WO2023061432A1 - 嘧啶三并环衍生物及其在药学上的应用 - Google Patents

嘧啶三并环衍生物及其在药学上的应用 Download PDF

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WO2023061432A1
WO2023061432A1 PCT/CN2022/125047 CN2022125047W WO2023061432A1 WO 2023061432 A1 WO2023061432 A1 WO 2023061432A1 CN 2022125047 W CN2022125047 W CN 2022125047W WO 2023061432 A1 WO2023061432 A1 WO 2023061432A1
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compound
alkyl
optionally
pharmaceutically acceptable
compounds
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PCT/CN2022/125047
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English (en)
French (fr)
Inventor
罗云富
张国利
李韶龙
戈伟智
陈曙辉
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正大天晴药业集团股份有限公司
南京明德新药研发有限公司
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Priority to MX2024004273A priority Critical patent/MX2024004273A/es
Priority to CN202280067441.6A priority patent/CN118234734A/zh
Priority to AU2022365972A priority patent/AU2022365972A1/en
Priority to CA3233865A priority patent/CA3233865A1/en
Priority to EP22880368.0A priority patent/EP4417613A1/en
Priority to JP2024521310A priority patent/JP2024537262A/ja
Priority to KR1020247013652A priority patent/KR20240074807A/ko
Publication of WO2023061432A1 publication Critical patent/WO2023061432A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/16—Peri-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/16—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present application relates to a pyrimidine trihecyclic derivative and its application in pharmacy, in particular to the compound represented by formula (I), its stereoisomer and its pharmaceutically acceptable salt.
  • Soluble guanylate cyclase widely exists in the cytosol of mammals and is a heterodimer composed of ⁇ and ⁇ subunits. Soluble guanylate cyclase is a key signal transduction enzyme in the NO-sGC-cGMP signaling pathway. After sGC is activated in vivo, it will catalyze the conversion of guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP).
  • GTP guanosine triphosphate
  • cGMP cyclic guanosine monophosphate
  • cGMP is an important secondary messenger molecule, which activates a variety of downstream effector molecules, such as cGMP-dependent protein kinase G and cGMP-gated ion channels, etc., and then triggers a series of downstream cascade reactions. It plays important physiological functions in the gastrointestinal system, cardiovascular system and central nervous system, such as promoting the relaxation of blood vessels and smooth muscles, inhibiting platelet aggregation, vascular remodeling, apoptosis and inflammation, and participating in neurotransmission, etc.
  • the NO/cGMP system can be inhibited, which can lead to, for example, hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, arteriosclerosis, angina pectoris, heart failure, myocardial infarction, thrombosis, stroke and sexual dysfunction, etc.
  • sGC-mediated signaling pathways is also closely related to the occurrence of fibrotic diseases such as chronic kidney disease and systemic sclerosis.
  • sGC stimulators have a dual mechanism of action: they can not depend on NO, but need to rely on the heme prosthetic group containing Fe 2+ to directly activate the sGC-cGMP signaling pathway; they can also enhance the sensitivity of sGC to endogenous NO and thus interact with NO produce synergy. Therefore, the sGC stimulator is a heme-dependent, NO-independent sGC stimulator. Stimulating sGC to generate more cGMP can regulate a variety of important physiological processes: promoting vascular smooth muscle relaxation, inhibiting platelet aggregation, etc. At the same time, activating sGC can also regulate other signaling pathways, such as TGF- ⁇ , to exert anti-fibrosis and anti-tumor effects. Therefore sGC stimulators can be used as potential therapeutic means for the treatment of cardiovascular diseases (heart failure, pulmonary hypertension, angina pectoris, myocardial infarction) and fibrotic diseases (renal fibrosis, systemic sclerosis).
  • Such compounds can be used as stimulators of soluble guanylate cyclase, have excellent in vitro stimulating activity on soluble guanylate cyclase, and have good pharmacokinetic properties.
  • R 1 is selected from H, -OH, C 1-3 alkyl, C 1-3 alkoxy or C 1-3 alkylamino;
  • R 2 is selected from benzyl or C 1-8 alkyl, said benzyl or C 1-8 alkyl is optionally substituted by 1, 2, 3, 4 or 5 halogen atoms;
  • R 3 and R 4 are each independently selected from H or C 1-3 alkyl
  • R 3 and R 4 and their joint connected atoms form a C 3-6 cycloalkyl group
  • R 1 is selected from H, -OH, C 1-3 alkyl or C 1-3 alkoxy, and other variables are as defined in the present application.
  • R is selected from H, -OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy or isopropoxy, Other variables are as defined herein.
  • R 1 is selected from H, -OH, methyl or methoxy, and other variables are as defined in the present application.
  • R 2 is selected from benzyl or C 1-6 alkyl, and the benzyl or C 1-6 alkyl is optionally substituted by 1, 2, 3, 4 or 5 halogen atoms , and other variables are as defined in this application.
  • R is selected from benzyl or C 1-4 alkyl, and the benzyl or C 1-4 alkyl is optionally substituted by 1, 2, 3, 4 or 5 halogen atoms , and other variables are as defined in this application.
  • R 2 is selected from benzyl or C 1-4 alkyl, and the benzyl or C 1-4 alkyl is optionally replaced by 1, 2, 3, 4 or 5 F, Cl Or Br atom substitution, other variables are as defined in the present application.
  • R 2 is selected from benzyl or C 1-4 alkyl, and the benzyl or C 1-4 alkyl is optionally substituted by 1, 2, 3, 4 or 5 F atoms , and other variables are as defined in this application.
  • R 2 is selected from C 1-4 alkyl, and the C 1-4 alkyl is optionally substituted by 1, 2, 3, 4 or 5 halogen atoms, and other variables are as in the present application defined.
  • R 2 is selected from C 1-4 alkyl, and the C 1-4 alkyl is optionally substituted by 1, 2, 3, 4 or 5 F, Cl or Br atoms, other Variables are as defined herein.
  • R 2 is selected from C 1-4 alkyl, and the C 1-4 alkyl is optionally substituted by 1, 2, 3, 4 or 5 F atoms, and other variables are as in the present application defined.
  • R 2 is selected from benzyl or n-butyl, the benzyl is substituted by 1 fluorine, the n-butyl is substituted by 5 fluorines, and other variables are as defined in the present application.
  • R 3 and R 4 are each independently selected from H, methyl, ethyl, n-propyl or isopropyl, or R 3 and R 4 and their joint connected atoms form cyclopropane basis, other variables are as defined in this application.
  • R 3 and R 4 are each independently selected from H or methyl, or R 3 and R 4 and their joint connected atoms form a cyclopropanyl group, and other variables are as defined in the present application.
  • the present application also provides the following compounds, their stereoisomers or pharmaceutically acceptable salts thereof, which are selected from:
  • the present application also provides the following compounds, their stereoisomers or pharmaceutically acceptable salts thereof, which are selected from:
  • the present application provides a pharmaceutical composition comprising the compound of the present application, its stereoisomer or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the present application further includes pharmaceutically acceptable excipients.
  • the present application provides the application of the above compound, its stereoisomer or pharmaceutically acceptable salt thereof, or its pharmaceutical composition in the preparation of medicines for treating diseases related to sGC agonists or stimulators.
  • the present application provides a method for treating diseases associated with sGC agonists or stimulators in mammals, comprising administering a therapeutically effective amount of the compound of the present application, its stereoisomer or its A pharmaceutically acceptable salt, or a pharmaceutical composition thereof.
  • the present application provides the application of the compound of the present application, its stereoisomer or pharmaceutically acceptable salt, or its pharmaceutical composition in the treatment of diseases related to sGC agonists or stimulators.
  • the present application provides the compound of the present application, its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating diseases related to sGC agonists or stimulators.
  • the disease associated with the sGC agonist or stimulator is selected from heart failure, or hypertension.
  • the compound of the present application can effectively stimulate sGC, significantly increase the level of cGMP, have good apparent distribution volume and half-life, have good heart distribution and have no risk of entering the brain.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to the salts of the compounds of the present application, which are prepared from the compounds with specific substituents found in the present application and relatively non-toxic acids or bases.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent.
  • Certain specific compounds of the present application contain basic and acidic functional groups and thus can be converted into either base or acid addition salts.
  • the pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods. In general, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present application may exist in particular geometric or stereoisomeric forms.
  • This application contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the application.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this application.
  • enantiomer or “optical isomer” refer to stereoisomers that are mirror images of each other.
  • cis-trans isomers or “geometric isomers” arise from the inability to rotate freely due to the double bond or the single bond of the carbon atoms forming the ring.
  • diastereoisomer refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of the molecules.
  • keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key or straight dotted key
  • the terms “enriched in an isomer”, “enriched in an isomer”, “enriched in an enantiomer” or “enantiomerically enriched” refer to one of the isomers or enantiomers
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • the terms “isomer excess” or “enantiomeric excess” refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
  • Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present application is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
  • the compounds of the present application may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
  • compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • heavy hydrogen can be used to replace hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in isotopic composition of the compounds of the present application, whether radioactive or not, are included within the scope of the present application.
  • substituted means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable.
  • Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically realizable basis.
  • any variable eg, R
  • its definition is independent at each occurrence.
  • said group may optionally be substituted with up to two R, with independent options for each occurrence of R.
  • substituents and/or variations thereof are permissible only if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • a substituent can be bonded to any atom on a ring when the bond of a substituent can cross-link two or more atoms on the ring, e.g., structural unit It means that the substituent R can be substituted at any position on cyclohexyl or cyclohexadiene. When the enumerated substituent does not indicate which atom it is connected to the substituted group, this substituent can be bonded through any atom, for example, pyridyl as a substituent can be connected to any atom on the pyridine ring. The carbon atom is attached to the group being substituted.
  • linking group listed does not indicate its linking direction
  • its linking direction is arbitrary, for example,
  • the connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right
  • any one or more sites of the group can be linked to other groups through chemical bonds.
  • connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group.
  • the chemical bonds that the site connects with other groups can use straight solid line bonds Straight dotted key or tilde express.
  • the straight-shaped solid-line bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group;
  • the straight dotted line bond indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy lines in indicate that the 1 and 2 carbon atoms in the phenyl group are connected to other groups;
  • alkyl is used to represent a straight-chain or branched saturated hydrocarbon group.
  • the alkyl group is a C 1-8 alkyl group; in other embodiments , the alkyl is C 1-4 alkyl; in other embodiments, the alkyl is C 1-3 alkyl. It can be monosubstituted (such as -CH 2 F) or polysubstituted (such as -CF 3 ), and can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl base and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.
  • C 1-4 alkyl is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 4 carbon atoms.
  • the C 1-4 alkyl group includes C 1-2 , C 1-3 and C 2-3 alkyl groups, etc.; it may be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • Examples of C 1-4 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl , s-butyl and t-butyl) and so on.
  • C 1-3 alkyl is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n - propyl and isopropyl), and the like.
  • C 1-3 alkylamino means -NH-C 1-3 alkyl.
  • C 3-6 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 6 carbon atoms, which is a monocyclic and bicyclic system, and the C 3-6 cycloalkyl includes C 3-5 , C 4-5 and C 5-6 cycloalkyl, etc.; it may be monovalent, divalent or multivalent.
  • Examples of C 3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • halogen or halogen by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.
  • the compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art In an equivalent alternative, preferred implementations include but are not limited to the examples of the present application.
  • the structure of the compounds of the present application can be confirmed by conventional methods known to those skilled in the art. If the application involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuK ⁇ radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
  • SXRD single crystal X-ray diffraction
  • ACN stands for acetonitrile
  • EtOAc stands for ethyl acetate
  • EtOH stands for ethanol
  • MeOH stands for methanol
  • HPLC high performance liquid chromatography
  • LCMS liquid chromatography-mass spectrometry
  • Hour mL stands for milliliter
  • mM stands for millimole per liter
  • mmol stands for millimole
  • ⁇ mol stands for micromole
  • HNMR hydrogen nuclear magnetic resonance spectrum
  • MS stands for mass spectrum
  • min stands for minute ; stands for trimethylaluminum
  • TFA stands for trifluoroacetic acid
  • DMSO stands for dimethylsulfoxide.
  • treating means administering a compound or formulation described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:
  • prevention means administering a compound or formulation described herein to prevent a disease or one or more symptoms associated with the disease, and includes: preventing a disease or disease state from occurring in a mammal, especially when Such mammals are susceptible to the disease state, but have not been diagnosed as having the disease state.
  • terapéuticaally effective amount means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying The amount of a compound of the application for the onset of one or more symptoms of a particular disease, condition or disorder described herein.
  • the amount of a compound of the present application that constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by a person skilled in the art according to its own knowledge and this disclosure.
  • Step 1 Synthesis of compound 001_2
  • Step 2 Synthesis of Compound 001_3
  • compound 001_2 (30g, 73.34mmol) was added to methanol (100mL) and N,N-dimethylformamide (300mL), and cyclopenta-2,4-dien-1-yl ( Diphenyl)phosphine dichloromethane dichloropalladium ferrocene (3.76g, 5.13mmol) and triethylamine (29.68g, 293.35mmol, 40.83mL).
  • Step 3 Synthesis of the hydrochloride salt of compound 001_4
  • ammonium chloride 28.22g, 527.54mmol was dispersed in toluene (360mL), a toluene solution of trimethylaluminum (2M, 253.22mL) was added, and compound 001_3 (36g , 105.51mmol), stirred at 80°C for 30 minutes, then raised the temperature to 110°C for 1.5 hours, cooled to 25°C and added methanol (48.68g, 1.52mol, 61.48mL) dropwise, keeping the temperature not higher than 40°C, and then added Hydrochloric acid (3M, 675.25 mL), keep the temperature not higher than 40°C, raise the temperature to 80°C and stir for 10 minutes, then cool down to 0°C and stir for 30 minutes.
  • methanol 48.68g, 1.52mol, 61.48mL
  • Hydrochloric acid 3M, 675.25 mL
  • compound 001_5 (5 g, 56.71 mmol) was dissolved in dry toluene (20 mL), and bromoacetonitrile (7.48 g, 62.38 mmol) was added. The reaction system was stirred at 25°C for 12 hours. The reaction system gradually changed from clear to white solid. After the reaction was complete, the suspension was filtered, the filter cake was washed with toluene (50 mL), and the solid was collected and dried under reduced pressure. Compound 001_6 was obtained.
  • Step 5 Synthesis of Compound 001_8
  • compound 001_7 (2.7g, 18.73mmol) was dissolved in dichloromethane (50mL), and after cooling to 0°C, N,N-diisopropylethylamine (7.26g, 56.20mmol), tri-n-propyl Cyclic phosphoric anhydride (50% ethyl acetate solution) (17.88g, 28.10mmol) and compound 001_6 (4.29g, 20.61mmol). The reaction system was stirred at 25°C for 12 hours. After the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (100 mL), and extracted with ethyl acetate (50 mL ⁇ 3).
  • the compound 001_8 (8.0g, 31.58mmol) was dissolved in methanol (500mL) and water (50mL), and potassium monopersulfate (58.25g, 94.74mmol) was added, and the reaction system was stirred at 25°C for 12 hours .
  • the reaction solution was poured into saturated aqueous sodium sulfite solution (50 mL), and extracted with ethyl acetate (20 mL ⁇ 3).
  • the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated.
  • Compound 001_9 was obtained.
  • Step 7 Synthesis of Compound 001_10
  • Step 8 Synthesis of Compound 001_11
  • Step 9 Synthesis of compounds 001_12 and 001_13
  • Step 10 Synthesis of Compound 001_14
  • the mixture of 001_12 and 001_13 (500mg, 920.10 ⁇ mol) was dissolved in toluene (5mL), and a toluene solution (2M, 1.38mL) of trimethylaluminum was added to the reaction system, and the temperature of the reaction system was raised to 75 °C for 5 hours. After the reaction was completed, the reaction system was cooled to room temperature, and dilute hydrochloric acid (1N, 10 mL) was added to the reaction system to quench the reaction.
  • Step 11 Synthesis of Compounds 001 and 002
  • Step 1 Synthesis of compound 003_1
  • compound 003_2 500 mg, 944.48 ⁇ mol was added into anhydrous methanol (10 mL) to dissolve, bis(trifluoroacetic acid) iodobenzene (2.03 g, 4.72 mmol) was added, and stirred at 50° C. for 6 hours.
  • the reaction solution was poured into saturated sodium bicarbonate water (50 mL), and extracted with ethyl acetate (30 mL ⁇ 3).
  • the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated.
  • Compound 003_3 was obtained.
  • Dissolve compound 003_3 (150 mg, 268.13 ⁇ mol) in anhydrous toluene (2 mL) at room temperature under nitrogen protection, add trimethylaluminum toluene solution (2M, 429.02 ⁇ L) to the reaction system, and heat the reaction system to 80 ° C Stirring was continued for 6 hours. After the reaction was completed, the reaction system was cooled to room temperature, and dilute hydrochloric acid (1N, 10 mL) was slowly added to the reaction system to quench the reaction, extracted with 2-methyltetrahydrofuran (50 mL ⁇ 4), and the organic phases were combined.
  • Step 5 Synthesis of Compounds 003 and 004
  • Step 1 Synthesis of compound 005_2
  • compound 005_3 (3.2 g, 12.11 mmol) was added to chloroform (16 mL) and ethanol (16 mL), followed by the addition of 2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate Acetate diethyl ester (4.60g, 18.16mmol), and the reaction solution was stirred at 50°C for 12 hours.
  • the reaction solution was poured into water (30 mL), extracted with dichloromethane (30 mL ⁇ 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue.
  • Step 4 Synthesis of Compounds 005_5 and 005_6
  • Step 1 Synthesis of Compounds 006_1 and 008_1
  • compound 005 (660mg, 1.32mmol) was added to tetrahydrofuran (6mL) and acetonitrile (3mL), followed by methanol (169.39mg, 5.29mmol), potassium monopersulfate (1.63g, 2.64mmol) , potassium dihydrogen phosphate (359.73mg, 2.64mmol) and cuprous bromide (37.92mg, 264.33 ⁇ mol), and the reaction solution was stirred at 80°C for 2 hours. After the reaction was completed, water (10 mL) was added to the reaction solution, extracted with ethyl acetate (10 mL ⁇ 2), and the organic phases were combined.
  • Step 2 Synthesis of Compounds 006 and 007
  • Step 3 Synthesis of Compounds 008 and 009
  • compound 005_1 (50g, 247.28mmol) was dissolved in toluene (500mL), to which was added malononitrile (16.34g, 247.28mmol), ⁇ -alanine (660.92mg, 7.42mmol) and acetic acid (14.85 g, 247.28mmol), the reaction system was heated to 130°C and stirred for 12 hours. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (500 mL), and the layers were separated. The aqueous phase was extracted with ethyl acetate (100 mL ⁇ 3).
  • Step 3 Synthesis of a mixture of compounds 010_3 and 012_1
  • LCMS analysis method column type: Luna 5 ⁇ m C18 (2*50mm); mobile phase A: H 2 O + 0.05% (v/v) TFA; mobile phase B: ACN + 0.05% (v/v) TFA, gradient: B%: 10%-100%, 6min.
  • compound 001_1 (300g, 1.14mol) was added to N,N-dimethylformamide (3L), followed by addition of o-fluorobenzyl chloride (164.91g, 1.14mol, 135.17mL) and cesium carbonate (408.81g, 1.25mol), and the reaction solution was stirred at 80°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and filtered to obtain a 3L N,N-dimethylformamide solution of compound 014_1 (423 g), which was used directly.
  • ammonium chloride (4.41g, 82.44mmol) was dispersed in toluene (50mL), trimethylaluminum toluene solution (2M, 39.57mL) was added, heated to 80°C and compound 014_2 (5g , 16.49mmol) into the reaction system, stirred at 80°C for 30 minutes, then raised the temperature to 110°C for 1.5 hours, cooled down to 25°C and added methanol (7.61g, 237.42mmol) dropwise, keeping the temperature not higher than 40°C, and then added Hydrochloric acid (3M, 105.52 mL), keep the temperature not higher than 40°C, raise the temperature to 80°C and stir for 10 minutes, then cool down to 0°C and stir for 30 minutes. After the reaction was completed, the filter cake was collected by filtration. The filter cake was rinsed with water (100 mL) to obtain the hydrochloride of compound 014_3.
  • compound 005_4 (6.58g, 24.71mmol) was added in tert-butanol (70mL), followed by addition of compound 014_3 hydrochloride (5g, 15.45mmol) and potassium carbonate (5.34g, 38.61mmol) , and the reaction solution was stirred at 85° C. for 12 hours.
  • reaction solution was cooled to room temperature, water (100 mL) was added, extracted with dimethyltetrahydrofuran (100 mL ⁇ 2), the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and reduced Concentrate under reduced pressure to obtain a mixture of compounds 014_4 and 014_5.
  • compound 014_6 (500mg, 1.08mmol) was dissolved in THF (5mL) and H 2 O (5mL), and potassium monopersulfate (1.33g, 2.17mmol), potassium dihydrogen phosphate (294.94 mg, 2.17mmol) and cuprous bromide (31.09mg, 216.72 ⁇ mol), the reaction solution was stirred at 80°C for 2 hours. After the reaction was completed, water (10 mL) was added to the reaction solution, ethyl acetate (10 mL ⁇ 2) was separated, and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue.
  • Step 1 Synthesis of compounds 017_1 and 017_2
  • the intermediate 017_2 (700.00mg, 1.38mmol) was dissolved in toluene (10mL), the nitrogen was replaced three times, AlMe 3 (2M, 2.07mL) was added to the reaction system, and the temperature of the reaction system was raised to 75°C Stir for 12 hours. After the reaction was completed, the reaction system was cooled to room temperature. Pour the reaction system into water (100 mL), add dilute hydrochloric acid (1N) to adjust the pH to 3-4, add 2-methyltetrahydrofuran (50 mL) to dilute, separate the layers, and collect the organic phase.
  • the aqueous phase was extracted with 2-methyltetrahydrofuran (50 mL ⁇ 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain a crude product.
  • HPLC analysis method column type: Kinetex 5 ⁇ m C18 (2.1*50mm); mobile phase A: H 2 O+0.04% (v/v) TFA; mobile phase B: ACN+0.02% (v/v) TFA, gradient: B%: 10%-80%, 6min.
  • compound 001_11 (9.04g, 36.14mmol) was added to tert-butanol (100mL), then the hydrochloride (5g) and potassium carbonate (3.87g, 38.61mmol) of compound 014_3 were added, and the reaction solution was heated at 80 Stir at °C for 12 hours.
  • water 500 mL was added to the reaction solution, extracted with ethyl acetate (200 mL ⁇ 3), and the organic phases were combined. The organic phase was washed with saturated brine (100 mL ⁇ 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue.
  • the residue was added to methyl tert-butyl ether (70 mL) for slurry at room temperature, filtered to collect the filter cake, and the filter cake was concentrated under reduced pressure to obtain compound 021_1.
  • compound 021_1 (4.5g, 8.90mmol) was dissolved in anhydrous toluene (130mL), and trimethylaluminum toluene solution (2M, 13.35mL) was slowly added dropwise, and the reaction system was stirred at 75°C for 12 Hour. After the reaction is complete, cool the reaction system to room temperature and slowly pour it into water (100mL), add 3M hydrochloric acid to adjust the pH to 3-4, add dimethyltetrahydrofuran (100mL ⁇ 3) for extraction, combine the organic phases, anhydrous sodium sulfate Dry, filter, and concentrate the filtrate under reduced pressure to obtain a residue.
  • the mixture of compound 023_1 (5g, 10.17mmol) was added to anhydrous methanol (100mL), followed by bis(trifluoroacetic acid) iodobenzene (10.94g, 25.43mmol), and the reaction solution was heated at 50°C Stir for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure and diluted with ethyl acetate (100 mL), washed with saturated sodium sulfite (50 mL ⁇ 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue.
  • the compound 023_2 (0.8g, 1.53mmol) was dissolved in anhydrous toluene (15mL), and trimethylaluminum toluene solution (2M, 2.45mL) was slowly added dropwise. Stirring was continued for 12 hours. After the reaction was completed, the reaction system was cooled to room temperature, slowly poured into water (20 mL), and 3M hydrochloric acid was added to adjust the pH to 3-4, and dimethyltetrahydrofuran (20 mL ⁇ 3) was added for extraction, and the organic phases were combined.
  • compound 024_2 and compound 024_3 were dispersed in anhydrous methanol (100 mL) and bistrifluoroacetoxyiodobenzene (12.14 g, 28.23 mmol) was added.
  • the reaction solution was stirred in an oil bath at 50°C for 2 hours. After the reaction was completed, it was lowered to room temperature, and concentrated to remove the solvent to obtain an oily residue.
  • Isopropyl ether (100 mL) was added to the residue, and after stirring at room temperature for 30 min, a white solid precipitated out, which was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product.
  • compound 024_4 (0.3 g, 534.34 ⁇ mol) was dissolved in anhydrous toluene (6 mL) and trimethylaluminum toluene solution (2M, 854.95 ⁇ L) was added.
  • the reaction system was stirred at 50°C for 12 hours, then heated to 90°C and stirred for 12 hours. Cool down to room temperature after the reaction.
  • the reaction solution was poured into water (100 mL), dilute hydrochloric acid (1N) was added to pH 3-4, and extracted with ethyl acetate (50 mL ⁇ 3).
  • the compound 024_1 (2.99g, 11.86mmol) was dissolved in tert-butanol (150mL), and the hydrochloride (2.4g, 7.41mmol) and potassium bicarbonate (1.86g, 18.53mmol) of the compound 014_3 were added ), stirred at 85°C for 12 hours.
  • 2-methyltetrahydrofuran 100 mL
  • water 100 mL
  • the aqueous phase was extracted with 2-methyltetrahydrofuran (100 mL ⁇ 3), and the organic phases were combined.
  • the organic phase was washed with saturated brine (100 mL ⁇ 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a mixture of intermediates 026_1 and 026_2.
  • compound 026_3 (350 mg, 668.59 ⁇ mol) was dissolved in anhydrous toluene (10 mL) and trimethylaluminum toluene solution (2M, 1.34 mL) was added. The reaction system was stirred at 75°C for 5 hours. After the reaction, it was lowered to room temperature. Pour the reaction solution into water (50 mL), add 1N dilute hydrochloric acid to pH 3-4, and extract with ethyl acetate (50 mL ⁇ 3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product.
  • cGMP-D2 D2-labeled cyclic guanosine monophosphate
  • anti-cGMP cryptate (Eu 3+ cryptate-labeled anti-cyclic guanosine monophosphate antibody) to 1 mL lysis buffer (lysis buffer) and mix well.
  • LNCap medium RPMI1640+10% fetal bovine serum+1% double antibody.
  • cGMP standard curve use Graphpad prism to make a standard curve according to the ratio of the concentration of cGMP to 665/615.
  • HTRF homogeneous time-resolved fluorescence technique
  • MEC Minimum effective concentration to stimulate cGMP production (greater than three-fold basal value) in lnCap cells.
  • the compound of the application can effectively stimulate sGC and significantly increase the level of cGMP.
  • the purpose of this study is to determine the pharmacokinetic parameters of the compound in male SD rats.
  • the project used 4 male SD rats, a group of 2 SD rats were administered intravenously, the dosage was 0.3mg/kg, and the concentration was 0.15mg/mL; another group of 2 SD rats were administered orally Administration, the dosage is 1mg/kg, the administration concentration is 0.2mg/mL; plasma samples are collected at 0.083 (intravenous group only), 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24h after administration , and then perform LC-MS/MS analysis on the collected samples and collect the data.
  • the collected analytical data was calculated with Phoenix WinNonlin 6.3 software to calculate relevant pharmacokinetic parameters.
  • the compound of this application has no risk of entering the brain and has a good heart distribution.

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Abstract

一种嘧啶三并环衍生物及其在药学上的应用，具体公开了式(I)所示化合物、其立体异构体及其药学上可接受的盐。

Description

嘧啶三并环衍生物及其在药学上的应用
本申请主张如下优先权
本申请要求1)于2021年10月13日向中国国家知识产权局提交的第2021111941776号中国专利申请的优先权和权益，2)于2021年12月20日向中国国家知识产权局提交的第2021115653068号中国专利申请的优先权和权益，和3)于2022年9月22日向中国国家知识产权局提交的第2022111609592号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本申请涉及一种嘧啶三并环衍生物及其在药学上的应用，具体涉及式(I)所示的化合物、其立体异构体及其药学上可接受的盐。
背景技术
可溶性鸟苷酸环化酶(sGC)广泛存在于哺乳动物的细胞溶质内，是由α和β两种亚基组成的异源二聚体。可溶性鸟苷酸环化酶是NO-sGC-cGMP信号通路中关键的信号转导酶，sGC在体内被激活后会催化三磷酸鸟苷(GTP)转化为环磷酸鸟苷(cGMP)。cGMP是一种重要的二级信使分子，通过激活其下游的多种效应分子，如cGMP依赖蛋白激酶G及cGMP门控离子通道等，进而引发下游一系列级联反应。在胃肠系统、心血管系统和中枢神经系统中发挥重要的生理功能，如促进血管和平滑肌舒张，抑制血小板凝聚、血管重塑、细胞凋亡和炎症发生以及参与神经传递等。在病理生理学条件下，NO/cGMP系统可被抑制，这可导致例如高血压、血小板激活、增加的细胞增生、内皮机能障碍、动脉硬化、心绞痛、心力衰竭、心肌梗死、血栓形成、中风和性功能障碍等。近两年又有研究显示，sGC介导的信号通路异常还与慢性肾脏疾病、系统性硬化病等纤维化疾病的发生有着密切的关系。
sGC刺激剂具有双重作用机制：既可以不依赖于NO，但需要依赖含有Fe 2+的血红素辅基直接激活sGC-cGMP信号通路；也可以增强sGC对内源性NO的敏感性从而与NO产生协同作用。因此sGC刺激剂是一种血红素依赖型，NO非依赖型sGC刺激剂。刺激sGC生成更多的cGMP可以调控多种重要生理过程：促进血管平滑肌舒张，抑制血小板凝集等，同时激活sGC还可以调节其他信号通路，如TGF-β，发挥抗纤维化和抗肿瘤效果。因此sGC刺激剂可以作为治疗心血管疾病(心力衰竭，肺动脉高压，心绞痛，心肌梗死)和纤维化疾病(肾纤维化，系统性硬化病)的潜在治疗手段。
针对目前市场和临床未满足的对此类可溶性鸟苷酸环化酶刺激剂的需求，本申请提供了一类新化合物。此类化合物可作为可溶性鸟苷酸环化酶的刺激剂，对可溶性鸟苷酸环化酶具有优良的体外刺激活性，并具有较好的药代动力学性质。
发明内容
本申请提供了式(I)所示化合物、其立体异构体或其药学上可接受的盐，
Figure PCTCN2022125047-appb-000001
其中，
R 1选自H、-OH、C 1-3烷基、C 1-3烷氧基或C 1-3烷氨基；
R 2选自苄基或C 1-8烷基，所述苄基或C 1-8烷基任选被1、2、3、4或5个卤素原子取代；
R 3、R 4各自独立地选自H或C 1-3烷基；
或者R 3和R 4以及它们共同相连的原子形成C 3-6环烷基；
条件是所述化合物不选自以下结构、其立体异构体或其药学上可接受的盐：
Figure PCTCN2022125047-appb-000002
在本申请的一些方案中，上述R 1选自H、-OH、C 1-3烷基或C 1-3烷氧基，其他变量如本申请所定义。
在本申请的一些方案中，上述R 1选自H、-OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基或异丙氧基，其他变量如本申请所定义。
在本申请的一些方案中，上述R 1选自H、-OH、甲基或甲氧基，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自苄基或C 1-6烷基，所述苄基或C 1-6烷基任选被1、2、3、4或5个卤素原子取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自苄基或C 1-4烷基，所述苄基或C 1-4烷基任选被1、2、3、4或5个卤素原子取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自苄基或C 1-4烷基，所述苄基或C 1-4烷基任选被1、2、3、4或5个F、Cl或Br原子取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自苄基或C 1-4烷基，所述苄基或C 1-4烷基任选被1、2、3、4或5个F原子取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自C 1-4烷基，所述C 1-4烷基任选被1、2、3、4或5个卤素原子取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自C 1-4烷基，所述C 1-4烷基任选被1、2、3、4或5个F、Cl或Br原子取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自C 1-4烷基，所述C 1-4烷基任选被1、2、3、4或5个F原子取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自苄基或正丁基，所述苄基被1个氟取代，所述正丁基被5个氟取代，其他变量如本申请所定义。
在本申请的一些方案中，上述R 2选自
Figure PCTCN2022125047-appb-000003
其他变量如本申请所定义。
在本申请的一些方案中，上述R 3、R 4各自独立地选自H、甲基、乙基、正丙基或异丙基，或者R 3和R 4以及它们共同相连的原子形成环丙烷基，其他变量如本申请所定义。
在本申请的一些方案中，上述R 3、R 4各自独立地选自H或甲基，或者R 3和R 4以及它们共同相连的原子形成环丙烷基，其他变量如本申请所定义。
在本申请的一些方案中，上述结构单元
Figure PCTCN2022125047-appb-000004
选自
Figure PCTCN2022125047-appb-000005
其他变量如本申请所定义。
本申请还有一些方案是由上述各变量任意组合而来。
本申请还提供下述化合物、其立体异构体或其药学上可接受的盐，其选自：
Figure PCTCN2022125047-appb-000006
本申请还提供下述化合物、其立体异构体或其药学上可接受的盐，其选自：
Figure PCTCN2022125047-appb-000007
Figure PCTCN2022125047-appb-000008
另一方面，本申请提供药物组合物，其包含本申请的化合物、其立体异构体或其药学上可接受的盐。在一些实施方案中，本申请的药物组合物还包括药学上可接受的辅料。
另一方面，本申请提供上述化合物、其立体异构体或其药学上可接受的盐、或其药物组合物在制备治疗sGC激动剂或刺激剂相关疾病的药物上应用。
另一方面，本申请提供治疗哺乳动物sGC激动剂或刺激剂相关疾病的方法，包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的本申请的化合物、其立体异构体或其药学上可接受的盐、或其药物组合物。
另一方面，本申请提供本申请的化合物、其立体异构体或其药学上可接受的盐、或其药物组合物在治疗sGC激动剂或刺激剂相关疾病上的应用。
另一方面，本申请提供治疗sGC激动剂或刺激剂相关疾病的本申请的化合物、其立体异构体或其药学上可接受的盐、或其药物组合物。
在本申请的一些方案中，所述sGC激动剂或刺激剂相关疾病选自心力衰竭、或高血压。
技术效果
本申请化合物能够有效刺激sGC，显著提高cGMP水平，具有较好的表观分布容积和半衰期，有较好的心脏分布且无入脑风险。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本申请化合物的盐，由本申请发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本申请的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。当本申请的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。本申请的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本申请的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
除非另有说明，术语“异构体”意在包括几何异构体、顺反异构体、立体异构体、对映异构体、旋光异构体、非对映异构体和互变异构体。
本申请的化合物可以存在特定的几何或立体异构体形式。本申请设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本申请的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本申请的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2022125047-appb-000009
和楔形虚线键
Figure PCTCN2022125047-appb-000010
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2022125047-appb-000011
和直形虚线键
Figure PCTCN2022125047-appb-000012
表示立体中心的相对构型，用波浪线
Figure PCTCN2022125047-appb-000013
表示楔形实线键
Figure PCTCN2022125047-appb-000014
或楔形虚线键
Figure PCTCN2022125047-appb-000015
或用波浪线
Figure PCTCN2022125047-appb-000016
表示直形实线键
Figure PCTCN2022125047-appb-000017
或直形虚线键
Figure PCTCN2022125047-appb-000018
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本申请某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本申请的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本申请的化合物的所有同位素组成的变换，无论放射性与否，都包括在本申请的范围之内。
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，取代基可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当一个取代基数量为0时，表示该取代基是不存在的，比如-A-(R) 0表示该结构实际上是-A。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基的键可以交叉连接到一个环上的两个以上原子时，这种取代基可以与这个环上的任意原子相键合，例如，结构单元
Figure PCTCN2022125047-appb-000019
表示其取代基R可在环己基或者环己二烯上的任意一个位置发生取代。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2022125047-appb-000020
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2022125047-appb-000021
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2022125047-appb-000022
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化 学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2022125047-appb-000023
直形虚线键
Figure PCTCN2022125047-appb-000024
或波浪线
Figure PCTCN2022125047-appb-000025
表示。例如-OCH 3中的直形实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2022125047-appb-000026
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
Figure PCTCN2022125047-appb-000027
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连；
Figure PCTCN2022125047-appb-000028
表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括
Figure PCTCN2022125047-appb-000029
Figure PCTCN2022125047-appb-000030
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2022125047-appb-000031
仍包括
Figure PCTCN2022125047-appb-000032
这种连接方式的基团，只是在连接1个化学键时，该位点的H会对应减少1个变成相应的一价哌啶基。
除非另有规定，术语“烷基”用于表示直链或支链的饱和的碳氢基团，在一些实施方案中，所述烷基为C 1-8烷基；在另一些实施方案中，所述烷基为C 1-4烷基；在另一些实施方案中，所述烷基为C 1-3烷基。其可以是单取代(如-CH 2F)或多取代的(如-CF 3)，可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。烷基的实例包括但不限于甲基(Me)，乙基(Et)，丙基(包括n-丙基和异丙基)，丁基(包括n-丁基，异丁基，s-丁基和t-丁基)，戊基(包括n-戊基，异戊基和新戊基)、己基等。
除非另有规定，术语“C 1-4烷基”用于表示直链或支链的由1至4个碳原子组成的饱和碳氢基团。所述C 1-4烷基包括C 1-2、C 1-3和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-4烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)等。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1- 3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，术语“C 1-3烷氨基”表示-NH-C 1-3烷基。
除非另有规定，“C 3-6环烷基”表示由3至6个碳原子组成的饱和环状碳氢基团，其为单环和双环体系，所述C 3-6环烷基包括C 3-5、C 4-5和C 5-6环烷基等；其可以是一价、二价或者多价。C 3-6环烷基的实例包括，但不限于，环丙基、环丁基、环戊基、环己基等。
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。
本申请的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本申请的实施例。
本申请的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本申请涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2022125047-appb-000033
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
本申请所使用的溶剂可经市售获得。本申请采用下述缩略词：ACN代表乙腈；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；HPLC代表高效液相色谱；LCMS代表液相色谱-质谱联用；℃代表摄氏度；h代表小时；mL代表毫升；mM代表毫摩尔每升；mmol代表毫摩尔；μmol代表微摩尔；HNMR代表核磁共振氢谱；MS代表质谱；min代表分钟；pH代表氢离子摩尔浓度负对数；AlMe 3代表三甲基铝；TFA代表三氟乙酸；DMSO代表二甲基亚砜。
术语“治疗”意为将本申请所述化合物或制剂进行给药以改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)抑制疾病或疾病状态，即遏制其发展；
(ii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
术语“预防”意为将本申请所述化合物或制剂进行给药以预防疾病或与所述疾病相关的一个或多个症状，且包括：预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时。
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请化合物的用量。构成“治疗有效量”的本申请化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
具体实施方式
下面通过实施例对本申请进行详细描述，但并不意味着对本申请任何不利限制。本文已经详细地描述了本申请，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本申请精神和范围的情况下针对本申请具体实施方式进行各种变化和改进将是显而易见的。本申请所使用的所有试剂是市售的，无需进一步纯化即可使用。
实施例1和2
Figure PCTCN2022125047-appb-000034
合成路线：
Figure PCTCN2022125047-appb-000035
步骤1：化合物001_2的合成
室温下，化合物001_1(100g,380.21mmol)加入到N,N-二甲基甲酰胺(1L),之后加入1,1,1,2,2-五氟 -4-碘丁烷(520.83g,1.90mol)和碳酸钾(131.37g,950.53mmol)，反应体系在80℃搅拌2小时。反应完毕，将反应液降温至室温，过滤，滤液减压浓缩。残余物经层析柱分离(洗脱剂:石油醚/乙酸乙酯＝1/0–15/1,体积比)，得到化合物001_2。
步骤2：化合物001_3的合成
室温和氮气条件下，将化合物001_2(30g,73.34mmol)加入甲醇(100mL)和N,N-二甲基甲酰胺(300mL)中，加入环戊–2,4–二烯–1–基(二苯基)膦二氯甲烷二氯钯二茂铁(3.76g,5.13mmol)和三乙胺(29.68g,293.35mmol,40.83mL)。反应混合物在一氧化氮环境下(15Psi)，80℃条件下搅拌12小时。反应完成后，反应液降温至室温，减压浓缩。残余物经层析柱分离(洗脱剂:石油醚/乙酸乙酯＝1/0–10/1,体积比)，得到化合物001_3。
步骤3：化合物001_4的盐酸盐的合成
室温和氮气条件下，将氯化铵(28.22g,527.54mmol)分散于甲苯(360mL)中，加入三甲基铝的甲苯溶液(2M,253.22mL)，加热至80℃后加入化合物001_3(36g,105.51mmol)，于80℃搅拌30分钟，然后升温至110℃反应1.5小时，降温至25℃滴加甲醇(48.68g,1.52mol,61.48mL)，保持温度不高于40℃，然后再加入盐酸(3M,675.25mL)，保持温度不高于40℃，升温至80℃搅拌10分钟，然后降温至0℃搅拌30分钟。反应完毕，将反应液过滤，收集滤饼，滤饼用水(200mL)淋洗后减压浓缩除去溶剂。得到化合物001_4的盐酸盐。 1H NMR(400MHz,DMSO-d 6)δppm 9.49(br d,J＝11.2Hz,4H)8.87(s,1H)8.53(dd,J＝8.8,2.4Hz,1H)4.94(t,J＝6.8Hz,2H)2.97-3.18(m,2H)。
步骤4：化合物001_6的合成
室温和氮气保护下，将化合物001_5(5g,56.71mmol)溶解到干燥的甲苯(20mL)中，加入溴乙腈(7.48g,62.38mmol)。反应体系在25℃下搅拌12小时。反应体系逐渐由澄清变成有白色固体析出。反应完毕，悬浊液过滤，用甲苯(50mL)洗涤滤饼，收集固体，减压干燥。得到化合物001_6。
步骤5：化合物001_8的合成
室温下，将化合物001_7(2.7g,18.73mmol)溶于二氯甲烷(50mL)，降温至0℃后加入N,N-二异丙基乙胺(7.26g,56.20mmol)，三正丙基环磷酸酐(50％乙酸乙酯溶液)(17.88g,28.10mmol)和化合物001_6(4.29g,20.61mmol)。反应体系在25℃搅拌12小时。反应结束后，反应液倒入饱和碳酸氢钠水溶液中(100mL)中，乙酸乙酯(50mL×3)萃取。有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。残余物通过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/1–0/1,体积比)纯化，得到化合物001_8。
步骤6：化合物001_9的合成
室温和氮气保护下，将化合物001_8(8.0g,31.58mmol)溶于甲醇(500mL)和水(50mL)，加入单过硫酸氢钾(58.25g,94.74mmol)，反应体系在25℃搅拌12小时。反应结束后反应液倒入饱和亚硫酸钠水溶 液(50mL)中，乙酸乙酯(20mL×3)萃取。有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。残余物通过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–4/1,体积比)纯化。得到化合物001_9。
步骤7：化合物001_10的合成
室温和氮气保护下，将化合物001_9(5g,26.86mmol)溶于无水甲醇(50mL)中，向其中加入丙二腈(2.13g,32.23mmol)和醋酸铵(4.14g,53.72mmol)。在氮气保护下将反应体系升温至60℃搅拌4小时。反应液倒入饱和氯化铵水溶液(70mL)中，乙酸乙酯(20mL×3)萃取。有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。残余物通过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–5/1,体积比)纯化得到化合物001_10。
步骤8：化合物001_11合成
在氮气保护下，将化合物001_10(1.1g,4.70mmol)溶于四氢呋喃(10mL)，降温至0℃，滴加甲基溴化镁的甲苯溶液(3M,3.13mL)。滴加完毕后搅拌15分钟。反应结束后，将其倒入饱和氯化铵水溶液(50mL)中，乙酸乙酯(20mL×3)萃取。有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。残余物通过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–5/1,体积比)得到化合物001_11。
步骤9：化合物001_12和001_13合成
室温和氮气保护下，将化合物001_4(1g,2.76mmol,盐酸盐)溶解于叔丁醇(20mL)中，向其中加入化合物001_11(1g,4.00mmol)和碳酸氢钾(692.06mg,6.91mmol)，反应体系升温至85℃搅拌12小时。反应结束后，反应液降至室温倒入水(70mL)中，乙酸乙酯(50mL×3)萃取。有机相经饱和食盐水(50mL)洗涤，无水硫酸钠干燥后过滤浓缩。残余物通过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–1/1,体积比)纯化。得到001_12和001_13的混合物。
步骤10：化合物001_14合成
室温和氮气保护下，将001_12和001_13的混合物(500mg,920.10μmol)溶解于甲苯(5mL)中，向反应体系中加入三甲基铝的甲苯溶液(2M,1.38mL)，反应体系升温到75℃拌5小时。反应完毕后，将反应体系降温到室温，向反应体系中加入稀盐酸(1N,10mL)淬灭反应。加乙酸乙酯(20mL)稀释，分液，收集有机相，水相用乙酸乙酯(20mL×3)萃取，合并有机相。有机相经无水硫酸钠干燥，过滤，滤液减压浓缩除去溶剂。残余物先通过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–1/1,体积比)，再经制备HPLC分离(柱型:Phenomenex luna C18(80mm*30mm I.D.,3μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％:30％-70％,8min)，得到化合物001_14。MS–ESI m/z:512.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.52(s,1H),11.29(s,1H),8.81–8.68(m,2H),4.91(t,J＝6.7Hz,2H),3.00(tt,J＝6.6,19.1Hz,2H),1.60–1.48(m,1H),1.44(s,3H),1.18–1.08(m,1H),0.81–0.64(m,2H)。
步骤11：化合物001和002的合成
将化合物001_14手性柱分离(柱型:DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[EtOH(含0.1％NH 3H 2O)]％:35％-35％,7min)。得到化合物001和002。
SFC分析方法：柱型:Chiralpak AD(50*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[EtOH(含0.1％IPAm)]，梯度：B％:5％-50％,3min。
001(保留时间:0.900min):MS–ESI m/z:512.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.52(s,1H),11.34–11.24(m,1H),8.82–8.68(m,2H),4.91(t,J＝6.5Hz,2H),3.09–2.91(m,2H),1.55(ddd,J＝4.4,6.0,10.0Hz,1H),1.44(s,3H),1.20–1.08(m,1H),0.81–0.64(m,2H)；
002(保留时间:1.017min):MS–ESI m/z:511.9[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.61–11.46(m,1H),11.28(s,1H),8.78–8.70(m,2H),4.91(t,J＝6.7Hz,2H),3.10–2.88(m,2H),1.55(ddd,J＝4.0,6.4,10.0Hz,1H),1.44(s,3H),1.19–1.04(m,1H),0.82–0.66(m,2H)。
实施例3和4
Figure PCTCN2022125047-appb-000036
合成路线:
Figure PCTCN2022125047-appb-000037
步骤1：化合物003_1的合成
在室温和氮气保护下，将化合物001_10(1.5g,6.40mmol)加入氯仿(10mL)和乙醇(10mL)溶解,将2,6-二甲基-1,4-二氢-3,5-吡啶二羧酸二乙酯(2.43g,9.61mmol)加入到反应体系中。反应液在氮气保护下25℃搅拌12小时。反应结束后，将反应液倒入水(100mL)中，乙酸乙酯(50mL×3)萃取。合并有机相经饱和食盐水(50mL)洗涤，无水硫酸钠干燥后过滤浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–1/1,体积比)。得到化合物003_1。
步骤2：化合物003_2的合成
在室温和氮气保护下，将化合物001_4(1.5g,4.15mmol,盐酸盐)溶解于叔丁醇(30mL)中，向其中加入化合物003_1(2g,8.47mmol)和碳酸氢钾(1.04g,10.37mmol)，反应体系升温至85℃搅拌12小时。反应液降至室温后倒入水(70mL)中，2-甲基四氢呋喃(30mL×3)萃取。合并的有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–1/1,体积比)。得到化合物003_2。
步骤3：化合物003_3的合成
在室温和氮气保护下，加入化合物003_2(500mg,944.48μmol)加入无水甲醇10mL)溶解，加入双(三氟乙酸)碘代苯(2.03g,4.72mmol)，在50℃下搅拌6小时。反应液倒入饱和碳酸氢钠水(50mL)中，乙酸乙酯(30mL×3)萃取。有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–1/1,体积比)。得到化合物003_3。
步骤4：化合物003_4的合成
在室温和氮气保护下，将化合物003_3(150mg,268.13μmol)溶解于无水甲苯(2mL)中，向反应体系中加入三甲基铝甲苯溶液(2M,429.02μL)，反应体系升温到80℃下搅拌6小时。反应完毕后，反应体系降温到室温，向反应体系中缓慢加入稀盐酸(1N,10mL)淬灭反应，用2-甲基四氢呋喃(50mL×4)萃取，合并有机相。有机相依次经半饱和食盐水(10mL×2)洗涤，饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/2-甲基四氢呋喃＝1/0–1/1,体积比)和制备HPLC分离(柱型:Phenomenex luna C18(80mm*30mm I.D.,3μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％:30％-55％,8min)，得到化合物003_4。MS–ESI m/z:527.9[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.77(s,1H),11.48(s,1H),8.85–8.65(m,2H),4.93(t,J＝6.8Hz,2H),3.19(s,3H),3.10–2.92(m,2H),1.63(ddd,J＝4.2,6.8,9.6Hz,1H),1.43–1.34(m,1H),1.00–0.84(m,2H)。
步骤5：化合物003和004的合成
将化合物003_4(40mg,75.85μmol)手性柱分离(柱型:DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相：A：CO 2，B：[Neu-IPA]，梯度：B％:25％-25％,10min)。得到化合物003和004。
SFC分析方法：柱型:Chiralcel OX-3(50*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[0.1％IPAm IPA]，梯度：B％:5％-50％,3min。
003(保留时间:0.997min):MS–ESI m/z:527.9[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.77(s,1H),11.47(s,1H),8.82–8.70(m,2H),4.93(t,J＝6.8Hz,2H),3.19(s,3H),3.02–2.95(m,2H),1.66–1.59(m,1H),1.42–1.35(m,1H),1.00–0.84(m,2H)；
004(保留时间:1.075min):MS–ESI m/z:528.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.88–11.70(m,1H),11.46(br s,1H),8.83–8.69(m,2H),4.93(t,J＝6.8Hz,2H),3.19(s,3H),3.10–2.92(m,2H),1.68–1.58(m,1H),1.43–1.34(m,1H),0.99–0.84(m,2H)。
实施例5
Figure PCTCN2022125047-appb-000038
合成路线:
Figure PCTCN2022125047-appb-000039
步骤1：化合物005_2的合成
室温下，叔丁醇钾四氢呋喃溶液(1M,276.95mL)加入到甲苯(2L)中，之后加入化合物005_1(50g,247.28mmol,45.87mL)，最后加入碘甲烷(228.14g,1.61mol,100.06mL)和十八冠醚-6(6.54g,24.73mmol)加料完毕，反应液在25℃下搅拌12小时。将反应液加入25％的氨水500mL中淬灭反应，加入1L水。用乙酸乙酯(1L×2)萃取，合并有机相。有机相用无水硫酸钠干燥，过滤，减压浓缩。残余物经层析柱分离纯化(洗脱剂:石油醚/2-甲基四氢呋喃＝1/0–1/1,体积比)得到化合物005_2。
MS–ESI m/z:216.9[M+H] +。
步骤2：化合物005_3的合成
室温下，将化合物005_2(20g,92.49mmol)和丙二睛(24.44g,369.98mmol)加入乙醇(200mL)中，之后加入吡啶(36.58g,462.47mmol,37.33mL)。反应液在70℃下搅拌12小时。将反应液减压浓缩后加入乙酸乙酯(200mL)溶解，用3M的盐酸水溶液调节pH至5-6，分液。水相用乙酸乙酯(100mL×2)萃取，合并有机相。有机相经无水硫酸钠干燥，过滤，减压浓缩，得到残余物。残余物经层析柱分离(洗脱剂：石油醚/乙酸乙酯＝1/0-6/1，体积比)。得到化合物005_3。 1H NMR(400MHz,CDCl 3)δ:4.42(q,J＝7.2Hz,2H),4.25(q,J＝7.2Hz,2H),1.61-1.66(m,6H),1.39-1.47(m,6H)。
步骤3：化合物005_4的合成
室温下，将化合物005_3(3.2g,12.11mmol)加入到氯仿(16mL)和乙醇(16mL)中，随后加入2,6-二甲基-1,4-二氢-3,5-吡啶二羧酸二乙酯(4.60g,18.16mmol)，反应液在50℃下搅拌12小时。将反应液倒入到水中(30mL)，加入二氯甲烷(30mL×3)萃取，合并有机相。有机相经无水硫酸钠干燥，过滤减压浓缩，得到残余物。残余物经层析柱分离纯化(洗脱剂：石油醚/乙酸乙酯＝1/0–95/5，体积比)。得到化合物005_4。MS–NEG m/z:265.2[M-H] -
步骤4：化合物005_5和005_6的合成
室温下，将化合物001_4(2.12g,5.87mmol,盐酸盐)和005_4(2.50g,9.39mmol)加入叔丁醇(30mL)中，然后加入碳酸钾(2.03g,14.67mmol)。反应混合物在85℃下搅拌12小时。反应完毕，将反应液降温至室温后用水(30mL)稀释，用二甲基四氢呋喃(30mL×3)萃取，合并有机相。有机相用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，得到残余物。残余物中加入30mL甲基叔丁基醚，在20℃下搅拌1小时，有明显固体产生，将反应液过滤，滤饼用甲基叔丁基醚(10mL)淋洗，得到005_5和005_6的混合物。MS–ESI m/z:546.1[M+H] +。
步骤5：化合物005的合成
室温和氮气保护下，将005_5和005_6的混合物(2.36g,4.33mmol)加入到甲苯(25mL)中，之后加入三甲基铝的甲苯溶液(2M,6.92mL)，升温至80℃并搅拌12小时。将反应液降温至室温，用3M HCl稀盐酸调节pH至5～6，用乙酸乙酯(25mL×3)萃取，合并有机相，无水硫酸钠干燥，过滤，减压浓缩得到残余物。向残余物中加入甲基叔丁基醚(10mL)，在25℃下搅拌1小时，过滤，减压浓缩得到化合物005。MS–ESI m/z:500.2[M+H] +； 1H NMR(400MHz,DMSO-d 6)δ:11.50(s,1H),11.10-11.19(m,1H),8.75(dd,J＝2.58,1.58Hz,1H),8.72(dd,J＝8.7,2.8Hz,1H),4.90(t,J＝6.8Hz,2H),4.0(s,1H),2.89-3.09(m,2H),1.48(s,3H),0.79(s,3H)。
实施例6,7,8和9
Figure PCTCN2022125047-appb-000040
合成路线:
Figure PCTCN2022125047-appb-000041
步骤1：化合物006_1和008_1的合成
室温和氮气保护下，将化合物005(660mg,1.32mmol)加入四氢呋喃(6mL)和乙腈(3mL)中，随后加入甲醇(169.39mg,5.29mmol)，单过硫酸氢钾(1.63g,2.64mmol)，磷酸二氢钾(359.73mg,2.64mmol)和溴化亚铜(37.92mg,264.33μmol)，反应液在80℃下搅拌2小时。反应完毕，向反应液中加入水(10mL)，用乙酸乙酯(10mL×2)萃取，合并有机相。有机相用无水硫酸钠干燥，过滤，减压浓缩，得到残余物。残余物经制备HPLC分离(柱型:Phenomenex luna C18(80mm*30mm I.D.,3μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％:25％-50％,8min)。得到化合物006_1和008_1。
化合物006_1：MS–ESI m/z:530.0[M+H] +1H NMR(400MHz,DMSO-d 6)δ:11.85(s,1H),11.33,(s,1H)8.77(dd,J＝2.8,1.6Hz,1H),8.71(dd,J＝8.4,2.8Hz,1H),4.92(t,J＝6.8Hz,2H),3.15(s,3H),3.00(tt,J＝19.2,6.8Hz,2H),1.35(s,3H)0.79(s,3H)；
化合物008_1：MS–ESI m/z:516.2[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.49(s,1H),11.19(s,1H),8.76(dd,J＝2.8,1.6Hz,1H),8.70(dd,J＝8.8,2.8Hz,1H),6.63(br s,1H),4.86-4.98(m,2H),3.00(tt,J＝19.2,6.4Hz,2H),1.38(s,3H),0.77(s,3H)。
步骤2：化合物006和007的合成
将化合物006_1(40mg,75.85μmol)手性柱分离(柱型:REGIS(S,S)WHELK-O1(250mm*25mm,10μm)；流动相：A：CO 2，B：[IPA(含0.1％NH 3H 2O)]，梯度：B％:25％-25％,7min)。得到化合物006和007。
SFC分析方法：柱型:Chiralcel(S,S)-Whelk-O1(100*4.6mm,I.D.,3.5μm)；流动相:[IPA(含0.1％IPAm)]％:10％-50％,3min。
006(保留时间:1.518min):MS–ESI m/z:530.2[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.33(br s,1H)11.70-12.03(m,1H)8.77(dd,J＝2.4,1.6Hz,1H)8.71(dd,J＝8.8,2.8Hz,1H),4.93(t,J＝6.72Hz,2H),3.16(s,3H),3.00(tt,J＝19.2,7.2Hz,2H),1.35(s,3H),0.79(s,3H)；
007(保留时间:1.644min):MS–ESI m/z:530.2[M+H] +1H NMR(400MHz,DMSO-d 6)δ:8.76-8.79(m,1H)8.70(br d,J＝8.8Hz,1H),4.93(t,J＝6.8Hz,2H),3.15(s,3H),2.93-3.09(m,2H),1.35(s,3H),0.79(s,3H)。
步骤3：化合物008和009的合成
将化合物008_1(70mg,135.83μmol)手性柱分离(柱型:Phenomenex-Cellulose-2(250mm*30mm,10μm)；流动相：A：CO 2，B：[MeOH(含0.1％NH 3H 2O)]；梯度：B％:30％-30％,10min)。得到化合物008和009。
SFC分析方法：柱型:Chiralcel OD-3(50mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[MeOH(含0.1％IPAm)]；梯度：B％:5％-50％,3min。
008(保留时间:1.008min):MS–ESI m/z:516.2[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.48(s,1H),11.19(s,1H),8.77(dd,J＝2.8,1.6Hz,1H),8.70(dd,J＝8.4,2.8Hz,1H),6.62(s,1H),4.87-4.97(m,2H),2.93-3.09(m,2H),1.38(s,3H),0.77(s,3H)；
009(保留时间:1.198min):MS–ESI m/z:516.2[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.34-11.71(m,1H),11.09-11.30(m,1H),8.76(s,1H),8.70(dd,J＝8.4,2.8Hz,1H),6.62(s,1H),4.92(br t,J＝6.4Hz,2H),2.92-3.12(m,2H),1.38(s,3H),0.77(s,3H)。
实施例10，11，12和13
Figure PCTCN2022125047-appb-000042
合成路线:
Figure PCTCN2022125047-appb-000043
步骤1：化合物010_1的合成
室温下，将化合物005_1(50g,247.28mmol)溶于甲苯(500mL)中，向其中加入丙二腈(16.34g,247.28mmol)，β-氨基丙酸(660.92mg,7.42mmol)和乙酸(14.85g,247.28mmol)，将反应体系升温至130℃搅拌12小时。反应液倒入饱和碳酸氢钠水溶液中(500mL)中，分液。水相用乙酸乙酯(100mL×3)萃取。有机相经饱和食盐水(100mL)洗涤，无水硫酸钠干燥后过滤浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–5/1,体积比)。得到化合物010_1。
步骤2：化合物010_2的合成
将化合物010_1(7g,27.97mmol)溶解于THF(15mL)中，置换氮气三次，反应体系降温到0℃，缓慢滴加甲基溴化镁的乙醚溶液(3M,13.99mL)，反应体系在0℃下搅拌15分钟。将反应体系倒入饱和氯化铵溶液(100mL)，加入乙酸乙酯(50mL×3)萃取，合并有机相。有机相经半饱和食盐水(10mL×2)洗涤，饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–5/1,体积比)。得到化合物010_2。
步骤3：化合物010_3和012_1的混合物的合成
将001_4(3.5g,9.68mmol,盐酸盐)溶解于叔丁醇(50mL)中，向其中加入化合物010_2(4.4g,16.52mmol)和碳酸氢钾(2.42g,24.19mmol)，反应体系升温至85℃搅拌12小时。反应液倒入水(100mL)中， 乙酸乙酯(50mL×3)萃取。有机相经饱和食盐水(50mL)洗涤，无水硫酸钠干燥后过滤浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–1/1,体积比)。得到化合物010_3和012_1的混合物。
步骤4：化合物010_4和010_6的合成
将上述混合物(800.00mg,1.47mmol)溶解于甲苯(10mL)中，置换氮气三次，向反应体系中加入三甲基铝的甲苯溶液(2M,2.20mL)，反应体系升温到75℃搅拌5小时。反应完毕后，将反应体系降温到室温，向反应体系中加入稀盐酸(1N 10mL)淬灭反应，加乙酸乙酯(50mL)稀释，分液，收集有机相，水相用乙酸乙酯(50mL×3)萃取，合并有机相。有机相经无水硫酸钠干燥，过滤，滤液减压浓缩除去溶剂得到粗产物。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–2/1,体积比)和制备HPLC分离(柱型:Phenomenex luna C18(250mm*50mm I.D.,10μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％:30％-60％,10min)得到化合物010_4和010_6。
LCMS分析方法：柱型:Luna 5μm C18(2*50mm)；流动相A:H 2O+0.05％(v/v)TFA；流动相B:ACN+0.05％(v/v)TFA，梯度：B％：10％-100％,6min。
010_4(保留时间:2.789min)MS–ESI m/z:500.1[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.55(s,1H),11.24(s,1H),8.75(dd,J＝1.6,2.8Hz,1H),8.70(dd,J＝2.8,8.4Hz,1H),4.91(t,J＝6.8Hz,2H),3.00(tt,J＝6.4,19.2Hz,2H),2.80(q,J＝7.2Hz,1H),1.41(s,3H),0.79(d,J＝7.2Hz,3H)；
010_6(保留时间：2.848min)MS–ESI m/z:500.0[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.42(s,1H),11.09(s,1H),8.82–8.65(m,2H),4.91(t,J＝6.6Hz,2H),3.09–2.89(m,3H),1.25(t,J＝3.4Hz,6H)。
步骤5：化合物010和011的合成
将化合物010_4(保留时间:2.789min)(720mg,1.27mmol)经手性柱分离(柱型:DAICEL CHIRALPAK IE(250mm*30mm,10μm)；流动相:A：CO 2，B：[EtOH(含0.1％NH 3H 2O)]；梯度：B％:38％-38％,6min)。得到化合物010和011。
SFC分析方法：柱型:Chiralpak AD-3(50mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[EtOH(含0.1％IPAm)]；梯度：B％:5％-50％,3min。
化合物010(保留时间:1.00min):MS–ESI m/z:500.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.41(br s,1H),11.09(br s,1H),8.82–8.59(m,2H),4.90(t,J＝6.8Hz,2H),3.13–2.87(m,3H),1.25(t,J＝3.2Hz,6H)；
化合物011(保留时间:1.311min):MS–ESI m/z:500.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.42(s,1H),11.09(s,1H),8.75(dd,J＝1.6,2.8Hz,1H),8.71(dd,J＝2.8,8.6Hz,1H),4.91(t,J＝6.8Hz,2H),3.13–2.91(m,3H),1.31–1.19(m,6H)。
步骤6：化合物012和013的合成
将化合物010_6(保留时间：2.848min)(270mg,540.68μmol)经手性柱分离(柱型:DAICEL  CHIRALPAK IE(250mm*30mm,10μm)；流动相：A：CO 2，B：[EtOH(含0.1％NH 3H 2O)]；梯度：B％:33％-33％,7min)。得到化合物012和013。
SFC分析方法：柱型:Chiralpak IE-3(50mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[EtOH(含0.1％IPAm)]，梯度：B％:5％-50％,3min。
化合物012(保留时间:1.087min):MS–ESI m/z:499.9[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.54(br s,1H),11.23(s,1H),8.75(dd,J＝1.6,2.8Hz,1H),8.70(dd,J＝2.8,8.8Hz,1H),4.91(t,J＝6.8Hz,2H),3.00(tt,J＝6.4,19.1Hz,2H),2.85–2.76(m,1H),1.41(s,3H),0.80(d,J＝7.2Hz,3H)；
化合物013(保留时间:1.193min):MS–ESI m/z:499.9[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.53(br s,1H),11.23(br s,1H),8.78–8.66(m,2H),4.91(t,J＝6.8Hz,2H),2.99(tt,J＝6.8,19.2Hz,2H),2.80(q,J＝7.2Hz,1H),1.41(s,3H),0.80(d,J＝7.2Hz,3H)。
实施例14和15
Figure PCTCN2022125047-appb-000044
合成路线:
Figure PCTCN2022125047-appb-000045
步骤1：化合物014_1的合成
在室温和氮气保护下，将化合物001_1(300g,1.14mol)加入到N,N-二甲基甲酰胺(3L),随后加入邻氟苄氯(164.91g,1.14mol,135.17mL)和碳酸铯(408.81g,1.25mol)，反应液在80℃搅拌2小时。反应完毕，将反应液降温至室温，过滤，得到化合物014_1(423g)的3L N,N-二甲基甲酰胺溶液，直接使用。
步骤2：化合物014_2的合成
在室温和氮气保护下，将化合物014_1(70g,188.62mmol)加入到1L N,N-二甲基甲酰胺溶液，加入甲醇(300mL)，加入环戊–2,4–二烯–1–基(二苯基)膦二氯甲烷二氯钯二茂铁(6.90g,9.43mmol)和三乙胺(76.34g,754.47mmol,105.01mL)，在一氧化碳(15psi)氛围下80℃搅拌12小时。反应完毕，将反应液减压浓缩，得到残余物。残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–15/1,体积比)得到化合物014_2。
步骤3：化合物014_3的盐酸盐的合成
在室温和氮气保护下，将氯化铵(4.41g,82.44mmol)分散于甲苯(50mL)中，加入三甲基铝甲苯溶液(2M,39.57mL)，加热至80℃然后将化合物014_2(5g,16.49mmol)加入反应体系，于80℃搅拌30分钟,然后升温至110℃反应1.5小时，降温至25℃滴加甲醇(7.61g,237.42mmol)，保持温度不高于40℃，然后再加入盐酸(3M,105.52mL)，保持温度不高于40℃，升温至80℃搅拌10分钟，然后降温至0℃搅拌30分钟。反应完毕，过滤收集滤饼。滤饼用水(100mL)淋洗，得到化合物014_3的盐酸盐。
步骤4：化合物014_4和014_5的合成
在室温和氮气保护下，将化合物005_4(6.58g,24.71mmol)加入叔丁醇(70mL)中，依次加入化合物014_3的盐酸盐(5g,15.45mmol)和碳酸钾(5.34g,38.61mmol)，反应液在85℃下搅拌12小时。反应完毕，将反应液降温至室温，加入水(100mL)，用二甲基四氢呋喃(100mL×2)萃取，合并有机相，用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，得到化合物014_4和014_5的混合物。
步骤5：化合物014_6的合成
在室温和氮气保护下，将化合物014_4和014_5的混合物(7g,13.79mmol)，加入甲苯(100mL)溶解。缓慢滴加三甲基铝的甲苯溶液(2M,28.97mL)，在80℃下搅拌12小时。将反应液降温至室温，缓慢倒入到水(100mL)中，加入3M的盐酸，将pH调至5-6，加入二甲基四氢呋喃(100mL×3)萃取，合并有机相。有机相经无水硫酸钠干燥，过滤减压浓缩，得到残余物。残余物加入甲基叔丁基醚(50mL)，在25℃下搅拌1小时过滤，得到化合物014_6。
步骤5：化合物014_7的合成
在室温和氮气保护下将化合物014_6(4.24g,9.19mmol)加入甲醇(80mL)溶解，加入双(三氟乙酸)碘代苯(7.90g,18.38mmol)，在50℃下搅拌12小时。反应完毕，将反应液降温至室温，加硅藻土过滤，得到滤液，减压浓缩，得到残余物。残余物经制备HPLC分离(柱型:Welch Xtimate C18(250mm*70mm I.D.,10μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％:35％-70％,20min)得到化合物014_7。MS–ESI m/z:492.0[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.81(br s,1H),11.26–11.39(m,1H),8.77(d,J＝1.00Hz,1H),8.71(dd,J＝8.8,2.8Hz,1H),7.35–7.43(m,1H),7.27–7.32(m,1H),7.20–7.26(m,1H),7.14–.20(m,1H),5.86(s,2H),3.14(s,3H),1.34(s,3H),0.77(s,3H)。
步骤6：化合物014和015的合成
将化合物014_7(200mg,406.96μmol)手性柱分离(柱型:REGIS(s,s)WHELK-O1(250mm*30mm,5μm)；流动相:[0.1％NH 3H 2O MeOH]％:32％-32％,16.5min)，得到化合物014和015。
SFC分析方法：柱型:Chiralpak IE-3(50mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[EtOH(含0.1％IPAm)]，梯度：B％:5％-50％,3min。
014(保留时间:2.612min)MS–ESI m/z:492.0[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.73–11.96(m,1H),11.31(s,1H),8.77(dd,J＝2.8,1.6Hz,1H),8.71(dd,J＝8.8,2.8Hz,1H),7.35–7.42(m,1H),7.27–7.32(m,1H),7.20–7.26(m,1H),7.14–7.20(m,1H),5.86(s,2H),3.14(s,3H),1.34(s,3H),0.77(s,3H)；
015(保留时间:2.749min)MS–ESI m/z:492.0[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.75–11.89(m,1H),11.28–11.36(m,1H),8.77(d,J＝2.63Hz,1H),8.71(dd,J＝8.66,2.76Hz,1H),7.35–7.42(m,1H),7.27–7.32(m,1H),7.20–7.26(m,1H),7.14–7.20(m,1H),5.86(s,2H),3.14(s,3H),1.34(s,3H),0.77(s,3H)。
实施例16
Figure PCTCN2022125047-appb-000046
合成路线:
Figure PCTCN2022125047-appb-000047
步骤1：化合物016的合成
室温和氮气保护下，将化合物014_6(500mg,1.08mmol)溶解于THF(5mL)和H 2O(5mL)中，加入单过硫酸氢钾(1.33g,2.17mmol)，磷酸二氢钾(294.94mg,2.17mmol)和溴化亚铜(31.09mg,216.72μmol)，反应液在80℃下搅拌2小时。反应完毕，向反应液中加入水(10mL)，乙酸乙酯(10mL×2)分液，合并有机相。有机相经无水硫酸钠干燥，过滤，减压浓缩，得到残余物。残余物经制备HPLC分离(Waters Xbridge Prep OBD C18(150mm*40mm I.D.,10μm)；流动相：A：ACN，B：[H 2O(含10mmol NH 4HCO 3+0.05％NH 3.H 2O)]，梯度：B％:40％-60％,8min)得到化合物016。MS–ESI m/z:478.2[M+H] +。 1H NMR(400MHz,DMSO-d 6)δ11.38–11.54(m,1H)11.17(br s,1H)8.76(s,1H)8.70(dd,J＝8.4,2.8Hz,1H)7.33–7.43(m,1H)7.20–7.31(m,2H)7.15–7.20(m,1H)6.61(s,1H)5.85(d,J＝3.2Hz,2H)1.37(s,3H)0.75(s,3H)。
实施例17、18、19和20
Figure PCTCN2022125047-appb-000048
合成路线:
Figure PCTCN2022125047-appb-000049
步骤1：化合物017_1和017_2的合成
室温和氮气保护下，将中间体014_3的盐酸盐(2.76g)加入到叔丁醇(40mL)中，之后依次加入中间体010_2(2.72g,10.21mmol)和碳酸氢钾(2.13g,21.28mmol)，反应液在80℃下搅拌12小时。反应结束后将反应液降温至室温，加入水(100mL)，二甲基四氢呋喃(100mL×2)分液，合并有机相。有机相用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩。所得残余物经过柱层析分离纯化(洗脱剂:石油醚/乙酸乙酯＝1/0–4/1,体积比)，得到中间体017_1和中间体017_2。
步骤2：化合物017_3和017_4的合成
室温和氮气保护下将中间体017_1(500.00mg,985.24μmol)溶解于甲苯(5mL)中，向反应体系中加入AlMe 3(2M,1.48mL)，反应体系升温到75℃下搅拌12小时。反应完毕后，将反应体系降温到室温。将反应液倒入水(50mL)，加入稀盐酸(1N)调pH 3～4，加2-甲基四氢呋喃(50mL)分液，收集有机相。水相用2-甲基四氢呋喃(20mL×3)萃取，合并有机相。有机相经无水硫酸钠干燥，过滤，滤液减压浓缩除去 溶剂得到粗产物。所得残余物经过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–3/1,体积比)和制备HPLC分离(柱型:Phenomenex luna C18(80mm*40mm I.D.,3μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％:33％-49％,7min)得到化合物017_3。
室温和氮气保护下，将中间体017_2(700.00mg,1.38mmol)溶解于甲苯(10mL)中，置换氮气三次，向反应体系中加入AlMe 3(2M,2.07mL)，反应体系升温到75℃下搅拌12小时。反应完毕后，将反应体系降温到室温。将反应体系倒入水(100mL)，加入稀盐酸(1N)调pH 3～4，加2-甲基四氢呋喃(50mL)稀释，分液，收集有机相。水相用2-甲基四氢呋喃(50mL×3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩除去溶剂得到粗产物。所得残余物依次经过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–3/1,体积比)和制备HPLC分离(流动相:乙腈/水；盐酸体系:0.04％HCl)和制备HPLC分离(柱型:Phenomenex luna C18(75mm*30mm I.D.,3μm)；流动相：A：ACN，B：[H 2O(含10mmol NH 4HCO 3+0.05％NH 3.H 2O)]，梯度：B％:35％-55％,8min)，得到化合物017_4。
HPLC分析方法：柱型:Kinetex 5μm C18(2.1*50mm)；流动相A:H 2O+0.04％(v/v)TFA；流动相B:ACN+0.02％(v/v)TFA，梯度：B％：10％-80％,6min。
017_3(保留时间:3.482min)MS–ESI m/z:462.2[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ11.53(s,1H),11.22(s,1H),8.81–8.62(m,2H),7.42–7.33(m,1H),7.30–7.14(m,3H),5.85(s,2H),2.78(q,J＝7.2Hz,1H),1.39(s,3H),0.78(d,J＝7.4Hz,3H)。
017_4(保留时间:3.574min)MS–ESI m/z:462.2[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ11.14–10.77(m,2H),8.76–8.73(m,1H),8.72–8.67(m,1H),7.41–7.34(m,1H),7.28–7.20(m,2H),7.19–7.13(m,1H),5.84(s,2H),3.09–2.99(m,1H),1.28–1.19(m,6H)。
步骤3：化合物017和018的合成
将化合物017_3(30mg,65.02μmol)经手性柱分离(柱型:DAICEL CHIRALPAK IE(250mm*30mm,10μm)；流动相:[0.1％NH 3H 2O MeOH；20％-45％,18min)。得到化合物017和018。
SFC分析方法：柱型:Chiralpak AS-3(50mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[MeOH(含0.1％IPAm)]；梯度：B％:5％-50％,3min。
化合物017(保留时间:1.138min):MS–ESI m/z:462.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ11.65–11.40(m,1H),11.23(br s,1H),8.81–8.61(m,2H),7.43–7.34(m,1H),7.31–7.14(m,3H),5.85(s,2H),2.79(d,J＝7.4Hz,1H),1.40(s,3H),0.78(d,J＝7.4Hz,3H)；
化合物018(保留时间:1.422min):MS–ESI m/z:462.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ11.71–11.01(m,2H),8.83–8.64(m,2H),7.42–7.35(m,1H),7.31–7.13(m,3H),5.85(s,2H),2.78(q,J＝7.3Hz,1H),1.39(s,3H),0.78(d,J＝7.4Hz,3H)。
步骤4：化合物019和020的合成
将化合物017_4(150mg,325.08μmol)经手性柱分离(柱型:DAICEL CHIRALPAK IE(250mm*30mm,10μm)；流动相：[0.1％NH 3H 2O EtOH]；梯度：B％:50％,12min)，得到化合物019和020。
SFC分析方法：柱型:(S,S)-WHELK-O1(50mm*4.6mm I.D.,3.5μm)；流动相：A：CO 2，B：[EtOH(含0.1％IPAm)]，梯度：B％:5％-50％,3min。
化合物019(保留时间:1.669min):MS–ESI m/z:462.31[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.38(br s,1H),11.07(s,1H),8.77–8.66(m,2H),7.41–7.32(m,1H),7.29–7.13(m,3H),5.84(s,2H),3.04(q,J＝7.1Hz,1H),1.27–1.20(m,6H)；
化合物020(保留时间:1.808min):MS–ESI m/z:462.34[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.38(br s,1H),11.08(br s,1H),8.76–8.65(m,2H),7.37(q,J＝7.1Hz,1H),7.28–7.12(m,3H),5.84(s,2H),3.04(q,J＝6.9Hz,1H),1.29–1.17(m,6H)。
实施例21和22
Figure PCTCN2022125047-appb-000050
合成路线:
Figure PCTCN2022125047-appb-000051
步骤1：化合物021_1的合成
室温和氮气保护下，将化合物001_11(9.04g,36.14mmol)加入叔丁醇(100mL)，随后加入化合物014_3的盐酸盐(5g)和碳酸钾(3.87g,38.61mmol)，反应液在80℃下搅拌12小时。反应完毕，向反应液中加入水(500mL)，用乙酸乙酯(200mL×3)萃取，合并有机相。有机相用饱和食盐水(100mL×3)洗涤，无水硫酸钠干燥，过滤，减压浓缩，得到残余物。残余物加入甲基叔丁基醚(70mL)室温打浆，过滤，收集滤饼，滤饼减压浓缩，得到化合物021_1。
步骤2：化合物021_2的合成
室温和氮气保护下，将化合物021_1(4.5g,8.90mmol)溶解于无水甲苯中(130mL)，缓慢滴加三甲基铝甲苯溶液(2M,13.35mL)，反应体系在75℃下搅拌12小时。反应完毕，将反应体系降温至室温后缓慢倒入水(100mL)中,加入3M盐酸将pH调至3～4，加入二甲基四氢呋喃(100mL×3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩得到残余物。残余物经制备HPLC分离(柱型:Phenomenex C18(250mm*70mm I.D.,10μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％:40％-70％,20min)得到化合物021_2。 1H NMR(400MHz,DMSO-d 6)δ:11.50(s,1H),11.28(s,1H),8.78–8.68(m,2H),7.44–7.34(m,1H),7.30–7.19(m,2H),7.20–7.12(m,1H),5.85(s,2H),1.58–1.49(m,1H),1.44(s,3H),1.18–1.08(m,1H),0.82–0.62(m,2H).
步骤3：化合物021和022的合成
将化合物021_2(200mg,422.45μmol)手性柱分离(柱型:DAICEL CHIRALPAK AD(250mm*30mm,10μm)；流动相:[0.1％NH 3H 2O EtOH]％:40％-40％,8min)，得到化合物021和022。
SFC分析方法：柱型:Chiralpak AD-3(50mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[EtOH(含0.1％IPAm)]，梯度：B％:5％-50％,3min。
021(保留时间:1.253min)MS–ESI m/z:474.2.0[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.50(s,1H),11.28(s,1H),8.78(s,2H),7.43–7.32(m,1H),7.30–7.20(m,2H),7.19–7.13(m,1H),5.85(s,2H),1.58–1.50(m,1H),1.43(s,3H),1.15–1.10(m,1H)0.77–0.67(m,2H)；
022(保留时间:1.367min)MS–ESI m/z:474.2.0[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.54–11.46(m,1H),11.28(s,1H),8.78–8.70(m,2H),7.44–7.34(m,1H),7.30–7.20(m,2H),7.20–7.14(m,1H),5.86(s,2H),1.60–1.50(m,1H),1.44(s,3H),1.13(ddd,J＝9.2,7.2,4.0Hz,1H)0.80–0.66(m,2H)。
实施例23
Figure PCTCN2022125047-appb-000052
合成路线:
Figure PCTCN2022125047-appb-000053
步骤1：化合物023_1的合成
室温和氮气保护下，将化合物014_3的盐酸盐(10g,30.89mmol)加入叔丁醇(150mL)，随后加入化合物003_1(14.59g,61.78mmol)和碳酸钾(7.73g,77.23mmol)，反应液在80℃下搅拌12小时。反应完毕，向反应液中加入水(500mL)，用乙酸乙酯(200mL×3)萃取，合并有机相。有机相用饱和食盐水(100mL×3)洗涤，无水硫酸钠干燥，过滤，减压浓缩，得到残余物。残余物加入甲基叔丁基醚(50mL)打浆，过滤，收集滤饼，滤饼减压浓缩，得到化合物023_1。
步骤2：化合物023_2的合成
室温和氮气保护下，将化合物023_1的混合物(5g,10.17mmol)加入无水甲醇(100mL)，随后加入双(三氟乙酸)碘代苯(10.94g,25.43mmol)，反应液在50℃下搅拌12小时。反应完毕,反应液减压浓缩后用乙酸乙酯(100mL)稀释，经饱和亚硫酸钠(50mL×3)洗涤，有机相经无水硫酸钠干燥后过滤，减压浓缩得到残余物。残余物经制备HPLC分离(柱型:Phenomenex luna C18(250mm*100mm I.D.,15μm)；流动相：A：ACN，B：[H 2O(含0.1％TFA)]，梯度：B％:35％-65％,20min)得到化合物023_2。
步骤3：化合物023的合成
室温和氮气保护下，将化合物023_2(0.8g,1.53mmol)溶解于无水甲苯(15mL)中，缓慢滴加三甲基铝甲苯溶液(2M,2.45mL)，加料完毕，反应液在100℃下搅拌12小时。反应完毕，将反应体系降温到室温，缓慢倒入到水(20mL)中,加入3M盐酸将pH调至3～4，加二甲基四氢呋喃(20mL×3)萃取，合并有机相。有机相用饱和食盐水(20mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到残余物。所得残余物经过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–3/1,体积比)和制备HPLC分离(柱型:Phenomenex luna C18(80mm*40mm I.D.,3μm)；流动相：A：ACN，B：[H 2O(含0.04％HCl)]，梯度：B％: 45％-65％,7min))得到化合物023。MS–ESI m/z:490.0[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ:11.75(br s,1H),11.46(s,1H),8.82–8.68(m,2H),7.44–7.34(m,1H),7.32–7.14(m,3H),5.80–5.94(m,2H),3.17(s,3H),1.58–1.68(m,1H),1.32–1.44(m,1H),0.82–0.98(m,2H).
实施例24和25
Figure PCTCN2022125047-appb-000054
合成路线:
Figure PCTCN2022125047-appb-000055
步骤1：化合物024_1的合成
在室温和氮气保护下，将化合物010_1(10g,39.96mmol)加入到氯仿(40mL)和乙醇(40mL)中,随后加入2,6-二甲基-1,4-二氢-3,5-吡啶二羧酸二乙酯(15.18g,59.94mmol)，反应液在25℃搅拌12小时。反应完毕，将反应液过滤，滤液减压浓缩得到残余物。所得残余物经过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–4/1,体积比)得到化合物024_1。
步骤2：化合物024_2的合成
在室温和氮气保护下，将中间体001_4的盐酸盐(8.5g)溶解于叔丁醇(150mL)中，加入中间体024_1(9.49g,37.60mmol)和碳酸氢钾(5.88g,58.75mmol)。反应液在90℃下搅拌12小时。反应结束后，将反应液加入水(500mL)中，有固体析出。混合液过滤，用甲叔醚(20mL)洗涤滤饼，收集滤饼，减压干燥。得到化合物024_2和化合物024_3的混合物。
步骤3：化合物024_4的合成
在室温和氮气保护下，将化合物024_2和化合物024_3(5g,9.41mmol)分散于无水甲醇(100mL)中加入双三氟乙酰氧基碘苯(12.14g,28.23mmol)。反应液在50℃油浴中搅拌2小时。反应结束后降至室温，浓缩除去溶剂得到油状剩余物。异丙醚(100mL)加入该剩余物中，室温搅拌30min后有白色固体析出，过滤，滤液减压浓缩得到粗品。残余物通过柱层析分离(洗脱剂:石油醚/2-甲基四氢呋喃＝1/0–1/1,体积比)纯化，得到中间体024_4。
步骤4：化合物024_5的合成
在室温和氮气保护下，将化合物024_4(0.3g,534.34μmol)溶于无水甲苯(6mL)加入三甲基铝甲苯溶液(2M,854.95μL)。反应体系在50℃下搅拌12小时，升温至90℃搅拌12小时。反应结束后降至室温。将反应液倒入水(100mL)中，加入稀盐酸(1N)至pH 3～4,乙酸乙酯(50mL×3)萃取。有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩得到粗品。残余物经制备HPLC分离(柱型:Phenomenex C18(75mm*30mm I.D.,3μm)；流动相：A：ACN，B：[H 2O(含10mmol NH 4HCO 3)]，梯度：B％:35％-55％,8min),得到化合物024_5。MS–ESI m/z:516.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ11.95–11.71(m,1H),11.38(br s,1H),8.82–8.66(m,2H),4.93(t,J＝6.6Hz,2H),3.19(s,3H),3.01(tt,J＝6.8,19.1Hz,2H),2.89(q,J＝7.6Hz,1H),0.82(d,J＝7.5Hz,3H)。
步骤5：化合物024和025的合成
将化合物024_5(100mg,153.29μmol)手性柱分离(柱型:DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％NH 3H 2O EtOH]％:10％-10％,12min)，得到化合物024和025。
SFC分析方法：柱型:Chiralpak AS-3(150mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[EtOH(含0.1％IPAm)]，梯度：B％:10％-50％,3min。
024(保留时间:1.496min)MS–ESI m/z:515.9[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.85(br s,1H),11.38(br s,1H),8.77(dd,J＝1.6,2.8Hz,1H),8.71(dd,J＝2.8,8.6Hz,1H),4.93(t,J＝6.6Hz,2H),3.19(s,3H),3.09–2.94(m,2H),2.90(q,J＝7.6Hz,1H),0.95–0.74(m,3H)；
025(保留时间:1.789min)MS–ESI m/z:516.2[M+H] +1H NMR(400MHz,DMSO-d 6)δ12.09–11.16(m,2H),8.88–8.61(m,2H),4.93(t,J＝6.8Hz,2H),3.19(s,3H),3.01(tt,J＝6.6,19.2Hz,2H),2.90(q,J＝7.6Hz,1H),0.82(d,J＝7.6Hz,3H)。
实施例26和27
Figure PCTCN2022125047-appb-000056
合成路线:
Figure PCTCN2022125047-appb-000057
步骤1：化合物026_1的合成
室温和氮气保护下，将化合物024_1(2.99g,11.86mmol)溶解于叔丁醇(150mL)中，加入化合物014_3的盐酸盐(2.4g,7.41mmol)和碳酸氢钾(1.86g,18.53mmol)，在85℃下搅拌12小时。反应结束后，向反应液加入2-甲基四氢呋喃(100mL)和水(100mL)，分液，水相用2-甲基四氢呋喃(100mL×3)萃取，合并有机相。有机相用饱和食盐水(100mL×3)洗涤，无水硫酸钠干燥后过滤，减压浓缩得到中间体026_1和026_2的混合物。
步骤2：化合物026_3的合成
在室温和氮气保护下，将化合物026_1和化合物026_2的混合物(1g,2.03mmol)分散于无水甲醇(20mL)中加入双三氟乙酰氧基碘苯(1.74g,4.05mmol)。反应液在50℃油浴中搅拌12小时。反应结束后降至室温，浓缩除去溶剂得到油状剩余物。残余物通过柱层析分离(洗脱剂:石油醚/乙酸乙酯＝1/0–7/3,体积比)纯化，得到化合物026_3。
步骤3：化合物026_4的合成
在室温和氮气保护下，将化合物026_3(350mg,668.59μmol)溶于无水甲苯(10mL)加入三甲基铝甲苯溶液(2M,1.34mL)。反应体系在75℃下搅拌5小时。反应结束后，降至室温。将反应液倒入水(50mL)中,加入1N稀盐酸至pH 3～4,乙酸乙酯(50mL×3)萃取。有机相经饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤浓缩得到粗品。残余物经制备HPLC分离(柱型:Waters Xbridge BEH C18(100mm*30mm I.D.,10μm)；流动相：A：ACN，B：[H 2O(含10mmol NH 4HCO 3)]，梯度：B％:30％-60％,8min))，得到化合物026_4。
步骤4：化合物026和027的合成
将化合物026_4(50mg,104.73μmol)手性柱分离(柱型:DAICEL CHIRALPAK IC(250mm*30mm,10μm)；流动相:[0.1％NH 3H 2O IPA]％:45％-45％,9min)，得到化合物026和027。
SFC分析方法：柱型:Chiralpak IC-3(50mm*4.6mm I.D.,3μm)；流动相：A：CO 2，B：[IPA(含0.1％IPAm)]，梯度：B％:5％-50％,3min。
026(保留时间:1.369min)MS–ESI m/z:478.2[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.82(br s,1H),11.37(br s,1H),8.77(dd,J＝1.6,2.4Hz,1H),8.71(dd,J＝2.8,8.8Hz,1H),7.41-7.36(m,1H),7.31-7.16(m,3H),5.86(s,2H),3.17(s,3H),2.88(q,J＝7.6Hz,1H),0.80(d,J＝7.6Hz,3H)；
027(保留时间:1.499min)MS–ESI m/z:478.2[M+H] +1H NMR(400MHz,DMSO-d 6)δ11.82(br s,1H),11.37(br s,1H),8.78-8.69(m,2H),7.41-7.37(m,1H),7.31-7.16(m,3H),5.86(s,2H),3.17(s,3H),2.88(q,J＝7.6Hz,1H),0.80(d,J＝7.6Hz,3H)。
生物测试
实验例1：体外活性测试
一、基于lnCap细胞的cGMP表达测试
1.实验步骤
1)溶液配制
● 10％ BSA(牛血清蛋白)
将10g BSA溶解于100mL双蒸水(ddH 2O)得到10％BSA。
● 5mM DETA(二乙烯三胺)-NO
称取10mg DETA-NO溶解于12.2mL双蒸水(ddH 2O)得到5mM DETA-NO，分装并冻存于-20℃冰箱。
● 洗涤缓冲液(Washing Buffer,50mL)
Figure PCTCN2022125047-appb-000058
● 分析缓冲液(Assay Buffer,50mL)
Figure PCTCN2022125047-appb-000059
● 检测缓冲液(Detection Buffer)
a)将50μL cGMP-D2(D2标记的环单磷鸟苷)加入到1mL裂解液(lysis buffer)混合均匀。
b)将50μL anti-cGMP cryptate(Eu 3+穴状化合物标记的抗环单磷鸟苷抗体)加入到1mL裂解液(lysis buffer)混合均匀。
2)化合物稀释
(1)用DMSO将化合物稀释至5mM。转移10μL化合物到Echo用浅孔板中。
(2)用Echo对化合物进行梯度稀释，将每个化合物稀释10个浓度梯度并分别加50nL到384微孔板中。
3)准备LNCap细胞
(1)LNCap培养基：RPMI1640+10％胎牛血清+1％双抗。
(2)将细胞传代过程中用到的磷酸盐缓冲液、胰酶、培养基放到37℃水浴锅中预热。
(3)从37℃5％CO 2培养箱中取出细胞(第14代)，用移液器吸去培养瓶中的旧培养液。
(4)吸取5mL磷酸盐缓冲液加入到培养瓶中漂洗细胞，然后弃去液体。
(5)吸取3mL胰酶加入培养瓶，摇晃后弃去液体，将培养瓶放入培养箱中。
(6)约2分钟后取出培养瓶，观察细胞都已分离后，吸取9mL培养基加入培养瓶并反复吹打几次，将细胞悬液转移至50mL离心管中。
(7)吸取0.7mL细胞悬液加入计数杯，在ViCell XR上计数。剩余细胞，1000rpm离心5min，并去上清。
(8)加入10mL洗涤缓冲液(washing buffer)清洗细胞，1000rpm离心5min，并去上清。
(9)加入分析缓冲液(assay buffer)并调整细胞浓度至1.25×10 6/mL。8μL/孔加入微孔板中。
4)DETA-NO配制和添加
(1)取10μL 5mM的DETA-NO分别加入到1240μL和1657μL分析缓冲液(assay buffer)中，得40μM和30μM的DETA-NO。
(2)用Bravo转移2μL/孔的DETA-NO到384微孔板中。
(3)1500rpm离心5min。将微孔板于37℃孵育30min。
5)准备cGMP标准曲线
(1)将1mM的cGMP存储液用试验缓冲液(assay buffer)稀释至10μM。然后4倍梯度稀释11个浓度梯度。
(2)将稀释好的cGMP加10μL/孔至微孔板中。
6)加检测试剂并读板
(1)用Bravo转移5μL/孔的cGMP-D2到384微孔板中。1500rpm离心1min。
(2)用Bravo转移5μL/孔的anti-cGMP cryptate到384微孔板中。1500rpm离心1min。
(3)常温孵育1h。
(4)用envision读取665/615。
7)数据分析
(1)cGMP标准曲线：根据cGMP的浓度与665/615的比值用Graphpad prism做标准曲线。
(2)HTRF(均相时间分辨荧光技术)比值(665/615)转换成cGMP浓度：在Graphpad prism中，将HTRF比值(665/615)复制到cGMP标准曲线的比值列中，运行分析“Log inhibitor vs response-variable  slope”，选择“interpolate”，将HTRF比值(665/615)转换成cGMP浓度。
(3)化合物激活曲线：根据转换的cGMP浓度与化合物的浓度用Graphpad prism中“Log agonist vs response-variable slope”分析方法做曲线。
本申请化合物对sGC刺激活性的MEC值如表1所示：
表1本申请化合物对sGC刺激活性的MEC值
化合物编号 MEC(nM) 化合物编号 MEC(nM) 化合物编号 MEC(nM)
001 71.4 008 729 024 64.5
002 74.1 010 62.6 025 128.4
003 240 011 56.3 026 9.2
004 673 012 101 027 10.2
006 214 013 140.5    
007 548 016 19.3    
MEC:在lnCap细胞中刺激cGMP产生(大于基础数值三倍)的最小有效浓度。
结论：本申请化合物能够有效刺激sGC，显著提高cGMP水平。
实验例2：体内药代动力学性质研究
实验目的：该研究的目的是为了测定化合物在雄性SD大鼠中的药代动力学参数。
实验材料：
Sprague Dawley大鼠(雄性,200-300g,7～9周龄，上海斯莱克)
实验方法：
该项目使用4只雄性SD大鼠，一组2只SD大鼠进行静脉注射给药，给药剂量为0.3mg/kg，给药浓度0.15mg/mL；另外一组2只SD大鼠进行口服给药，给药剂量为1mg/kg，给药浓度0.2mg/mL；收集给药后0.083(仅静脉组)、0.25、0.5、1、2、4、6、8、12、24h的血浆样品，然后对收集的样品进行LC-MS/MS分析并采集数据。采集的分析数据用Phoenix WinNonlin 6.3软件计算相关药代动力学参数。
实验结果见表2。
表2体内药代动力学实验结果
Figure PCTCN2022125047-appb-000060
Figure PCTCN2022125047-appb-000061
结论：本申请化合物具有较好的表观分布容积和半衰期。
实验例3：组织分布测试：动物体内组织分布研究
实验目的：测试在口服给药的情况下，化合物在血浆，心脏，脑脊液和脑组织中的分布比例。
实验材料：
Sprague Dawley大鼠(雄性,200-300g,7～9周龄，上海斯莱克)
实验方法：
该项目使用6只雄性SD大鼠，进行口服给药，给药剂量为1mg/kg，给药浓度0.2mg/mL；收集给药后2、6、12h的脑脊液，脑，心脏组织和血浆样品，然后对收集的样品进行LC-MS/MS分析并采集数据。采集的分析数据用Phoenix WinNonlin 6.3软件计算相关药代动力学参数。
实验结果见表3。
表3体内组织分布结果
Figure PCTCN2022125047-appb-000062
ND:低于检测限.
结论：本申请化合物没有入脑风险并有较好的心脏分布。

Claims (9)

  1. 式(I)所示化合物、其立体异构体或其药学上可接受的盐，
    Figure PCTCN2022125047-appb-100001
    其中，
    R 1选自H、-OH、C 1-3烷基、C 1-3烷氧基或C 1-3烷氨基；
    R 2选自苄基或C 1-8烷基，所述苄基或C 1-8烷基任选被1、2、3、4或5个卤素原子取代；
    R 3、R 4各自独立地选自H或C 1-3烷基；
    或者R 3和R 4以及它们共同相连的原子形成C 3-6环烷基；
    条件是所述化合物不选自以下结构、其立体异构体或其药学上可接受的盐：
    Figure PCTCN2022125047-appb-100002
  2. 根据权利要求1所述化合物、其立体异构体或其药学上可接受的盐，其中，R 1选自H、-OH、C 1-3烷基或C 1-3烷氧基；任选地，R 1选自H、-OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基或异丙氧基；任选地，R 1选自H、-OH、甲基或甲氧基。
  3. 根据权利要求1所述化合物、其立体异构体或其药学上可接受的盐，其中，R 2选自苄基或C 1-6烷基，所述苄基或C 1-6烷基任选被1、2、3、4或5个卤素原子取代；任选地，R 2选自苄基或C 1-4烷基，所述苄基或C 1-4烷基任选被1、2、3、4或5个卤素原子取代；任选地，R 2选自苄基或C 1-4烷基，所述苄基或C 1-4烷基任选被1、2、3、4或5个F、Cl或Br原子取代；任选地，R 2选自苄基或C 1-4烷基，所述苄基或C 1-4烷基任选被1、2、3、4或5个F原子取代；任选地，R 2选自
    Figure PCTCN2022125047-appb-100003
  4. 根据权利要求1所述化合物、其立体异构体或其药学上可接受的盐，其中，R 3、R 4各自独立地选自H、甲基、乙基、正丙基或异丙基，或者R 3和R 4以及它们共同相连的原子形成环丙烷基；任选地，R 3、R 4各自独立地选自H或甲基，或者R 3和R 4以及它们共同相连的原子形成环丙烷基。
  5. 根据权利要求1所述化合物、其立体异构体或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2022125047-appb-100004
    选自
    Figure PCTCN2022125047-appb-100005
    Figure PCTCN2022125047-appb-100006
  6. 下式化合物、其立体异构体或其药学上可接受的盐：
    Figure PCTCN2022125047-appb-100007
    Figure PCTCN2022125047-appb-100008
  7. 下式化合物、其立体异构体或其药学上可接受的盐：
    Figure PCTCN2022125047-appb-100009
    Figure PCTCN2022125047-appb-100010
    Figure PCTCN2022125047-appb-100011
  8. 一种药物组合物，其包含权利要求1～7任意一项所述的化合物、其立体异构体或其药学上可接受的盐。
  9. 权利要求1～7任意一项所述的化合物、其立体异构体或其药学上可接受的盐、或权利要求8所述的药物组合物在制备治疗sGC激动剂或刺激剂相关疾病的药物上的应用；任选地，所述sGC激动剂或刺激剂相关疾病选自心力衰竭、或高血压。
PCT/CN2022/125047 2021-10-13 2022-10-13 嘧啶三并环衍生物及其在药学上的应用 WO2023061432A1 (zh)

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CN107964018A (zh) * 2016-10-19 2018-04-27 中国人民解放军军事医学科学院毒物药物研究所 取代嘌呤酮类衍生物及其医药用途
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