, the heterocyclic group is non-aromatic. The 3-20-membered heterocyclic group may be further selected from the following groups:

[0079]

[0080] Unless otherwise indicated, heterocyclic or heteroaryl includes all possible isomeric forms thereof, such as positional isomers thereof. Thus, for some illustrative non-limiting examples, pyridyl or pyridinylene includes pyridine-2-yl, pyridine-2-ylene, pyridine-3-yl, pyridine-3-ylene, pyridine-4-ylene and pyridine-4-ylene; thienyl or thienylene includes thien-2-yl, thien-2-ylene, thien-3-ylene and thien-3-ylene.

[0081] In any method for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any related molecule. This can be achieved by conventional protecting groups, such as those described in textbooks or reference books in this area. Protecting groups can be removed at a convenient subsequent stage using methods known in the art. Those skilled in the art will recognize that, depending on the specific protecting group, other reagents may be used for the deprotection step, including but not limited to Pd / C, Pd(OH) , PdCl , Pd(OAc) / Et SiH, Raney nickel, appropriately selected acid, appropriately selected alkali, fluoride, etc.

[0082] The target compound can be isolated according to known methods, for example by extraction, filtration, column chromatography, FCC or preparative HPLC.

[0083] According to their molecular structure, the compounds of the present invention may be chiral and therefore may exist in various enantiomeric forms. These compounds may thus exist in racemic form or in optically active form. The compounds of the present invention or their intermediates may be separated into enantiomeric compounds by chemical or physical methods known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are prepared from the mixture by reaction with optically active resolution agents. Examples of suitable resolution agents are optically active acids, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g. N-benzoylproline or N-phenylsulfonylproline) or various optically active camphorsulfonic acids in the R and S forms. Chromatographic enantiomer resolution may also be advantageously performed with the aid of optically active resolution agents (e.g. dinitrobenzoylphenylglycine, cellulose triacetate or other carbohydrate derivatives or chiral derivatized methacrylate polymers immobilized on silica gel). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, for example hexane / isopropanol / acetonitrile.

[0084] Those skilled in the art will appreciate that, since nitrogen needs to have an available lone pair of electrons for being oxidized to oxides, not all nitrogen-containing heterocycles can form N-oxides; those skilled in the art will recognize nitrogen-containing heterocycles that can form N-oxides. Those skilled in the art will also recognize that tertiary amines can form N-oxides. The synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, and the synthetic methods include oxidizing heterocycles and tertiary amines with peroxyacids such as peracetic acid and metachloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate and dioxirane such as dimethyl dioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature.

[0085] Pharmaceutically acceptable salts may be acid addition salts of compounds of the invention having a nitrogen atom in a chain or ring which are sufficiently basic, such as acid addition salts formed with inorganic acids such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid, or hydrogen sulfate, or acid addition salts formed with organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, diglucoside. Sugar 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, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0086] In addition, another suitable pharmaceutically acceptable salt of the compound of the present invention with sufficient acidity is an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., calcium salt or magnesium salt), an ammonium salt, or a salt formed with an organic base that provides a physiologically acceptable cation, such as a salt formed with the following substances: sodium ion, potassium ion, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trishydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. As an example, the pharmaceutically acceptable salts include salts of the group -COOH formed with the following substances: sodium ion, potassium ion, calcium ion, magnesium ion, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trishydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol.

[0087] In addition, basic nitrogen-containing groups can be quaternized with 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 bromides, etc. As examples, pharmaceutically acceptable salts include hydrochlorides, sulfates, nitrates, bisulfates, hydrobromides, acetates, oxalates, citrates, methanesulfonates, formates or meglumine salts, etc.

[0088] Since the compounds of the present invention may have multiple salt-forming sites, the "pharmaceutically acceptable salts" include not only salts formed at one of the salt-forming sites of the compounds of the present invention, but also salts formed at 2, 3 or all of the salt-forming sites. For this reason, the molar ratio of the compound of formula (I) to the radical ion (anion) of the acid or the cation of the base required for salt formation in the "pharmaceutically acceptable salt" may vary within a wide range, for example, it may be 4:1 to 1:4, such as 3:1, 2:1, 1:1, 1:2, 1:3, etc.

[0089] According to the present invention, pharmaceutically acceptable anions include anions selected from inorganic acids or organic acids ionized. 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, hexanoic 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, persulfate, 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, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0090] Depending on the position and properties of the different substituents, the compounds of the present invention may also contain one or more asymmetric centers. Asymmetric carbon atoms may exist in (R) or (S) configurations, with only one asymmetric center resulting in a racemic mixture and multiple asymmetric centers resulting in a diastereomeric mixture. In some cases, asymmetry may also exist due to hindered rotation around a particular bond, such as the central bond connecting two substituted aromatic rings of a particular compound. Furthermore, the substituents may also exist in cis or trans isomeric forms.

[0091] The compounds of the present invention also include all possible stereoisomers thereof, in the form of a single stereoisomer or any mixture of any proportion of said stereoisomers (e.g., R-isomers or S-isomers, or E-isomers or Z-isomers). The separation of a single stereoisomer (e.g., a single enantiomer or a single diastereomer) of a compound of the present invention can be achieved by any suitable prior art method (e.g., chromatography, in particular, for example, chiral chromatography).

[0092] The term "tautomer" refers to functional group isomers resulting from the rapid movement of an atom in a molecule between two positions. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Prototropic tautomers arise from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to separate a single tautomer usually produce a mixture whose physicochemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0093] In the present invention, the compounds involved also include isotopically labeled compounds, which are the same as those shown in Formula I, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually occurring in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of H, C, N, O, S, F and Cl, such as 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 32 P. 35 S. 18 F and 36 Cl. Compounds of the invention, prodrugs thereof, or pharmaceutically acceptable salts of said compounds or said prodrugs containing the above-mentioned isotopes and / or other isotopes of other atoms are within the scope of the invention. Certain isotopically labeled compounds of the invention, for example, those incorporating radioactive isotopes (such as 3 H and 14 C) compounds can be used in drug and / or substrate tissue distribution assays. 3 H) and carbon 14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. 2 H) substitution may provide certain therapeutic advantages (e.g., increased in vivo half-life or reduced dosage requirements) derived from greater metabolic stability and may therefore be preferred in certain circumstances. The compounds of the invention as claimed in the claims may be specifically defined as substituted with deuterium or tritium. In addition, the presence of hydrogen in a substituent without the term deuterium or tritium being separately listed does not exclude deuterium or tritium, but may also contain deuterium or tritium.

[0094] The term "effective amount" or "therapeutically effective amount" refers to an amount of the compound of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases as defined below). The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition to be treated, such as the weight and age of the subject, the severity of the disease condition, and the mode of administration, which can be easily determined by a person of ordinary skill in the art. The specific dosage will vary depending on the following factors: the specific compound selected, the dosage regimen relied on, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.

[0095] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of excipient types include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. Excipients can enhance the handling characteristics of a pharmaceutical formulation, i.e., make the formulation more suitable for direct compression by increasing fluidity and / or adhesion. Examples of typical pharmaceutically acceptable carriers suitable for the above-mentioned preparations 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 carboxymethylcellulose, ethyl cellulose and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinyl pyrrolidone; 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; ethylene glycol polymers; fatty alcohols; and cereal hydrolyzed solids and other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants and other excipients commonly used in pharmaceutical preparations.

[0096] The term "solvate" refers to those forms of the compounds of the present invention, which form complexes in a solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates, in which the coordination is with water. In the present invention, preferred solvates are hydrates. Further, pharmaceutically acceptable solvates (hydrates) of compounds of formula I of the present invention refer to cocrystals and inclusion compounds formed by compound I with one or more molecules of water or other solvents in stoichiometry. Solvents that can be used for solvates include, but are not limited to, water, methanol, ethanol, ethylene glycol and acetic acid.

[0097] The term "prodrug" or "drug precursor" refers to a compound that is converted in vivo into a compound represented by the aforementioned general formula or specific compound. Such conversion is affected by the hydrolysis of the prodrug in the blood or the conversion of the prodrug into the parent structure by enzymes in the blood or tissues. The prodrug of the present invention can be an ester. Among the esters that can be used 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 in the present invention contains a hydroxyl / carboxyl group, which can be acylated to obtain a compound in the form of a prodrug. Other prodrug forms include phosphate esters, such as these phosphate ester compounds that are obtained by phosphorylation of the hydroxyl group on the parent.

[0098] The "cancer" mentioned in the present invention includes, but is not limited to, bladder cancer, breast cancer (such as ductal carcinoma), cervical cancer (such as squamous cell carcinoma), colorectal cancer (such as adenocarcinoma), esophageal cancer (such as squamous cell carcinoma), gastric cancer (such as adenocarcinoma, medulloblastoma, colon cancer, choriocarcinoma, squamous cell carcinoma), head and neck cancer, blood cancer (such as acute lymphocytic anemia, acute myeloid leukemia, acute lymphocytic leukemia B cell, anaplastic large cell lymphoma, B cell lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic eosinophilic leukemia / hypereosinophilic syndrome, chronic myeloid leukemia, Hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma , T-cell acute lymphoblastic leukemia), lung cancer (such as bronchoalveolar adenocarcinoma, mesothelioma, mucoepidermoid carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma), liver cancer (such as hepatocellular carcinoma), lymphoma, nervous system cancer (such as glioblastoma, neuroblastoma, glioma), ovarian cancer (such as adenocarcinoma), pancreatic cancer (such as ductal carcinoma), prostate cancer (such as adenocarcinoma), kidney cancer (such as renal cell carcinoma, renal clear cell carcinoma), sarcoma (such as chondrosarcoma, Ewing sarcoma, fibrosarcoma, multifocal sarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma), skin cancer (such as melanoma, epidermoid carcinoma, squamous cell carcinoma), thyroid cancer (such as medullary carcinoma) and uterine cancer, etc.

[0099] As used herein, "autoimmune disease" or "autoimmune disorder" refers to an immune-mediated condition due to an attack on one's own tissues, 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 diseases, ulcerative colitis, Crohn's disease, rheumatoid arthritis, polyarthritis, local and systemic scleroderma, systemic lupus erythematosus, discoid lupus erythematosus, cutaneous erythematosus, cutaneous lupus erythematosus (including pernio lupus erythematosus, lupus nephritis, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, dermatomyositis, polymyositis, idiopathic edema, chronic thyroiditis, Guillain-Barré syndrome, Graves' disease, myasthenia gravis, Sjögren's syndrome, panarteritis nodosa, autoimmune enteropathy, uveitis, autoimmune oophoritis, chronic immune thrombocytopenic purpura, colitis, diabetes, psoriasis, pemphigus vulgaris, proliferative glomerulonephritis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, chronic arthritis, inflammatory chronic sinusitis, colitis, celiac disease, inflammatory bowel disease, Barlow's esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis and autoimmune-mediated blood diseases), etc. DETAILED DESCRIPTION

[0100] The present invention is further described below in conjunction with specific embodiments. The present invention includes but is not limited to the following embodiments.

[0101] The experimental methods in the following examples are conventional methods unless otherwise specified; the obtained compounds were determined by Varian Mercury-plus 400 nuclear magnetic resonance spectrometer and Waters Q-TOF-Ultima mass spectrometer. 1 H NMR spectra and mass spectra; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

[0102] Abbreviations used in the following examples and elsewhere herein are described:

[0103]

[0104]

[0105]

[0106] Example 1.

[0107] Preparation of intermediate A-1 (3-(N-methyl-N-trifluoroacetylamino)-thiophene[2,3-b]pyridine-2-carboxylic acid):

[0108]

[0109] Reagents and reaction conditions:

[0110] a) Add potassium thioacetate (57.11 g, 0.50 mol) and anhydrous DMF (250 mL) to a reaction flask (500 mL), add tert-butyl bromoacetate 1a-1 (97.53 g, 0.50 mol) dropwise at rt with stirring, and continue to react at rt for 30 min after the addition. The reaction solution is concentrated under reduced pressure at 80°C to remove the solvent, and after cooling, water (150 mL) is added to dissolve, and chloroform (150 mL) is extracted twice. The chloroform layer is washed twice with a saturated NaCl solution (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain an orange-red liquid 1a-2 (95.01 g, yield 99.9%).

[0111] b) Add tert-butyl 2-acetylthioacetate 1a-2 (10.46 g, 55 mmol), 2-chloro-3-cyanopyridine 1a-3 (6.93 g, 50 mmol) and anhydrous DMF (100 mL) to a reaction flask (250 mL), cool to 0-5 ° C, and add NaOMe (3.24 g, 60 mmol) in batches. Heat to rt and continue the reaction for 1 h. Pour the reaction solution into water (1.2 L) with stirring, and a large amount of light yellow solid precipitates. Filter, wash with water, and recrystallize from ethanol-water to obtain tert-butyl 3-aminothieno[2,3-b]pyridine-2-carboxylate (1a-4, 10.26 g, yield 82%).

[0112] c) 1a-4 (10.01 g, 40 mmol), NaHCO3 (6.72 g, 80 mmol) and anhydrous chloroform (80 mL) were added to a reaction flask (250 mL). (Tfac)2O 6.8 mL (48 mmol) was added dropwise under stirring at rt. After the addition was complete, the reaction was continued at rt for 30 min. Water (40 mL) was added to the reaction solution, and the mixture was stirred at rt until no gas was generated. The chloroform layer was separated, and the aqueous layer was extracted twice with chloroform (40 mL). The chloroform layers were combined, washed twice with saturated NaCl solution (80 mL), dried over anhydrous Na2SO4, and filtered to obtain tert-butyl 3-N-trifluoroacetylaminothieno[2,3-b]pyridine-2-carboxylate (1a-5, 13.85 g, yield 100%).

[0113] d) Add 1a-5 (13.85 g, 40 mmol) and anhydrous DMF (70 mL) to a reaction flask (250 mL), cool to 0-5 ° C, add NaH (1.92 g, 48 mmol, content 60%), and add MeI (3.24 mL, 52 mmol) / DMF (10 ml) dropwise until no gas is released. After the addition is complete, heat to rt and react for 2 h. The reaction solution is adjusted to pH 7 with acetic acid, water (50 mL) is added, and chloroform (50 mL) is extracted three times. The chloroform layers are combined, washed twice with saturated NaCl solution (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The concentrate is recrystallized from CHCl3-PE to obtain N-methyl-N-trifluoroacetyl-3-aminothieno[2,3-b]pyridine-2-carboxylic acid tert-butyl ester (1a-6, 12.84 g, yield 89%).

[0114] e) 1a-6 (2.88 g, 8 mmol), dry DCM (20 mL) and TFA (10 mL) were added to a reaction flask (50 mL) and reacted at 40°C overnight. DCM and TFA were removed by concentration under reduced pressure to obtain a yellow colloid. The mixture was passed through a silica gel column with a gradient elution of CHCl3:MeOH=50:1,7:3 to obtain N-methyl-N-trifluoroacetyl-3-aminothieno[2,3-b]pyridine-2-carboxylic acid (A-1, 2.38 g, yield 98%). A-1: ​​LC-MS (+ESI): m / z 305 ([M+H] + ); 1 H NMR (400MHz, DMSO-d6) δ8.70(dd,J=4.6,1.3Hz,1H),8.26(d,J=8.1Hz,1H),7.53(dd,J=8.1,4.6Hz,1H),3.28(s,3H).

[0115] Using appropriate synthetic precursors, the Example Intermediates A-2 to A-26 in the following Table 1 were synthesized according to the reagents and reaction conditions described above for Example 1 (Intermediate A-1).

[0116] Table 1:

[0117]

[0118]

[0119]

[0120] Example 2.

[0121] Preparation of intermediate A-27 (3-N-ethylamino-6-methylthiophene[2,3-b]pyridine-2-carboxylic acid):

[0122]

[0123] Reagents and reaction conditions:

[0124] a) Add tert-butyl 2-acetylthioacetate 1a-2 (10.46 g, 55 mmol), 2-chloro-3-cyano-6-methylpyridine 2a-1 (7.63 g, 50 mmol) and anhydrous DMF (100 mL) to a reaction flask (250 mL), cool to 0-5 ° C, and add NaOMe (3.24 g, 60 mmol) in batches. Heat to rt and continue to react for 1 h. Pour the reaction solution into water (1.2 L) with stirring, and a large amount of light yellow solid precipitates. Filter, wash with water, and recrystallize from ethanol-water to obtain tert-butyl 3-amino-6-methylthieno[2,3-b]pyridine-2-carboxylate 2a-2 (10.57 g, 80%).

[0125] b) Add 2a-2 (10.57 g, 40 mmol), acetaldehyde (2.81 mL, 50 mmol), acetonitrile (80 mL), and Et3SiH (19.14 mL, 120 nmol) to a reaction flask (250 mL); add (Tfac)2O (16.90 mL, 120 mmol) dropwise at rt with stirring, and continue to react at rt for 18 h. The reaction solution was concentrated under reduced pressure to remove the solvent and residual reagents, and purified by silica gel column chromatography (PE-EA gradient elution) to obtain tert-butyl 3-N-ethylamino-6-methylthieno[2,3-b]pyridine-2-carboxylate 2a-3 (10.64 g, 91%).

[0126] c) Add 2a-3 (5.85 g, 20 mmol), dry dichloromethane (20 mL) and trifluoroacetic acid (20 mL) to a reaction flask (50 mL) and react at 40°C overnight. Concentrate under reduced pressure to remove the solvent and residual reagents, and purify by silica gel column chromatography (CHCl3-MeOH gradient elution) to obtain 3-N-ethylamino-6-methylthieno[2,3-b]pyridine-2-carboxylic acid A-27 (4.49 g, 95%), (+)-ESI-MS: m / z 237 ([M+H] + ); 1 H NMR (400MHz, CDCl3) δ8.31 (d, J = 8.5Hz, 1H), 7.14 (d, J = 8.5Hz, 1H), 3.45 (m, 2H), 2.67 (s, 3H), 1.33 (t, J = 7.2Hz, 3H).

[0127] Example intermediates A-28-A-29 in Table 2 below were synthesized according to the procedure outlined above for Example 2 (Intermediate A-27) using appropriate synthetic precursors.

[0128] Table 2:

[0129]

[0130]

[0131] Example 3.

[0132] Preparation of intermediate A-30 (3-(N-methyl-N-trifluoroacetylamino-6-hydroxymethylthiophene[2,3-b]pyridine-2-carboxylic acid):

[0133]

[0134] Reagents and conditions:

[0135] a) Add tert-butyl 2-acetylthioacetate 1a-2 (10.64 g, 56 mmol), 2-chloro-3-cyano-6-acetoxymethylpyridine 3a-1 (10.53 g, 50 mmol) and anhydrous DMF (100 mL) to a reaction flask (250 mL), cool to 0-5 ° C, and add NaOMe (3.24 g, 60 mmol) in batches. Heat to rt and continue to react for 1 h. Pour the reaction solution into water (1.2 L) with stirring, and a large amount of light yellow solid precipitates. Filter, wash with water, and recrystallize from ethanol-water to obtain tert-butyl 3-amino-6-acetoxymethylthieno[2,3-b]pyridine-2-carboxylate 3a-2 (13.54 g, 84%).

[0136] b) Add 3a-2 (13.54 g, 42 mmol), NaHCO3 (6.72 g, 80 mmol) and anhydrous chloroform (80 mL) to a reaction flask (250 mL), add (Tfac)2O (6.8 mL, 48 mmol) dropwise under stirring at rt, and continue to react at rt for 30 min after the addition is complete. Add water (40 mL) to the reaction solution, stir at rt until no gas is generated, separate the chloroform layer, and extract the water layer twice with chloroform (40 mL). Combine the chloroform layers, wash twice with saturated NaCl solution (80 mL), dry with anhydrous Na2SO4, and filter to obtain tert-butyl 3-N-trifluoroacetylamino-6-acetoxymethylthieno[2,3-b]pyridine-2-carboxylate 3a-3 (17.22 g, 98%).

[0137] c) Add 3a-3 (16.73 g, 40 mmol) and anhydrous DMF (70 mL) to a reaction flask (250 mL), cool to 0-5 ° C, add NaH (1.92 g, 48 mmol, content 60%), and add MeI (3.24 mL, 52 mmol) / DMF (10 ml) dropwise until no gas is released. After the addition is complete, heat to rt and react for 2 h. The reaction solution is adjusted to pH 7 with acetic acid, water (50 mL) is added, and chloroform (50 mL) is extracted three times. The chloroform layers are combined, washed twice with saturated NaCl solution (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The concentrate is recrystallized from CHCl3-PE to obtain N-methyl-N-trifluoroacetyl-3-aminothieno[2,3-b]pyridine-2-carboxylic acid tert-butyl ester 3a-4 (12.84 g, 89%).

[0138] d) 3a-4 (3.35 g, 8 mmol), dry dichloromethane (20 mL) and trifluoroacetic acid (10 mL) were added to a reaction bottle (50 mL) and reacted at 40°C overnight. The mixture was concentrated under reduced pressure to remove dichloromethane and residual trifluoroacetic acid, and purified by silica gel column with a gradient elution of CHCl3-MeOH 20:1→3:1 to give 3-N-methyl-N-trifluoroacetylamino-6-hydroxymethylthieno[2,3-b]pyridine-2-carboxylic acid A-30 (2.54 g, 95%), (+)-ESI-MS: m / z 335 ([M+H] + ); 1 H NMR (400MHz, DMSO-d6) δ 8.50 (d, J = 8.1 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 4.91 (s, 2H), 3.28 (s, 3H).

[0139] Example 4.

[0140] Preparation of intermediate B-1 (tert-butyl 3-(4-(2-aminoethyl)phenyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate):

[0141]

[0142] Reagents and conditions:

[0143] a) Add p-bromophenylethylamine 1b-1 (10.00 g, 50 mmol), KI (0.41 g, 2.5 mmol), K2CO3 (16.58 g, 120 mmol) and acetonitrile (100 mL) to a reaction flask (250 mL), heat to reflux, and add BnCl (20.89 g, 165 mmol) dropwise. After the addition is complete, reflux for 2 h. Filter the reaction solution to remove inorganic salts, concentrate the filtrate under reduced pressure to remove acetonitrile, add chloroform (200 mL), wash with saturated NaCl solution (100 mL × 2), dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a crude product. The crude product is concentrated under reduced pressure to remove excess BnCl and by-product benzyl alcohol to obtain 1b-2 (light yellow liquid 18.39 g, 97%).

[0144] b) Add 1b-3 (4.25 g, 20 mmol), 1b-2 (9.13 g, 24 mmol), Pd(OAc)2 (449 mg, 2 mmol), X-phos (953 mg, 2 mmol), Cs2CO3 (13.03 g, 40 mmol) and toluene (80 mL) to a reaction flask (250 mL), replace N2 with vacuum, heat to 100°C, and react for 18 h. Filter the insoluble matter from the reaction solution, concentrate the filtrate under reduced pressure, and pass the concentrate through a silica gel column with a gradient elution of PE:EA=19:1,9:1 to obtain 1b-4 (8.59 g, 84%).

[0145] c) 1b-4 (8.15 g, 15.9 mmol), HCO2NH4 (20.09 g, 318.5 mmol), Pd(OH)2 / C (2.26 g, containing 15% Pd) and MeOH (65 mL) were added to a reaction flask (250 mL), and the N2 was replaced by vacuum. The temperature was raised to 60°C and the reaction was allowed to proceed overnight. The insoluble matter was filtered out from the reaction solution, and the filtrate was concentrated under reduced pressure to remove methanol. Chloroform (200 mL) was added to the concentrate, which was washed with saturated NaCl solution (50 mL×3), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a crude product. The crude product was passed through a silica gel column and eluted with a gradient of CHCl3-MeOH 20:1→8:2 to obtain B-1 (5.15 g, 93%), (+)-ESI-MS: m / z 332 ([M+H] + ); 1 H 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).

[0146] Using appropriate synthetic precursors, Example Intermediates B-2 to B-26 in Table 3 below were synthesized according to the reagents and reaction conditions described above for Example 4 (Intermediate B-1).

[0147] Table 3:

[0148]

[0149]

[0150] Embodiment 5:

[0151] Preparation of intermediate B-21 (tert-butyl 4-(4-aminoethylphenyl)piperidine-1-carboxylate):

[0152]

[0153] Reagents and reaction conditions

[0154] a) 1b-2 (1.66 g, 4.35 mmol), tert-butyl 4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,6-tetrahydropyridine-1-carboxylate (2.69 g, 8.70 mmol), Pd(dppf)Cl2 (0.32 g, 0.44 mmol), potassium carbonate (1.20 g, 8.68 mmol), ethanol (10 mL) and water (2 mL) were added to a 50 mL microwave tube. The mixture was heated at 130 ° C under microwave irradiation for 1 h. The residual solid was filtered off and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with EA-PE gradient elution to give 2b-1 (419 mg, 20%) as a yellow solid.

[0155] b) Add 2b-1 (400 mg, 0.83 mmol), HCO2NH4 (1.05 g, 16.65 mmol), Pd(OH)2 / C (115 mg, containing 15% Pd) and MeOH (5 mL) to a reaction bottle (25 mL), replace N2 with vacuum, heat to 60°C, and react overnight. The reaction solution was filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure to remove methanol. The concentrate was added with chloroform (10 mL), washed with saturated NaCl solution (10 mL) three times, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a crude product. The crude product was passed through a silica gel column and eluted with a gradient of CHCl3-MeOH 20:1→5:1 to obtain B-21 (227 mg, 90%), (+)-ESI-MS: m / z 305 ([M+H] + ).

[0156] Example Intermediates B-22 to B-24 in Table 4 below were synthesized according to the procedure outlined above for Example 5 (Intermediate B-21) using appropriate synthetic precursors.

[0157] Table 4:

[0158]

[0159] Example 6

[0160] Preparation of intermediate B-25 (tert-butyl 3-(4-(1-amino-isopropyl)phenyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate):

[0161]

[0162] Reagents and reaction conditions

[0163] a) Add p-bromoacetophenone 3b-1 (3.38 g, 17.0 mmol), tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (3.00 g, 14.1 mmol), potassium carbonate (5.86 g, 42.4 mmol), and HMPA (30 mL) to a 100 mL round-bottom flask. The resulting solution was stirred overnight at 70 ° C in an oil bath, then cooled to rt and quenched with water (30 mL). The resulting solution was extracted three times with EA (30 mL), the organic layers were combined, concentrated under reduced pressure, and purified by silica gel column chromatography (EA-PE 1:5 elution) to obtain a brown oil 3b-2 (1.68 g, 30%), ESI-MS (m / z): 331 [M+H] + .

[0164] b) 3b-2 (1.65 g, 5 mmol), potassium tert-butoxide (1.13 g, 13.0 mmol), TosMIC (1.46 g, 7.5 mmol), tert-butanol (20 mL) and DME (20 mL) were added to a 100 mL round-bottom flask. The resulting solution was stirred overnight in an oil bath at 90°C, cooled after the reaction, and quenched with water (20 mL). The reaction solution was extracted 3 times with EA (20 mL), and the combined organic layers were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (eluted with EA-PE 1:5) to obtain a brown oil 3b-3 (1.13 g, 66%), ESI-MS (m / z): 342 [M+H] + .

[0165] c) Add 3b-3 (0.96 g, 2.8 mmol), NH3 / MeOH (7M, 20 mL), and Raney nickel (500 mg) to a 100 mL round-bottom flask maintained under nitrogen purge. The reaction mixture was continuously purged with hydrogen and stirred at rt for 2 h. The reaction solution was filtered to remove the solid, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluted with dichloromethane-methanol 10:1) to obtain a yellow oil B-25 (790 mg, 82%), ESI-MS (m / z): 346 [M+H] + .

[0166] Example 7

[0167] Preparation of intermediate B-26 ((S)-1-(4-bromophenyl)-2-amino-3-methoxypropane):

[0168]

[0169] Reagents and reaction conditions

[0170] a) Add 4b-1 (12.25 g, 50 mmol) and anhydrous alcohol (30 mL) to a 100 mL round-bottom flask, cool to 0 ° C, stir, slowly drop SOCl2 (5.45 mL, 75 mmol), raise to rt after the dropwise addition, and stir overnight. After the reaction, concentrate under reduced pressure to remove the solvent, add water (30 mL), extract with EA (30 mL) three times, combine the organic layers, and concentrate under reduced pressure to obtain 4b-2 (11.70 g, 86%).

[0171] b) Add 4b-2 (9.52 g, 35 mmol), BnCl (11.5 mL, 100 mmol), KI (8.3 g, 50 mmol), K2CO3 (6.91 g, 50 mmol) and MeCN (30 mL) to a 100 mL round-bottom flask and stir at 60 °C for 4 h. After the reaction, cool and add water (30 mL) to quench, extract with EA (30 mL) three times, combine the organic layers, concentrate under reduced pressure, and purify by silica gel column chromatography (EA-PE 1:5 elution) to obtain 4b-3 (14.25, 90%), ESI-MS (m / z): 452 [M+H] + .

[0172] c) Add 4b-3 (13.57 g, 30 mmol), NaBH4 (2.27 g, 60 mmol) and THF (30 mL) to a 100 mL round-bottom flask kept under nitrogen purge, and stir at rt for 2 h. Filter the reaction solution to remove the solid, add 20 mL of saturated potassium carbonate solution to the filtrate, separate the organic layer, concentrate under reduced pressure, and purify by silica gel column chromatography (eluted with dichloromethane-methanol 20:1) to obtain 4b-4 (9.85 g, 80%), ESI-MS (m / z): 410 [M+H] + .

[0173] d) Add 4b-4 (0.92 g, 4 mmol), anhydrous THF (5 mL) and NaH (0.192 g, 4.8 mmol, content 60%) to a reaction bottle (25 mL), stir at rt for 30 min, and then add MeI (0.33 mL, 5.2 mmol) / DMF (1 ml) dropwise. After the addition is complete, react for 2 h. The reaction solution is adjusted to pH 7 with acetic acid, water (5 mL) is added, and chloroform (5 mL) is extracted three times. The chloroform layers are combined, washed twice with saturated NaCl solution (5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The concentrate is recrystallized from CHCl3-PE to obtain B-26 (859 mg, yield 88%), ESI-MS (m / z): 244 [M+H] + .

[0174] Example 8

[0175] Preparation of intermediate B-27 (tert-butyl 3-(4-(2-amino-3-ethoxypropyl)phenyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate):

[0176]

[0177] Reagents and reaction conditions

[0178] Reactions a and b are the same as a and b in Example 7.

[0179] c) 1b-3 (4.25 g, 20 mmol), 4b-3 (9.04 g, 20 mmol), Pd(OAc)2 (449 mg, 2 mmol), X-phos (953 mg, 2 mmol), Cs2CO3 (13.03 g, 40 mmol) and toluene (80 mL) were added to a reaction flask (250 mL), the atmosphere was replaced with N2 by vacuum, the temperature was raised to 100°C, and the reaction was continued for 18 h. The insoluble matter was filtered off from the reaction solution, the filtrate was concentrated under reduced pressure, and the concentrate was passed through a silica gel column with a gradient elution of PE:EA = 19:1 → 9:1 to obtain 5b-1 (9.34 g, 82%).

[0180] d) Add 5b-1 (8.15 g, 15.9 mmol), HCO2NH4 (20.09 g, 318.5 mmol), Pd(OH)2 / C (2.26 g, containing 15% Pd) and MeOH (65 mL) to a reaction flask (250 mL), replace N2 with vacuum, raise the temperature to 60°C, and react overnight. The reaction solution was filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure to remove methanol. Chloroform (200 mL) was added to the concentrate, washed with saturated NaCl solution (50 mL×3), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a crude product. The crude product was passed through a silica gel column and eluted with a gradient of CHCl3-MeOH 20:1→8:2 to obtain B-27 (4.57 g, 91%). (+)-ESI-MS: m / z 390 ([M+H] + ).

[0181] Example intermediates B-28 to B-30 in Table 5 below were synthesized according to the procedures outlined above for Examples 7 and 8 (Intermediates B-26 and B-27) using appropriate synthetic precursors.

[0182] Table 5:

[0183]

[0184]

[0185] Example 9

[0186] Preparation of compound I-1 (N-4-(3,8-diazabicyclo[3.2.1]octane-3-yl)phenethyl-6-methyl-3-methylaminothiophene[2,3-b]pyridine-2-carboxamide) and its hydrochloride

[0187]

[0188] a) Add A-2 (382 mg, 1.2 mmol), B-1 (398 mg, 1.2 mmol), EDCI (276 mg, 1.44 mmol), HOBt (178 mg, 1.32 mmol) and dry DMF (4 mL) to a reaction bottle (10 mL), add DIEA (611 uL, 3.6 mmol), and heat to 60 ° C for 2 h. The reaction solution was concentrated under reduced pressure at 80 ° C, and the concentrate was passed through a silica gel column with a gradient elution of PE: EA = 7:3 → 6:4 to obtain 624 mg of amide (yield 82%).

[0189] b) Add amide (604 mg, 0.96 mmol) and dichloromethane (4 mL) to a reaction flask (10 mL), add trifluoroacetic acid (745 uL, 10 mmol), and heat to 40 °C to react overnight. After the reaction is complete, remove dichloromethane and trifluoroacetic acid under reduced pressure to obtain a de-Boc product.

[0190] c) MeOH (4 mL) and K2CO3 (553 mg, 4 mmol) were added to the de-Boc product and stirred at rt for 30 min. The reaction solution was extracted with water (10 mL) and chloroform (10 mL×3), the chloroform layers were combined, washed with saturated NaCl solution (10 mL×2), dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain the de-Tfac product I-1 (238 mg, 57%), (+)-ESI-MS: m / z 436; 1 H NMR (400MHz, CDCl3) δ8.30(d,J=8.5Hz,1H),8.10(q,J=5.7Hz,1H),7.15(d,J=8.6H z,2H),7.12(d,J=8.5Hz,1H),6.80(d,J=8.6Hz,2H),5.59(t,J=5.6Hz,1H),3.78(br s,2H),3.60(td,J=6.9,5.6Hz,2H),3.47(dd,J=11.4,2.3Hz,2H),3.32(d,J=5.7H z,3H),3.02(dd,J=11.3,1.5Hz,2H),2.83(t,J=6.9Hz,2H),2.67(s,3H),1.94(br s,4H).

[0191] d) I-1 (237 mg) was dissolved in MeOH (8 mL), and 36% hydrochloric acid (120 uL) was added under stirring at rt, and a large amount of orange-yellow solid precipitated. It was filtered and washed with a small amount of MeOH to remove free HCl, to obtain I-1 hydrochloride (253 mg, 100%), (+)-ESI-MS: m / z 436; 1 H NMR(400MHz, DMSO-d6)δ9.50-9.46(m,2H),8.49(d,J=8.5Hz,1H),7.87(t,J=4.9Hz,1H),7 .30(d,J=8.5Hz,1H),7.09(d,J=8.6Hz,2H),6.84(d,J=8.6Hz,2H),6.53(brs,3H),4.09(br s,2H),3.55(dd,J=11.2,1.6Hz,2H),3.36(tt,J=7.8,4.8Hz,2H),3.17(s,3H),3.0 8(d,J=12.8Hz,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.

[0192] Using appropriate synthetic precursors, Example compounds I-2 to I-82 in the following Table 6 were synthesized according to the reagents and reaction conditions described above for Example 9 (Compound I-1).

[0193] Table 6:

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208] Example 10

[0209] Preparation of compound I-83 (N-4-(3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl-6-amino-3-methylaminothiophene[2,3-b]pyridine-2-carboxamide)

[0210]

[0211] Reagents and reaction conditions:

[0212] a) I-82a (111 mg, 0.2 mmol), tert-butyl carbamate (117 mg, 1.0 mmol), Cs2CO3 (326 mg, 1.0 mmol) and 1,4-dioxane (1 ml) were added to a 25 mL flask, stirred, and X-Phos Pd (II) (39.5 mg, 0.05 mmol) was added. Nitrogen was bubbled through the reaction mixture for 2 minutes, and then the reaction was heated to 90 ° C overnight. After the reaction was completed, it was cooled to rt, the solid was filtered off, the filtrate was decompressed to remove the solvent, and purified by silica gel column chromatography (EA-PE 1:5→2:3 gradient elution) to obtain the amination product I-83a (42 mg, 33%) as a light yellow solid, (+)-ESI-MS: m / z 637.

[0213] b) Add I-83a (21.2 mg, 0.033 mmol) and DCM (1 mL) to a reaction bottle (5 mL), add TFA (100 uL, 1.535 mmol), and heat to 40 °C to react overnight. After the reaction, remove DCM and TFA under reduced pressure to obtain I-83 (7.92 mg, 55%), (+)-ESI-MS: m / z 437; 1 H NMR (400MHz, DMSO-d6) δ7.91(d,J=8.4Hz,1H),7.87(t,J=5.0Hz,1H),7.10(d,J=8.6Hz,2H),6.84(d,J=8.6Hz,2H),6.55(br s,2H),6.52(br s,1H),6.45(d,J=8.6Hz,1H),4.10(br s,2H),3.56(br d,J=11.2Hz,2H),3.37(td,J=7.2,5.0Hz,2H),3.18(s,3H),3.09(br d,J=11.5Hz,2H),2.73(t,J=7.2Hz,2H),2.60(s,3H),1.90-2.00(m,4H).

[0214] Using appropriate synthetic precursors, Example compounds I-84 to I-85 in the following Table 7 were synthesized according to the reagents and reaction conditions described above for Example 10 (Compound I-83).

[0215] Table 7:

[0216]

[0217]

[0218] Example 11.

[0219] The activity of USP28 was determined by the ubiquitin-rhodamine 110 method.

[0220] Purified USP28 and the substrate Ubiquitin-Rhodamine 110 (Ubiquitin-Rhodamine 110) used to determine the activity of DUBs were both from R&D Systems. The test compound was first dissolved in DMSO to prepare a 10mM stock solution, and then diluted to the required concentration (DMSO content ≤0.5%) with a buffer solution [containing 20mM Tris-HCl (pH 8.0), 2mM CaCl2, 3mM BME, 0.01% Prionex, 0.01% Triton X-100], pre-mixed with USP28 (final concentration 4nM) in a 96-well plate and incubated at room temperature for 30 minutes, and then the substrate (Ubiquitin-Rhodamine 110) was added to 125nM. The final volume of the entire reaction was 20μL. Immediately after adding the substrate, the released fluorescence was detected on the microplate reader (excitation wavelength 485nm, emission wavelength 535nm). The inhibition rate of the test compound on USP25 was calculated according to the following formula:

[0221] Inhibition % = 1 - [(fluorescence value of test compound + substrate - fluorescence value of test compound (without substrate)) / average fluorescence value of DMSO control group - fluorescence value of test compound (without substrate)]

[0222] According to the inhibition rate of the test compound on USP28 at different concentrations, its IC 50 value

[0223] Example 12.

[0224] The activity of USP25 was determined by the ubiquitin-rhodamine 110 method.

[0225] Purified USP25 and the substrate Ubiquitin-Rhodamine 110 (Ubiquitin-Rhodamine 110) used to determine the activity of DUBs were both from R&D Systems. The test compound was first dissolved in DMSO to prepare a 10mM stock solution, and then diluted to the required concentration (DMSO content ≤ 0.5%) with a buffer solution [containing 20mM Tris-HCl (pH 8.0), 2mM CaCl2, 3mM BME, 0.01% Prionex, 0.01% Triton X-100], pre-mixed with USP25 (final concentration 15nM) in a 96-well plate and incubated at room temperature for 30 minutes, and then the substrate (ubiquitin-Rhodamine 110) was added to 125nM. The final volume of the entire reaction was 20μL. Immediately after adding the substrate, the released fluorescence (excitation wavelength 485 nm, emission wavelength 535 nm) was detected on a microplate reader, and the inhibition rate of the test compound on USP25 was calculated according to the following formula:

[0226] Inhibition % = 1 - [(fluorescence value of test compound + substrate - fluorescence value of test compound (without substrate)) / average fluorescence value of DMSO control group - fluorescence value of test compound (without substrate)]

[0227] As in Example 11, the IC 50 value

[0228] Table 8 Inhibitory activity (IC) of the compounds of the present invention against USP28 and USP25 50 )

[0229]

[0230] The symbols in Table 8 above correspond to the ICs represented 50 The range is as follows:

[0231]

[0232] Embodiment 13

[0233] Referring to Example 11 and Example 12, the inhibitory activity (IC) of USP28 and USP25 was measured for the representative compounds of the present invention and the following comparative compound structures. 50 ) test, the results are shown in Table 9 below

[0234] Table 9: Inhibitory activities (IC 50 ,nM) comparison

[0235]

[0236] The above experimental results show that the example compounds of the present invention have very significant improvements in the inhibitory activity against USP28 and / or USP25 compared to the prior art compounds before structural optimization. As shown in Table 9, the inhibitory activity of the improved compounds against USP28 and USP25 is more than 15 times that of the prior art compounds, indicating that the monoalkyl (or substituted alkyl) group of 3-NH2 of the general formula I of the present invention is a key site for the inhibitory activity against USP28 and USP25 of this type of compounds.

[0237] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A compound of formula I and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt: in: X is C-R5; n is 1 or 2; m is 1, 2, or 3; R1, R2, R5 are the same or different and are independently selected from hydrogen, halogen, hydroxyl, amino and the following groups which are optionally unsubstituted or substituted: (C1-C6) alkyl, (C1-C6) alkyloxy, halo (C1-C6) alkyl, halo (C1-C6) alkyloxy; Each R4 is the same or different and is independently selected from hydrogen, halogen, hydroxy, amino, and (C1-C6) alkyl, (C1-C6) alkyloxy, halo-(C1-C6) alkyl, halo-(C1-C6) alkyloxy; R3 is methyl or ethyl; Y is selected from Wherein, Z is nitrogen hydrogen (NH), oxygen (O), sulfur (S) or methylene (CH2); q is selected from 1, 2 or 3; each R8 is the same or different and is independently selected from hydrogen, halogen; or Y is selected from wherein p is selected from 0, 1, 2 or 3, and each R6 is the same or different and is independently selected from (C1-C6) alkyloxy, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; R7 is selected from the following groups:

2. The compound of formula (I) according to claim 1, and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt, characterized in that: R4 and R8 are selected from hydrogen.

3. The compound of formula I according to claim 1 or 2, and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt, characterized in that: R1, R2, and R5 are the same or different and are independently selected from hydrogen, halogen, hydroxy, amino, and (C1-C6) alkyl.

4. The compound of formula I according to claim 1 or 2, and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt, characterized in that: R1, R2, and R5 are the same or different and are independently selected from hydrogen or (C1-C6) alkyl.

5. The compound of formula I according to claim 1 or 2, and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt, characterized in that: The compound of formula I is selected from the following structures:

6. A method for preparing the compound of formula I according to any one of claims 1 to 5 and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt, characterized in that The following steps are involved: The intermediate carboxylic acid A and the intermediate amine B are reacted with a peptide coupling reagent under alkaline conditions to form an amide, and then the protecting group trifluoroacetyl (Tfac) is removed to obtain the target compound I: Wherein, R'4 and Y' are respectively R4 and Y of Formula I, or R4 and Y whose hydroxyl group and amino group are protected by Boc.

7. The preparation method according to claim 6, characterized in that: The reagents and reaction conditions in the steps include: a) amide coupling reaction: the coupling reagent is selected from EDCI-HOBt, BOP, HATU, the base is selected from DEA, TEA, EDCI or DMAP; the solvent is selected from DCM or DMF; b) Tfac removal reaction: the base is selected from K2CO3 or NaOMe, and the solvent is selected from MeOH; c) Boc removal reaction: dilute hydrochloric acid-methanol.

8. The preparation method according to claim 6, characterized in that The intermediate carboxylic acid A can be prepared by the following steps: In this step, the reagents and reaction conditions are: a) DMF; b) NaOMe / DMF; c) (Tfac)2O / NaHCO3 / CHCl3; d) NaH / DMF, R3-I; e) TFA / DCM.

9. The preparation method according to claim 6, characterized in that The intermediate amine B is selected from B-Ia or B-Ib: R'4, R'6, R'7 and R'8 are respectively R4, R6, R7 and R8 of formula I according to any one of claims 1 to 5 or R4, R6, R7 and R8 in which the hydroxyl group and the amino group are protected by Boc.

10. The preparation method according to claim 6, characterized in that: The intermediate amine B is selected from B-II and is prepared by the following steps: wherein R'4 and R'6 are respectively R4 and R6 of formula I according to any one of claims 1 to 5 or R4 and R6 whose hydroxyl group or amino group is protected by Boc, In this step, the reagents and reaction conditions are: a) BnCl, KI, K2CO3 / MeCN; b) H-R7-Boc, Pd(OAc)2, X-phos, Cs(CO3)2, toluene; c) HCO2NH4, Pd(OH)2 / C, MeOH.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound of formula I as described in any one of claims 1 to 5 and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt.

12. Use of the compound of formula I according to any one of claims 1 to 5 and its racemate, stereoisomer, tautomer, isotope-labeled substance or pharmaceutically acceptable salt or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating a disease or disorder associated with the inhibition of USP28 and / or USP25.

13. The use according to claim 12, characterized in that The diseases or disorders associated with USP28 and / or USP25 are cancer, inflammation, autoimmune diseases, viral infections and bacterial infections.

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