Compound and application thereof in treatment of chronic hepatitis B, hepatic fibrosis and liver cancer

Abstract

The invention relates to a compound and application thereof in treatment of chronic hepatitis B, hepatic fibrosis and liver cancer. Specifically provided are a compound represented by formula I, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate of the compound, # imgabs0 # of the compound, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate of the compound.

Description

Compound and its application in the treatment of chronic hepatitis B, liver fibrosis and liver cancer

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202310416237.7 and application date April 18, 2023, and claims its priority. The content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present invention relates to the field of medicine, and in particular to a new class of compounds and their application in the treatment of chronic hepatitis B, liver fibrosis and liver cancer. Background Art

[0004] Chronic hepatitis B virus (HBV) infection poses a serious threat to human health. According to the World Health Organization, approximately 2 billion people worldwide have been infected with the hepatitis B virus, of whom 350 million are chronic HBV carriers. Approximately 1 million people die annually from liver failure, cirrhosis, and hepatocellular carcinoma (HCC) caused by HBV infection. my country is a high-incidence region for HBV infection. Although the rate of new HBV infections has been significantly reduced with the widespread use of hepatitis B vaccines and the development of technologies to prevent mother-to-child transmission, a large epidemiological study of over 2 million cases in 2016 showed that the HBsAg positivity rate among men aged 21-49 in rural areas of my country remains as high as 6%. This suggests that HBV infection will remain a significant challenge for my country's public health system for a considerable period of time.

[0005] HBV is a partially double-stranded circular DNA (rcDNA) virus that is tropic for hepatocellular cells. Its genome is approximately 3.2 kb in length and consists of four partially overlapping open reading frames (ORFs), including the S, C, P, and X gene regions. HBV replication involves a complex series of processes that transmit the viral genetic information from parental DNA to offspring, including transcription of mRNA using covalently closed circular DNA (cccDNA) as an initial template, translation, and reverse transcription using mRNA as a template to synthesize viral negative-strand DNA and viral positive-strand DNA. HBV cccDNA is stably present in the nucleus of infected hepatocytes and serves as a viral transcription template to produce viral gene products. It is the original replication template for HBV. The presence of HBV cccDNA is the root cause of viral replication and infection. Inhibiting or eliminating HBV cccDNA is key to curing chronic hepatitis B.

[0006] Anti-HBV treatment has always been one of the hot spots and difficulties in infectious science research. Most patients with chronic hepatitis B require long-term or lifelong treatment, and there is currently no treatment option that can completely eradicate the hepatitis B virus in the host body. Currently, clinically approved antiviral drugs mainly include two categories: nucleoside (acid) analogues and interferons. The target of nucleoside (acid) analogue drugs is to inhibit the activity of HBV DNA polymerase and reverse transcriptase, thereby inhibiting the replication of HBV; interferon exerts its antiviral effect by acting on multiple links in the HBV replication cycle. It mainly includes activating the host immune response, inhibiting the transcription of HBVcccDNA, inhibiting the formation of viral nucleocapsids or increasing their degradation. Although these drugs can effectively inhibit HBV replication, they are unable to eliminate or silence cccDNA in the nucleus of hepatocytes, and there are problems such as large drug side effects (especially interferon) and high drug resistance.

[0007] In clinical practice, it is difficult to achieve seroconversion of HBV surface antigen (HBsAg) in the treatment of chronic hepatitis B patients with these two types of drugs, and it is impossible to achieve functional cure. Therefore, in response to the shortcomings of existing antiviral drugs, different types of new antiviral drugs under development have been discovered, mainly including five major categories: viral entry inhibitors, capsid inhibitors, nucleic acid interfering drugs, HBsAg inhibitors and immunomodulators. Although many antiviral drugs under development have certain therapeutic effects, large-scale clinical trials are still needed to further verify their antiviral effects. Therefore, the search for new antiviral drugs is still necessary.

[0008] Summary of the Invention

[0009] Calcium ions (Ca 2+ ) as an important second messenger in cells can interact with a variety of cellular proteins, regulate a variety of physiological processes and participate in the progression of a variety of diseases, including HBV infection. In recent years, many studies have shown that different Ca2+ molecules on the plasma membrane, endoplasmic reticulum and mitochondria are involved in the regulation of HBV infection. 2+ channels, HBV can increase cytoplasmic Ca 2+ Furthermore, in HBV-infected cells, Ca 2+ Activation of Ca signaling can promote viral replication through multiple molecular mechanisms. 2+ Signaling will be an effective method for treating HBV infection.

[0010] Transient receptor potential (TRP) channel superfamily and intracellular Ca 2+TRP channels are closely related to homeostasis regulation. They were first discovered in the visual system of Drosophila. To date, the 28 subtypes found in mammals are divided into 7 subfamilies: TRPA, TRPC, TRPM, TRPML, TRPN, TRPP and TRPV. Transient receptor potential vanilloid 4 (TRPV4) is a member of the TRPV subfamily and is widely distributed in the heart, brain, kidney, lung, liver, bone tissue and skin. It is sensitive to Ca 2+ It is selectively permeable and its activation causes Ca 2+ influx, thereby increasing intracellular free Ca 2+ It is involved in many physiological and pathological processes and is a potential therapeutic target for many diseases.

[0011] The present invention first discovered that overexpression of TRPV4 can promote HBV replication and transcription, and silencing of TRPV4 can inhibit HBV replication and transcription, and further discovered that TRPV4 inhibitors have a significant effect of inhibiting the expression of HBV core protein (HBcAg).

[0012] The inventors have discovered that a class of new compounds for which protection is sought in this application has good activity and has a good therapeutic effect on liver diseases such as chronic hepatitis B, liver fibrosis, and liver cancer, and these molecules have good drugability (solubility, oral bioavailability, etc.) and safety.

[0013] To this end, in the first aspect of the present invention, the present invention provides a compound represented by the following general formula I:

[0014] or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof,

[0015] in:

[0016] R 1 is selected from a fused heteroaryl group, wherein the fused heteroaryl group is formed by fusion of a six-membered ring and a five-membered ring, wherein the six-membered ring contains one heteroatom or no heteroatom, and the five-membered ring contains two heteroatoms; the heteroatom is selected from N, O or S atoms, preferably N atoms; optionally, the six-membered ring (preferably a six-membered ring containing no heteroatoms) is substituted by 1, 2, 3 or 4 -CN, -NO2, or halogen, preferably by -CN;

[0017] R 2 Selected from

[0018] R a 、R b 、R cEach independently selected from C1-C6 alkyl;

[0019] Z is selected from O and S;

[0020] R 3 Selected from C1-C6 alkyl.

[0021] In some embodiments, R 1 Selected from:

[0022] In some embodiments, R 1 Selected from:

[0023] In some embodiments, R a 、R b 、R c Each is independently selected from methyl and ethyl.

[0024] In some embodiments, Z is selected from O.

[0025] In some embodiments, R 2 Selected from

[0026] In some embodiments, R 2 In 3-position.

[0027] In some embodiments, R 3 It is a methyl group.

[0028] In some embodiments, the compound is selected from the group consisting of:

[0029] In a second aspect of the present invention, the present invention provides a method for preparing the aforementioned compound, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, comprising:

[0030] make and A nucleophilic substitution reaction occurs to obtain a compound shown in formula I

[0031] Among them, R 1 、R 2 、R 3 The definition of is the same as that in the above formula I;

[0032] M is selected from halogen.

[0033] In some embodiments, M is selected from Cl, Br.

[0034] In some embodiments, the nucleophilic substitution reaction is performed under basic conditions.

[0035] In some embodiments, the nucleophilic substitution reaction is carried out in the presence of DMEDA and potassium carbonate.

[0036] In some embodiments, the nucleophilic substitution reaction is performed in the presence of a catalyst.

[0037] In some embodiments, the nucleophilic substitution reaction is performed in the presence of CuI.

[0038] In the third aspect of the present invention, the present invention provides a pharmaceutical composition comprising the aforementioned compound, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; optionally, further comprising a pharmaceutically acceptable excipient.

[0039] In some embodiments, the pharmaceutical composition further comprises other drugs.

[0040] In some embodiments, the other drug is used to treat and / or prevent diseases or infections caused by HBV or to maintain liver homeostasis.

[0041] In some embodiments, the disease or infection caused by HBV is a liver disease.

[0042] In some embodiments, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.

[0043] In some embodiments, the other drugs include but are not limited to nucleotide drugs (such as entecavir, telbivudine, tenofovir disoproxil, adefovir disoproxil or lamivudine), interferon (such as interferon α2a, interferon α1b or interferon α2b), therapeutic vaccines, Toll-like receptor agonists, cell entry inhibitors, RNA interference drugs, cccDNA targeted drugs, or any combination thereof.

[0044] In the fourth aspect of the present invention, the present invention provides the use of the aforementioned compound, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating and / or preventing diseases or infections caused by HBV or for maintaining liver homeostasis.

[0045] In some embodiments, the disease or infection caused by HBV is a liver disease.

[0046] In some embodiments, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.

[0047] In the fifth aspect of the present invention, the present invention provides the aforementioned compound, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, or the aforementioned pharmaceutical composition, which is used to treat and / or prevent diseases or infections caused by HBV or to maintain liver homeostasis.

[0048] In some embodiments, the disease or infection caused by HBV is a liver disease.

[0049] In some embodiments, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.

[0050] In a sixth aspect, the present invention provides a method for treating and / or preventing a disease or infection caused by HBV or for maintaining liver homeostasis, comprising:

[0051] An effective amount of the aforementioned compound, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the aforementioned pharmaceutical composition is administered to a subject in need thereof.

[0052] In some embodiments, the disease or infection caused by HBV is a liver disease.

[0053] In some embodiments, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1-1 shows the changes in HBV DNA and HBcAg expression after silencing TRPV4 expression or overexpressing TRPV4 in HepG2.2.15 and HepAD38 cells;

[0055] Figures 1-2 show the changes in cccDNA after silencing TRPV4 or overexpressing TRPV4 in HepAD38 cells;

[0056] Figures 1-3 show the changes in pgRNA and totalRNA after silencing TRPV4 or overexpressing TRPV4 in HepG2.2.15 cells; and the changes in pgRNA, total RNA, and precore after silencing TRPV4 or overexpressing TRPV4 in HepGAD38 cells.

[0057] Figure 2-1 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting after the addition of test compounds UTU011001 and UTU011002;

[0058] Figure 2-2 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting after adding the test compounds UTU011003 and UTU011004;

[0059] Figure 2-3 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting after adding the test compounds UTU011005 and UTU011006;

[0060] Figure 2-4 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compounds UTU011007 and UTU011008. DETAILED DESCRIPTION

[0061] It should be understood that the terminology used herein is intended to describe specific embodiments and is not intended to be limiting. In addition, although any method, device, and material similar or equivalent to those described herein may be used for implementing or testing the present invention, preferred methods, devices, and materials are now described.

[0062] In the present invention, unless otherwise explicitly stated, the description method "... are independently selected from" used throughout this document can mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, and can also mean that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0063] The substituents of the compounds of the present invention are disclosed by group class or range. It is specifically noted that the present invention includes each independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0064] The term "alkyl" refers to a saturated aliphatic hydrocarbon group including branched and straight chains having a specified number of carbon atoms. For example, "C1-C6 alkyl" refers to C1, C2, C3, C4, C5, and C6. In addition, for example, "C1-C6 alkyl" refers to an alkyl group having 1 to 6 carbon atoms, preferably "C1-C4 alkyl", and more preferably "C1-C3 alkyl". Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.

[0065] The term "substituted" means that any one or more hydrogens on the designated atom or group are replaced with the selection of the designated group, provided that the normal valence of the designated atom is not exceeded.

[0066] The heteroatom is N, O, S or P.

[0067] Halogen refers to fluorine, chlorine, bromine or iodine.

[0068] The term "heteroaryl" refers to substituted and unsubstituted aromatic 5- or 6-membered monocyclic groups, 8-, 9- or 10-membered bicyclic groups and 11- to 14-membered tricyclic groups having at least one heteroatom (N, O, S or P) in at least one ring, the heteroatom-containing ring optionally further having 1, 2 or 3 heteroatoms selected from N, O, S or P. Among them, substituted and unsubstituted aromatic 8-, 9- or 10-membered bicyclic groups and 11- to 14-membered tricyclic groups having at least one heteroatom (N, O, S or P) in at least one ring are "fused heteroaryl". For heteroaryl groups that are bicyclic or tricyclic, a bicyclic or tricyclic overall structure is required to form an aromatic system. The heteroaryl group may be attached on any available nitrogen or carbon atom of any ring. It will be appreciated by those skilled in the art that two adjacent atoms (preferably carbon atoms) are shared between every two rings in the fused ring.

[0069] Exemplary fused heteroaryl groups include, but are not limited to, indolyl, 5-azaindolyl, pyrrolo[2,3-d]pyrimidinyl, 5,6-diazaindolyl, 6-azaindolyl, 7-azaindolyl, pyrazolo[3,4-b]pyridinyl, pyrrolo[2,3-c]pyridazinyl, thieno[2,3-d]imidazolyl, thieno[2,3-d]imidazolyl, pyrazolo[3,4-c]pyridinyl, benzothiazolyl, benzimidazolyl, benzoxazolyl, benzothienyl, quinolinyl, isoquinolinyl, benzofuranyl, indolizinyl, quinoxalinyl, indazolyl, pyrrolopyrimidinyl, furopyridinyl, isoindolyl, and the like.

[0070] In the present invention, the fused heteroaryl group is optionally substituted by 1, 2, 3 or 4 groups selected from cyano, nitro and halogen, wherein the 2, 3 or 4 substituents may be the same or different. For example, when the fused heteroaryl group is substituted by two groups selected from cyano, nitro and halogen, the two substituents may both be cyano, or may be cyano, nitro, or a combination of cyano and halogen.

[0071] As used herein, "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. This effect can be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptom to regress.

[0072] In the present invention, "subject" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

[0073] In the present invention, an "effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired therapeutic or preventive effect. The "therapeutically effective amount" of the substance / molecule of the present invention may vary according to factors such as the individual's disease state, age, sex and weight, and the ability of the substance / molecule to elicit the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance / molecule outweigh any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired preventive effect. Usually, but not necessarily, since a prophylactic dose is used for a subject before the onset of the disease or in the early stages of the disease, the prophylactic effective amount will be lower than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent.

[0074] The pharmaceutical composition of the present invention may contain pharmaceutically acceptable excipients, including but not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, etc.

[0075] The pharmaceutical composition of the present invention can be prepared into various forms according to different routes of administration. For example, the pharmaceutical composition can be administered by any of the following methods: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or administration via an explanted reservoir. Oral, intraperitoneal, or intravenous administration is preferred.

[0076] The compounds of the present invention may optionally be used in combination with one or more other active ingredients, and the dosage and ratio of each can be adjusted by those skilled in the art according to the specific disease and patient conditions and clinical needs.

[0077] As used herein, unless otherwise indicated, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the present invention. Prodrugs become active compounds only through this reaction under biological conditions, or they are inactive in their unreactive form. Prodrugs can generally be prepared using known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, 5th edition).

[0078] When the stereoisomers in the compounds described herein are specifically designated as (R)- or (S)-isomers in the chemical name, it should be understood that the predominant configuration is the (R)-isomer or the (S)-isomer, respectively. Any asymmetric carbon atom may be present in the (R)-, (S)-, or (R, S)-configuration, preferably in the (R)- or (S)-configuration.

[0079] "Solvate" or "solvate" are used interchangeably to refer to a compound that exists in combination with a solvent molecule. The combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; for example, methanol or ethanol may form an "alcoholate," which may also be stoichiometric or non-stoichiometric. As used herein, the term "solvate" refers to a solid form, i.e., a compound in solution in a solvent, which may be solvated but is not a solvate as the term is used herein.

[0080] The present invention is further explained below with reference to specific examples. Unless otherwise specified, all reagents or raw materials can be purchased commercially.

[0081] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker ASCEND TM -400 NMR spectrometer, the determination solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0082] MS was determined using Agilent 6110, Agilent 1100, Agilent 6120, and Agilent G6125B liquid chromatography-mass spectrometers.

[0083] HPLC determination was performed using a Shimadzu HPLC-2010C high pressure liquid chromatograph (XBRIDGE 2.1*50 mm, 3.5 μm column).

[0084] Chiral HPLC analysis was performed using THARSFC X5.

[0085] The thin layer chromatography silica gel plate used was Yantai Qingdao GF254 silica gel plate. The specifications of the silica gel plate used in thin layer chromatography (TLC) were 0.15 mm to 0.2 mm, and the specifications used for thin layer chromatography separation and purification products were 0.4 mm to 0.5 mm.

[0086] Column chromatography generally uses Qingdao Marine Silica Gel 200-300 mesh silica gel as the carrier.

[0087] High performance liquid phase preparation was performed using Waters 2767, Waters 2545, and Chuangxin Hengtong LC3000 preparative chromatographs.

[0088] Chiral preparative column chromatography used Shimadzu LC-20AP and THARSFC PREP 80.

[0089] The CombiFlash rapid preparation instrument used was Combiflash Rf200 (TELEDYNE ISCO).

[0090] The pressurized hydrogenation reaction used a Beijing Jiawei Kechuang Technology GCD-500G hydrogen generator.

[0091] A Biotage initiator+ microwave reactor was used for the microwave reaction.

[0092] Unless otherwise specified in the experimental examples, all reactions were carried out under argon or nitrogen atmosphere.

[0093] Example 1 Synthesis of Compounds

[0094] 1. Target compound UTU011001

[0095] Diethyl (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011001)

[0096] (2R,3R)-7-Methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dodec-6-ene(3)

[0097] To a solution of (1R,2R)-1,2-diphenyl-1,2-ethanediol 2 (150 g, 699 mmol) and 3-methyl-2-cyclohexene-1-one 1 (77 g, 699 mmol) in cyclohexane (1398 mL) was added PPTS (4.39 g, 17.48 mmol) and the mixture was reacted for 16 hours. Ether (500 mL) was added for dilution and the mixture was washed with saturated NaHCO3 solution. The layers were separated and the organic phase was dried over anhydrous MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate = 5:1 to give compound (2R,3R)-7-methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dodec-6-ene (3).

[0098] (1S,4'R,5'R,6R)-6-methyl-4',5'-diphenylspiro[bicyclo[4.1.0]heptane-2,2'-[1,3]-dioxetane](4)

[0099] In a 500 mL round-bottom flask, zinc copper powder (5 g), n-hexane (500 mL), (2R, 3R)-7-methyl-2,3-diphenyl-1,4-dioxirano[4.5]dodec-6-ene 3, diiodomethane (150 mL, 1865 mmol), diethylzinc (500 mL, 1 mol / L in Hexane, 500 mmol) was reacted. After the reaction was completed as monitored by LCMS, it was quenched with saturated Na2CO3 (800 mL), then filtered through celite, the filter cake was washed with Et2O (2 L), the filtrate was washed with saturated brine (1 L), dried over anhydrous MgSO4, filtered and the organic phase was concentrated, and the resulting residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate = 5:1 to obtain compound (1S, 4'R, 5'R, 6R)-6-methyl-4', 5'-diphenylspiro[bicyclo[4.1.0]heptane-2,2'-[1,3]-dioxolane] (4).

[0100] (2R,3R,7S)-7-(Bromomethyl)-7-methyl-2,3-diphenyl-1,4-dioxacyclo[4.5]-decane (5)

[0101] (1S, 4R, 5R, 6R)-6-methyl-4', 5'-diphenylspiro[bicyclo[4.1.0]heptane-2,2'-[1,3]dioxolane] (137 g, 428 mmol) was dissolved in methanol (1993 mL), and hydrobromic acid (145 mL, 1283 mmol) was added to react. The mixture was concentrated under reduced pressure to obtain a yellow residue. Hexane (1 L) was added to the residue, stirred, and concentrated under reduced pressure to obtain the compound (2R, 3R, 7S)-7-(bromomethyl)-7-methyl-2,3-diphenyl-1,4-dioxacyclo[4.5]-decane (5).

[0102] 2-((2R,3R,7S)-7-Methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dec-7-yl)-methyl)-isoindole-1,3-dione (7)

[0103] In a 1 L round-bottom flask, (2R, 3R, 7S)-7-(bromomethyl)-7-methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]decane 5 (172 g, 428 mmol) and potassium phthalimide (399 g, 2140 mmol) were added to react and filtered, and H2O (3 L) was added for dilution. The mixture was extracted with EA (3 x 500 mL). The organic layers were combined, washed with saturated brine (2 x 300 mL), dried over anhydrous MgSO4, filtered and concentrated. The crude product was eluted by silica gel column chromatography to obtain crude compound 2-((2R, 3R, 7S)-7-methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dec-7-yl)-methyl)-isoindole-1,3-dione (7), which was used directly in the next step.

[0104] ((2R,3R,7S)-7-Methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dec-7-yl)methanamine (8)

[0105] 2-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dec-7-yl)-methyl)-isoindole-1,3-dione (7) was added to a 3 L round-bottom flask and dissolved with ethanol (2.25 L). Hydrazine hydrate (40.3 mL, 1284 mmol) was then added to react and filtered. The filter cake was washed with ethanol (100 mL*2). The filtrate was concentrated under reduced pressure. Tetrahydrofuran (500 mL) was added to the obtained residue and stirred. The white solid was filtered off and the filtrate was concentrated. The obtained residue was dissolved in hexane (500 mL). The solution was heated and stirred, then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to obtain the compound ((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dec-7-yl)methylamine (8).

[0106] 3-({[(2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxabicyclo[4.5]dodecyl-7-yl]methyl]-amino)-4-nitrobenzonitrile (10)

[0107] To a solution of ((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxabicyclo[4.5]dec-7-yl)methanamine 8 (134 g, 0.40 mol) and 3-fluoro-4-nitrobenzonitrile 9 (133 g, 0.8 mol) in ethanol (1 L) was added TEA (100 g, 1 mol) and allowed to react for 16 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (PE / EA = 10:1) to give compound 3-({[(2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxabicyclo[4.5]dodec-7-yl]methyl]amino)-4-nitrobenzonitrile (10).

[0108] 4-Amino-3-(((((((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxacyclo[4.5]dec-7-yl)methyl)amino)benzonitrile (11)

[0109] To a solution of 3-({[(2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxacyclo[4.5]dodec-7-yl]methyl]-amino)-4-nitrobenzonitrile 10 (100 g, 0.21 mol) in MeOH / H2O (900 mL, 1:1) was added NH4Cl (54 g, 1 mol) and the mixture was allowed to react for 16 hours. The mixture was quenched by the addition of H2O (1 L) and extracted with EA (3×2 L). The organic layers were combined, washed with saturated brine (2×2 L), dried over anhydrous MgSO4, filtered, and concentrated. The resulting residue was purified to afford compound 4-amino-3-(((((((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxacyclo[4.5]dec-7-yl)methyl)amino)benzonitrile (11).

[0110] 1-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxopyrrolo[4.5]dec-7-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (12)

[0111] 4-Amino-3-(((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxacyclo[4.5]dec-7-yl)methyl)amino)benzonitrile 11 (55 g, 0.12 mol) was added to trimethyl orthoformate (500 mL), p-toluenesulfonic acid was added, and the mixture was heated to 100°C and stirred for 16 hours. The mixture was quenched with H2O (1 L) and extracted with EA (3 x 1 L). The organic layers were combined, washed with saturated brine (2 x 1 L), dried over MgSO4, filtered, and concentrated. The resulting residue was purified and eluted to give compound 1-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxacyclo[4.5]dec-7-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (12).

[0112] (S)-1-((1-methyl-3-oxocyclohexyl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (13)

[0113] 1-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dec-7-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile 12 (30 g, 0.06 mol) was added to HCOOH (50 mL) and reacted for 16 hours. The mixture was concentrated under reduced pressure and the resulting residue was purified by silica gel chromatography to afford compound (S)-1-((1-methyl-3-oxocyclohexyl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (13).

[0114] 1-((3S,5S)-5-methyl-1-oxospiro[2.5]octan-5-yl)methyl)-1H-benzo[d]-imidazole-6-carbonitrile (14)

[0115] To a 2 L flask were added DMSO (604 mL), 1-{[(1S)-1-methyl-3-oxocyclohexyl]methyl}-1H-benzimidazole-6-carbonitrile 13, potassium tert-butoxide (3 g, 0.03 mol), and trimethylsulfoxide iodide (10 g, 0.05 mol). The mixture was reacted for 16 hours. The mixture was diluted with 1 L of water and 500 mL of DCM. The mixture was separated, extracted, and the organic phases were combined, washed, dried, filtered, and concentrated. The resulting residue was purified and eluted to give the compound trans-epoxide (1-{[(3S, 5S)-5-methyl-1-oxospiro[2.5]octane-5-yl]methyl}-1H-benzimidazole-6-carbonitrile) (14a) and cis-epoxide (1-{[(3R, 5S)-5-methyl-1-oxospiro[2.5]octane-5-yl]methyl}-1H-benzimidazole-6-carbonitrile) (14b).

[0116] 1-((5S,7S)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-7-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (15)

[0117] To a solution of 1-((3S,5S)-5-methyl-1-oxapiro[2.5]octan-5-ylmethyl)-1H-benzo[d]-imidazole-6-carbonitrile 14a (2 g, 7.1 mmol) in NMP (20 mL) was added ethyl carbamate (1.9 g, 21.3 mmol) and KOtBu (1.6 g, 14.2 mmol) and allowed to react for 16 h. The mixture was quenched with H2O (60 mL) and extracted with EA (3 x 30 mL). The combined organic layers were washed with saturated brine (2 x 30 mL), dried over anhydrous MgSO4, filtered, and concentrated. The resulting residue was purified and eluted to afford compound 1-((5S,7S)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-7-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (15).

[0118] Diethyl (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011001)

[0119] To a solution of 1-((5S,7S)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]decan-7-yl)methyl)-1Hbenzo[d]imidazole-6-carbonitrile 15 in 1,4-dioxane (10 mL) were added diethyl (6-chloropyridin-3-yl)phosphonate (6 mg, 0.024 mmol), DMEDA (2 mg, 0.024 mmol), CuI (2 mg, 0.024 mmol) and K2CO3 ( The mixture was reacted with 4-nitro-2-nitro-4-nitropropene (3.5 mg, 0.024 mmol), quenched with H2O (2 mL), extracted with EA (3x5 mL), the organic layers were combined and concentrated under reduced pressure, and the residue was purified to give the compound diethyl (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011001.

[0120] UTU011001: Mass(m / z):538.2(M+H+). 1H NMR (400MHz, CD3OD) δ8.68(dd,J=6.6,1.4Hz,1H),8.45(s,1H),8.33(dd,J=8.8,2.0Hz,1H),8.22(s,1 H),8.09(ddd,J=12.2,8.8,2.2Hz,1H),7.84(d,J=8.4Hz,1H),7.61(dd,J=8.4,1.4Hz,1H),4.22(s,2H ),4.17(m,4H),4.02(m,2H),2.05(s,1H),1.97(d,J=14.4Hz,1H),1.88(m,1H),1.77(d,J=14.0Hz,2H) ,1.60(m,2H),1.51(dd,J=12.8,3.5Hz,1H),1.36(t,J=7.2Hz,6H),1.31(d,J=3.8Hz,2H),1.22(s,3H).

[0121] 2. Target compound UTU011002

[0122] Ethyl hydrogen (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011002)

[0123] Ethyl (5-(diethoxyphosphoryl)pyridin-2-yl)carbamate (3)

[0124] To a solution of ethyl (5-bromopyridin-2-yl)carbamate (400 mg, 1.64 mmol) in 1,4-dioxane (10 mL) were added diethyl phosphate (226 mg, 1.64 mmol), Pd(OAc)2 (26 mg, 0.11 mmol), Xantphos (190 mg, 0.33 mmol) and K3PO4 (1 g, 4.92 mmol) and the mixture was allowed to react for 16 hours. The mixture was quenched by the addition of water (20 mL) and extracted with EA (3 x 20 mL). The organic layers were combined, washed with saturated brine (2 x 100 mL), dried over anhydrous MgSO4, filtered and concentrated. The resulting residue was purified by silica gel chromatography to give ethyl (5-(diethoxyphosphoryl)pyridin-2-yl)carbamate (3).

[0125] Ethyl hydrogen (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011002)

[0126] To a solution of ethyl 5-(diethoxyphosphoryl)pyridin-2-yl)carbamate (50 mg, 0.17 mmol) in NMP (10 mL) were added 1-((3S,5S)-5-methyl-1-oxapyrrolo[2.5]octan-5-yl)methyl)-1Hbenzo[d]imidazole-6-carbonitrile (48 mg, 0.17 mmol) and potassium tert-butoxide (19 mg, 0.17 mmol) and the mixture was reacted. Water (20 mL) was added to quench the mixture, and the mixture was extracted with EA (3×20 mL). The organic layers were combined and concentrated under reduced pressure. The residue was purified to give the compound ethyl hydrogen (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011002.

[0127] UTU011002:Mass(m / z):509.6(M+H + ). 1 H NMR (400MHz, CDCl3) δ9.70(s,1H),8.54(d,J=6.6Hz,1H),8.08(m,3H),7.92(ddd,J=11.8,8.8,2.0Hz,1H),7.81(dd,J=8.6,1.0Hz, 1H),4.28(m,2H),3.92(m,4H),2.07(d,J=15.6Hz,1H),1.99(m,2H),1.81(m,3H),1.45(m,2H),1.20(d,J=7.2Hz,3H),1.14(s,3H).

[0128] 3. Target compound UTU011003

[0129] 1-((5S,7S)-7-methyl-3-(5-(methylsulfonyl)pyridin-2-yl)-2-oxo-1-oxo-3-azaspiro[4.5]decan-7-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (UTU011003)

[0130] To a solution of 1-((5S,7S)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]decan-7-yl)methyl)-1Hbenzo[d]imidazole-6-carbonitrile (20 mg, 0.06 mmol) in 1,4-dioxane (10 mL) were added 2-bromo-5-(methylsulfonyl)pyridine (14 mg, 0.06 mmol), Pd2(dba)3 (10 mg, 0.012 mmol), and Xantphos (7 mg, 0.012 mmol) and the reaction was carried out for 16 hours. Water (20 mL) was added to quench the mixture, and the mixture was extracted with EA (3×20 mL). The organic layers were combined and concentrated under reduced pressure. The residue was purified to give compound 1-((5S,7S)-7-methyl-3-(5-(methylsulfonyl)pyridin-2-yl)-2-oxo-1-oxo-3-azaspiro-[4.5]decan-7-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile UTU011003.

[0131] UTU011003:Mass(m / z):480.2(M+H + ). 1 H NMR (400MHz, DMSO-d6) δ8.82(dd,J=2.2,0.8Hz,1H),8.48(s,1H),8.37(d,J=0.8Hz,1H),8.29(m,2H),7.83(d,J=8.4Hz,1H),7.59(dd,J=8 .4,1.4Hz,1H),4.16(s,2H),3.95(m,2H),3.28(s,3H),1.93(m,2H),1.70(m,3H),1.51(m,2H),1.35(dd,J=12.8,8.8Hz,1H),1.07(s,3H).

[0132] 4. Target compound UTU011004

[0133] Ethyl (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)(methyl)phosphonate (UTU011004)

[0134] To a solution of 1-((5S,7S)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]decan-7-yl)methyl)-1Hbenzo[d]imidazole-6-carbonitrile in 1,4-dioxane (10 mL) were added ethyl (6-chloropyridin-3-yl)(methyl)phosphonate (6 mg, 0.024 mmol), DMEDA (2 mg, 0.024 mmol), CuI (2 mg, 0.024 mmol) and K2CO3 (3.5 mg, 0.024 mmol) to react. Water (20 mL) was added to quench the mixture, and the mixture was extracted with EA (3×20 mL). The organic layers were combined and concentrated under reduced pressure. The residue was purified to give the compound ethyl (6-((5S,7S)-7-((6-cyano-1H-benzo[d]imidazol-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)(methyl)phosphonate UTU011004.

[0135] UTU011004:Mass(m / z):508.2(M+H + ). 1 H NMR(400MHz,DMSO-d6)δ8.64(m,1H),8.56(s,1H),8.41(s,1H),8.20(m,1H),8.13 (m,1H),7.84(s,1H),7.59(d,J=6.8Hz,1H),4.17(s,2H),3.92(dt,J=10.8,5.6Hz, 3H),3.78(dd,J=12.2,5.2Hz,1H),1.90(m,2H),1.74(s,0.7H),1.68(d,J=14.8Hz, 3H), 1.64 (s, 1.3H), 1.50 (m, 2H), 1.32 (m, 2H), 1.19 (t, J = 7.0Hz, 3H), 1.06 (s, 3H).

[0136] 5. Target compounds UTU011005 & UTU011006

[0137] Diethyl (6-((5S,7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011005)

[0138] Diethyl (6-((5R,7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011006)

[0139] 2-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4.5]dec-7-yl)methyl)-2H-pyrazolo[3,4-c]pyridine (3)

[0140] To a solution of 1H-pyrazolo[3,4-c]pyridine (583 mg, 4.9 mmol) in DMF (30 mL) was added NaH (196 mg, 4.9 mmol) and (2R,3R,7S)-7-(bromomethyl)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4.5]dec-7-yl)methyl)-2H-pyrazolo[3,4-c]pyridine (3). The reaction mixture was quenched with water (200 mL) and extracted with EA (3 x 200 mL). The organic layers were combined, washed with saturated brine (3 x 100 mL), dried over anhydrous MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel chromatography to afford compound 2-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4.5]dec-7-yl)methyl)-2H-pyrazolo[3,4-c]pyridine (3).

[0141] (S)-3-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-3-methylcyclohexane-1-one (4)

[0142] 2-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4.5]dec-7-yl)methyl)-2H-pyrazolo[3,4-c]pyridine (1.2 g, 2.7 mmol) was dissolved in HCOOH (20 mL) and reacted for 16 hours. The mixture was concentrated under reduced pressure and the resulting residue was purified by silica gel chromatography to give compound (S)-3-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-3-methylcyclohexane-1-one (4).

[0143] 2-(((5S)-5-Methyl-1-oxospiro[2.5]octan-5-yl)methyl)-2H-pyrazolo[3,4-c]pyridine (5)

[0144] (S)-3-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-3-methylcyclohexane-1-one (500 mg, 2.1 mmol) was dissolved in dimethyl sulfoxide (10 mL), and trimethylsulfoxide iodide (428 mg, 2.1 mmol) and potassium tert-butoxide (235 mg, 2.1 mmol) were added sequentially to react. Water (50 mL) was added to quench the reaction, and the mixture was extracted with EA (3x50 mL). The organic layers were combined, washed with saturated brine (2x50 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to give compound 2-(((5S)-5-methyl-1-oxospiro[2.5]octan-5-yl)methyl)-2H-pyrazolo[3,4-c]pyridine (5).

[0145] (7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-1-oxa-3-azaspiro[4.5]decan-2-one (6)

[0146] To a solution of 2-(((5S)-5-methyl-1-oxospiro[2.5]octan-5-yl)methyl)-2H-pyrazolo[3,4-c]pyridine (190 mg, 0.74 mmol) in dimethyl sulfoxide (10 mL) were added potassium tert-butoxide (136 mg, 1.2 mmol) and ethyl carbamate (197 mg, 2.22 mmol) in sequence and the mixture was allowed to react for 16 hours. The mixture was quenched by the addition of water (50 mL) and extracted with EA (3 x 50 mL). The organic layers were combined, washed with saturated brine (2 x 50 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to afford compound (7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-1-oxa-3-azaspiro[4.5]decan-2-one (6).

[0147] Diethyl (6-((5S,7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011005)

[0148] Diethyl (6-((5R,7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011006)

[0149] To a solution of (7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-1-oxa-3-azaspiro[4.5]decan-2-one (200 mg, 0.66 mmol) in 1,4-dioxane (10 mL) was added diethyl (6-chloropyridin-3-yl)phosphonate (160 mg, 0.66 mmol), tetramethylethylenediamine (6 mg, 0.066 mmol), CuI (6 mg, 0.066 mmol), and K2CO3 (9 mg, 0.066 mmol). The mixture was quenched by addition of water (20 mL), extracted with EA (3 x 20 mL), and the organic layers were combined and concentrated under reduced pressure. The resulting residue was purified to give diethyl (6-((5S,7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011005 and diethyl (6-((5R,7S)-7-((2H-pyrazolo[3,4-c]pyridin-2-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011006, respectively.

[0150] UTU011005:Mass(m / z):514.2(M+H + ). 1 H NMR(400MHz,CD3OD)δ9.07 (s,1H),8.56(ddd,J=6.6,2.2,0.8Hz,1H),8.32(s,1H),8.21(ddd,J=8.8,2.8,0.8Hz,1H),7.97(ddd,J=12.2,8.8,2.2Hz,2H),7.67(d,J=5.4H z,1H),4.32(s,2H),4.04(m,4H),3.93(q,J=10.8Hz,2H),1.85(m,4H),1.61(m,1H),1.45(m,3H),1.24(t,J=7.2Hz,6H),1.07(d,J=7.8Hz,3H).

[0151] UTU011006:Mass(m / z):514.2(M+H + ). 1HNMR (400MHz, CD3OD) δ9.01 (s, 1H), 8.57 (ddd, J = 6.6, 2.2, 0.8Hz, 1H), 8.28 (m, 2H), 8.01 (d dd,J=12.4,8.8,2.2Hz,1H),7.94(d,J=6.2Hz,1H),7.61(d,J=5.8Hz,1H),4.69(d,J=13.8H z,1H),4.55(d,J=13.8Hz,1H),4.05(m,5H),3.91(d,J=10.8Hz,1H),2.01(d,J=14.8Hz,3H) ,1.64(m,4H),1.31(s,0.5H),1.25(q,J=7.4Hz,6H),1.18(d,J=10.2Hz,0.5H),0.89(s,3H).

[0152] 6. Target compounds UTU011007 & UTU011008

[0153] Diethyl (6-((5S,7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011007

[0154] Diethyl (6-((5R,7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011008

[0155] 1-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4.5]dec-7-yl)methyl)-1H-pyrazolo[3,4-c]pyridine (3)

[0156] To a solution of 1H-pyrazolo[3,4-c]pyridine (583 mg, 4.9 mmol) in DMF (30 mL) was added NaH (196 mg, 4.9 mmol) and (2R,3R,7S)-7-(bromomethyl)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4.5]-decane (2 g, 4.9 mmol) at 0°C and allowed to react for 16 hours. The mixture was quenched by addition of water (200 mL) and extracted with EA (3 x 200 mL). The combined organic layers were washed with saturated brine (3 x 100 mL), dried over anhydrous MgSO4, filtered, and concentrated. The resulting residue was purified and eluted to afford compound 1-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4.5]dec-7-yl)methyl)-1H-pyrazolo[3,4-c]pyridine (3).

[0157] (S)-3-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-3-methylcyclohexane-1-one (4)

[0158] 1-((2R,3R,7S)-7-methyl-2,3-diphenyl-1,4-dioxaspiro[4,5]dec-7-yl)methyl)-1H-pyrazolo[3,4-c]pyridine (1.2 g, 2.7 mmol) was dissolved in HCOOH (20 mL) and reacted for 16 hours. The mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel chromatography to afford compound (S)-3-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-3-methylcyclohexane-1-one (4).

[0159] 1-(((5S)-5-Methyl-1-oxapyrrolo[2.5]octan-5-yl)methyl)-1H-pyrazolo[3,4-c]pyridine (5)

[0160] (S)-3-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-3-methylcyclohexane-1-one (500 mg, 2.1 mmol) was dissolved in dimethyl sulfoxide (10 mL), and trimethylsulfoxide iodide (428 mg, 2.1 mmol) and potassium tert-butoxide (235 mg, 2.1 mmol) were added in sequence and reacted for 16 hours. Water (30 mL) was added to quench the mixture, and the mixture was extracted with EA (3x30 mL). The organic layers were combined, washed with saturated brine (2x30 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to give compound 1-(((5S)-5-methyl-1-oxapyrrolo[2.5]octan-5-yl)methyl)-1H-pyrazolo[3,4-c]pyridine (5).

[0161] (7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-1-oxa-3-azaspiro[4.5]decan-2-one (6)

[0162] To a solution of 1-((5S)-5-methyl-1-oxaspiro[2.5]octan-5-yl)methyl)-1H-pyrazolo[3,4-c]pyridine (190 mg, 0.74 mmol) in dimethyl sulfoxide (10 mL) were added potassium tert-butoxide (136 mg, 1.2 mmol) and ethyl carbamate (197 mg, 2.22 mmol) in sequence. The mixture was quenched with water (30 mL) and extracted with EA (3x30 mL). The organic layers were combined, washed with saturated brine (2x30 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to give compound (7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-1-oxa-3-azaspiro[4.5]dec-2-one (6).

[0163] Diethyl (6-((5S,7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011007)

[0164] Diethyl (6-((5R,7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate (UTU011008)

[0165] To a solution of (7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-1-oxa-3-azaspiro-2-one (200 mg, 0.66 mmol) in 1,4-dioxane (10 mL) was added diethyl (6-chloropyridin-3-yl)phosphonate (160 mg, 0.66 mmol), DMEDA (6 mg, 0.066 mmol), CuI (6 mg, 0.066 mmol), and KCO (9 mg, 0.066 mmol). The mixture was quenched by addition of water (20 mL), extracted with EA (3 x 20 mL), and the organic layers were combined and concentrated under reduced pressure. The resulting residue was purified to give diethyl (6-((5S,7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011007 and diethyl (6-((5R,7S)-7-((1H-pyrazolo[3,4-c]pyridin-1-yl)methyl)-7-methyl-2-oxo-1-oxo-3-azaspiro[4.5]dec-3-yl)pyridin-3-yl)phosphonate UTU011008, respectively.

[0166] UTU011007:Mass(m / z):514.2(M+H + ). 1 HNMR (400MHz, CD3OD) δ9.17(s,1H),8.68(m,1H),8.33(m,1H),8.22(s,1H),8.22(d,J=1.4Hz,1H),8.09(ddd,J=12.4,8.8,2.2Hz,1H),7.83( d,J=5.4Hz,1H),4.42(s,2H),4.16(dtd,J=10.2,7.2,3.2Hz,4H),4.03(m,2H),1.95(m,4H),1.62(s,4H),1.36(t,J=7.2Hz,6H),1.19(s,3H).

[0167] UTU011008:Mass(m / z):514.2(M+H + ). 1H NMR (400MHz, CD3OD) δ9.15 (s, 1H), 8.70 (ddd, J=6.6, 2.2, 0.8Hz, 1H), 8.38 (ddd, J=8.8, 2.8, 0.8Hz ,1H),8.23(d,J=5.4Hz,1H),8.20(s,1H),8.12(ddd,J=12.4,8.8,2.2Hz,1H),7.81(d,J=5.6Hz,1H ),4.86(d,J=14.8Hz,1H),4.56(d,J=14.6Hz,1H),4.17(m,5H),4.05(d,J=10.8Hz,1H),2.25(d,J= 14.4Hz,1H),2.08(m,2H),1.79(m,4H),1.36(t,J=7.2Hz,6H),1.30(d,J=10.0Hz,1H),0.95(s,3H).

[0168] In vitro efficacy evaluation of the compound of Example 2

[0169] 1. Experimental Methods

[0170] 1. Culture and treatment of HepG2.2.15 and HepAD38 cells:

[0171] 1.1 Cell recovery

[0172] 1) Preparation: Expose the clean bench to ultraviolet light for 30 minutes in advance; open a water bath and adjust the temperature to 37°C; prepare 70% alcohol;

[0173] 2) After removing the cells from the liquid nitrogen tank, quickly place them in a 37°C water bath and slowly shake the cryovials to thaw.

[0174] 3) When the cells in the cryovial are completely thawed, disinfect the outer wall of the cryovial with 70% alcohol. Then, transfer the thawed cells to a 15 ml centrifuge tube containing 2 ml of fresh culture medium in a clean bench.

[0175] 4) Centrifuge at 800 rpm for 3 min at room temperature;

[0176] 5) Remove the centrifuge tube and discard the supernatant. Add 2 ml of PBS to completely resuspend the cells. Centrifuge at 800 rpm for 3 minutes at room temperature.

[0177] 6) After completely discarding the supernatant, resuspend the cells in 2 ml of fresh culture medium, transfer the cell suspension to a 25 cm2 cell culture flask, and then add 3 ml of fresh culture medium;

[0178] 7) Shake the culture flask crosswise to evenly distribute the cells at the bottom of the flask, and then place it in a 37°C cell culture incubator for long-term culture.

[0179] 1.2 Cell culture medium replacement

[0180] The HepG2.2.15 and HepAD38 cell lines grow relatively slowly. When the color of the culture medium changes from red to light yellow, it means that the nutrients in the culture medium are exhausted and fresh culture medium needs to be replaced in time.

[0181] The steps for changing the cell culture medium are: discard the old culture medium, then add 2 to 4 ml of PBS to the cell culture flask or culture dish, gently shake the culture flask or culture dish, discard the PBS, add enough fresh culture medium and place it in the cell culture incubator to continue culturing.

[0182] 1.3 Cell passaging

[0183] The growth cycle of HepG2.2.15 and HepAD38 cells is approximately 3 days. After 3 days of cell growth, if the cell growth density is greater than 90% under a microscope, it is time to subculture the cells. The steps for cell subculture are as follows:

[0184] 1) Preparation: Expose the clean bench to ultraviolet light for 30 minutes in advance. Remove the fresh culture medium, trypsin, and PBS from the 4°C refrigerator and bring to room temperature.

[0185] 2) Remove the cells to be passaged from the cell culture incubator, discard the old culture medium, and then add 2-4 ml of PBS, shake slowly, and discard. Repeat this step once.

[0186] 3) Add 1 ml of trypsin to the culture dish and shake it to spread the trypsin all over the bottom of the dish. Discard any excess trypsin and place the dish in a cell culture incubator for 1 minute to digest the cells.

[0187] 4) Remove the culture dish and observe the degree of cell digestion under a microscope. Well-digested cells will have larger intercellular spaces. Then, add 2 ml of fresh culture medium to the dish to terminate the trypsin digestion.

[0188] 5) Use a 1ml pipette to gently pipette the cells to form a single-cell suspension, and then transfer the cell suspension to a 15ml centrifuge tube;

[0189] 6) Centrifuge at 800 rpm for 3 min at room temperature;

[0190] 7) Remove the centrifuge tube and a clear cell pellet is visible. Discard the supernatant, add 2 ml of PBS to completely resuspend the cells, and centrifuge at 800 rpm for 3 minutes at room temperature. You can repeat this step once more.

[0191] 8) After completely discarding the supernatant, resuspend the cells in 2 ml of fresh culture medium and divide the cell suspension into 2 to 3 culture dishes for further culture.

[0192] 1.4 Cell cryopreservation

[0193] If you need to freeze cells, you need to select cells that are in good growth condition. If the cells are round, with a clean background and a cell confluence of approximately 80% under a microscope, you can freeze the cells. The steps for cell freezing are as follows:

[0194] 1) Preparation: Add an appropriate amount of isopropanol to the cryostat box for cryopreservation of cells; prepare freezing solution (90% FBS + 10% DMSO), making sure to prepare the solution immediately before use.

[0195] 2) Remove the cells to be frozen from the cell culture incubator, discard the old culture medium, and then add 2-4 ml of PBS, shake slowly, and discard. Repeat this step once.

[0196] 3) Add 1 ml of trypsin to the culture dish and shake it to spread the trypsin all over the bottom of the dish. Discard any excess trypsin and place the dish in a cell culture incubator for 1 minute to digest the cells.

[0197] 4) After centrifugation of the digested cells, discard the supernatant, then wash twice with PBS and centrifuge at 800 rpm for 3 minutes at room temperature;

[0198] 5) After discarding as much supernatant as possible, resuspend the cells in the prepared freezing solution, mix thoroughly, and transfer the cell suspension to a cryovial. Be sure to clearly label the tube wall with the cell name and date.

[0199] 6) Place the cryovial in a programmed cooling box and immediately place it in a -80°C freezer. After 24 hours, place the frozen cells in a liquid nitrogen tank for long-term storage.

[0200] 1.5 Cell transfection

[0201] The cell transfection reagent used in this experiment was Transfection kit. Next, we will describe the cell transfection steps in detail using a six-well plate as an example.

[0202] 1) Seeding cells in a six-well plate: Evenly seed well-growing cells into each well of the six-well plate at a density of 1.5–2.5 x 10^5 cells per well. Add 2 ml of fresh culture medium and culture in a cell culture incubator.

[0203] 2) After 24 hours of cell growth, observe under a microscope that the cells are in the exponential growth phase and the cell density is between 60% and 80%, indicating optimal transfection efficiency. At this point, we can proceed with transfection.

[0204] 3) The cell transfection type involved in this study is mainly transfection of exogenous overexpression plasmids. The preparation method is as follows (taking the transfection amount per well of a six-well plate as an example):

[0205] 2 μg target plasmid + 200 μl jetPRIME Buffer + 5 μl jetPRIME Reagent;

[0206] 4) Prepare the transfection system according to the above preparation method. First, add a certain amount of target plasmid or siRNA to jetPRIME Buffer, vortex thoroughly to mix, and centrifuge at low speed. Then, add an appropriate amount of jetPRIME Reagent to the system. Vortex again to mix, centrifuge, and incubate at room temperature for 15 minutes before adding the transfection system dropwise to each well of a six-well plate.

[0207] 5) During the transfection incubation, replace the culture medium in the six-well plate with fresh culture medium, 2 ml per well. After the transfection incubation is complete, slowly add the transfection mixture dropwise to the six-well plate, 200 μl per well.

[0208] 6) After adding the transfection mixture, gently shake the six-well plate and then place it in a cell culture incubator for continued incubation. At a specific time (for RNA level testing, generally 24 hours is sufficient; for protein level testing, generally 48 hours is sufficient), collect the cells for subsequent experiments.

[0209] 2. Q-PCR detection of HBV RNA expression

[0210] 2.1 Extraction of total cell RNA

[0211] HepG2.2.15 and HepAD38 cells were treated with the test compounds, and total RNA was extracted 48 hours later. This experiment was performed strictly according to the Total RNA Kit instructions. The steps are as follows (Note: This experiment was performed on ice throughout, and RNase-free pipette tips were used throughout):

[0212] 1) Remove the 12-well cell culture plate from the incubator, discard the cell culture medium, and wash the cells twice with 1 mL of 1× PBS. Aspirate the PBS using a vacuum aspirator.

[0213] 2) Prepare 350 μL of TRK Lysis Buffer per well (mixture: 1 mL TRK Lysis Buffer + 20 μL β-mercaptoethanol) as needed and vortex to mix thoroughly. Add 350 μL to each well of a 12-well cell culture plate and incubate at room temperature for 5 minutes to allow cells to fully lyse.

[0214] 3) Prepare 70% ethanol per well as needed and vortex to mix thoroughly. Add 350 μL to each well of a 12-well cell culture plate, shake to mix thoroughly, and transfer to a spin column (assembled with a spin column and collection tube).

[0215] 4) Centrifuge at 4°C, 12,000 g, 60 sec. Discard the waste liquid and retain the centrifuge column.

[0216] 5) Add 500 μL RNA Wash Buffer I and centrifuge at 4°C, 12,000 g, for 60 seconds. Discard the waste liquid and retain the collection column.

[0217] 6) Add 500 μL RNA Wash Buffer II (make sure ethanol has been added) and centrifuge at 4°C, 12,000 g, for 60 seconds. Discard the waste liquid and retain the collection column.

[0218] 7) Repeat step 6) once more, centrifuge at 4°C, 12000g, 90 seconds, discard the waste liquid after centrifugation, and retain the collection column;

[0219] 8) Centrifuge at 4°C, 12000 g, 120 sec to ensure the spin column is fully dried;

[0220] 9) Insert the dried spin column into a new 1.5 mL EPDM tube and add 30 μL of DEPC water to the membrane in the center of the spin column. Be careful not to touch the cap of the EPDM tube during this step. Let it sit at room temperature for 5 minutes to ensure that the RNA is fully dissolved in the DEPC water. Centrifuge at 4°C, 12,000 g, for 60 seconds, and retain the EPDM tube.

[0221] 10) Detect RNA concentration using NanoDrop ultra-micro-volume spectrophotometer;

[0222] 2.2 RNA reverse transcription to cDNA: According to PrimeScript TM RT reagent Kit (Perfect Real Time) instructions, the specific experimental steps are as follows:

[0223] 2.2.1 Prepare the reverse transcription reaction system (operate on ice):

[0224] Table 2-1 Reverse transcription reaction system

[0225] 2.2.2 Reverse transcription reaction conditions are as follows:

[0226] Table 2-2 Reverse transcription reaction conditions

[0227] 2.2.3 The resulting product is cDNA, which can be used directly in subsequent Q-PCR experiments or stored at -20°C.

[0228] 2.3 Q-PCR detection of HBV RNA expression: According to Power Green PCR Master Mix instructions, specific steps are as follows:

[0229] 2.3.1 Prepare the Q-PCR reaction system according to the instructions as follows:

[0230] Table 2-3 Q-PCR reaction system

[0231] 2.3.2 Using ABI The 7500 system uses the following two-step standard amplification procedure:

[0232] Table 2-4 Q-PCR reaction conditions

[0233] After the reaction was completed, the amplification curve and melting curve of Q-PCR were confirmed and the relative quantification method (2 - ΔΔC T Analyze the experimental results.

[0234] 2.3.3 The primer sequences used are as follows:

[0235] Table 2-5 Primer sequences used in Q-PCR experiments

[0236] 3. HBV DNA extraction and detection

[0237] 3.1 Extraction of genomic DNA from cells

[0238] 1) Remove the six-well plate from the cell culture incubator, discard the culture medium, and then add an appropriate amount of PBS to each well and shake the culture plate to wash the cells; this process needs to be repeated once.

[0239] 2) Cell Harvesting: Add an appropriate amount of trypsin to each well. Shake the culture plate to ensure the trypsin covers the entire bottom of the dish. After standing at room temperature for 1-2 minutes, add 1 ml of culture medium to each well to terminate digestion. Collect the cell suspension into a 1.5 ml EP tube. Centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and wash twice with PBS, discarding as much supernatant as possible.

[0240] 3) Add 100 μl of LB2 solution to each EP tube and mix thoroughly by pipetting slowly;

[0241] 4) Add 20 μl of RNase A to each sample and incubate at room temperature for 2 minutes to remove RNA from the sample;

[0242] 5) After incubation, add 20 μl of Proteinase K to each sample and incubate at room temperature for 2 minutes to remove proteins from the sample.

[0243] 6) After incubation, add 500 μl of BB2 solution to each sample, vortex immediately for 5 seconds, and then incubate at room temperature for 10 minutes;

[0244] 7) After incubation, transfer all the liquid in the EP tube to a centrifuge column and centrifuge at 12,000 rpm for 30 seconds. Discard the liquid in the collection tube.

[0245] 8) Add 500 μl of CB2 solution to each sample, centrifuge at 12,000 rpm for 30 seconds, and discard the liquid in the collection tube;

[0246] 9) Repeat step 8) once;

[0247] 10) Add 500 μl of WB2 solution to each sample (add a certain amount of anhydrous ethanol before use), centrifuge at 12,000 rpm for 30 seconds, and discard the liquid in the collection tube;

[0248] 11) Repeat step 10) once;

[0249] 12) Insert the spin column into the collection tube and centrifuge at 12,000 rpm for 2 minutes to completely remove the residual WB2 solution;

[0250] 13) Transfer the spin column to a new 1.5 ml EP tube. Add 50 μl of preheated EB (65°C) to the center of the spin column. Let stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to elute the DNA. Store the eluted DNA at -20°C until needed.

[0251] 3.2 Extraction of HBV Hirt DNA

[0252] 1) Remove the six-well plate from the cell culture incubator, discard the culture medium, and then add an appropriate amount of PBS to each well and shake the culture plate to wash the cells; this process needs to be repeated once.

[0253] 2) Add 750 μl of TE buffer (10 mM Tris, 10 mM EDTA, pH 7.5) to each well, followed by 50 μl of 10% SDS solution. The plate was then gently shaken on a horizontal shaker at room temperature for 30 min to allow complete cell lysis.

[0254] 3) Transfer all the liquid in each well to a 2 ml EP tube, then add 200 μl of 5 M NaCl to each sample. After slowly inverting the tube several times, place the sample on a 4°C shaker. This process requires overnight processing.

[0255] 4) Remove the sample from the 4°C shaker and centrifuge at 4°C, 12,000 rpm for 30 min;

[0256] 5) After centrifugation, transfer the supernatant to a new 2 ml EP tube;

[0257] 6) Phenol extraction: Add an equal volume of phenol to each sample, slowly invert the tube twice, and centrifuge at 12,000 rpm at 4°C for 10 min.

[0258] 7) After centrifugation, remove the supernatant (following the principle of "less is better than more") and add an equal volume of phenol for secondary extraction. Centrifuge at 4°C and 12,000 rpm for 10 minutes and remove the supernatant.

[0259] 8) Phenol / chloroform / isopropanol extraction: add an equal volume of phenol / chloroform / isopropanol to each sample, slowly invert the tube twice, and centrifuge at 12,000 rpm at 4°C for 10 min.

[0260] 9) Transfer the supernatant to a new 2 ml EP tube and add twice the volume of anhydrous ethanol as that of the sample. This process requires standing at room temperature overnight.

[0261] 10) Centrifuge the sample at 12,000 rpm for 30 min at 4°C.

[0262] 11) Discard the supernatant, add 800 μl of 70% ethanol to each sample, and centrifuge at 12,000 rpm for 10 min at 4°C.

[0263] 12) Discard the supernatant and repeat step 11) once;

[0264] 13) Use a vacuum aspirator to remove as much supernatant as possible, then place the EP tube in a fume hood with the lid open and let it dry for 10 minutes.

[0265] 14) Add 20 μl of nuclease-free water to each sample to dissolve Hirt DNA and store at -20°C until use. The total HBV Hirt DNA extracted in this process is a mixture of DP-rcDNA and cccDNA.

[0266] 3.3 Isolation and purification of HBV cccDNA

[0267] Hirt DNA was extracted from the HBV stable cell line as described above. Next, we will isolate and purify cccDNA:

[0268] 1) Transfer 19 μl of Hirt DNA to a 200 μl EP tube and dilute with 23 μl of nuclease-free water to a total volume of 42 μl.

[0269] 2) Heat the sample in a metal bath at 85°C for 5 minutes. This denatures the DP-rcDNA into single-stranded linear DNA, while leaving cccDNA (covalently closed circular double-stranded DNA) unaffected.

[0270] 3) PSAD (Plasmid-safe ATP-dependent DNase) enzyme is added to the sample to digest the single-stranded linear DNA. The specific reaction system is shown in Table 3-1 below: The reaction conditions are 37°C for 16 hours.

[0271] Table 3-1 PSAD enzyme digestion reaction system for single-stranded linear DNA

[0272] 4) PSAD enzyme inactivation: reaction conditions are 70°C, 30 min.

[0273] 5) The final sample needs to be purified using a DNA clean & concentrator kit. The following are the experimental steps for cccDNA purification:

[0274] a) Add 7 volumes of DNA Binding Buffer (350 μl) to the 50 μl sample obtained in the previous step and vortex to mix.

[0275] b) Transfer the mixed liquid to a Zymo-Spin™ column and centrifuge at 16,000 g for 1 min.

[0276] c) Discard the waste solution, add 200 μl DNA Washing Buffer to the column, and centrifuge at 16,000 g for 1 min.

[0277] d) discard the waste liquid and repeat step c) once;

[0278] e) Transfer the Zymo-Spin™ column to a new 1.5 ml EP tube and add 20 μl of DNA Elution Buffer to the center of the column. Incubate at room temperature for 2 minutes. Centrifuge at 16,000 g for 1 minute to elute the purified cccDNA. Store the eluted cccDNA at -20°C until further use.

[0279] 3.4 Detection of HBV DNA and cccDNA

[0280] In this study, the relative quantification of HBV DNA and cccDNA in HBV stable cell lines was detected using real-time fluorescence quantitative PCR technology.

[0281] 1) HBV total DNA qPCR: Follow the instructions of the FastStart Essential DNA Probes Master as follows:

[0282] a) Prepare the HBV total DNA qPCR reaction system as shown in Table 3-2:

[0283] Total DNA upstream primer: 5'-CCGTCTGTGCCTTGTCATCTG-3'

[0284] Total DNA downstream primer: 5'-AGTCCAAGAGTYCTCTTATGYAAGACCTT-3'

[0285] Probes: 5'-FAM-CCGTGTGCACATGGCTTCACCTCTGC-TAMRA-3'

[0286] Table 3-2 HBV total DNA qPCR reaction system

[0287] b) Detection was performed using the Roche LightCycler 480 II real-time fluorescence quantitative PCR instrument. The reaction conditions were as shown in Table 3-3:

[0288] Table 3-3 HBV total DNA qPCR reaction conditions

[0289] c) After the reaction is completed, the data are analyzed and processed using a relative quantitative method (2-ΔΔCT method).

[0290] 2) HBV cccDNA qPCR: Follow the instructions of FastStart Essential DNA Probes Master.

[0291] The specific operation is as follows: FastStart Universal SYBR Green Master (ROX)

[0292] a) Prepare the HBV cccDNA DNA qPCR reaction system as shown in Table 3-4:

[0293] cccDNA upstream primer: 5'-TCATCTGCCGGACCGTGTAC-3'

[0294] cccDNA downstream primer: 5'-TCCGGATACAGAGCTGAGGCG-3'

[0295] Probes: 5'-FAM-TTCAAGCCTCCAAGCTGTGCCTTGCCTGGC-TAMRA-3'

[0296] Table 3-4 HBV cccDNA qPCR reaction system

[0297] b) Detection was performed using a Roche LightCycler 480 II real-time fluorescence quantitative PCR instrument, and the reaction conditions were as shown in Table 3-5;

[0298] Table 3-5 HBV cccDNA qPCR reaction conditions

[0299] 4. Western blotting to detect protein expression

[0300] 4.1 Total Cell Protein Extraction: Remove cells, discard the original culture medium, add 1-2 mL 1× PBS, wash twice, trypsinize, discard the supernatant, gently flick the cells at the bottom of the tube, add 1 mL PBS to wash cells twice again, centrifuge at 1200 rpm for 5 minutes, completely discard the supernatant, and flick the cells at the bottom of the tube. Depending on the cell number, add 50-100 μL of protein lysis buffer containing protease inhibitors and phosphatase inhibitors, mix thoroughly by pipetting, and lyse the cells on ice for 30 minutes. Centrifuge at 12,000 rcf at 4°C for 15 minutes. Aspirate the supernatant and transfer it to a new EP tube. The obtained supernatant is the total cell protein. The protein concentration can be directly determined later or stored at -80°C.

[0301] 4.2 Protein concentration determination by BCA assay: Mix BCA reagent solution A and solution B at a ratio of 50:1 to prepare a working solution. Take 5 μL of the sample to be tested, add 95 μL of deionized water, mix thoroughly, and dilute 20-fold. Take 20 μL of the diluted sample to be tested and add it to a 96-well plate. After adding 200 μL of the working solution, place the 96-well plate in a 37°C incubator for 30 min and then analyze the plate.

[0302] 4.3 Protein denaturation: Take 100 μg of sample respectively, make up the volume of each sample with protein lysis buffer, add 1 / 3 of the total volume of 4× loading buffer, mix thoroughly, denature at 99℃ for 10 min, centrifuge and store at -80℃.

[0303] 4.4 Protein electrophoresis: Install the precast gel into the electrophoresis tank, add 1×MOPS electrophoresis fluid, unplug the comb on the top of the precast gel, and gently blow on the sample well to remove the residual gel in the well; carefully add the pre-stained protein Maker and protein samples of equal weight into the gel well; adjust the voltage to 60V~80V, and after 30 minutes, adjust the voltage to 110V~120V. When bromophenol blue runs to the groove at the bottom of the gel, disconnect the power supply and stop electrophoresis. The entire electrophoresis process takes about 2h~2.5h.

[0304] 4.5 Electrotransfer: Immerse the PVDF membrane in anhydrous methanol to activate it for 1 minute; place the transfer clip with the black side facing down, and place the sea surface-filter paper-PAGE gel-PVDF membrane-filter paper-sponge in order from bottom to top, carefully expelling the bubbles between the gel / membrane and the filter paper. Fasten the transfer clip and place it in the transfer tank with the PAGE gel facing the negative electrode and the PVDF membrane facing the positive electrode; place it in an ice box and pour in pre-cooled 1× electrotransfer solution; adjust the power supply to 100V and transfer the membrane for 1 to 1.5 hours.

[0305] 4.6 Blocking: After transfer, remove the PVDF membrane and wash it several times with 1×TBST to remove the residual electrotransfer solution on the membrane. Then, immerse it in 5% skim milk for blocking at room temperature for 2 hours or at 4°C overnight.

[0306] 4.7 Incubation with primary antibodies: Dilute primary antibodies GAPDH and HCBP6 at 1 / 1000. Seal the PVDF membrane with the above antibodies at the corresponding positions in a hybridization bag according to the molecular weight of the proteins and incubate overnight at 4°C.

[0307] 4.8 Wash the membrane: Cut open the hybridization bag, recover the primary antibody, remove the PVDF membrane, place it in 1×TBST, and wash the membrane three times on a shaker, each time for 10 minutes.

[0308] 4.9 Incubation with secondary antibody: Dilute the secondary antibody at a ratio of 1 / 5,000 to 1 / 10,000. Immerse the PVDF membrane in the corresponding secondary antibody and incubate on a shaker at room temperature for 45-60 minutes.

[0309] 4.10 Membrane washing: Recover the secondary antibody, remove the PVDF membrane, place it in 1×TBST, and wash the membrane three times on a shaker, each time for 10 minutes. When detecting phosphorylated proteins, the number of washes and the washing time should be appropriately reduced.

[0310] 4.11 Exposure and color development: Place the membrane in the dark box of the Fusion Solo imager, add ECL exposure solution, and develop.

[0311] 5. Detection of HBsAg and HBeAg in Cell Culture Supernatant

[0312] In this study, HBsAg and HBeAg were detected using the HBV surface antigen diagnostic kit and the HBV e antigen detection kit (ELISA method).

[0313] 1) Collect 500 μl of cell culture supernatant (HepG2.2.15 and HepAD38 cell lines) and centrifuge at 3000 rcf for 10 min to remove cell debris.

[0314] 2) Take the kit out of the 4°C refrigerator and let it stand at room temperature for 30 minutes before use;

[0315] 3) Prepare the washing solution by diluting the 20× concentrated washing solution with ddH2O to make a 1× washing solution for later use;

[0316] 4) One blank control well (no sample or enzyme conjugate added), two negative control wells (add 50 μl of negative control), two positive control wells (add 50 μl of positive control), and several sample test wells (add 50 μl of the sample to be tested to each well) are required.

[0317] 5) Add 50 μl of enzyme conjugate to each well, seal the plate with a sealing film, gently shake to mix, and incubate in a 37°C incubator for 60 min.

[0318] 6) Washing: After incubation, discard the reaction solution and add 300 μl of diluted 1× wash solution to each well. Incubate at room temperature for 30 seconds and discard the wash solution.

[0319] 7) Repeat step 6) five times. After washing, pat the plate dry as much as possible.

[0320] 8) Add 50 μl of substrate solution A and solution B to each well, seal the reaction plate with a sealing film, gently shake to mix, and incubate at 37°C for 15 minutes. Protect from light during the incubation process;

[0321] 9) Add 50 μl of stop solution to each well and gently shake to stop the reaction;

[0322] 10) On-machine detection: Use Varioskan Flash microplate reader to detect and save the processed data.

[0323] 6. Extraction of HBV replication intermediates

[0324] 1) Remove the cells to be tested, remove the culture medium, wash twice with PBS, and digest with trypsin for counting;

[0325] 2) Take the same number of cells, collect the cells at 2000 rpm for 3 min, and wash twice with PBS;

[0326] 3) Add 500 μL of replication intermediate lysate, mix thoroughly by pipetting, and incubate at 37°C for 15 min;

[0327] 4) Centrifuge at 16,000 g for 5 min, transfer the supernatant to a new EP tube, and take 10 μL for Western blotting to detect the internal reference β-actin.

[0328] 5) Add 4 μL 1 M MgCl and 5 μL 5 U / μL DNase I to the remaining sample and incubate at 37°C for 4 h.

[0329] 6) 9000g × 5 min, remove the supernatant, add 200 μL 35% PEG8000, vortex to mix, and incubate on ice for 1 h;

[0330] 7) 12000g × 5min, remove the supernatant, add 477.5μL proteinase K digestion solution and 12.5μL proteinase K (20mg / mL), mix thoroughly, and incubate in a 45℃ water bath overnight;

[0331] 8) Remove overnight samples, extract with phenol-chloroform three times after flash separation, precipitate with isopropanol, and wash with 70% ethanol;

[0332] 9) After sufficient drying, dissolve in 10 μL of sterile water and store in a -20°C refrigerator.

[0333] 7. Southern blot

[0334] 1) Prepare 30 mL of 0.9% agarose gel:

[0335] a) In a clean 200 mL Erlenmeyer flask, weigh 0.27 g of agarose and add 30 mL of 1× TAE.

[0336] b) Microwave until fully dissolved, and fill to 30 mL with 1×TAE depending on evaporation.

[0337] c) After mixing, pour it onto the plastic sheet, insert the comb, and solidify at room temperature for 30 minutes.

[0338] 2) Sample preparation: Take out the sample to be tested, mix thoroughly, add 2 μL 6× loading buffer, and mix thoroughly;

[0339] 3) Sample loading and electrophoresis: Add the sample to the sample loading well, turn on the power supply, and perform electrophoresis separation at 90V for about 2 hours;

[0340] 4) Transfer membrane preparation: two layers of large filter paper, glue, NC membrane slightly smaller than the glue, and two layers of small filter paper slightly smaller than the membrane;

[0341] 5) Remove the Southern blot gel and place it in 30 mL of alkaline denaturing solution. Shake at room temperature for 30 minutes. Repeat once.

[0342] 6) Soak the NC membrane in RNase-free ddH2O and 20×SSC for 10 min respectively;

[0343] 7) Transfer: Place the transfer tray, two large filter papers, gel, membrane, two small filter papers, absorbent paper, and a 1 kg weight in the order from bottom to top. Add an appropriate amount of 20× SSC to the transfer tray and incubate at room temperature for 16-20 hours.

[0344] 8) Prehybridization: Remove the membrane containing the target nucleic acid, fix it by UV crosslinking, and soak it in 10× SSC for 1 minute. Then, place it in 20 mL of preheated prehybridization solution and place it in a hybridization oven at 68°C for 1 hour.

[0345] 9) Hybridization:

[0346] 10) Take out the hybridization solution containing HBV DNA probe and dissolve it at room temperature;

[0347] 11) Incubate in a 68°C water bath for 10 minutes, then immediately cool on ice for 10 minutes.

[0348] 12) Remove the prehybridization solution, add hybridization solution, and place in a hybridization oven at 68°C overnight;

[0349] 13) Washing the membrane: Wash twice with 2×SSC / 0.1% SDS and twice with 1×SSC / 0.1% SDS at 68°C for 20 min each time; then wash with Washing Buffer at 37°C for 5 min.

[0350] 14) Blocking: Add 20 mL of Blocking Buffer and incubate at 37°C for 30 min.

[0351] 15) Incubation with digoxigenin-labeled secondary antibody: Remove the digoxigenin-labeled secondary antibody and incubate at 10,000 g for 5 min. Remove the upper layer and add it to new Blocking Buffer at a ratio of 1:10,000 and incubate at 37°C for 30 min.

[0352] 16) Place in 20 mL Detection Buffer at 37°C for 5 min.

[0353] 17) Remove the membrane and place it in a dark folder. Add CSPD dropwise. Collect the signal with X-ray film and take photos to analyze the results.

[0354] 8. In vitro nucleocapsid opening assay

[0355] Also known as in vitro endogenous DNA polymerase chain extension reaction, take cells cultured in a 10cm cell culture dish as an example.

[0356] 1) Discard the cell culture medium and add 2 mL of coreDNA lysis buffer to lyse the cells on ice for 15-20 min.

[0357] 2) Harvest the cell lysate and centrifuge at 16,000 g for 10 min in a centrifuge precooled to 4°C.

[0358] 3) Remove the supernatant, add 3 mL of 20% sucrose solution to a 5 mL ultracentrifuge tube, and carefully add 2 mL of the collected lysate. Centrifuge at 46,000 rpm (Beckman, SW55) at 4°C for 3.5 h.

[0359] 4) Remove and discard the supernatant. Resuspend the pellet in 200 μL of TNE buffer. Aliquot 40 μL into 1.5 mL centrifuge tubes and store in a 4°C refrigerator.

[0360] 5) Mix 40 μL of the purified nucleocapsid lysate with 50 μL of 2× reaction buffer. Add the compound to be studied and a control (DMSO, etc.) to the reaction and add nuclease-free water to a total volume of 100 μL.

[0361] 6) After incubation at 37°C for 16 hours, the cells were pre-digested with or without DNase I at 37°C for 30 minutes.

[0362] 7) Extract viral DNA.

[0363] 8) Southern blot was used to detect viral DNA to indirectly determine whether the viral nucleocapsid was intact.

[0364] 9. Nucleocapsid electrophoresis experiment

[0365] Take cells cultured in a 10cm cell culture dish as an example. The specific steps are as follows:

[0366] 1) Discard the cell culture medium, add 2 mL of coreDNA lysis buffer and lyse the cells for 15-20 minutes.

[0367] 2) Harvest the cell lysate and centrifuge at 16,000 g for 10 min in a centrifuge precooled to 4°C.

[0368] 3) Remove the supernatant, add 3 mL of 20% sucrose solution to a 5 mL ultracentrifuge tube, and carefully add 2 mL of the collected lysate. Centrifuge at 46,000 rpm (Beckman, SW55) at 4°C for 3.5 h.

[0369] 4) Remove and discard the supernatant. Resuspend the pellet in 200 μL of TNE buffer. Aliquot 40 μL into 1.5 mL centrifuge tubes and store in a 4°C refrigerator.

[0370] 5) Mix 40 μL of the purified nucleocapsid lysate with 50 μL of 2× reaction buffer. Add the compound to be studied and a control (DMSO, etc.) to the reaction and add nuclease-free water to a total volume of 100 μL.

[0371] 6) After incubation at 37°C for 6 hours, take 20 μL of the reaction product and add loading buffer for electrophoresis on a 1.8% agarose gel at 72V and room temperature for 8 hours.

[0372] 7) After electrophoresis, transfer the membrane using TNE buffer. The transfer conditions and procedures are the same as those for Southern blot.

[0373] 8) After transfer, remove the membrane, fix it with 4% paraformaldehyde (prepared in PBS) at room temperature for 15 minutes, and rinse with running water.

[0374] 9) Fix with pre-cooled 50% methanol (prepared in PBS) for 30 minutes and rinse with running water.

[0375] 10) Block the plate with blocking solution (containing 5% (w / v) skim milk powder, 1× TBS, 0.1% Tween-20) at room temperature for 1-2 hours.

[0376] 11) Apply anti-HBc (CST, catalog number sc-52406) antibody at 4°C overnight.

[0377] 12) Wash with wash buffer (containing 1×TBS, 0.1% Tween-20) at room temperature for 3×5 min, apply goat anti-mouse secondary antibody at room temperature for 1 hour, and then wash with wash buffer (containing 1×TBS, 0.1% Tween-20) at room temperature for 3×5 min.

[0378] 13) Use a Bio-rad scanner or a Li-COR scanner to expose the nucleocapsid signal.

[0379] 2. Experimental steps and results

[0380] (a) In vitro efficacy evaluation - the effect of TRPV4 on HBV RNA, HBV DNA, cccDNA, and HBV core protein (HBcAg)

[0381] HepG2.2.15 and HepAD38 cells were passaged and plated normally. After 12 hours of adherent growth, they were transiently transfected with exogenous TRPV4 overexpression plasmids or siRNA. Cells were harvested 48 hours later for total RNA extraction, DNA extraction using the Hirt method, and cccDNA isolation and purification. Intracellular HBV RNA (total RNA, pgRNA, precore RNA), HBV DNA, and cccDNA were detected by Q-PCR. Intracellular HBV core protein (HBcAg) expression was assessed by Western blotting. The results are shown in Figures 1-1 to 1-3.

[0382] The results showed that silencing TRPV4 in HepG2.2.15 and HepAD38 cells decreased HBV DNA and HBcAg expression, while overexpressing TRPV4 significantly increased HBV DNA and HBcAg expression (Figure 1-1). In HepAD38 cells, silencing TRPV4 downregulated cccDNA after 12 days of induction, while overexpressing TRPV4 upregulated cccDNA (Figure 1-2). In HepG2.2.15 cells, silencing TRPV4 downregulated both pgRNA and total RNA, while overexpressing TRPV4 upregulated both pgRNA and total RNA. In HepGAD38 cells, silencing TRPV4 downregulated pgRNA, total RNA, and precore, while overexpressing TRPV4 upregulated pgRNA, total RNA, and precore (Figure 1-3). These results indicate that TRPV4 promotes HBV replication and transcription, while silencing TRPV4 inhibits HBV replication and transcription.

[0383] (b) In vitro efficacy evaluation - expression of HBV core protein (HBcAg)

[0384] HepG2.2.15 and HepAD38 cells were passaged and plated as normal. After 12 hours of adherent growth, test compounds were added at varying concentrations (10 nM, 20 nM, 50 nM, 100 nM, and 500 nM). After 48 hours, cells were harvested, protein was extracted, and intracellular HBV core protein (HBcAg) expression was determined by Western blotting. The results are shown in the table below or in Figures 2-1 to 2-4.

[0385] Table 4: Western blotting grayscale scan results Note: “-” means not tested.

[0386] The results showed that most of the compounds of the present invention could inhibit the expression of HBV core protein (HBcAg) in HepG2.2.15 and HepAD38 cells to varying degrees ( Figures 2-1 to 2-4 , Table 4 ).

Claims

1. The compound represented by the following general formula I: or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, in: R 1 is selected from fused heteroaryl, wherein the fused heteroaryl is formed by fusion of a six-membered ring and a five-membered ring, and the six-membered ring contains 1 heteroatom or no heteroatom, and the five-membered ring contains 2 heteroatoms; the heteroatom is selected from N, O or S atoms, preferably N atoms; optionally, the six-membered ring (preferably a six-membered ring without heteroatoms) is substituted by 1, 2, 3 or 4 -CN, -NO2, halogen, preferably substituted by -CN; Preferably, R 1 Selected from: More preferably, R 1 Selected from: R 2 Selected from R a , R b , R c Each independently selected from C1-C6 alkyl; Preferably, R a , R b , R c Each independently selected from methyl and ethyl; Z is selected from O, S, preferably O; Preferably, R 2 Selected from Preferably, the above R 2 In 3-position; R 3 Selected from C1-C6 alkyl; Preferably, R 3 It is methyl.

2. The compound according to claim 1, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein: The compound is selected from the following:

3. A method for preparing the compound according to any one of claims 1 to 2, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, comprising: make and Nucleophilic substitution reaction occurs to obtain Compound Among them, R 1 , R 2 , R 3 As defined in claim 1, M is selected from halogen, M is preferably selected from Cl, Br; Preferably, the nucleophilic substitution reaction is carried out under alkaline conditions; More preferably, the nucleophilic substitution reaction is carried out in the presence of DMEDA and potassium carbonate; Preferably, the nucleophilic substitution reaction is carried out in the presence of a catalyst; More preferably, the nucleophilic substitution reaction is carried out in the presence of CuI.

4. A pharmaceutical composition comprising the compound according to any one of claims 1 to 2, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; optionally, further comprising a pharmaceutically acceptable excipient.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutical composition further comprises other drugs; Preferably, the other drug is used to treat and / or prevent diseases or infections caused by HBV or to maintain liver homeostasis; Preferably, the disease or infection caused by HBV is a liver disease; Preferably, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof; Preferably, the other drugs include but are not limited to nucleotide drugs (such as entecavir, telbivudine, tenofovir disoproxil, adefovir dipivoxil or lamivudine), interferons (such as interferon α2a, interferon α1b or interferon α2b), therapeutic vaccines, Toll-like receptor agonists, cell entry inhibitors, RNA interference drugs, cccDNA targeted drugs, or any combination thereof.

6. Use of the compound according to any one of claims 1 to 2, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the pharmaceutical composition according to any one of claims 4 to 5 in the preparation of a medicament for treating and / or preventing a disease or infection caused by HBV or for maintaining liver homeostasis; Preferably, the disease or infection caused by HBV is a liver disease; Preferably, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.

7. A compound according to any one of claims 1 to 2, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or a pharmaceutical composition according to any one of claims 4 to 5, for use in treating and / or preventing a disease or infection caused by HBV or for maintaining liver homeostasis; Preferably, the disease or infection caused by HBV is a liver disease; Preferably, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.

8. A method for treating and / or preventing a disease or infection caused by HBV or for maintaining liver homeostasis, comprising: Administering an effective amount of the compound of any one of claims 1 to 2, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the pharmaceutical composition of any one of claims 4 to 5 to a subject in need thereof; Preferably, the disease or infection caused by HBV is a liver disease; Preferably, the liver disease is selected from hepatitis B (such as chronic hepatitis B), liver fibrosis, liver failure, cirrhosis, liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.

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