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WO2024217429A1 - 化合物及其在慢性乙型肝炎、肝纤维化、肝癌的治疗中的应用 - Google Patents

化合物及其在慢性乙型肝炎、肝纤维化、肝癌的治疗中的应用 Download PDF

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Publication number
WO2024217429A1
WO2024217429A1 PCT/CN2024/088156 CN2024088156W WO2024217429A1 WO 2024217429 A1 WO2024217429 A1 WO 2024217429A1 CN 2024088156 W CN2024088156 W CN 2024088156W WO 2024217429 A1 WO2024217429 A1 WO 2024217429A1
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methyl
compound
liver
pharmaceutically acceptable
mmol
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PCT/CN2024/088156
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English (en)
French (fr)
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高杰
成军
芦红萍
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河北乌图药业有限公司
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Publication of WO2024217429A1 publication Critical patent/WO2024217429A1/zh

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  • 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.
  • HBV infection seriously threatens human health.
  • WHO World Health Organization
  • HBV chronic hepatitis B virus
  • hepatitis B virus of which 350 million are chronic HBV carriers. Every year, about 1 million people die from liver failure, cirrhosis and primary hepatocellular carcinoma (HCC) caused by HBV infection.
  • HCC primary hepatocellular carcinoma
  • my country is a high-incidence area for HBV infection.
  • HBV infection rate of HBV has been greatly reduced with the popularization of hepatitis B vaccine and the development of mother-to-child transmission prevention technology
  • a large-sample epidemiological study of more than 2 million cases in 2016 showed that the HBsAg positive rate of men aged 21-49 in rural areas of my country is still as high as 6%. It can be seen that HBV infection will still be a severe challenge facing my country's public health system for a long time.
  • HBV is a partially double-stranded circular DNA (rcDNA) virus that is tropic to hepatocellular cells. Its genome is about 3.2 kb in length and consists of four partially overlapping open reading frames (ORFs), including the S gene region, the C gene region, the P gene region, and the X gene region.
  • HBV replication is a complex process that transfers the genetic information of the virus from parental DNA to offspring, including transcription of mRNA using covalently closed circular DNA (cccDNA) as the initial template, translation, and reverse transcription synthesis of viral negative-strand DNA and viral positive-strand DNA using mRNA as a template.
  • cccDNA covalently closed circular DNA
  • HBV ccc DNA 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 of HBV.
  • the existence of HBV cccDNA is the root cause of viral replication and infection. Inhibiting or eliminating HBV cccDNA is the key to curing chronic hepatitis B.
  • nucleoside (acid) analogs mainly include two categories: nucleoside (acid) analogs and interferons.
  • the target of nucleoside (acid) analog 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.
  • HBVcccDNA mainly includes activating the host immune response, inhibiting the transcription of HBVcccDNA, inhibiting the formation of viral nucleocapsids or increasing their degradation.
  • these drugs can effectively inhibit HBV replication, they cannot 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.
  • HBV surface antigen HBV surface antigen (HBsAg)
  • HBsAg HBV surface antigen
  • Ca 2+ Calcium ions 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.
  • HBV can increase the level of Ca 2+ in the cytoplasm, depending on different Ca 2+ channels on the plasma membrane, endoplasmic reticulum and mitochondria.
  • the activation of Ca 2+ signals can promote viral replication through a variety of molecular mechanisms. Therefore, existing evidence suggests that targeting Ca 2+ signals will be an effective method for treating HBV infection.
  • TRP transient receptor potential
  • TRPV4 Transient receptor potential vanilloid 4
  • the present invention firstly discovered that overexpression of TRPV4 can promote the replication and transcription of HBV, and silencing of TRPV4 can inhibit the replication and transcription of HBV, and further discovered that TRPV4 inhibitors have a significant inhibitory effect on the expression of HBV core protein (HBcAg).
  • HBV core protein HBV core protein
  • liver diseases such as chronic hepatitis B, liver fibrosis, liver cancer, and these molecules have good drugability (solubility, oral bioavailability, etc.) and safety.
  • the present invention provides a compound represented by the following general formula O:
  • X is selected from N, CH;
  • R1 is selected from 8-10 membered fused heteroaryl groups, and each monocyclic ring of the fused heteroaryl group contains at least one heteroatom;
  • R2 is selected from C1-C6 alkyl, C1-C6 alkyl substituted with 1-3 hydroxyl groups,
  • R a and R b are each independently selected from C1-C6 alkyl
  • Z is selected from O and S
  • R 2 is substituted at carbon atom No. 2, No. 3 or No. 5;
  • R3 is selected from C1-C6 alkyl.
  • R 1 is selected from fused heteroaryl, wherein each monocyclic ring contains at least The fused heteroaryl group contains one less heteroatom, and the fused heteroaryl group is formed by fusion of a six-membered ring and a five-membered ring.
  • R 1 is selected from a fused heteroaryl group, in which each monocyclic ring contains at least one heteroatom, the fused heteroaryl group is formed by fusion of a six-membered ring and a five-membered ring, and the fused heteroaryl group contains three heteroatoms in total, and the heteroatom is preferably a N atom.
  • R 1 is selected from:
  • R is selected from
  • R2 is selected from C1-C6 alkyl substituted with 1-3 hydroxyl groups
  • R2 is selected from C1-C6 alkyl substituted with 1 hydroxyl group
  • R2 is selected from
  • Ra and Rb are both methyl.
  • R2 is substituted at carbon atom No. 3.
  • R 3 is methyl
  • the compound has a structure shown in Formula I-1 or Formula I-2,
  • X is selected from N, CH;
  • Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , and Y 7 are each independently selected from CH and N, and Y 3 , Y 4 , Y 5 , and Y 6 are not CH at the same time;
  • R2 is selected from C1-C6 alkyl, C1-C6 alkyl substituted with 1-3 hydroxyl groups,
  • R a and R b are each independently selected from C1-C6 alkyl
  • Z is selected from O and S.
  • Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , and Y 7 are each independently selected from CH and N, and Y 3 , Y 4 , Y 5 , and Y 6 are not CH at the same time, and the following conditions are satisfied:
  • any two of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 are N, and the rest are CH,
  • any two of Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are N, and the rest are CH.
  • R2 is selected from C1-C6 alkyl substituted with 1-3 hydroxyl groups
  • R2 is selected from C1-C6 alkyl substituted with 1 hydroxyl group
  • R2 is selected from
  • Ra and Rb are both methyl.
  • the compound is selected from the group consisting of:
  • 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:
  • R 1 and R 3 are as defined above, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are as defined above, X and R 2 are as defined above, and M is selected from halogen.
  • M is selected from Cl, Br.
  • the nucleophilic substitution reaction is performed under basic conditions.
  • the nucleophilic substitution reaction is performed in the presence of DMEDA and potassium carbonate.
  • the nucleophilic substitution reaction is performed in the presence of a catalyst.
  • the nucleophilic substitution reaction is performed in the presence of CuI.
  • the present invention provides a pharmaceutical composition
  • 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.
  • the pharmaceutical composition further comprises other drugs.
  • the other drug is used to treat and/or prevent a disease or infection caused by HBV or to maintain liver homeostasis.
  • the disease or infection caused by HBV is a liver disease.
  • 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.
  • hepatitis B such as chronic hepatitis B
  • liver fibrosis liver failure
  • cirrhosis liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.
  • the other drugs include but are not limited to nucleotide drugs (such as entecavir, telbivudine, tenofovir disoproxil, adefovir dipivoxil or lamivudine), interferon (such as interferon ⁇ 2a, interferon ⁇ 1b or interferon ⁇ 2b), therapeutic vaccines, Toll-like receptor agonists, cell entry inhibitors agents, RNA interference drugs, cccDNA targeted drugs, or any combination thereof.
  • nucleotide drugs such as entecavir, telbivudine, tenofovir disoproxil, adefovir dipivoxil or lamivudine
  • interferon such as interferon ⁇ 2a, interferon ⁇ 1b or interferon ⁇ 2b
  • therapeutic vaccines such as interferon ⁇ 2a, interferon ⁇ 1b or interferon ⁇ 2b
  • Toll-like receptor agonists such as
  • 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 a disease or infection caused by HBV or for maintaining liver homeostasis.
  • the disease or infection caused by HBV is a liver disease.
  • 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.
  • hepatitis B such as chronic hepatitis B
  • liver fibrosis liver failure
  • cirrhosis liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.
  • 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.
  • the disease or infection caused by HBV is a liver disease.
  • 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.
  • hepatitis B such as chronic hepatitis B
  • liver fibrosis liver failure
  • cirrhosis liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.
  • the present invention provides a method for treating and/or preventing a disease or infection caused by HBV or for maintaining liver homeostasis, comprising:
  • 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.
  • the disease or infection caused by HBV is a liver disease.
  • 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.
  • hepatitis B such as chronic hepatitis B
  • liver fibrosis liver failure
  • cirrhosis liver cancer (such as primary hepatocellular carcinoma), or any combination thereof.
  • Figure 1-1 shows the changes in HBV DNA and HBcAg expression in HepG2.2.15 and HepAD38 cells after silencing TRPV4 expression or overexpressing TRPV4;
  • FIG. 1-2 shows that after silencing TRPV4 or overexpressing TRPV4 in HepAD38 cells, cccDNA changes in
  • Figures 1-3 show the changes in pgRNA and totalRNA after silencing TRPV4 or overexpressing TRPV4 in HepG2.2.15 cells; the changes in pg RNA, total RNA, and precore after silencing TRPV4 or overexpressing TRPV4 in HepGAD38 cells;
  • FIG 2-1 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compounds UTU011025 and UTU011026;
  • FIG 2-2 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compounds UTU011027 and UTU011028;
  • FIG. 2-3 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compounds UTU011029 and UTU011030;
  • FIG 2-4 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compounds UTU011031 and UTU011032;
  • FIG. 2-5 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compounds UTU011033 and UTU011034;
  • Figure 2-6 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compounds UTU011035 and UTU011036.
  • Figure 2-7 shows the expression of HBV core protein (HBcAg) in cells detected by Western blotting method after adding the test compound UTU011037.
  • C1-C6 alkyl specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl.
  • alkyl refers to a branched and straight chain saturated aliphatic hydrocarbon group including a specified number of carbon atoms.
  • C1-C6 alkyl refers to C1, C2, C3, C4, C5 and C6.
  • C1-C6 alkyl refers to an alkyl group having 1 to 6 carbon atoms, preferably “C1-C4 alkyl", and more preferably "C1-C3 alkyl”.
  • 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), etc.
  • C1-C6 alkyl substituted by 1-3 hydroxyl groups means that 1, 2 or 3 hydrogen atoms in the aforementioned C1-C6 alkyl are replaced by hydroxyl groups. In some embodiments, "C1-C6 alkyl substituted by 1 hydroxyl group” is preferred. Examples of “C1-C6 alkyl substituted by 1-3 hydroxyl groups” include -C(OH) 3 , -CH(OH) 2 , -CH 2 (OH), -CH 2 CH 2 (OH), -CH 2 CH(OH) 2 , -CH 2 C(OH) 3 or Preferably wherein the alkyl group is as defined above.
  • substituted means that any one or more hydrogens on the designated atom or group are replaced with the selection of designated groups, provided that the normal valence of the designated atom is not exceeded.
  • the heteroatom is N, O or S.
  • Halogen refers to fluorine, chlorine, bromine or iodine.
  • 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 (O, N or S) in at least one ring, and the heteroatom-containing ring optionally further has 1, 2 or 3 heteroatoms selected from O, N or S.
  • substituted and unsubstituted aromatic 8-, 9- or 10-membered bicyclic groups and 11- to 14-membered tricyclic groups having at least one heteroatom (O, N or S) in at least one ring are "fused heteroaryl".
  • a bicyclic or tricyclic overall structure is required to form an aromatic system.
  • the heteroaryl group can be attached to any available nitrogen or carbon atom of any ring.
  • Exemplary monocyclic heteroaryl rings include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, and the like.
  • bicyclic 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, quinolyl, isoquinolyl, benzofuranyl, indolizinyl, quinoxalinyl, indazolyl, pyrrolopyrimidinyl, furopyridinyl, isoindolyl, and the like
  • R 2 is substituted at carbon atom No. 2, No. 3 or No. 5, which means that the structural formula of the compound represented by formula O can be
  • treatment generally refers to obtaining the desired pharmacological and/or physiological effect.
  • the effect can be preventive, based on the complete or partial prevention of the disease or its symptoms; and/or can be therapeutic, based on the partial or complete stabilization or cure of the disease and/or side effects caused by the disease.
  • treatment covers any treatment of a patient's disease, including: (a) preventing the disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) inhibiting the symptoms of the disease, i.e., preventing its development; or (c) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.
  • a 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.
  • a mammal refers to a human.
  • an effective amount refers to an amount that is effective in achieving the desired therapeutic or preventive effect at the necessary dosage and time.
  • the “therapeutically effective amount” of the substance/molecule of the present invention can be determined based on factors such as the individual's disease state, age, The amount of 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 dose and time to achieve the desired prophylactic effect. Usually, but not necessarily, since the prophylactic dose is used for the 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.
  • a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor volume; inhibit (i.e., slow down to a certain extent, preferably stop) cancer cell infiltration 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.
  • the pharmaceutical composition of the present invention may contain pharmaceutically acceptable excipients, including but not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human 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, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, etc.
  • pharmaceutically acceptable excipients including but not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphates, glycerol, sorbic
  • the pharmaceutical composition of the present invention can be prepared into various forms according to different administration routes.
  • the pharmaceutical composition can be administered in any of the following ways: 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.
  • the compounds of the present invention may optionally be used in combination with one or more other active ingredients, and the respective dosages and ratios thereof may be adjusted by those skilled in the art according to specific symptoms, patient conditions, clinical needs, etc.
  • 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 under biological conditions through this reaction, 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).
  • stereoisomers in the compounds described herein are specifically designated as (R)- or (S)-isomers in the chemical name, it should be understood that the major configuration is the (R)-isomer or (S)-isomer, respectively.
  • Any asymmetric carbon atom may exist in the (R)-, (S)- or (R, S)-configuration, preferably in the (R)- or (S)-configuration.
  • Solvate or “solvate” are used interchangeably and refer to a compound that exists in combination with certain solvent molecules.
  • the combination may include a stoichiometric amount of a certain solvent, such as a monohydrate or a 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.
  • solvate refers to a solid form, i.e., a compound in a solution of a solvent, although it may be solvated, it is not a solvate as the term is used herein.
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • NMR shifts ( ⁇ ) are given in units of 10 -6 (ppm).
  • NMR measurements were performed using a Bruker ASCEND TM -400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD) as the measuring solvent, and tetramethylsilane (TMS) as the internal standard.
  • DMSO-d 6 deuterated dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS tetramethylsilane
  • MS was determined using Agilent 6110, Agilent 1100, Agilent 6120, and Agilent G6125B liquid chromatography-mass spectrometers.
  • HPLC determination was performed using a Shimadzu HPLC-2010C high pressure liquid chromatograph (XBRIDGE 2.1*50mm, 3.5um chromatographic column).
  • the thin layer chromatography silica gel plate used was Yantai Qingdao GF254 silica gel plate.
  • the silica gel plate used in thin layer chromatography (TLC) had a specification of 0.15 mm to 0.2 mm, and the specification used for thin layer chromatography separation and purification products was 0.4 mm to 0.5 mm.
  • High performance liquid phase preparation used Waters 2767, Waters 2545, and Chuangxin Hengtong LC3000 preparative chromatographs.
  • Chiral preparative columns used were Shimadzu LC-20AP and THARSFC PREP 80.
  • the CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).
  • the pressurized hydrogenation reaction used a Beijing Jiawei Kechuang Technology GCD-500G hydrogen generator.
  • Microwave reactions were performed using a Biotage initiator+ microwave reactor.
  • PPTS (4.39 g, 17.48 mmol) was added to a cyclohexane (1398 mL) 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) and reacted for 16 hours.
  • Ether 500 mL was added to dilute the mixture and the mixture was washed with saturated NaHCO3 solution. The organic phase was dried over anhydrous MgSO4, filtered and concentrated.
  • the crude product was eluted by silica gel column chromatography to obtain a crude compound 2-((2R, 3R, 7S)-7-methyl-2,3-diphenyl-1,4-dioxanaphtho[4.5]dec-7-yl)-methyl)-isoindole-1,3-dione, which was directly used in the next step.
  • N4-((2R, 3R, 7S)-7-methyl-2,3-diphenyl-1,4-dioxopyrrolo[4.5]dec-7-yl)methyl)pyridine-3,4-diamine 600 mg, 1.4 mmol was dissolved in trimethyl orthoformate (10 mL), and p-toluenesulfonic acid was added to react for 16 hours. Water (50 mL) was added to quench, and EA (3x50 mL) was used for extraction. The organic layers were combined, washed, dried, filtered and concentrated.
  • Trimethylsulfoxide iodide (428 mg, 2.1 mmol) and potassium tert-butoxide (235 mg, 2.1 mmol) were added in sequence and stirred at 25°C. The mixture was stirred and reacted for 16 hours. Water (100 mL) was added to quench the mixture, and the mixture was extracted with EA (3 x 50 mL). The organic layers were combined and concentrated under reduced pressure. The residue was purified by silica gel chromatography and eluted to obtain compound 2-(((5S)-5-methyl-1-oxaspiro[2.5]octan-5-yl)methyl)-2H-pyrazolo[3,4-b]pyridine (5).
  • 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.
  • the steps for changing the cell medium are: discard the old culture medium, then add 2 to 4 ml of PBS to the cell culture bottle or culture dish, gently shake the culture bottle or culture dish, discard the PBS, add enough fresh culture medium and place it in the cell culture incubator to continue culturing.
  • the growth cycle of HepG2.2.15 and HepAD38 cells is about 3 days. After 3 days of cell growth, if the cell growth density is greater than 90% under a microscope, cell subculture is required. The steps of cell subculture are as follows:
  • pancreatic enzyme Add 1 ml of pancreatic enzyme to the culture dish, shake the dish to make the pancreatic enzyme spread all over the bottom of the dish, discard the excess pancreatic enzyme, and place the dish in a cell culture incubator for 1 minute to digest the cells;
  • the cell transfection reagent used in this experiment was Transfection kit. Next, we will The steps of cell transfection are described in detail using a six-well plate as an example.
  • Inoculate cells in a six-well plate Evenly seed cells with good growth status into each well of the six-well plate, with the inoculation amount of 1.5-2.5x10 ⁇ 5 cells per well, and add 2 ml of fresh culture medium to culture in a cell culture incubator;
  • 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):
  • the culture medium in the six-well plate can be replaced with fresh culture medium, 2 ml per well; after the transfection system is incubated, the transfection mixture is slowly added to the six-well plate, 200 ⁇ l per well;
  • RNA level detection generally 24 hours of treatment is sufficient
  • protein level detection generally 48 hours of treatment is sufficient
  • the tested compounds were added to HepG2.2.15 and HepAD38 cells, and total RNA was extracted from the cells 48 hours later. This experiment was carried out strictly in accordance with the instructions of the Total RNA Kit. The steps are as follows (Note:
  • step 6) Repeat step 6) once, centrifuge at 4°C, 12000g, 90 sec, discard the waste liquid after centrifugation, and retain the collection column;
  • RNA reverse transcription to cDNA according to PrimeScript TM RT reagent Kit (Perfect Real Time)
  • the obtained product is cDNA, which can be directly used in subsequent Q-PCR experiments or stored at -20°C.
  • the 7500 system uses the following two-step standard amplification procedure:
  • Collect cells Add an appropriate amount of trypsin to each well, shake the culture plate to cover the entire bottom of the dish, let it stand at room temperature for 1-2 minutes, add 1 ml of culture medium to each well to stop digestion, and then collect the cell suspension into a 1.5 ml EP tube. Centrifuge at 2000 rpm for 5 minutes, discard the supernatant, wash twice with PBS and discard as much supernatant as possible;
  • Phenol extraction add an equal volume of phenol to each sample, slowly invert twice, and centrifuge at 4°C and 12,000 rpm for 10 min;
  • Phenol/chloroform/isoprophanol extraction add equal volumes of phenol/chloroform/isopropanol to each sample, slowly invert upside down twice, and centrifuge at 4°C, 12,000 rpm for 10 min;
  • Hirt DNA was extracted from the HBV stable cell line as described above. Next we will isolate and purify cccDNA:
  • reaction conditions are 70°C, 30 min.
  • HBV total DNA qPCR follows the instructions of FastStart Essential DNA Probes Master. The specific steps are as follows:
  • HBV cccDNA qPCR follows the instructions of FastStart Essential DNA Probes Master. The specific steps are as follows: FastStart Universal SYBR Green Master (ROX)
  • Protein denaturation Take 100 ⁇ g 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°C for 10 min, and store at -80°C after centrifugation.
  • Electroblotting 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 facing the negative electrode.
  • the membrane faces the positive electrode; put 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.
  • HBsAg and HBeAg were detected using HBV surface antigen diagnostic kit and HBV e antigen detection kit (ELISA method).
  • step 6) Repeat step 6) five times; after washing the plate, pat the plate dry as much as possible;
  • Transfer preparation two layers of large filter paper, glue, NC membrane slightly smaller than the glue, two layers of small filter paper slightly smaller than the membrane Paper;
  • Prehybridization Take out the membrane containing the target nucleic acid, fix it by UV crosslinking, soak it in 10 ⁇ SSC for 1 min, then put it into 20 mL of preheated prehybridization solution, and place it in a hybridization oven at 68°C for 1 h;
  • Blocking Add 20 mL Blocking Buffer, 37°C, 30 min;
  • Southern blot method is used to detect viral DNA to indirectly determine whether the viral nucleocapsid is intact.
  • TNE buffer After electrophoresis, use TNE buffer to transfer the membrane.
  • the transfer conditions and procedures are consistent with the Southern blot method.
  • Block with blocking solution (containing 5% (w/v) skimmed milk powder, 1 ⁇ TBS, 0.1% Tween-20) at room temperature for 1 to 2 hours.
  • HepG2.2.15 and HepAD38 cells were passaged and plated normally. After 12 hours of adherent growth, they were transiently transfected with TRPV4 exogenous overexpression plasmid or siRNA. After 48 hours, the cells were collected, and total RNA was extracted, DNA was extracted by Hirt method, cccDNA was separated and purified, and total intracellular protein was detected by Q-PCR method. The expression of HBV RNA (total RNA, pgRNA, precore RNA), HBV DNA and cccDNA in cells was detected by Western blotting. The results are shown in Figures 1-1 to 1-3.
  • HepG2.2.15 and HepAD38 cells were passaged and plated normally. After 12 hours of adherent growth, different concentrations (10nM, 20nM, 50nM, 100nM, 500nM) of the test compound were added. After 48 hours, the cells were collected, proteins were extracted, and the expression of HBV core protein (HBcAg) in the cells was detected by Western blotting. The results are shown in the table below or Figures 2-1 to 2-7.

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Abstract

涉及化合物及其在慢性乙型肝炎、肝纤维化、肝癌的治疗中的应用。具体提供了式O所示化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,

Description

化合物及其在慢性乙型肝炎、肝纤维化、肝癌的治疗中的应用
相关申请的交叉引用
本申请是以CN申请号为202310414071.5,申请日为2023年4月18日的申请为基础,并主张其优先权,该CN申请的内容在此作为整体引入本申请中。
技术领域
本发明涉及医药领域,具体涉及一类新化合物及其在慢性乙型肝炎、肝纤维化、肝癌的治疗中的应用。
背景技术
慢性乙肝病毒(Hepatitis B virus,HBV)感染严重威害着人类的健康。据世界卫生组织报道,全球约有20亿人曾感染过乙型肝炎病毒,其中3.5亿人为慢性HBV感染者。每年约有100万死于HBV感染所致的肝衰竭、硬化和原发性肝细胞癌(HCC)。我国是HBV感染的高发地区,尽管随着乙肝疫苗的普及化及母婴阻断技术的发展,HBV的新感染率大大降低,但2016年的一项200余万例的大样本流行病学研究表明,我国21-49岁的农村地区男性HBsAg的阳性率仍高达6%,可见在相当长的时间内HBV感染仍然是我国公共卫生系统面临的严峻挑战。
HBV是一种嗜肝细胞的部分双链的环状DNA(rcDNA)病毒。其基因组全长约3.2kb,由四个部分重叠的开放读码框(ORFs)组成,包括S基因区、C基因区、P基因区和X基因区。HBV的复制是将病毒的遗传信息从亲代DNA传递给子代一系列复杂过程,包括以共价闭合环状DNA(Covalently Closed Circular DNA,cccDNA)为初始模板转录mRNA、翻译、以mRNA为模板,反转录合成病毒负链DNA和病毒正链DNA等复杂过程。而HBV ccc DNA稳定存在于被感染的肝细胞核中,并充当病毒转录模板以产生病毒基因产物,是HBV的原始复制模板,HBV cccDNA的存在是病毒复制及感染得以维持的根源,抑制或清除HBV cccDNA是治愈慢性乙型肝炎的关键。
抗HBV的治疗方法一直是感染科学研究的热点及难点之一。慢性乙型肝炎患者大多数需要长期或终生治疗,目前没有一种治疗方案可以将宿主体内的乙肝病毒彻底根除。目前临床上批准的抗病毒治疗药物主要包括核苷(酸)类似物和干扰素两大类。核苷(酸)类似物药物的作用靶点是抑制HBV DNA多聚酶和逆转录酶的活性,进而抑制HBV的复制;干扰素通过作用于HBV复制周期的多个环节来发挥抗病毒作用。主要包括激活宿主免疫应答、抑制HBVcccDNA的转录、抑制病毒核衣壳的形成或增加其降解。这些药物虽然能够有效地抑制HBV复制,但是存在无法消除或沉默肝细胞核内cccDNA,且存在药物副作用较大(尤其是干扰素)、耐药性较高等问题。
在临床上用这两类药物来治疗慢性乙型肝炎患者很难实现HBV表面抗原(HBsAg)的血清学转换,无法达到功能性治愈水平。因此,针对于现有抗病毒药物的不足,现今发现了不同类型的新型在研抗病毒药物,主要包括病毒进入抑制剂、衣壳抑制剂、核酸干扰药物、HBsAg抑制剂及免疫调节剂五大类。虽然诸多在研抗病毒药物都有一定的治疗效果,但是还需要大型临床试验进一步验证其抗病毒效果。因此,寻找新的抗病毒药物仍然是必要的。
发明内容
钙离子(Ca2+)作为细胞内重要的第二信使可以与多种细胞蛋白相互作用,调节多种生理过程并参与多种疾病进展,包括HBV感染。近年来,多项研究表明,依赖于质膜、内质网和线粒体上不同的Ca2+通道,HBV可以提高胞质中Ca2+水平。进一步,在HBV感染的细胞中,Ca2+信号的激活可以通过多种分子机制促进病毒复制。因此,现有证据表明靶向Ca2+信号将是治疗HBV感染的有效方法。
瞬时性受体电位(transient receptor potential,TRP)通道超家族与细胞内Ca2+稳态调控密切相关,TRP通道最早发现于果蝇的视觉系统,迄今为止,在哺乳动物发现的28个亚型被划分到7个亚家族:TRPA,TRPC,TRPM,TRPML,TRPN,TRPP和TRPV。瞬时受体电位香草酸亚型4(transient receptor potential vanilloid 4,TRPV4)是TRPV亚家族的成员,广泛分布于心脏、大脑、肾脏、肺 脏、肝脏、骨组织及皮肤等,对Ca2+具有选择通透性,其激活会引起Ca2+内流,从而增加细胞内游离Ca2+的浓度,参与许多生理、病理过程,是多种疾病的潜在治疗靶点。
本发明首先发现过表达TRPV4可以促进HBV的复制和转录,沉默TRPV4可以抑制HBV的复制和转录,进而发现TRPV4抑制剂具有明显抑制HBV核心蛋白(HBcAg)表达的作用。
本发明人发现,本发明申请请求保护的一类新化合物活性好,对慢性乙型肝炎、肝纤维化、肝癌等肝脏疾病有较好的治疗效果,且这些分子具有较好的成药性(溶解度、口服生物利用度等)和安全性。
为此,在本发明的第一方面,本发明提供了以下通式O所示化合物:
或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,
其中:
X选自N、CH;
R1选自8-10元稠杂芳基,且所述稠杂芳基中,每个单环含有至少一个杂原子;
R2选自C1-C6烷基、被1-3个羟基取代的C1-C6烷基、
Ra、Rb各自独立地选自C1-C6烷基;
Z选自O、S;
R2在2号、3号或5号碳原子处进行取代;
R3选自C1-C6烷基。
在一些实施方案中,R1选自稠杂芳基,所述稠杂芳基中,每个单环含有至 少一个杂原子,且所述稠杂芳基由六元环和五元环稠合形成。
在一些实施方案中,R1选自稠杂芳基,所述稠杂芳基中,每个单环含有至少一个杂原子,所述稠杂芳基由六元环和五元环稠合形成,且所述稠杂芳基共含有三个杂原子,所述杂原子优选为N原子。
在一些实施方案中,R1选自:
在一些实施方案中,R1选自
在一些实施方案中,R2选自被1-3个羟基取代的C1-C6烷基、
在一些实施方案中,R2选自被1个羟基取代的C1-C6烷基、
在一些实施方案中,R2选自
在一些实施方案中,Ra、Rb均为甲基。
在一些实施方案中,R2在3号碳原子处进行取代。
在一些实施方案中,R3为甲基。
在一些实施方案中,所述化合物具有式I-1或式I-2所示的结构,
其中:
X选自N、CH;
Y1、Y2、Y3、Y4、Y5、Y6、Y7各自独立地选自CH、N,且Y3、Y4、Y5、Y6不同时为CH;
R2选自C1-C6烷基、被1-3个羟基取代的C1-C6烷基、
Ra、Rb各自独立地选自C1-C6烷基;
Z选自O、S。
在一些实施方案中,Y1、Y2、Y3、Y4、Y5、Y6、Y7各自独立地选自CH、N,Y3、Y4、Y5、Y6不同时为CH,且满足以下条件:
(a)式I-1中,Y1、Y2、Y3、Y4、Y5和Y6中,任意两个为N,其余为CH,
(b)式I-2中,Y2、Y3、Y4、Y5、Y6和Y7中,任意两个为N,其余为CH。
在一些实施方案中,作为整体,选自以下:
在一些实施方案中,作为整体,选自
在一些实施方案中,作为整体,选自
在一些实施方案中,作为整体,选自
在一些实施方案中,R2选自被1-3个羟基取代的C1-C6烷基、
在一些实施方案中,R2选自被1个羟基取代的C1-C6烷基、
在一些实施方案中,R2选自
在一些实施方案中,Ra、Rb均为甲基。
在一些实施方案中,所述化合物选自以下:

在本发明的第二方面,本发明提供了制备前述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物的方法,其包括:
使发生亲核取代反应,获得式O所示化合物
或者,
使发生亲核取代反应,获得式I-1所示化合物
或者,
使发生亲核取代反应,获得式I-2所示化合物
其中,R1、R3的定义如前所述,Y1、Y2、Y3、Y4、Y5、Y6、Y7的定义如前所述,X、R2的定义如前所述,M选自卤素。
在一些实施方案中,M选自Cl、Br。
在一些实施方案中,所述亲核取代反应是在碱性条件下进行的。
在一些实施方案中,所述亲核取代反应是在DMEDA和碳酸钾存在的条件下进行的。
在一些实施方案中,所述亲核取代反应是在催化剂存在的条件下进行的。
在一些实施方案中,所述亲核取代反应是在CuI存在的条件下进行的。
在本发明的第三方面,本发明提供了药物组合物,其包含前述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物;任选地,还包含药学上可接受的辅料。
在一些实施方案中,所述药物组合物还包含其他药物。
在一些实施方案中,所述其他药物用于治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态。
在一些实施方案中,所述由HBV引发的疾病或感染为肝脏疾病。
在一些实施方案中,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、肝硬化、肝癌(如原发性肝细胞癌),或其任意组合。
在一些实施方案中,所述其他药物包括但不限于核苷酸类药物(如恩替卡韦、替比夫定、替诺福韦酯、阿德福韦酯或拉米夫定)、干扰素(如干扰素α2a、干扰素α1b或干扰素α2b)、治疗性疫苗、Toll样受体激动剂、入胞抑制 剂、RNA干扰药物、cccDNA靶向药物,或其任意组合。
在本发明的第四方面,本发明提供了前述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,或者前述的药物组合物在制备用于治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态的药物中的用途。
在一些实施方案中,所述由HBV引发的疾病或感染为肝脏疾病。
在一些实施方案中,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、肝硬化、肝癌(如原发性肝细胞癌),或其任意组合。
在本发明的第五方面,本发明提供了前述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,或者前述的药物组合物,其用于治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态。
在一些实施方案中,所述由HBV引发的疾病或感染为肝脏疾病。
在一些实施方案中,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、肝硬化、肝癌(如原发性肝细胞癌),或其任意组合。
在本发明的第六方面,本发明提供了一种治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态的方法,其包括:
给予有需要的受试者有效量的前述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,或者前述的药物组合物。
在一些实施方案中,所述由HBV引发的疾病或感染为肝脏疾病。
在一些实施方案中,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、肝硬化、肝癌(如原发性肝细胞癌),或其任意组合。
附图说明
图1-1表示在HepG2.2.15和HepAD38细胞中,沉默TRPV4表达或过表达TRPV4后,HBV DNA和HBcAg的表达变化情况;
图1-2表示在HepAD38细胞中沉默TRPV4或过表达TRPV4后,cccDNA 的变化情况;
图1-3表示在HepG2.2.15细胞中沉默TRPV4或过表达TRPV4后,pgRNA及totalRNA的变化情况;在HepGAD38细胞中沉默TRPV4或过表达TRPV4后,pg RNA及total RNA、precore的变化情况;
图2-1表示加入待测化合物UTU011025、UTU011026后,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量情况;
图2-2表示加入待测化合物UTU011027、UTU011028后,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量情况;
图2-3表示加入待测化合物UTU011029、UTU011030后,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量情况;
图2-4表示加入待测化合物UTU011031、UTU011032后,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量情况;
图2-5表示加入待测化合物UTU011033、UTU011034后,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量情况;
图2-6表示加入待测化合物UTU011035、UTU011036后,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量情况。
图2-7表示加入待测化合物UTU011037后,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量情况。
具体实施方式
应该理解,此处采用的术语目的在于描述具体的实施方案,并非意在限制。此外,尽管类似或者等价于此处描述的任何方法、装置和材料均可用于实施或者测试本发明,但是现在描述的是优选的方法、装置和材料。
本发明中,除非以其他方式明确指出,在本文中通篇采用的描述方式“…各自独立地选自”既可以是指在不同基团中,相同或不同的符号之间所表达的具体选项之间互相不影响,也可以表示在相同的基团中,相同或不同的符号之间所表达的具体选项之间互相不影响。
本发明化合物的取代基按照基团种类或范围公开。特别指出,本发明包括这 些基团种类和范围的各个成员的每一个独立的次级组合。例如,术语“C1-C6烷基”特别指独立公开的甲基、乙基、C3烷基、C4烷基、C5烷基和C6烷基。
术语“烷基”是指包括具有指定碳原子数的支链和直链饱和脂肪族烃基。例如,“C1-C6烷基”是指C1、C2、C3、C4、C5和C6。另外,例如“C1-C6烷基”是指具有1至6个碳原子的烷基,优选“C1-C4烷基”,更优选“C1-C3烷基”。烷基的实例包括但不限于甲基、乙基、丙基(例如正丙基和异丙基)、丁基(例如,正丁基、异丁基、叔丁基)、戊基(例如正戊基、异戊基、新戊基)等。
术语“被1-3个羟基取代的C1-C6烷基”,是指前述C1-C6烷基中1个、2个或3个氢原子被羟基替代,在一些实施方案中,优选为“被1个羟基取代的C1-C6烷基”。“被1-3个羟基取代的C1-C6烷基”的实例如-C(OH)3、-CH(OH)2、-CH2(OH)、-CH2CH2(OH)、-CH2CH(OH)2、-CH2C(OH)3优选为其中,烷基的定义如前所述。
术语“被取代的”是指指定原子或基团上的任一或多个氢被指定基团的选择替代,条件为不超过指定原子的正常价态。
杂原子为N、O或S。
卤素指氟、氯、溴或碘。
术语“杂芳基”是指在至少一个环中具有至少一个杂原子(O、N或S)的被取代的和未被取代的芳香族5-元或6-元单环基团、8-元、9-元或10-元二环基团和11-元至14-元三环基团,该含杂原子环任选还具有1个、2个或3个选自O、N或S的杂原子。其中,在至少一个环中具有至少一个杂原子(O、N或S)的被取代的和未被取代的芳香族8-元、9-元或10-元二环基团和11-元至14-元三环基团即为“稠杂芳基”。为二环或三环的杂芳基,需要二环或三环整体结构形成芳香体系。杂芳基可在任一环的任一可用氮或碳原子上连接。
示例性单环杂芳环包括但不限于:吡咯基、吡唑基、咪唑基、噁唑基、异噁唑基、噻唑基、噻二唑基、异噻唑基、呋喃基、噻吩基、恶二唑基、吡啶基、吡嗪基、嘧啶基、哒嗪基、三嗪基、三氮唑基等。
示例性二环杂芳基包括但不限于:吲哚基、5-氮杂吲哚基、吡咯并[2,3-d]嘧啶基、5,6-二氮杂吲哚基、6-氮杂吲哚基、7-氮杂吲哚基、吡唑并[3,4-b]吡啶基、 吡咯并[2,3-c]哒嗪基、噻吩并[2,3-d]咪唑基、噻吩并[2,3-d]咪唑基、吡唑并[3,4-c]吡啶基、苯并噻唑基、苯并咪唑基、苯并噁唑基、苯并噻吩基、喹啉基、异喹啉基、苯并呋喃基、吲嗪基、喹喔啉基、吲唑基、吡咯并嘧啶基、呋喃并吡啶基、异吲哚基等。
本发明中,式O所示的化合物中,R2在2号、3号或5号碳原子处进行取代,其表示,式O所示化合物的结构式可以为
本发明中,“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状,可以是预防性的;和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用,可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗,包括:(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状;(b)抑制疾病的症状,即阻止其发展;或(c)缓解疾病的症状,即,导致疾病或症状退化。
本发明中,“受试者”指脊椎动物。在某些实施方案中,脊椎动物指哺乳动物。哺乳动物包括,但不限于,牲畜(诸如牛)、宠物(诸如猫、犬、和马)、灵长类动物、小鼠和大鼠。在某些实施方案中,哺乳动物指人。
本发明中,“有效量”指在必需的剂量和时间上有效实现期望的治疗或预防效果的量。本发明的物质/分子的“治疗有效量”可根据诸如个体的疾病状态、年龄、 性别和体重及该物质/分子在个体中引发期望应答的能力等因素而变化。治疗有效量还涵盖该物质/分子的治疗有益效果胜过任何有毒或有害后果的量。“预防有效量”指在必需的剂量和时间上有效实现期望的预防效果的量。通常而非必然,由于预防剂量是在疾病发作之前或在疾病的早期用于受试者的,因此预防有效量会低于治疗有效量。在癌症的情况中,药物的治疗有效量可减少癌细胞数;缩小肿瘤体积;抑制(即一定程度的减缓,优选停止)癌细胞浸润到周围器官中;抑制(即一定程度的减缓,优选停止)肿瘤转移;一定程度的抑制肿瘤生长;和/或一定程度的减轻与癌症有关的一种或多种症状。
本发明涉及的药物组合物可以包含药学上可接受的辅料,辅料包括但不限于:离子交换剂,氧化铝,硬脂酸铝,卵磷脂,血清蛋白如人血白蛋白,缓冲物质如磷酸盐,甘油,山梨酸,山梨酸钾,饱和植物脂肪酸的部分甘油酯混合物,水,盐或电解质,如硫酸鱼精蛋白,磷酸氢二钠,磷酸氢钾,氯化钠,锌盐,胶态氧化硅,三硅酸镁,聚乙烯吡咯烷酮,纤维素物质,聚乙二醇,羧甲基纤维素钠,聚丙烯酸酯,蜂蜡,羊毛脂等等。
本发明所述药物组合物可以根据不同给药途径而制备成各种形式。例如,所述的药物组合物可以以下面的任意方式施用:口服、喷雾吸入、直肠用药、鼻腔用药、颊部用药、阴道用药、局部用药、非肠道用药如皮下、静脉、肌内、腹膜内、鞘内、心室内、胸骨内和颅内注射或输入、或借助一种外植储器用药。其中优选口服、腹膜内或静脉内用药方式。
本发明所述的化合物任选地还可与其它一种或多种活性成分联合使用,其各自用量和比例可由本领域技术人员根据具体病症和患者具体情况以及临床需要等而进行调整。
如本文所使用,除非另外说明,术语“前药”是指可以在生物学条件(体外或体内)下水解、氧化或进行其他反应以提供本发明的化合物的衍生物。前药仅在生物学条件下经过该反应成为活性化合物,或者它们在它们不反应的形式中不具有活性。通常可以使用公知的方法制备前药,例如Burger's Medicinal Chemistry and Drug Discovery(1995)172-178,949-982(Manfred E.Wolff编,第5版)中描述的那些方法。
本文所述的化合物中的立体异构体,当以化学名称特别指定为(R)-或(S)-异构体时,应分别理解为主要构型为(R)-异构体或(S)-异构体。任何不对称碳原子可以存在于(R)-、(S)-或(R、S)-构型中,优选以(R)-或(S)-构型存在。
“溶剂化物”或“溶剂合物”可以互换使用,指的是以与某种溶剂分子的组合存在的化合物。该组合可以包括化学计量的量的某种溶剂,例如一水合物或二水合物,或者可以包括任意量的水;又如,甲醇或乙醇可以形成“醇化物”,其也可以为化学计量的或非化学计量的。在本文中使用的术语“溶剂合物”指的是固体形式,即,在溶剂的溶液中的化合物虽然其可以为溶剂化的,但是它不是如本文中使用的术语的溶剂合物。
下面结合具体实施例对本发明进行进一步的解释说明。如无特别说明,所有试剂或原料均可以市购获得。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker ASCENDTM-400核磁仪,测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl3)、氘代甲醇(CD3OD),内标为四甲基硅烷(TMS)。
MS的测定用Agilent 6110,Agilent 1100,Agilent 6120,AgilentG6125B液相质谱联用仪。
HPLC的测定使用岛津HPLC-2010C高压液相色谱仪(XBRIDGE 2.1*50mm,3.5um色谱柱)。
手性HPLC分析测定使用THARSFC X5。
薄层层析硅胶板使用烟台青岛GF254硅胶板,薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm~0.2mm,薄层层析分离纯化产品采用的规格是0.4mm~0.5mm。
柱层析一般使用青岛海洋硅胶200~300目硅胶为载体。
高效液相制备使用Waters 2767、Waters 2545、和创新恒通LC3000制备型色谱仪。
手性制备柱层析使用,Shimadzu LC-20AP、THARSFC PREP 80。
CombiFlash快速制备仪使用Combiflash Rf200(TELEDYNE ISCO)。
加压氢化反应使用北京佳维科创科技GCD-500G型氢气发生器。
微波反应使用Biotage initiator+型微波反应器。
实验例中如无特殊说明,反应均在氩气氛或者氮气氛下进行。
实施例1化合物的合成
1.目标化合物UTU011025
(5S,7S)-7-((3H咪唑[4,5-c]吡啶-3-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011025)
(2R,3R)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]十二碳-6-烯(3)
向(1R,2R)-1,2-二苯基-1,2-乙二醇2(150g,699mmol)和3-甲基-2-环己烯-1-酮1(77g,699mmol)的环己烷(1398mL)溶液中加入PPTS(4.39g,17.48mmol)反应16小时,加入乙醚(500mL)稀释,用饱和NaHCO3溶液洗涤,分液,有机相用无水MgSO4干燥,过滤并浓缩,所得残余物经硅胶柱层析纯化,洗脱,得到化合物(2R,3R)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]十二碳-6-烯(3)。
(1S,4'R,5'R,6R)-6-甲基-4',5'-二苯基螺环[双环[4.1.0]庚烷-2,2'-[1,3]-二氧环烷](4)
在500mL的圆底烧瓶中加入锌铜粉(5g),正己烷(500mL),(2R,3R)-7-甲基-2,3-二苯基-1,4-二氧西罗[4.5]十二碳-6-烯(3),二碘甲烷(150mL,1865 mmol),二乙基锌(500mL,1mol/L in Hexane,500mmol)反应,LCMS监测反应结束后,用饱和Na2CO3(800mL)淬灭,然后通过硅藻土过滤,洗涤滤饼,滤液用饱和食盐水(1L)洗涤,无水MgSO4干燥,过滤并浓缩有机相,所得残余物经硅胶柱层析纯化,洗脱,得到化合物(1S,4'R,5'R,6R)-6-甲基-4',5'-二苯基螺环[双环[4.1.0]庚烷-2,2'-[1,3]-二氧环烷](4)。
(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧杂环[4.5]-癸烷(5)将(1S,4'R,5'R,6R)-6-甲基-4',5'-二苯基螺环[双环[4.1.0]庚烷-2,2'-[1,3]二氧戊环](137g,428mmol)溶解于甲醇(1993mL)中,加入氢溴酸(145mL,1283mmol)反应,减压浓缩,得到黄色残渣,向残渣中添加己烷(1L),搅拌,减压浓缩,得到化合物(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧杂环[4.5]-癸烷(5)。
3-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-3H-咪唑[4,5-c]吡啶(7)
在0℃下向3H咪唑并[4,5-c]吡啶(583mg,4.9mmol)的DMF(30mL)溶液中添加NaH(196mg,4.9mmol),(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸烷(2g,4.9mmol)反应16小时。加入H2O(150mL)淬灭,用EA(3x200mL)萃取,合并有机层,用饱和食盐水(200mL)洗涤,MgSO4干燥,过滤并减压浓缩,所得残余物经硅胶柱层析纯化,洗脱,得到化合物3-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-3H-咪唑[4,5-c]吡啶(7)。
(S)-3-((3H-咪唑并[4,5-c]吡啶-3-基)甲基)-3-甲基环己烷-1-酮(8)将3-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-3H-咪唑[4,5-c]吡啶(1.2g,2.7mmol)溶于HCOOH(15mL)中,反应16小时。减压浓缩反应溶液,所得残余物经硅胶柱层析纯化,洗脱,得到化合物(S)-3-((3H-咪唑并[4,5-c]吡啶-3-基)甲基)-3-甲基环己烷-1-酮(8)。3-((3S,5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-3H-咪唑[4,5-c]-吡啶(9)
将(S)-3-((3H-咪唑并[4,5-c]吡啶-3-基)甲基)-3-甲基环己烷-1-酮(500 mg,2.1mmol)溶于DMSO(10mL)中,加入三甲基碘化亚砜(428mg,2.1mmol),叔丁醇钾(235mg,2.1mmol)反应16小时。加入H2O(100mL)淬灭,并用EA(3x100mL)萃取,合并有机层,饱和食盐水(2x100mL)洗涤,无水MgSO4干燥,过滤并浓缩。所得残余物经硅胶柱层析纯化,洗脱,得到化合物3-((3S,5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-3H-咪唑[4,5-c]-吡啶(9)。
(7S)-7-((3H-咪唑并[4,5-c]吡啶-3-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(10)
向3-((3S,5S)-5-甲基-1-奥沙匹罗[2.5]辛烷-5-基)甲基)-3H咪唑-[4,5-c]吡啶(190mg,0.74mmol)的DMSO(10mL)溶液中添加叔丁醇钾(166mg,1.48mmol),氨基甲酸乙酯(197mg,2.22mmol),反应16小时。加入H2O(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,饱和食盐水(2x50mL)洗涤,无水MgSO4干燥,过滤并浓缩。所得残余物经硅胶柱层析纯化,洗脱,得到化合物(7S)-7-((3H-咪唑并[4,5-c]吡啶-3-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(10)。
(5S,7S)-7-((3H咪唑[4,5-c]吡啶-3-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011025)
向(7S)-7-((3H-咪唑并[4,5-c]吡啶-3-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(80mg,0.26mmol)的1,4-二氧六环(10mL)溶液中添加2-(5-氯吡嗪-2-基)丙烷-2-醇(45mg,0.26mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入H2O(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((3H咪唑[4,5-c]吡啶-3-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮UTU011025。
UTU011025:Mass(m/z):437.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.18(d,J=1.6Hz,1H),9.07(s,0.5H),8.95(s,0.5H),8.59(d,J=1.6Hz,1H),8.39(s,0.5H),8.35(s,0.5H),8.32(d,J=11.4Hz,1H),7.74(d,J=5.6Hz,0.5H),7.66 (d,J=5.6Hz,0.5H),5.42(s,1H),4.16(m,2H),3.85(m,2H),1.93(m,2H),1.72(m,2H),1.64(s,2H),1.44(d,J=1.8Hz,6H),1.37(m,2H),1.07(d,J=7.8Hz,3H).
2.目标化合物UTU011026
(5R,7S)-7-((1H-咪唑并[4,5-c]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011026)
2-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]癸-7-基)-甲基)-异吲哚-1,3-二酮(3)
在1L的圆底烧瓶中加入(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]癸烷5(172g,428mmol),邻苯二甲酰亚胺钾(399g,2140mmol)和NMP(900mL)反应,加入H2O(3L)稀释,EA萃取(3x500mL),合并有机层,用饱和食盐水(2x300mL)洗涤,无水MgSO4干燥,过滤并浓缩,所得粗产品经硅胶柱层析,洗脱,得到粗品化合物2-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]癸-7-基)-甲基)-异吲哚-1,3-二酮,直接用于下一步。
((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]癸-7-基)甲胺(4)在3L的圆底烧瓶中加入2-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]癸-7-基)-甲基)-异吲哚-1,3-二酮,用乙醇(2.25L)溶解,添加水合肼(40.3mL,1284mmol)反应,LCMS监测反应完成。用乙醇(100mL*2)清洗滤饼,减压浓缩滤液,所得残余物中加入四氢呋喃(500mL)并搅拌,浓缩滤液,将所得残余物溶解在己烷(500mL)中,加热搅拌溶液, 然后冷却至室温并过滤,减压浓缩滤液得到化合物((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂萘[4.5]癸-7-基)甲胺(4)。
N-(((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-3-硝基吡啶-4-胺(6)
将((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸烷-7-基)-甲酰胺(1g,2.9mmol)和4-氯-3-硝基吡啶(458mg,2.9mmol)溶于乙醇(10mL)中,加入TEA(587mg,5.8mmol)反应16小时。过滤,浓缩滤液,通过硅胶色谱法纯化残留物,得到化合物N-(((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-3-硝基吡啶-4-胺(6)。
N4-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)吡啶-3,4-二胺(7)
将N-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-3-硝基吡啶-4-胺(800mg,1.7mmol)溶于甲醇(20mL)中,添加氯化铵(459mg,8.5mmol),反应16小时。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,用饱和氯化钠(2x50mL)洗涤,无水MgSO4干燥,过滤并浓缩。通过硅胶色谱法纯化残留物,洗脱,得到化合物N4-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)吡啶-3,4-二胺(7)。
1-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-1H-咪唑[4,5-c]吡啶(8)
将N4-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)吡啶-3,4-二胺(600mg,1.4mmol)溶于原甲酸三甲酯(10mL),加入对甲苯磺酸反应16小时。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,洗涤,干燥,过滤并浓缩。通过硅胶色谱法纯化残留物,洗脱,得到化合物1-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-1H-咪唑[4,5-c]吡啶(8)。
(S)-3-((1H-咪唑[4,5-c]吡啶-1-基)甲基)-3-甲基环已烷-1-酮(9)
将1-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲 基)-1H-咪唑[4,5-c]吡啶(500mg,1.14mmol)溶于HCOOH(10mL)中反应16小时。减压浓缩,通过硅胶色谱法纯化残留物,洗脱,得到化合物(S)-3-((1H-咪唑[4,5-c]吡啶-1-基)甲基)-3-甲基环已烷-1-酮(9)。
1-((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-1H-咪唑[4,5-c]吡啶(10)将(S)-3-((1H-咪唑[4,5-c]吡啶-1-基)甲基)-3-甲基环已烷-1-酮(200mg,0.82mmol)溶于二甲基亚砜(10mL)中,依次加入三甲基碘化亚砜(167mg,0.82mmol),叔丁醇钾(92mg,0.82mmol)反应16小时。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,用饱和氯化钠(2x50mL)洗涤,无水MgSO4干燥,过滤并浓缩。通过硅胶色谱法纯化残留物,洗脱,得到化合物1-((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-1H-咪唑[4,5-c]吡啶(10)。
(7S)-7-((1H-咪唑并[4,5-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(11)
在1-((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-1H-咪唑[4,5-c]吡啶(170mg,0.66mmol)的DMSO(10mL)溶液中加入叔丁醇钾(148mg,1.32mmol),氨基甲酸乙酯(176mg,1.98mmol)反应。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,洗涤,干燥,过滤并浓缩。通过硅胶色谱法纯化残留物,洗脱,得到化合物(7S)-7-((1H-咪唑并[4,5-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(11)。
(5R,7S)-7-((1H-咪唑并[4,5-c]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011026)
将(7S)-7-((1H-咪唑并[4,5-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(80mg,0.26mmol)溶于1,4-二氧六环(10mL)中,然后添加2-(5-氯吡嗪-2-基)丙烷-2-醇(45mg,0.26mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物5R,7S)-7-((1H-咪唑并[4,5-c]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5] 癸-2-酮UTU011026。
UTU011026:Mass(m/z):437.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.18(d,J=1.6Hz,1H),8.95(s,1H),8.59(d,J=1.6Hz,1H),8.34(s,3H),7.74(d,J=5.8Hz,1H),5.42(s,1H),4.12(s,2H),3.86(d,J=15.8Hz,2H),1.98(d,J=12.6Hz,1H),1.88(d,J=14.6Hz,1H),1.71(d,J=14.2Hz,1H),1.61(t,J=m,4H),1.44(d,J=1.8Hz,6H),1.37(d,J=8.8Hz,1H),1.06(s,3H).
3.目标化合物(UTU011027)
(5S,7S)-7-((1H-吡唑[3,4-c]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011027)
1-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸-7-基)甲基)-1H-吡唑[3,4-c]吡啶(3)
向1H吡唑并[3,4-c]吡啶(583mg,4.9mmol)的DMF(30mL)溶液中加入NaH(196mg,4.9mmol),(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧杂环[4.5]-癸烷(2g,4.9mmol)反应。加入水(200mL)淬灭,用EA(3x200mL)萃取,合并有机层,用饱和食盐水(3x100mL)洗涤,无水MgSO4干燥,过滤并浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物1-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸-7-基) 甲基)-1H-吡唑[3,4-c]吡啶(3)。
(S)-3-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-3-甲基环已烷-1-酮(4)将1-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸-7-基)甲基)-1H-吡唑[3,4-c]吡啶(1.2g,2.7mmol)溶于HCOOH(15mL)反应。减压浓缩,所得残余物通过硅胶色谱法纯化,洗脱,得到化合物(S)-3-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-3-甲基环已烷-1-酮(4)。
1-(((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-1H-吡唑[3,4-c]吡啶(5)将(S)-3-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-3-甲基环已烷-1-酮(500mg,2.1mmol)溶于二甲基亚砜(10mL)中,依次加入三甲基碘化亚砜(428mg,2.1mmol),叔丁醇钾(235mg,2.1mmol)反应。加入水(30mL)淬灭,用EA(3x30mL)萃取,合并有机层,用饱和食盐水(2x30mL)洗涤,无水MgSO4干燥,过滤并减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物1-(((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-1H-吡唑[3,4-c]吡啶(5)。
(7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)
向1-((5S)-5-甲基-1-氧杂螺环[2.5]辛烷-5-基)甲基)-1H吡唑[3,4-c]吡啶(190mg,0.74mmol)的二甲基亚砜(10mL)溶液中依次加入叔丁醇钾(166mg,1.48mmol),氨基甲酸乙酯(197mg,2.22mmol)反应。加入水(30mL)淬灭,用EA(3x30mL)萃取,合并有机层,用饱和食盐水(2x30mL)洗涤,无水MgSO4干燥,过滤并减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物(7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)。
(5S,7S)-7-((1H-吡唑[3,4-c]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011027)
向(7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(80mg,0.26mmol)的1,4-二氧六环(10mL)的溶液中,添加2-(5-氯吡嗪-2-基)丙烷-2-醇(45mg,0.26mmol),DMEDA(4.6mg,0.05 mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。
加入水(20mL)淬灭,用EA(3x30mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((1H-吡唑[3,4-c]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸
-2-酮UTU011027。
UTU011027:Mass(m/z):437.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.26(s,1H),9.19(d,J=1.6Hz,1H),8.59(d,J=1.6Hz,1H),8.23(t,J=2.8Hz,2H),7.75(dd,J=5.6,1.2Hz,1H),5.41(s,1H),4.37(s,2H),3.89(m,2H),1.94(m,2H),1.82(d,J=14.4Hz,1H),1.67(m,2H),1.52(dd,J=13.0,9.2Hz,2H),1.45(d,J=2.8Hz,6H),1.39(s,1H),1.06(s,3H).
4.目标化合物UTU011028
(5S,7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(UTU011028)
2-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸-7-基)甲基)-2H-吡唑[3,4-c]吡啶(3)
向1H吡唑并[3,4-c]吡啶(583mg,4.9mmol)的DMF(30mL)溶液中加 入NaH(196mg,4.9mmol),(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧杂环[4.5]-癸烷(2g,4.9mmol)反应。加入水(200mL)淬灭,用EA(3x200mL)萃取,合并有机层,用饱和食盐水(3x100mL)洗涤,无水MgSO4干燥,过滤并浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物2-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸-7-基)甲基)-2H-吡唑[3,4-c]吡啶(3)。
(S)-3-((2H--吡唑并[3,4-c]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(4)将2-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸-7-基)甲基)-2H-吡唑[3,4-c]吡啶(1.2g,2.7mmol)溶解到HCOOH(15mL)中反应,减压浓缩,所得残余物通过硅胶色谱法纯化,洗脱,得到化合物(S)-3-((2H--吡唑并[3,4-c]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(4)。
2-(((5S)-5-甲基-1-氧螺[2.5]辛烷-5-基)甲基)-2H--吡唑[3,4-c]吡啶(5)将(S)-3-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(500mg,2.1mmol)溶于二甲基亚砜(10mL)中,加入三甲基碘化亚砜(428mg,2.1mmol),叔丁醇钾(235mg,2.1mmol)反应。加入水(50mL)淬灭,EA(3x50mL)萃取,合并有机层,用饱和食盐水(2x50mL)洗涤,无水MgSO4干燥,过滤并减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物2-(((5S)-5-甲基-1-氧螺[2.5]辛烷-5-基)甲基)-2H--吡唑[3,4-c]吡啶(5)。
(7S)-7-((2H--吡唑并[3,4-c]吡啶-2-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)
向2-(((5S)-5-甲基-1-氧螺[2.5]辛烷-5-基)甲基)-2H-吡唑[3,4-c]吡啶(190mg,0.74mmol)的二甲基亚砜(10mL)溶液中依次加入叔丁醇钾(166mg,1.48mmol),氨基甲酸乙酯(197mg,2.22mmol)反应。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,用饱和食盐水(2x50mL)洗涤,无水MgSO4干燥,过滤并减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物(7S)-7-((2H--吡唑并[3,4-c]吡啶-2-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)。
(5S,7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(UTU011028)
向(7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(80mg,0.26mmol)的1,4-二氧六环(10mL)的溶液中添加2-(5-氯吡嗪-2-基)丙烷-2-醇(45mg,0.26mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水(20mL)淬灭,用EA(3x20mL)萃取,合并有机层,减压浓缩。通过纯化残余物,得到化合物(5S,7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮UTU011028。
UTU011028:Mass(m/z):437.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.18(d,J=1.6Hz,1H),9.15(s,1H),8.59(d,J=1.6Hz,1H),8.48(d,J=0.8Hz,1H),8.06(d,J=5.8Hz,1H),7.66(dd,J=5.8,1.4Hz,1H),5.42(s,1H),4.37(s,2H),3.90(m,2H),1.97(d,J=13.4Hz,1H),1.83(t,J=14.8Hz,2H),1.68(m,2H),1.57(m,2H),1.45(d,J=2.6Hz,6H),1.39(d,J=12.8Hz,1H),1.05(s,3H).
5.目标化合物UTU011029
(5S,7S)-7-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011029)
5-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-5H吡咯[3,2-d]嘧啶(3)
向5H吡咯[3,2-d]嘧啶(583mg,4.9mmol)的DMF(30mL)溶液中添加NaH(196mg,4.9mmol),((2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸烷(2g,4.9mmol)反应。加入水(20mL)淬灭,用EA(3x20mL)萃取,合并有机层,减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物5-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-5H吡咯[3,2-d]嘧啶(3)。
(S)-3-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-3-甲基环已烷-1-酮(4)将5-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-5H吡咯[3,2-d]嘧啶(1.2g,2.7mmol)溶于HCOOH(15mL)中反应16小时。减压浓缩反应溶液,所得残余物通过硅胶色谱法纯化,洗脱,得到化合物(S)-3-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-3-甲基环已烷-1-酮(4)。5-((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-5H吡咯[3,2-d]嘧啶(5)将(S)-3-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-3-甲基环已烷-1-酮(500mg,2.1mmol)溶于DMSO(10mL)中,搅拌溶解,添加三甲基碘化亚砜(428mg,2.1mmol),叔丁醇钾(235mg,2.1mmol)反应。加入水(20mL)淬 灭,用EA(3x20mL)萃取,合并有机层,减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物5-((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-5H吡咯[3,2-d]嘧啶(5)。
(7S)-7-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)
将5-((5S)-5-甲基-1-氧杂吡咯[2.5]辛烷-5-基)甲基)-5H吡咯[3,2-d]嘧啶(190mg,0.74mmol)溶于DMSO(10mL)中,添加叔丁醇钾(166mg,1.48mmol),氨基甲酸乙酯(197mg,2.22mmol)反应。加入水(20mL)淬灭,用EA(3x20mL)萃取,合并有机层,减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物(7S)-7-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)。
(5S,7S)-7-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011029)
向(7S)-7-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(80mg,0.26mmol)的1,4-二氧六环(10mL)溶液中添加2-(5-氯吡嗪-2-基)丙烷-2-醇(45mg,0.26mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水(20mL)淬灭,用EA(3x20mL)萃取,合并有机层,减压浓缩。通过纯化残余物,得到化合物(5S,7S)-7-((5H吡咯[3,2-d]嘧啶-5-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮UTU011029。
UTU011029:Mass(m/z):437.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.15(d,J=1.6Hz,1H),9.09(s,1H),8.77(s,1H),8.55(d,J=1.6Hz,1H),7.82(d,J=3.2Hz,1H),6.60(m,1H),5.37(s,1H),4.10(s,2H),3.82(m,2H),1.67(m,7H),1.41(d,J=1.8Hz,6H),1.35(dd,J=13.0,4.2Hz,1H),1.02(s,3H).
6.目标化合物UTU011030
(5S,7S)-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡 嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸烷-2-酮(UTU011030)
7-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-7H吡咯[2,3-d]嘧啶(3)
向7H吡咯[2,3-d]嘧啶(583mg,4.9mmol)的DMF(30mL)溶液中添加NaH(196mg,4.9mmol),(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧杂螺环[4.5]癸烷(2g,4.9mmol)反应。加入水(100mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物7-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-7H吡咯[2,3-d]嘧啶(3)。
(S)-3-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-3-甲基环已烷-1-酮(4)将7-((2R,3R,7S)-7-甲基-2,3-二苯基-1,4-二氧杂环吡咯[4.5]癸-7-基)甲基)-7H吡咯[2,3-d]嘧啶(1.2g,2.7mmol)溶于HCOOH(15mL)中反应16小时。减压浓缩反应溶液,所得残余物通过硅胶色谱法纯化,洗脱,得到化合物(S)-3-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-3-甲基环已烷-1-酮(4)。7-((5S)-5-甲基-1-氧杂螺环[2.5]辛烷-5-基)甲基)-7H吡咯[2,3-d]嘧啶(5)将(S)-3-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-3-甲基环已烷-1-酮(500mg, 2.1mmol)溶于DMSO(10mL)中,搅拌溶解,添加三甲基碘化亚砜(428mg,2.1mmol),叔丁醇钾(235mg,2.1mmol)反应。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物7-((5S)-5-甲基-1-氧杂螺环[2.5]辛烷-5-基)甲基)-7H吡咯[2,3-d]嘧啶(5)。
(7S)-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)
向7-((5S)-5-甲基-1-氧杂螺环[2.5]辛烷-5-基)甲基)-7H吡咯[2,3-d]嘧啶(190mg,0.74mmol)的DMSO(10mL)溶液中添加叔丁醇钾(166mg,1.48mmol),氨基甲酸乙酯(197mg,2.22mmol)反应16小时。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩。所得残余物通过硅胶色谱法纯化,洗脱,得到化合物7-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(6)。
(5S,7S)-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸烷-2-酮(UTU011030)
向(7S)-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(80mg,0.26mmol)的1,4-二氧六环(10mL)溶液中添加2-(5-氯吡嗪-2-基)丙烷-2-醇(45mg,0.26mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水(20mL)淬灭,用EA(3x20mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-((7H吡咯[2,3-d]嘧啶-7-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸烷-2-酮UTU011030。
UTU011030:Mass(m/z):437.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.15(d,J=1.6Hz,1H),8.96(s,1H),8.75(s,1H),8.55(d,J=1.6Hz,1H),7.57(d,J=3.6Hz,1H),6.61(d,J=3.6Hz,1H),5.37(s,1H),4.08(d,J=3.2Hz,2H),3.87(d,J=10.2Hz,1H),3.77(d,J=10.4Hz,1H),1.93(d,J=13.0Hz,1H),1.79(m,2H),1.61(dd,J=15.0,3.8Hz,2H),1.52(dd,J=13.4,4.0Hz,1H),1.41
(d,J=1.4Hz,6H),1.36(m,2H),1.01(s,3H).
7.目标化合物UTU011031
(5S,7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011031)
向(7S)-7-((2H-1l4-吡唑并[5,4-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(50mg,0.11mmol,制备方法参见UTU011028)的1,4-二氧六环(10mL)溶液中添加(5-溴吡嗪-2-基)二甲基氧化膦(26mg,0.11mmol)、DMEDA(2mg,0.022mmol),CuI(2mg,0.022mmol)和K2CO3(6mg,0.044mmol)反应。加入水淬灭,用EA(3x20mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮UTU011031。
UTU011031:Mass(m/z):455.2(M+H+).1H NMR(400MHz,CD3OD)δ9.59(d,J=3.6Hz,1H),9.13(s,1H),8.78(d,J=1.4Hz,1H),8.41(s,1H),8.07(s,1H),7.74(d,J=5.6Hz,1H),4.43(s,2H),4.00(d,J=1.2Hz,2H),2.01(m,3H),1.87(dd,J=13.8,4.4Hz,6H),1.61(m,5H),1.22(s,3H).
8.目标化合物UTU011032
(5S,7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-(5-(二甲基硫代磷酰基)吡啶-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011032)
向(7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(100mg,0.33mmol,制备方法参见UTU011028)的1,4-二氧六环(10mL)溶液中加入(6-氯吡啶-3-基)二甲基硫化膦(68mg,0.33mmol),DMEDA(1mg,0.066mmol),CuI(6mg,0.066mmol)和K2CO3(20mg,0.14mmol)反应。加入水(20mL)淬灭,用EA(3x30mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((2H-吡唑并[3,4-c]吡啶-2-基)甲基)-3-(5-(二甲基硫代磷酰基)吡啶-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮UTU011032。
UTU011032:Mass(m/z):470.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.16(s,1H),8.85(d,J=6.8Hz,1H),8.48(s,1H),8.34(m,1H),8.18(d,J=8.8Hz,1H),8.06(d,J=5.8Hz,1H),7.66(d,J=6.0Hz,1H),4.37(s,2H),3.96(d,J=14.2Hz,2H),1.99(d,J=13.6Hz,6H),1.85(d,J=9.0Hz,2H),1.73(d,J=13.8Hz,2H),1.55(s,2H),1.45(m,2H),1.05(s,3H).
9.目标化合物UTU011033
(5S,7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-3-(5-(二甲基硫代磷酰基)吡啶-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011033)
向(7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(80mg,0.25mmol,制备方法参见UTU011027)的1,4-二氧六环(10mL)溶液中添加(6-氯吡啶-3-基)二甲基硫化膦(42mg,0.25mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水淬灭,用EA(3x30mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-3-(5-(二甲基硫代磷酰基)吡啶-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮UTU011033。
UTU011033:Mass(m/z):470.1(M+H+).1H NMR(400MHz,CD3OD)δ9.03(s,1H),8.75(d,J=7.0Hz,1H),8.20(dd,J=8.8,6.4Hz,2H),8.12(s,1H),8.09(s,1H),7.70(d,J=5.4Hz,1H),4.30(s,2H),3.91(d,J=11.6Hz,2H),1.89(d,J=13.6Hz,6H),1.78(m,2H),1.49(m,6H),1.07(d,J=8.4Hz,3H).
10.目标化合物UTU011034
(5R,7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011034)
向(7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸-2-酮(50mg,0.11mmol,制备方法参见UTU011027)的1,4-二氧六环(10mL)溶液中添加(5-溴吡嗪-2-基)二甲基氧化膦(26mg,0.11mmol),DMEDA(2mg,0.022mmol),CuI(2mg,0.022mmol)和K2CO3(6mg,0.044mmol)反应。加入水(20mL)淬灭,用EA(3x20mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5R,7S)-7-((1H-吡唑并[3,4-c]吡啶-1-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮UTU011034。
UTU011034:Mass(m/z):455.2(M+H+).1H NMR(400MHz,CD3OD)δ9.60(d,J=3.6Hz,1H),9.15(s,1H),8.78(d,J=1.4Hz,1H),8.22(dd,J=12.0,3.2Hz,2H),7.81(dd,J=5.8,1.2Hz,1H),4.43(s,2H),3.97(m,2H),2.06(m,2H),1.91(m,2H),1.86(dd,J=13.8,2.8Hz,6H),1.62(m,4H),1.22(s,3H).
11.目标化合物UTU011035
(5S,7S)-7-((1H-吡唑并[3,4-b]吡啶-1-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011035)
1-((2S,3S,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-1H-吡唑[3,4-b]吡啶(3)
向1H-吡唑并[3,4-b]吡啶(583mg,4.9mmol)的DMF(30mL)溶液中添加NaH(196mg,4.9mmol),(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧杂螺[4.5]癸烷(2g,4.9mmol)反应。加入水(100mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩,所得残余物用硅胶色谱法纯化,洗脱,得到化合物1-((2S,3S,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-1H-吡唑[3,4-b]吡啶(3)。
(S)-3-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(4)将1-((2S,3S,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-1H-吡唑[3,4-b]吡啶(1.2g,2.7mmol)溶于HCOOH(15mL)中反应16小时。减压浓缩反应溶液,所得残余物用硅胶色谱法纯化,洗脱,得到化合物(S)-3-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(4)。2-(((5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-2H吡唑[3,4-b]吡啶(5)将(S)-3-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-甲基环已烷-1-酮溶于DMSO(10mL)中,搅拌溶解,依次添加三甲基碘化亚砜(428mg,2.1mmol),叔丁醇钾(235mg,2.1mmol)反应16小时。加入水(100mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩,所得残余物用硅胶色谱法纯 化,洗脱,得到化合物2-(((5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-2H吡唑[3,4-b]吡啶(5)。
(7S)-7-((1H-吡唑并[3,4-b]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(6)
向2-(((5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-2H吡唑[3,4-b]吡啶(190mg,0.74mmol)的DMSO(10mL)溶液中添加叔丁醇钾(166mg,1.48mmol),氨基甲酸乙酯(197mg,2.22mmol),反应16小时。加入水(50mL)淬灭,用EA(3x30mL)萃取,合并有机层,减压浓缩,所得残余物用硅胶色谱法纯化,洗脱,得到化合物(7S)-7-((1H-吡唑并[3,4-b]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(6)。
(5S,7S)-7-((1H-吡唑并[3,4-b]吡啶-1-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011035)
向(7S)-7-((1H-吡唑并[3,4-b]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮的1,4-二氧六环(10mL)溶液中添加(5-溴吡嗪-2-基)二甲基氧化膦(54mg,0.31mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水(30mL)淬灭,用EA(3x30mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((1H-吡唑并[3,4-b]吡啶-1-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮UTU011035。
UTU011035:Mass(m/z):455.2(M+H+).1H NMR(400MHz,DMSO-d6)δ9.43(d,J=3.4Hz,1H),8.72(s,1H),8.56(dd,J=4.4,1.4Hz,1H),8.26(dd,J=8.0,1.4Hz,1H),8.19(s,1H),7.22(m,1H),4.31(s,2H),3.90(m,2H),1.93(m,4H),1.72(d,J=13.8Hz,6H),1.53(m,6H),1.07(s,3H).
12.目标化合物UTU011036
(5S,7S)-7-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011036)
2-((2S,3S,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-2H-吡唑[3,4-b]吡啶(3)
向1H-吡唑并[3,4-b]吡啶(583mg,4.9mmol)的DMF(30mL)溶液中添加NaH(196mg,4.9mmol),(2R,3R,7S)-7-(溴甲基)-7-甲基-2,3-二苯基-1,4-二氧螺[4.5]癸烷(2g,4.9mmol)反应16小时。加入水(100mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩,所得残余物用硅胶色谱法纯化,洗脱,得到化合物2-((2S,3S,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-2H-吡唑[3,4-b]吡啶(3)。
(S)-3-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(4)将2-((2S,3S,7S)-7-甲基-2,3-二苯基-1,4-二氧杂吡咯[4.5]癸-7-基)甲基)-2H-吡唑[3,4-b]吡啶(1.2g,2.7mmol)溶于HCOOH(15mL)中反应16小时。减压浓缩反应溶液,所得残余物用硅胶色谱法纯化,洗脱,得到化合物(S)-3-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(4)。2-(((5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-2H吡唑[3,4-b]吡啶(5)将(S)-3-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-甲基环已烷-1-酮(500mg,2.1mmol)溶于DMSO(10mL)中,搅拌溶解,依次添加三甲基碘化亚砜(428mg,2.1mmol),叔丁醇钾(235mg,2.1mmol),在25℃下搅 拌反应16小时。加入水(100mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩,所得残余物用硅胶色谱法纯化,洗脱,得到化合物2-(((5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-2H吡唑[3,4-b]吡啶(5)。
(7S)-7-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(6)
向2-(((5S)-5-甲基-1-氧杂螺[2.5]辛烷-5-基)甲基)-2H吡唑[3,4-b]吡啶(190mg,0.74mmol)的DMSO(10mL)溶液中依次添加叔丁醇钾(166mg,1.48mmol),氨基甲酸乙酯(197mg,2.22mmol)反应16小时。加入水(50mL)淬灭,用EA(3x50mL)萃取,合并有机层,减压浓缩,所得残余物用硅胶色谱法纯化,洗脱,得到化合物(7S)-7-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(6)。
(5S,7S)-7-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(UTU011036)
向(7S)-7-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮的1,4-二氧六环(10mL)溶液中添加(5-溴吡嗪-2-基)二甲基氧化膦(54mg,0.31mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水(20mL)淬灭,用EA(3x30mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((2H-吡唑并[3,4-b]吡啶-2-基)甲基)-3-(5-(二甲基磷酰基)吡嗪-2-基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮UTU011036。
UTU011036:Mass(m/z):455.2(M+H+).1H NMR(400MHz,CD3OD)δ9.55(d,J=3.6Hz,1H),8.74(d,J=1.2Hz,1H),8.56(s,1H),8.30(s,1H),8.23(dd,J=8.4,1.6Hz,1H),7.13(dd,J=8.4,4.4Hz,1H),4.32(s,2H),3.96(s,2H),1.99(m,4H),1.83(d,J=4.8Hz,3H),1.80(d,J=4.8Hz,3H),1.55(m,4H),1.17(s,3H).
13.目标化合物UTU011037
(5S,7S)-7-((1H-吡唑[3,4-b]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基) 吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮(UTU011037)
向(7S)-7-((1H-吡唑并[3,4-b]吡啶-1-基)甲基)-7-甲基-1-氧杂-3-氮杂螺环[4.5]癸烷-2-酮(80mg,0.26mmol,制备方法参见UTU011035)的1,4-二氧六环(10mL)溶液中依次添加2-(5-氯吡嗪-2-基)丙烷-2-醇(54mg,0.31mmol),DMEDA(4.6mg,0.05mmol),CuI(4.7mg,0.05mmol)和K2CO3(14mg,0.1mmol)反应。加入水(20mL)淬灭,用EA(3x30mL)萃取,合并有机层,减压浓缩,通过纯化残余物,得到化合物(5S,7S)-7-((1H-吡唑[3,4-b]吡啶-1-基)甲基)-3-(5-(2-羟基丙烷-2-基)吡嗪-2-基)-7-甲基-1-氧代-3-氮杂螺环[4.5]癸-2-酮UTU011037。
UTU011037:Mass(m/z):437.2(M+H+).1H NMR(400MHz,CD3OD)δ9.27(d,J=1.4Hz,1H),8.57(d,J=1.4Hz,1H),8.51(dd,J=4.6,1.4Hz,1H),8.21(dd,J=8.0,1.4Hz,1H),8.08(s,1H),7.20(dd,J=8.0,4.6Hz,1H),4.34(d,J=1.2Hz,2H),3.92(m,2H),1.91(m,4H),1.66(m,1H),1.57(s,1H),1.52(s,6H),1.47(d,J=3.6Hz,1H),1.13(s,3H).
实施例2化合物的体外药效评价
一、实验方法
1.HepG2.2.15和HepAD38细胞的培养及处理方法:
1.1细胞复苏
1)准备工作:提前将超净工作台紫外照射30min;打开水浴锅并将其温度 调至37℃;配制70%酒精备用;
2)将冻存于液氮罐里的细胞取出后,迅速放到37℃水浴锅中,然后缓慢摇动冻存管使其融化;
3)当冻存管中的细胞完全融化后,用70%酒精消毒冻存管外壁,然后在超净工作台内将融化的细胞转移到含有2ml新鲜培养基的15ml离心管内;
4)800rpm,室温离心3min;
5)取出离心管后可见明显的细胞沉淀,弃掉上清液,加入2ml PBS完全重悬细胞后,800rpm,室温离心3min;
6)完全弃掉上清液后,用2ml新鲜培养基重悬细胞后,将细胞悬液转移到25cm2的细胞培养瓶中,然后再补充3ml新鲜培养基;
7)将培养瓶十字晃动使细胞均匀分布在培养瓶底部,然后放入37℃细胞培养箱中长期培养。
1.2细胞换液
HepG2.2.15和HepAD38两株细胞系生长较为缓慢,当培养基的颜色由红色变为淡黄色时,说明此时培养基里的营养成分耗尽,需要及时更换新鲜培养基。
细胞换液的步骤是:弃掉旧的培养基,然后向细胞培养瓶或培养皿中加入2~4ml PBS,轻轻晃动培养瓶或培养皿后,弃掉PBS后,加入足量的新鲜培养基放入细胞培养箱中继续培养。
1.3细胞传代
HepG2.2.15和HepAD38细胞的生长周期大概是3d。细胞生长3d后,如果在显微镜下观察细胞的生长密度大于90%,这时候就需要进行细胞传代。细胞传代的步骤如下:
1)准备工作:提前将超净工作台紫外照射30min;将待用的新鲜培养基、胰酶和PBS提前从4℃冰箱取出后放在室温;
2)从细胞培养箱中取出需要传代的细胞,弃掉旧的培养基,然后加入2~4ml的PBS缓慢摇动后弃掉,此步骤需重复一次;
3)在培养皿中加入1ml的胰酶,然后晃动培养皿使胰酶铺满整个皿底,弃 掉多余的胰酶后,将培养皿放到细胞培养箱中孵育1min来消化细胞;
4)取出培养皿,显微镜下观察细胞的消化程度,消化良好的细胞会观察到细胞间隙变大。随后在培养皿中加入2ml的新鲜培养基来终止胰酶消化;
5)用1ml的移液器轻轻吹打细胞,使其变成单细胞悬液,然后将细胞悬液移至15ml离心管中;
6)800rpm,室温离心3min;
7)取出离心管后可见明显的细胞沉淀,弃掉上清液,加入2ml PBS完全重悬细胞后,800rpm,室温离心3min;可选择性的重复此步骤一次;
8)完全弃掉上清液后,用2ml新鲜培养基重悬细胞后,将细胞悬液分到2~3个培养皿中继续培养。
1.4细胞冻存
如果需要冻存细胞,需要选择生长状态良好的细胞进行冻存。如果显微镜下观察细胞形态圆润,背景干净,细胞融合度大约80%左右,即可进行细胞冻存。细胞冻存的步骤如下:
1)准备工作:将冻存细胞的程序降温盒内加入适量的异丙醇备用;配制冻存液(90%FBS+10%DMSO),遵循现配现用的原则;
2)从细胞培养箱中取出准备冻存的细胞,弃掉旧的培养基,然后加入2~4ml的PBS缓慢摇动后弃掉,此步骤需重复一次;
3)在培养皿中加入1ml的胰酶,然后晃动培养皿使胰酶铺满整个皿底,弃掉多于的胰酶后,将培养皿放到细胞培养箱中孵育1min来消化细胞;
4)消化好的细胞离心后,弃上清,然后用PBS洗两次,800rpm,室温离心3min;
5)尽可能的弃掉上清液后,用配好的冻存液重悬细胞,充分混匀后将细胞悬液移至冻存管中,需在管壁上标记清楚细胞名称及日期;
6)将冻存管放到程序降温盒内,立即放到-80℃冰箱内,24h后将冻存的细胞放到液氮罐里长期储存备用。
1.5细胞转染
本实验应用的细胞转染试剂为转染试剂盒。接下来,我们将以 六孔板为例详细描述细胞转染的步骤。
1)在六孔板里接种细胞:将生长状态良好的细胞平均种到六孔板的每一个孔里,接种细胞量为每孔1.5~2.5x10^5个细胞,并加入2ml新鲜培养基放到细胞培养箱培养;
2)细胞生长24h后,显微镜下观察细胞生长呈倍数增长期,并且细胞密度为60%到80%之间转染效率最优。此时我们可以进行转染;
3)本研究中涉及到的细胞转染类型主要是转染外源性过表达质粒。配制方法如下(以六孔板每孔转染量为例):
2μg目的质粒+200μl jetPRIME Buffer+5μl jetPRIME Reagent;
4)按照以上的配制方法配制转染体系。在配制过程中需注意,首先将一定量的目的质粒或siRNA加入jetPRIME Buffer中,充分震荡混匀后,低速离心后再将适量的jetPRIME Reagent加入体系内,再进行震荡混匀离心后,室温孵育15min即可将转染体系滴加到六孔板的每个孔内。
5)在转染体系孵育时,可先将六孔板里的培养基换成新鲜培养基,每孔2ml;转染体系孵育结束后,将转染混合液缓慢滴加到六孔板里,每孔200μl;
6)加入转染混合液后,轻轻晃动六孔板,然后将六孔板放到细胞培养箱内继续培养;在特定的时间(检测RNA水平的指标,一般处理24h即可;检测蛋白水平的指标,一般处理48h即可)收集细胞后进行后续的实验。
2.Q-PCR检测HBV RNA的表达
2.1细胞总RNA提取
HepG2.2.15和HepAD38细胞分别加入受测化合物,48h后提取细胞中的总RNA,本实验严格按照Total RNA Kit试剂盒说明书进行,步骤如下(注意:
本实验全程冰上进行,全程使用无RNA酶的专用枪头):
1)从孵箱中取出12孔细胞培养板,弃去细胞培养液,1×PBS 1mL清洗细胞2次,负压吸引装置吸干净PBS;
2)根据需要配制350μL TRK Lysis Buffer混合液/孔(混合液配制方法:1mL TRKLysis Buffer+20μL β-巯基乙醇),涡旋混匀。12孔细胞培养板每孔分别加入350μL,室温静置5min,使细胞充分裂解;
3)根据需要配制70%乙醇/孔,涡旋混匀。12孔细胞培养板每孔分别加入350μL,摇晃混匀,转移至离心柱(离心柱+收集管组装好)内;
4)4℃离心机,12000g,60sec,离心后弃废液,保留离心柱;
5)加入500μL RNA Wash Buffer Ⅰ,4℃离心机,12000g,60sec,离心后弃废液,保留收集柱;
6)加入500μL RNA Wash Buffer Ⅱ(确认已加乙醇),4℃离心机,12000g,60sec,离心后弃废液,保留收集柱;
7)重复步骤6)一遍,4℃离心机,12000g,90sec,离心后弃废液,保留收集柱;
8)4℃离心机,12000g,120sec,以保证离心柱充分干燥;
9)干燥后的离心柱插入新的1.5mL EP管中,在离心柱中心的膜上加入30μL DEPC水。注意该步骤不要触碰到EP管盖子。室温静置5min,以确保RNA被DEPC水充分溶解。4℃离心机,12000g,60sec,保留EP管;
10)NanoDrop超微量分光光度计检测RNA浓度;
2.2RNA逆转录为cDNA:按照PrimeScriptTM RT reagent Kit(Perfect Real Time)
说明书,具体实验步骤如下:
2.2.1配制逆转录反应体系(冰上操作):
表2-1逆转录反应体系
2.2.2逆转录上机反应条件如下:
表2-2逆转录反应条件
2.2.3所得产物即为cDNA,可直接用于后续Q-PCR实验或者-20℃保存。
2.3Q-PCR检测HBV RNA的表达:按照PowerGreen PCR Master Mix说明书,具体操作如下:
2.3.1根据说明书配制Q-PCR反应体系如下:
表2-3 Q-PCR反应体系
2.3.2使用ABI7500 system采用下列两步法标准扩增程序:
表2-4 Q-PCR反应条件
反应结束后,确认Q-PCR的扩增曲线和溶解曲线,采用相对定量法(2-ΔΔC T法)对实验结果进行数据分析。
2.3.3所用引物序列如下:
表2-5 Q-PCR实验所用引物序列
3.HBV DNA的提取与检测
3.1细胞内基因组DNA的提取
1)从细胞培养箱中取出待提取DNA的六孔板,弃掉培养基,然后每孔加入适量的PBS晃动培养板来清洗细胞;此过程需重复一次。
2)收集细胞:每孔加入适量的胰酶,晃动培养板使胰酶铺满整个皿底,室温静置1~2min后,每孔加入1ml的培养基终止消化,然后将细胞悬液收集到1.5ml的EP管里。2000rpm离心5min,弃上清,用PBS洗两遍后尽可能的弃干净上清液;
3)在每个EP管内加入100μl的LB2溶液,缓慢吹打,充分混匀;
4)在每个样品中加入20μl RNase A,室温孵育2min,目的是去除样本中的RNA;
5)孵育结束后,每个样品中加入20μl Proteinase K,室温孵育2min,目的是去除样本中的蛋白质;
6)孵育结束后,每个样品中加入500μl BB2溶液,需要立即涡旋5s,然后室温孵育10min;
7)孵育结束后,将EP管中的全部液体转移到离心柱中,12000rpm离心30s,弃掉收集管中的液体;
8)在每个样品中加入500μl CB2溶液,12000rpm离心30s,弃掉收集管中 的液体;
9)重复步骤8)一次;
10)在每个样品中加入500μl WB2溶液(使用前需加入一定量的无水乙醇),12000rpm离心30s,弃掉收集管中的液体;
11)重复步骤10)一次;
12)将离心柱插到收集管中,12000rpm离心2min,彻底去除残留的WB2溶液;
13)将离心柱转移到一个新的1.5ml的EP管中,在离心柱的中央加入50μl预热的EB(65℃),室温静置2min,12000rpm离心2min,洗脱DNA。洗脱出的DNA于-20℃储存备用。
3.2 HBV Hirt DNA的提取
1)从细胞培养箱中取出待提取DNA的六孔板,弃掉培养基,然后每孔加入适量的PBS晃动培养板来清洗细胞;此过程需重复一次。
2)在每个孔中加入750μl的TE缓冲液(10mM Tris,10mM EDTA,pH 7.5),然后每孔再加入50μl的10%SDS溶液,随后,在室温条件下将培养板放到水平摇床上缓慢摇动30min,促使细胞裂解完全;
3)将每孔的所有液体转移到2ml的EP管中,然后每个样本中加入200μl的5M NaCl,缓慢上下颠倒几次后,将样本放到4℃翻转摇床上,此过程需要过夜处理;
4)从4℃翻转摇床上取下样本,进行4℃、12000rpm离心30min;
5)离心结束后,将上清液转移到一个新的2ml的EP管内;
6)苯酚(phenol)萃取:向每个样本中加入与样本等体积的苯酚,缓慢上下颠倒两次,4℃、12000rpm离心10min;
7)离心后取上清(遵循宁少勿多的原则),再加入等体积的苯酚进行二次萃取,4℃、12000rpm离心10min后取上清;
8)苯酚/氯仿/异丙醇(Phenol/chloroform/isoprophanol)萃取:向每个样本中加入等体积的苯酚/氯仿/异丙醇,缓慢上下颠倒两次,4℃、12000rpm离心10min;
9)将上清转移到新的2ml的EP管内,加入样本两倍体积的无水乙醇,此过程需要室温静置过夜处理;
10)样本4℃、12000rpm离心30min;
11)弃上清,每个样本中加入800μl的70%乙醇,4℃、12000rpm离心10min;
12)弃上清,重复步骤11)一次;
13)用负压吸引器尽可能的吸干上清,将EP管开盖放到通风橱内静置干燥10min;
14)每个样本中加入20μl的无核酸酶水溶解Hirt DNA,-20℃储存备用。此过程提取的总的HBV Hirt DNA,是DP-rcDNA和cccDNA的混合物。
3.3 HBV cccDNA的分离纯化
从HBV稳定细胞系中提取Hirt DNA,方法如上所述。接下来我们将分离纯化cccDNA:
1)取19μl Hirt DNA到200μl的小EP管中,然后加入23μl无核酸酶水稀释,总体积为42μl;
2)将金属浴调至85℃,样本放至金属浴中加热5min。目的是将DP-rcDNA变性为单链线性DNA,而cccDNA(共价闭合环状双链DNA)不受影响;
3)样本中加入PSAD(Plasmid-safe ATP-dependent DNase)酶消化单链线性DNA,具体的反应体系如下表3-1:反应条件是37℃,16h。
表3-1 PSAD酶消化单链线性DNA反应体系
4)PSAD酶失活:反应条件是70℃,30min。
5)最终得到的样本需要用DNA clean&concentrator kit试剂盒进行cccDNA 的纯化,以下为cccDNA纯化的实验步骤:
a)在上述步骤中获得的50μl样本中加入7倍体积的DNA Binding Buffer 350μl,涡旋震荡混匀;
b)将混匀后的所有液体转移到Zymo-SpinTM柱子中,16000g离心1min;
c)弃废液,在柱子里加入200μl DNA Washing Buffer,16000g离心1min;
d)弃废液,重复步骤c)一次;
e)将Zymo-SpinTM柱子转移到一个新的1.5ml的EP管中,并在柱子中央加入20μl DNA Elution Buffer,室温静置2min,16000g离心1min,洗脱纯化的cccDNA。洗脱出的cccDNA于-20℃储存备用。
3.4 HBV DNA及cccDNA的检测
本研究中HBV稳定细胞系中的HBV DNA和cccDNA的相对定量,都是运用实时荧光定量PCR技术进行检测。
1)HBV total DNA qPCR:按照FastStart Essential DNA Probes Master说明书,具体操作如下:
a)配制HBV total DNA qPCR的反应体系如下表3-2:
total DNA上游引物:5’-CCGTCTGTGCCTTGTCATCTG-3’
total DNA下游引物:5’-AGTCCAAGAGTYCTCTTATGYAAGACCTT-3’
Probes:5’-FAM-CCGTGTGCACATGGCTTCACCTCTGC-TAMRA-3’
表3-2 HBV total DNA qPCR的反应体系
b)使用罗氏LightCycler480 II实时荧光定量PCR仪器检测,反应条件如下表3-3:
表3-3 HBV total DNA qPCR的反应条件
c)反应结束后,使用相对定量的方法(2-ΔΔCT法)分析处理数据。
2)HBV cccDNA qPCR:按照FastStart Essential DNA Probes Master说明书,具体操作如下:FastStart Universal SYBR Green Master(ROX)
a)配制HBV cccDNA DNA qPCR的反应体系如下表3-4:
cccDNA上游引物:5’-TCATCTGCCGGACCGTGTAC-3’
cccDNA下游引物:5’-TCCGGATACAGAGCTGAGGCG-3’
Probes:5’-FAM-TTCAAGCCTCCAAGCTGTGCCTTGCCTGGC-TAMRA-3’
表3-4 HBV cccDNA qPCR的反应体系
b)使用罗氏LightCycler480 II实时荧光定量PCR仪器检测,反应条件如下表3-5;
表3-5 HBV cccDNA qPCR的反应条件
4.蛋白免疫印迹检测蛋白水平表达
4.1细胞总蛋白提取:取出细胞,弃去原培养基,加入1~2mL1×PBS,清洗两次,胰酶消化,弃去上清,轻轻弹起管底细胞,再次加入1mL PBS洗涤细胞两次,1200rpm,离心5min,完全弃去上清,弹匀管底细胞,根据细胞量,加入50~100μL含有蛋白酶抑制剂和磷酸酶抑制剂的蛋白裂解液,充分吹打混匀细胞,置于冰上裂解30min后,4℃,12,000rcf,离心15min,吸取上清放入新的EP管中,所得即为细胞总蛋白。可直接进行后续蛋白浓度的测定或-80℃保存。
4.2 BCA法测蛋白浓度:将BCA试剂A液与B液按照50:1的比例混匀,制备工作液;取5μL待测样本,加入95μL去离子水混匀,稀释20倍,取20μL稀释后的待测样本加入到96孔板中;加入200μL工作液后,将96孔板置于37℃孵箱,孵育30min,上机检测。
4.3蛋白变性:分别取100μg样品,用蛋白裂解液将各样本体积补齐,加入1/3总体积的4×loading buffer,充分混匀,99℃变性10min,离心后-80℃保存。
4.4蛋白电泳:将预制胶安装入电泳槽中,加入1×MOPS电泳液,拔掉预制胶顶层梳子,轻轻吹打上样孔,去除孔内残余凝胶;将预染蛋白Maker及等质量的蛋白样品小心加入凝胶孔中;将电压调至60V~80V,30min后,调整电压为110V~120V,当溴酚蓝跑至凝胶底部凹槽处,断开电源,停止电泳,整个电泳过程约2h~2.5h。
4.5电转印:将PVDF膜浸入无水甲醇中激活1min;将转膜夹黑色面朝下,由下向上依次放置海面-滤纸-PAGE胶-PVDF膜-滤纸-海绵,小心赶出胶/膜和滤纸之间的气泡,扣紧转膜夹,放入转膜槽中,PAGE胶朝向负极,PVDF 膜朝向正极;放入冰盒,倒入预冷的1×电转液;将电源调至100V,转膜1~1.5h。
4.6封闭:转膜结束后,取出PVDF膜,放入1×TBST清洗数次,去除膜上残留的电转液后,浸入5%脱脂牛奶中封闭,室温2h,或4℃过夜。
4.7孵育一抗:按照1/1000稀释一抗GAPDH,HCBP6;按照蛋白分子量的大小,将相应位置的PVDF膜与上述抗体封闭于杂交袋中,4℃孵育过夜。
4.8洗膜:剪开杂交袋,回收一抗,取出PVDF膜,放入1×TBST中,置于摇床上洗膜三次,每次10min。
4.9孵育二抗:按照1/5,000~1/10,000的比例稀释二抗,将上述PVDF膜浸入相应二抗中,置于摇床上,室温孵育45-60min。
4.10洗膜:回收二抗,取出PVDF膜,放入1×TBST中,置于摇床上洗膜三次,每次10min;检测磷酸化蛋白时,需适当减少洗膜次数和洗膜时间。
4.11曝光显色:将膜置于置于Fusion Solo成像仪暗箱中,滴加ECL曝光液,进行显影。
5.细胞培养上清中HBsAg和HBeAg的检测
本研究中HBsAg和HBeAg的检测方法是用HBV表面抗原诊断试剂盒和HBV e抗原检测试剂盒(ELISA法)检测。
1)收集细胞(HepG2.2.15和HepAD38细胞系)培养上清500μl,3000rcf离心10min去除细胞残渣和碎片;
2)将试剂盒从4℃冰箱取出,需在室温静置30min后再使用;
3)配制洗液,将20×浓缩洗液用ddH 2O稀释成1×洗液备用;
4)需设置1个空白对照孔(不加样本和酶结合物)、2个阴性对照孔(加入阴性对照50μl)、2个阳性对照孔(加入阳性对照50μl)和若干个样本检测孔(每孔各加50μl待测样本);
5)每孔加入50μl酶结合物,然后用封板膜封住反应板,轻微振荡混匀,放置在37℃的恒温箱中孵育60min;
6)洗板:孵育结束后,弃掉反应液,每孔加入300μl稀释后的1×洗液,室温静置30s,弃洗液;
7)重复步骤6)五次;洗板结束后,尽可能的将板子内的洗液拍干;
8)每孔加入50μl底物A液和B液,用封板膜封住反应板,轻微振荡混匀,37℃孵育15min,孵育过程需要避光;
9)每孔加入50μl终止液,轻微振荡混匀终止反应;
10)上机检测:用Varioskan Flash酶标仪检测并保存处理数据。
6.HBV复制中间体提取
1)取出待检测细胞,去培养基,PBS洗两次,胰酶消化计数;
2)取相同细胞数细胞,2000rpm×3min收集细胞,PBS洗两次;
3)加入500μL复制中间体裂解液,吹打混匀,37℃孵育15min;
4)16000g×5min,转移上清至新EP管,取10μL用于Western blot检测内参β-actin;
5)在剩余样品中加入4μL 1M MgCl,5μL 5U/μL DNaseⅠ,37℃孵育4h;
6)9000g×5min,取上清,加入200μL35%PEG8000,涡旋震荡混匀,冰浴1h;
7)12000g×5min,去上清,加入477.5μL蛋白酶K消化液,12.5μL蛋白酶K(20mg/mL),充分混匀后,45℃水浴过夜;
8)取出过夜样品,瞬离后酚氯仿抽提3次,异丙醇沉淀,70%乙醇清洗;
9)充分干燥后,10μL灭菌水溶解,-20℃冰箱保存。
7.Southern blot
1)配制30mL 0.9%琼脂糖凝胶:
a)取干净的200mL锥形瓶,称取琼脂糖0.27g,加入30mL 1×TAE;
b)微波炉加入至充分溶解,根据蒸发情况用1×TAE补齐至30mL;
c)混匀后倒入制胶板,插入梳子,室温凝固30min。
2)样品准备:取出待检测样品,充分混匀,加入2μL 6×loading buffer,混匀;
3)上样、电泳:将样品加入上样孔,接通电源,90V电泳分离约2h;
4)转膜准备:两层大滤纸、胶、略小于胶的NC膜、略小于膜的两层小滤 纸;
5)取出Southern blot胶,置于30mL碱变性液中,室温摇床30min;重复一次;
6)将NC膜分别在RNase-Free ddH2O和20×SSC中浸泡10min;
7)转膜:按从下到上的顺序依次为转膜盘、两层大滤纸、胶、膜、两层小滤纸、吸水纸和1kg重物,在转膜盘中加入适量20×SSC,室温放置16-20h;
8)预杂交:取出含有目的核酸的膜,紫外交联固定后置于10×SSC浸泡1min,然后放入20mL预热的预杂交液中,置于杂交炉中68℃,1h;
9)杂交:
10)取出含HBV DNA探针的杂交液室温溶解;
11)68℃水浴10min,立即置于冰上冷却10min;
12)去除预杂交液,加入杂交液,置于杂交炉中68℃过夜;
13)洗膜:分别用2×SSC/0.1%SDS、1×SSC/0.1%SDS各洗膜两次,68℃,20min/次;Washing Buffer,37℃,5min;
14)封闭:加入20mLBlocking Buffer,37℃,30min;
15)孵育地高辛标记的二抗:取出地高辛标记的二抗,10000g,5min,取上层,按1:10000比例加入新的Blocking Buffer中,37℃,30min;
16)置于20mL Detection Buffer,37℃,5min;
17)取出膜置于暗夹中,滴加CSPD;X-ray胶片收集信号,拍照分析结果。
8.体外核衣壳打开实验
也称体外内源性DNA聚合酶链延伸反应,以10cm细胞培养皿培养的细胞为例。
1)弃去细胞培养基,加入2mL coreDNA裂解缓冲液冰上裂解细胞15-20min。
2)收获细胞裂解液,置于预冷至4℃的离心机16000g离心10min。
3)将上清取出,在5mL超速离心管内加入3mL20%蔗糖溶液,并小心加入收集的2mL裂解液。超速离心机46000rpm(Beckman,SW55)4℃ 离心3.5h。
4)取出,弃上清。将沉淀重悬于200μL TNE缓冲液。并每40μL等分至1.5mL离心管放于4℃冰箱。
5)将上述40μL纯化的核衣壳裂解液与50μL的2×Reaction buffer混合。然后将所要研究的化合物以及对照(DMSO等)加入反应中并补充Nuclease-free water使总体积为100μL。
6)37℃孵箱孵育16h后,DNase I预先在37℃消化或不消化30min。
7)提取病毒DNA。
8)Southern blot法检测病毒DNA来间接判定病毒核衣壳是否完整。
9.核衣壳电泳实验
以10cm细胞培养皿培养的细胞为例。具体步骤如下:
1)弃去细胞培养基,加入2mL coreDNA裂解缓冲液并上裂解细胞15-20min。
2)收获细胞裂解液,置于预冷至4℃的离心机16000g离心10min。
3)将上清取出,在5mL超速离心管内加入3mL 20%蔗糖溶液,并小心加入收集的2mL裂解液。超速离心机46000rpm(Beckman,SW55)4℃离心3.5h。
4)取出,弃上清。将沉淀重悬于200μL TNE缓冲液。并每40μL等分至1.5mL离心管放于4℃冰箱。
5)将上述40μL纯化的核衣壳裂解液与50μL的2×Reaction buffer混合。然后将所要研究的化合物以及对照(DMSO等)加入反应中并补充Nuclease-free water使总体积为100μL。
6)37℃孵箱孵育6h后,取20μL反应产物加入Loading buffer进行浓度为1.8%的琼脂糖凝胶电泳。电泳条件72V,室温8h。
7)电泳结束后使用TNE buffer转膜,转膜条件及程序与Southern blot法一致。
8)转膜结束,取出膜,用4%多聚甲醛(PBS配制)室温固定15min,用 流水润洗。
9)用预冷的50%甲醇(PBS配制)固定30min,用流水润洗。
10)用封闭液(含5%(w/v)脱脂奶粉,1×TBS,0.1% Tween-20)室温封闭1~2h。
11)敷anti-HBc(CST,货号sc-52406)抗体4℃过夜。
12)用Wash buffer(含1×TBS,0.1%Tween-20)室温清洗3×5min,敷山羊抗鼠二抗室温1h。再用Wash buffer(含1×TBS,0.1% Tween-20)室温清洗3×5min。
13)使用Bio-rad扫膜仪或Li-COR扫膜仪曝光核衣壳信号。
二、实验步骤和实验结果
(a)体外药效评价-TRPV4对HBV RNA、HBV DNA、cccDNA及HBV核心蛋白(HBcAg)的作用
HepG2.2.15和HepAD38细胞正常传代分板,贴壁生长12小时后,瞬时转染TRPV4外源性过表达质粒或siRNA,48小时后收集细胞,分别提取总RNA,Hirt法提取DNA,分离纯化cccDNA,以及细胞内总蛋白,应用Q-PCR方法检测细胞内的HBV RNA(total RNA、pgRNA、precore RNA)、HBV DNA和cccDNA;Western blotting检测细胞内的HBV核心蛋白(HBcAg)的表达量。结果参见图1-1~图1-3。
结果显示在HepG2.2.15和HepAD38细胞中,沉默TRPV4表达后,HBV DNA和HBcAg表达下降,而过表达TRPV4后,HBV DNA和HBcAg表达明显升高(图1-1);在HepAD38细胞中沉默TRPV4,诱导12天后cccDNA下调;过表达TRPV4,cccDNA上调(图1-2);HepG2.2.15细胞中沉默TRPV4,pgRNA及totalRNA均下调;过表达TRPV4,pgRNA及totalRNA均上调;在HepGAD38细胞中沉默TRPV4,pg RNA及total RNA、precore下调;过表达TRPV4,pg RNA及total RNA、precore上调(图1-3)。以上结果表明TRPV4可以促进HBV的复制和转录,而沉默TRPV4以后,可以抑制HBV的复制和转录。
(b)体外药效评价-HBV核心蛋白(HBcAg)的表达
HepG2.2.15和HepAD38细胞正常传代分板,贴壁生长12小时后,加入不同浓度(10nM、20nM、50nM、100nM、500nM)的待测化合物,48小时后收集细胞,提取蛋白,应用Western blotting方法检测细胞内的HBV核心蛋白(HBcAg)的表达量。结果参见下表或图2-1~图2-7。
表4:Western blotting灰度扫描结果
注:“-”表示未测试。
结果显示,本发明的大部分化合物可不同程度抑制HepG2.2.15和HepAD38细胞中HBV核心蛋白(HBcAg)的表达(图2-1~图2-7、表4)。

Claims (9)

  1. 以下通式O所示化合物:
    或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,
    其中:
    X选自N、CH;
    R1选自8-10元稠杂芳基,且所述稠杂芳基中,每个单环含有至少一个杂原子;
    优选地,R1选自稠杂芳基,所述稠杂芳基中,每个单环含有至少一个杂原子,且所述稠杂芳基由六元环和五元环稠合形成;
    更优选地,R1选自稠杂芳基,所述稠杂芳基中,每个单环含有至少一个杂原子,所述稠杂芳基由六元环和五元环稠合形成,且所述稠杂芳基共含有三个杂原子,所述杂原子优选为N原子;
    进一步优选地,R1选自:
    最优选地,R1选自
    R2选自C1-C6烷基、被1-3个羟基取代的C1-C6烷基、
    优选地,R2选自被1-3个羟基取代的C1-C6烷基、
    更优选地,R2选自被1个羟基取代的C1-C6烷基、
    最优选地,R2选自
    Ra、Rb各自独立地选自C1-C6烷基;
    优选地,Ra、Rb均为甲基;
    Z选自O、S;
    R2在2号、3号或5号碳原子处进行取代;
    优选地,R2在3号碳原子处进行取代;
    R3选自C1-C6烷基;
    优选地,R3为甲基。
  2. 根据权利要求1所述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,其中,所述化合物具有式I-1或式I-2所示的结构,
    其中:
    X选自N、CH;
    Y1、Y2、Y3、Y4、Y5、Y6、Y7各自独立地选自CH、N,且Y3、Y4、Y5、Y6不同时为CH;
    优选地,Y1、Y2、Y3、Y4、Y5、Y6、Y7各自独立地选自CH、N,Y3、Y4、Y5、Y6不同时为CH,且满足以下条件:
    (a)式I-1中,Y1、Y2、Y3、Y4、Y5和Y6中,任意两个为N,其余为CH,
    (b)式I-2中,Y2、Y3、Y4、Y5、Y6和Y7中,任意两个为N,其余为CH;
    更优选地,作为整体,选自以下:
    或者更优选地,作为整体,选自
    最优选地,作为整体,选自
    或者最优选地,作为整体,选自
    R2选自C1-C6烷基、被1-3个羟基取代的C1-C6烷基、
    优选地,R2选自被1-3个羟基取代的C1-C6烷基、
    更优选地,R2选自被1个羟基取代的C1-C6烷基、
    最优选地,R2选自
    Ra、Rb各自独立地选自C1-C6烷基;
    优选地,Ra、Rb均为甲基;
    Z选自O、S。
  3. 根据权利要求1-2任一项所述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,其中,所述化合物选自以下:

  4. 制备权利要求1-3任一项所述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物的方法,其包括:
    使发生亲核取代反应,获得式O所示化合物
    或者,
    使发生亲核取代反应,获得式I-1所示化合物
    或者,
    使发生亲核取代反应,获得式I-2所示化合物
    其中,R1、R3的定义如权利要求1所限定,Y1、Y2、Y3、Y4、Y5、Y6、Y7的定义如权利要求2所限定,X、R2的定义如权利要求1-2任一项所限定,M选自卤素,M优选选自Cl、Br;
    优选地,所述亲核取代反应是在碱性条件下进行的;
    更优选地,所述亲核取代反应是在DMEDA和碳酸钾存在的条件下进行的;
    优选地,所述亲核取代反应是在催化剂存在的条件下进行的;
    更优选地,所述亲核取代反应是在CuI存在的条件下进行的。
  5. 药物组合物,其包含权利要求1-3任一项所述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物;任选地,还包含药学上可接受的辅料。
  6. 根据要求5所述的药物组合物,其中,所述药物组合物还包含其他药物;
    优选地,所述其他药物用于治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态;
    优选地,所述由HBV引发的疾病或感染为肝脏疾病;
    优选地,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、肝硬化、肝癌(如原发性肝细胞癌),或其任意组合;
    优选地,所述其他药物包括但不限于核苷酸类药物(如恩替卡韦、替比夫定、替诺福韦酯、阿德福韦酯或拉米夫定)、干扰素(如干扰素α2a、干扰素α1b或干扰素α2b)、治疗性疫苗、Toll样受体激动剂、入胞抑制剂、RNA干扰药物、cccDNA靶向药物,或其任意组合。
  7. 权利要求1-3任一项所述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,或者权利要求5-6任一项所述的药物组合物在制备用于治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态的药物中的用途;
    优选地,所述由HBV引发的疾病或感染为肝脏疾病;
    优选地,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、肝硬化、肝癌(如原发性肝细胞癌),或其任意组合。
  8. 权利要求1-3任一项所述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,或者权利要求5-6任一项所述的药物组合物,其用于治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态;
    优选地,所述由HBV引发的疾病或感染为肝脏疾病;
    优选地,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、肝硬化、肝癌(如原发性肝细胞癌),或其任意组合。
  9. 一种治疗和/或预防由HBV引发的疾病或感染或者用于维持肝脏稳态的方法,其包括:
    给予有需要的受试者有效量的权利要求1-3任一项所述的化合物,或所述化合物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物,或者权利要求5-6任一项所述的药物组合物;
    优选地,所述由HBV引发的疾病或感染为肝脏疾病;
    优选地,所述肝脏疾病选自乙型肝炎(如慢性乙型肝炎)、肝纤维化、肝衰竭、 肝硬化、肝癌(如原发性肝细胞癌),或其任意组合。
PCT/CN2024/088156 2023-04-18 2024-04-17 化合物及其在慢性乙型肝炎、肝纤维化、肝癌的治疗中的应用 WO2024217429A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012174342A1 (en) * 2011-06-17 2012-12-20 Glaxosmithkline Llc Trpv4 antagonists
CN103732583A (zh) * 2011-06-17 2014-04-16 葛兰素史密斯克莱知识产权(第2号)有限公司 Trpv4拮抗剂
WO2018185701A1 (en) * 2017-04-06 2018-10-11 Glaxosmithkline Intellectual Property (No.2) Limited Trpv4 antagonists as antitussive agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012174342A1 (en) * 2011-06-17 2012-12-20 Glaxosmithkline Llc Trpv4 antagonists
CN103732583A (zh) * 2011-06-17 2014-04-16 葛兰素史密斯克莱知识产权(第2号)有限公司 Trpv4拮抗剂
WO2018185701A1 (en) * 2017-04-06 2018-10-11 Glaxosmithkline Intellectual Property (No.2) Limited Trpv4 antagonists as antitussive agents

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