WO2022253270A1 - 三嗪基甲基环烷基羧酸衍生物及其药物组合物和用途 - Google Patents

三嗪基甲基环烷基羧酸衍生物及其药物组合物和用途 Download PDF

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WO2022253270A1
WO2022253270A1 PCT/CN2022/096597 CN2022096597W WO2022253270A1 WO 2022253270 A1 WO2022253270 A1 WO 2022253270A1 CN 2022096597 W CN2022096597 W CN 2022096597W WO 2022253270 A1 WO2022253270 A1 WO 2022253270A1
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alkyl
alkoxy
halogenated
stereoisomer
pharmaceutically acceptable
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PCT/CN2022/096597
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English (en)
French (fr)
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胡斌
朱经峰
杨文�
谢婧
石晓永
张凯
赵志明
阚超
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上海海雁医药科技有限公司
扬子江药业集团有限公司
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Priority to CN202280038438.1A priority Critical patent/CN117425643A/zh
Priority to EP22815312.8A priority patent/EP4349816A1/en
Publication of WO2022253270A1 publication Critical patent/WO2022253270A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/42—One nitrogen atom
    • C07D251/46—One nitrogen atom with oxygen or sulfur atoms attached to the two other ring carbon atoms

Definitions

  • the invention relates to the technical field of medicine, in particular to a triazinylmethyl cycloalkyl carboxylic acid derivative, a pharmaceutically acceptable salt, a stereoisomer, a pharmaceutical composition and a pharmaceutical application thereof.
  • P2X purinergic receptors are a family of ion channels activated by extracellular adenosine triphosphate (ATP). Purinergic receptors are involved in a variety of biological functions, especially in relation to pain sensitivity.
  • the P2X3 receptor one of the members of this family, was originally cloned from the rat dorsal root ganglion (Chen et al., Nature, vol. 377, pp. 428-431 (1995)).
  • the nucleotide and amino acid sequences of both rat P2X3 and human P2X3 are now known (Lewis et al., Nature, Vol. 377, pp. 432-435 (1995); and Garcia-Guzman et al., Brain Res. Mol. BrainRes., Vol. 47, pp. 59-66 (1997)).
  • P2X3 is involved in the afferent pathway controlling the bladder volume reflex, therefore, inhibition of P2X3 can treat storage and voiding-related disorders such as overactive bladder (Cockayne et al., Nature, Vol. 407, pp. 1011-1015 (2000 )).
  • P2X3 is also selectively expressed on nociceptive neurons, small diameter sensory neurons (ie, neurons stimulated by pain or injury), consistent with its role in pain sensitivity.
  • small diameter sensory neurons ie, neurons stimulated by pain or injury
  • blocking P2X3 receptors has analgesic effects in animal models of chronic inflammatory and neuropathic pain (Jarvis et al., PNAS, 99, 17179-17184 (2002)). Therefore, methods of reducing P2X3 levels or activity can be used to modulate pain perception in painful subjects.
  • P2X3 is also capable of forming P2X2/3 heterodimers with P2X2, another member of the P2X purinergic ligand-gated ion channel family.
  • P2X2/3 is highly expressed on the terminals (central and peripheral) of sensory neurons (Chen et al., Nature, Vol. 377, pp. 428-431 (1995)). Recent findings also suggest that P2X2/3 is predominantly expressed in bladder sensory neurons (more than P2X3) and may play a role in bladder filling and nociceptive sensation (Zhong et al., Neuroscience, Vol. 120, pp. 667- 675 pages (2003)).
  • P2X2/3 receptor ligands especially antagonists.
  • the object of the present invention is to provide novel triazinylmethylcycloalkylcarboxylic acid derivatives, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, pharmaceutical compositions thereof and their use as P2X3 antagonists , this type of compound has higher inhibitory activity on P2X3, lower inhibitory activity on P2X2/3, and has significant inhibitory selectivity.
  • the first aspect of the present invention provides a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:
  • r is 1, 2 or 3;
  • W is a benzene ring or a 5- to 6-membered heteroaryl ring (preferably a pyridine ring); the benzene ring and a 5- to 6-membered heteroaryl ring (preferably a pyridine ring) are unsubstituted or replaced by or 4 substituents independently selected from the following group: deuterium, halogen (preferably fluorine, chlorine), cyano, hydroxyl, carboxyl, C 1-8 alkyl (preferably C 1-6 alkyl, more Preferably C 1-3 alkyl), C 1-8 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), C 2-4 alkenyl, C 2- 4 alkynyl, halogenated C 1-8 alkyl (preferably halogenated C 1-6 alkyl, more preferably halogenated C 1-3 alkyl), cyano substituted C 1-8 alkyl (preferably cyano C 1-6 alkyl, more preferably cyano substituted C 1-3 al
  • R a represents that the hydrogen on the benzene ring is replaced by n R a , n is 0, 1 or 2; each R a is the same or different and is independently cyano, hydroxyl, carboxyl, halogen (preferably fluorine or chlorine), C 1-8 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), halogen substituted C 1-8 alkyl (preferably halogen substituted C 1-6 alkyl , more preferably halogen substituted C 1-3 alkyl), cyano substituted C 1-8 alkyl (preferably cyano substituted C 1-6 alkyl, more preferably cyano substituted C 1-3 alkyl), C 1-8 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), -C (O) C 1-8 alkyl (preferably -C (O) C 1-6 alkyl, more preferably -C(O)C 1-3 alkyl), -C(O)OC 1
  • R c is hydrogen, C 1-8 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), C 3-8 cycloalkyl (preferably C 3-6 cycloalkyl) , C 1-8 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), cyano, hydroxyl, carboxyl or halogen (preferably fluorine or chlorine);
  • R b represents that the hydrogen on the benzene ring is replaced by m R b , m is 0, 1 or 2; each R b is the same or different and is independently cyano, hydroxyl, carboxyl, halogen (preferably fluorine or chlorine), C 1-8 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), halogenated C 1-8 alkyl (preferably halogenated C 1-6 alkyl , more preferably halogenated C 1-3 alkyl), cyano substituted C 1-8 alkyl (preferably cyano substituted C 1-6 alkyl, more preferably cyano substituted C 1-3 alkyl), C 1-8 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), -C (O) C 1-8 alkyl (preferably -C (O) C 1-6 alkyl, more preferably -C(O)C 1-3 alkyl), -C(O)OC 1-8 alky
  • R 0 is hydrogen or C 1-6 alkyl
  • R a1 and R b1 are each independently hydrogen or C 1-3 alkyl; or R a1 and R b1 form a 4 to 6-membered saturated monoheterocycle together with the connected nitrogen atom; the 4 to 6 member saturated monoheterocycle is unsubstituted or substituted by 1, 2 or 3 substituents each independently selected from the group consisting of deuterium, halogen, cyano, hydroxyl, carboxyl, C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-3 alkyl, halogenated C 1-3 alkoxy, -SO 2 C 1-3 alkyl, -S(O)C 1 -3 Alkyl, -C(O)NH 2 , -C(O)NH(C 1-3 Alkyl), -C(O)N(C 1-3 Alkyl) 2 , -C(O)OC 1-3 alkyl, -OC(O)C
  • R a0 and R b0 are each independently hydrogen, C 1-3 alkyl or acetyl; or R a0 and R b0 form a 4 to 6-membered saturated monoheterocyclic ring together with the connected nitrogen atom; the 4 to 6-membered saturated
  • the single heterocycle is unsubstituted or substituted by 1, 2 or 3 substituents each independently selected from the group consisting of deuterium, halogen, cyano, hydroxyl, carboxyl, C 1-3 alkyl, C 1-3 alkane Oxygen, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-3 alkyl, halogenated C 1-3 alkoxy, -SO 2 C 1-3 alkyl, -S(O )C 1-3 alkyl, -C(O)NH 2 , -C(O)NH(C 1-3 alkyl), -C(O)N(C 1-3 alkyl) 2 , -C( O)
  • r is 1.
  • W is (R d ) u means that the hydrogen on the pyridine ring is replaced by u R d , u is 0, 1 or 2; each R d is the same or different and is independently deuterium, halogen (preferably fluorine, chlorine), cyanide radical, hydroxyl, carboxyl, C 1-8 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), C 1-8 alkoxy (preferably C 1-6 alkoxy , more preferably C 1-3 alkoxy), C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-8 alkyl (preferably halogenated C 1-6 alkyl, more preferably halogenated C 1-3 alkyl), halogenated C 1-8 alkoxy (preferably halogenated C 1-6 alkoxy, more preferably halogenated C 1-3 alkoxy), NR a0 R b0 , -SO 2 C 1-3 alkyl, -S(O)C
  • u is 0 or 1;
  • Rd is fluorine, chlorine, cyano, hydroxyl, carboxyl, C 1-3 alkyl (preferably methyl, ethyl, n-propyl, isopropyl), C 1-3 alkoxy (preferably methoxy), halogenated C 1-3 alkyl (preferably monofluoromethyl, difluoromethyl, trifluoromethyl), halogenated C 1-3 alkoxy (preferably monofluoromethoxy, difluoromethoxy, trifluoromethoxy), NR a0 R b0 , -SO 2 C 1-3 alkyl, -S(O)C 1-3 alkyl, - C(O)NR a1 R b1 , -C(O)C 1-3 alkyl, -C(O)OC 1-3 alkyl or -OC(O)C 1-3 alkyl.
  • R d contains an electron-withdrawing group.
  • Rd is an electron withdrawing group
  • R d is a C 1-8 alkyl group substituted by an electron-withdrawing group; further, R d is a C 1-8 alkyl group substituted by a halogen.
  • u is 0 or 1
  • R d is halogen (preferably fluorine) or halogenated C 1-3 alkyl (preferably monofluoromethyl, difluoromethyl, trifluoromethyl); further Alternatively, Rd is fluorine.
  • W is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • W is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • m is 0 or 1;
  • R b is cyano, hydroxyl, carboxyl, fluorine, chlorine, C 1-3 alkyl, C 1-3 alkoxy, -C(O)C 1-3 Alkyl, -C(O)OC 1-3 alkyl, -OC(O)C 1-3 alkyl or -C(O)NR a1 R b1 .
  • R b is an electron withdrawing group.
  • m is 1, R b is halogen or cyano; further, R b is chlorine.
  • m is 1 and R b is a para substitution.
  • n is 0 or 1;
  • Ra is cyano, hydroxyl, carboxyl, fluorine, chlorine, C 1-3 alkyl, C 1-3 alkoxy, -C(O)C 1-3 Alkyl, -C(O)OC 1-3 alkyl, -OC(O)C 1-3 alkyl or -C(O)NR a1 R b1 .
  • n 0.
  • R is hydrogen, C 1-3 alkyl, C 3-6 cycloalkyl, C 1-3 alkoxy, cyano, hydroxyl, carboxyl, fluorine, chlorine, -C(O) NR a1 R b1 , -C(O)C 1-3 alkyl or -C(O)OC 1-3 alkyl.
  • R c is hydrogen
  • R 0 is hydrogen
  • R 0 is C 1-6 alkyl; further R 0 is C 1-4 alkyl.
  • the formula (I) is the structure shown in the formula (II):
  • R a , R c , R d , n, u are as defined above.
  • formula (I) is the structure shown in formula (II-1):
  • R a , R c , R d , n, u are as defined above.
  • formula (I) is the structure shown in formula (II-2):
  • R a , R c , R d , n, u are as defined above.
  • formula (I) is the structure shown in formula (II-3):
  • R a , R c , R d , n, u are as defined above.
  • formula (I) is the structure shown in formula (II-4):
  • R a , R c , R d , n, u are as defined above.
  • the compound of formula (I) is selected from specific compounds in the embodiments.
  • the compound of formula (I) is a compound selected from Table 1 or a stereoisomer thereof:
  • the compound of formula (I) is a compound selected from Table 2:
  • the compound of formula (I) is a compound selected from Table 3:
  • the second aspect of the present invention provides a pharmaceutical composition, comprising the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; and a pharmaceutically acceptable carrier.
  • the third aspect of the present invention provides a compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the preparation of a drug for treating diseases related to P2X3 activity or P2X2/3 activity application.
  • the fourth aspect of the present invention provides the application of the pharmaceutical composition described in the second aspect in the preparation of a medicament for treating diseases related to P2X3 activity or P2X2/3 activity.
  • the fifth aspect of the present invention provides a method for treating diseases related to P2X3 activity or P2X2/3 activity, comprising administering to a patient a therapeutically effective amount of the compound described in the first aspect of the present invention, or a pharmaceutically acceptable A salt, or a stereoisomer thereof, or the composition described in the second aspect of the present invention.
  • the disease associated with P2X3 activity or P2X2/3 activity is pain, urinary tract disorders, gastrointestinal diseases, cancer, immune-related diseases, cough, depression, anxiety or stress-related disorders .
  • the diseases related to P2X3 activity or P2X2/3 activity are P2X3 or P2X2/3 mediated pain, urinary tract disorders, gastrointestinal diseases, cancer, immune-related diseases, cough, depression, anxiety or stress-related disorders.
  • Fig. 1 is the single crystal structure figure of the carboxylate of intermediate V7;
  • Figure 2 shows the effect of different treatment groups on the threshold of paw withdrawal 2 hours after administration;
  • * and *** represent respectively P ⁇ 0.05 and P ⁇ 0.001 between the test compound group and the vehicle group;
  • Figure 3 shows the effect of different treatment groups on the paw withdrawal threshold 4 hours after administration; in Figure 3, ** indicates that the test compound group is P ⁇ 0.01 compared with the vehicle group.
  • this class of triazinylmethylcycloalkylcarboxylic acid derivatives has significant P2X3 inhibitory activity and lower P2X2/3 inhibitory activity, and has greater safety window. Therefore, this series of compounds is expected to be developed into drugs for regulating P2X3 and/or P2X2/3 to treat various diseases mediated by P2X3 and/or P2X2/3 (or otherwise related to P2X3 and/or P2X2/3) , including but not limited to exerting a good analgesic effect. Furthermore, the inventors also found that in the triazinylmethylcycloalkylcarboxylic acid derivatives, there are obvious activity differences among different stereoisomers.
  • Alkyl refers to straight and branched chain saturated aliphatic hydrocarbon groups.
  • C 1-8 alkyl refers to an alkyl group having 1 to 8 carbon atoms, preferably a C 1-6 alkyl group; more preferably a C 1-3 alkyl group; non-limiting examples of the alkyl group include: Methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethyl propyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-d
  • Cycloalkyl and “cycloalkyl ring” are used interchangeably and both refer to a saturated monocyclic, bicyclic or polycyclic cyclic hydrocarbon group, which may be fused with an aryl or heteroaryl group. Cycloalkyl rings can be optionally substituted. In certain embodiments, cycloalkyl rings contain one or more carbonyl groups, such as oxo groups.
  • C 3-8 cycloalkyl refers to a monocyclic cycloalkyl group with 3 to 8 carbon atoms
  • cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, Cycloheptyl, cyclooctyl, cyclobutanone, cyclopentanone, cyclopentane-1,3-dione, etc.
  • Heterocycloalkyl and “heterocycloalkyl ring” are used interchangeably and both refer to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen and sulfur, which may be combined with an aryl or heteroaryl fused. Heterocycloalkyl rings can be optionally substituted. In certain embodiments, heterocycloalkyl rings contain one or more carbonyl or thiocarbonyl groups, eg, groups comprising oxo and thioxo.
  • 3 to 6 membered heterocycloalkyl refers to a monocyclic cyclic hydrocarbon group having 3 to 6 ring atoms, wherein 1, 2 or 3 ring atoms are heteroatoms selected from nitrogen, oxygen and sulfur, preferably 3 One or two of the ring atoms in the 6-membered heterocycloalkyl group are heteroatoms selected from nitrogen, oxygen and sulfur.
  • Non-limiting examples of monocyclic heterocycloalkyl groups include aziridine, oxiranyl, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyrrolyl , oxazolidinyl, dioxolanyl, piperidinyl, piperazinyl, morpholinyl, dioxanyl, thiomorpholinyl, thiomorpholine-1,1-dioxide, tetra Hydropyranyl, azetidin-2-one, oxetane-2-one, dihydrofuran-2(3H)-one, pyrrolidin-2-one, pyrrolidin- 2,5-diketone, dihydrofuran-2,5-diketone, piperidin-2-one, tetrahydro-2H-pyran-2-one, piperazin-2-one, morphine Lin-3-
  • Heteroaryl and “heteroaryl ring” are used interchangeably and both refer to a monocyclic, bicyclic or polycyclic 4n+2 aromatic ring system having ring carbon atoms and ring heteroatoms (e.g., having A group of shared 6 or 10 ⁇ electrons) where each heteroatom is independently selected from nitrogen, oxygen and sulfur.
  • heteroaryl also includes ring systems in which the aforementioned heteroaryl ring is fused to one or more cycloalkyl rings, heterocycloalkyl rings, cycloalkenyl rings, heterocycloalkenyl rings or aromatic rings. Heteroaryl rings can be optionally substituted.
  • 5 to 6 membered heteroaryl refers to a monocyclic heteroaryl having 5 to 6 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms.
  • Non-limiting examples include thienyl, furyl, thiazolyl, isothiazolyl, imidazolyl, oxazolyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,3-triazolyl, 1,2 ,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, 1,2,3-oxadiazole Base, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, Pyrazinyl, triazinyl,
  • Heteroatom means nitrogen, oxygen or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Halogen means fluorine, chlorine, bromine or iodine.
  • Halo refers to a group in which one or more (eg 1, 2, 3, 4 or 5) hydrogens are replaced by a halogen.
  • Haloalkyl refers to an alkyl group substituted by one or more (eg 1, 2, 3, 4 or 5) halogens, wherein alkyl is as defined above. It is preferably a halogenated C 1-8 alkyl group, more preferably a halogenated C 1-6 alkyl group, and more preferably a halogenated C 1-3 alkyl group.
  • haloalkyl examples include, but are not limited to, monochloromethyl, dichloromethyl, trichloromethyl, monochloroethyl, 1,2-dichloroethyl, trichloroethyl, monobromoethyl, monochloroethyl, Fluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, etc.
  • Deuterated alkyl refers to an alkyl group substituted by one or more (eg 1, 2, 3, 4 or 5) deuterium atoms, wherein alkyl is as defined above.
  • deuterated C 1-8 alkyl more preferably deuterated C 1-6 alkyl, more preferably deuterated C 1-3 alkyl.
  • deuteroalkyl include, but are not limited to, monodeuteromethyl, monodeuteroethyl, dideuuteromethyl, diduterioethyl, trideuteromethyl, trideuteroethyl, and the like.
  • Alkoxy means -O-alkyl, wherein alkyl is as defined above. It is preferably C 1-8 alkoxy, more preferably C 1-6 alkoxy, most preferably C 1-3 alkoxy. Non-limiting examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, t-butoxy, isobutoxy, pentyloxy, and the like.
  • Cycloalkyloxy means an -O-cycloalkyl group in which cycloalkyl is as defined above. It is preferably C 3-8 cycloalkyloxy, more preferably C 3-6 cycloalkyloxy.
  • Non-limiting examples of cycloalkyloxy include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • Haloalkoxy means an alkoxy group substituted by one or more (eg 1, 2, 3, 4 or 5) halogens, wherein alkoxy group is as defined above. It is preferably a halogenated C 1-8 alkoxy group, more preferably a halogenated C 1-6 alkoxy group, and more preferably a halogenated C 1-3 alkoxy group.
  • Haloalkoxy includes, but is not limited to, trifluoromethoxy, trifluoroethoxy, monofluoromethoxy, monofluoroethoxy, difluoromethoxy, difluoroethoxy, and the like.
  • Amino means -NH 2
  • cyano means -CN
  • nitro means -NO 2
  • benzyl means -CH 2 -phenyl
  • oxo O
  • carboxy means -C(O)OH
  • acetyl means -C(O)CH 3
  • hydroxymethyl means -CH 2 OH
  • hydroxyethyl means -CH 2 CH 2 OH or -CHOHCH 3
  • ""Hydroxy” refers to -OH and "thiol” refers to -SH.
  • Substituted refers to one or more hydrogen atoms in the group, preferably 1 to 5 hydrogen atoms are independently replaced by the corresponding number of substituents, more preferably 1 to 3 hydrogen atoms are independently replaced by the corresponding number of substituents of substituents. It goes without saying that substituents are only in their possible chemical positions and that a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (eg, ethylenic) bond.
  • substituted substituents independently selected from " in the present invention means that when more than one hydrogen on the group is replaced by a substituent, the types of the substituents may be the same or different, so The selected substituents are each independent species.
  • any group herein may be substituted or unsubstituted.
  • the substituents are preferably 1 to 5 following groups, independently selected from deuterium, halogen (preferably fluorine, chlorine), cyano, hydroxyl, carboxyl, C 1-8 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), C 1-8 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), C 2 -4 alkenyl, C 2-4 alkynyl, halogenated C 1-8 alkyl (preferably halogenated C 1-6 alkyl, more preferably halogenated C 1-3 alkyl), cyano substituted C 1 -8 alkyl (preferably cyano substituted C 1-6 alkyl, more preferably cyano substituted C 1-3 alkyl), halogenated C 1-8 alkoxy (preferably halogenated C 1-6 alkane Oxygen, more preferably halogen
  • R a1 and R b1 are each independently hydrogen or C 1-3 alkyl; or R a1 and R b1 form a 4 to 6-membered saturated monoheterocycle together with the connected nitrogen atom; the 4 to 6 member saturated monoheterocycle is unsubstituted or substituted by 1, 2 or 3 substituents each independently selected from the group consisting of deuterium, halogen, cyano, hydroxyl, carboxyl, C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-3 alkyl, halogenated C 1-3 alkoxy, -SO 2 C 1-3 alkyl, -S(O)C 1 -3 Alkyl, -C(O)NH 2 , -C(O)NH(C 1-3 Alkyl), -C(O)N(C 1-3 Alkyl) 2 , -C(O)OC 1-3 alkyl, -OC(O)C
  • R a0 and R b0 are each independently hydrogen, C 1-3 alkyl or acetyl; or R a0 and R b0 form a 4 to 6-membered saturated monoheterocyclic ring together with the connected nitrogen atom; the 4 to 6-membered saturated
  • the single heterocycle is unsubstituted or substituted by 1, 2 or 3 substituents each independently selected from the group consisting of deuterium, halogen, cyano, hydroxyl, carboxyl, C 1-3 alkyl, C 1-3 alkane Oxygen, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-3 alkyl, halogenated C 1-3 alkoxy, -SO 2 C 1-3 alkyl, -S(O )C 1-3 alkyl, -C(O)NH 2 , -C(O)NH(C 1-3 alkyl), -C(O)N(C 1-3 alkyl) 2 , -C( O)
  • any two "preferred” may be independent of each other.
  • any two substituents may be the same or different.
  • it can be substituted by two identical or different halogens, and can be substituted by one halogen and one hydroxyl.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof can be administered in a suitable dosage form with one or more pharmaceutically acceptable carriers.
  • These dosage forms are suitable for oral, rectal, topical, buccal and other parenteral administration (eg, subcutaneous, intramuscular, intravenous, etc.).
  • dosage forms suitable for oral administration include capsules, tablets, granules, syrups and the like.
  • the compounds of the present invention contained in these formulations may be solid powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions and the like.
  • the above-mentioned dosage forms can be made from the active compound and one or more carriers or excipients through common pharmaceutical methods.
  • the aforementioned carriers need to be compatible with the active compound or other excipients.
  • commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like.
  • Carriers for liquid preparations include water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like.
  • the active compounds can form solutions or suspensions with the above-mentioned carriers.
  • “Pharmaceutically acceptable carrier” means a non-toxic, inert, solid, semi-solid substance or liquid filler, diluent, encapsulating material or auxiliary preparation or excipient of any type which is compatible with the patient, preferably breastfeeding An animal, more preferably a human, is suitable for delivering an active agent to a target of interest without terminating the activity of the agent.
  • compositions of the present invention are formulated, dosed and administered in a manner consistent with medical practice.
  • the "therapeutically effective amount" of a compound to be administered is determined by factors such as the particular condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • “Therapeutically effective amount” refers to the amount of a compound of the present invention that will elicit a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease, etc.
  • the therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof contained in the pharmaceutical composition or the pharmaceutical composition of the present invention is preferably 0.1 mg/kg to 5 g/kg (weight).
  • Patient refers to an animal, preferably a mammal, more preferably a human.
  • mammal refers to warm-blooded vertebrate mammals including, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs and humans.
  • Treating means alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or condition (eg, cancer). Treatment also includes curing, preventing its development, or alleviating to some extent one or more symptoms of a disease or disorder.
  • the “pharmaceutically acceptable salt” includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to a salt formed with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects.
  • “Pharmaceutically acceptable base addition salts” include, but are not limited to, salts with inorganic bases and salts with organic bases.
  • the compounds of the present invention may contain one or more chiral centers and exist in different optically active forms. When a compound contains one chiral center, the compound contains enantiomers. When compounds contain more than one chiral center, diastereomers may exist.
  • the present invention includes these two stereoisomers as well as mixtures of these two stereoisomers, such as racemates, diastereomeric mixtures and the like.
  • the compound of formula (I) of the present invention may also exist cis-trans isomers due to the presence of a cycloalkyl structure, wherein the asymmetric carbon atom of the compound of formula (I) that may exist may be in (R) configuration, (S) configuration , (R, R) configuration, (S, S) configuration, (S, R) configuration or (R, S) configuration exists.
  • the present invention includes cis-trans isomers and mixtures thereof, such as cis-isomer mixtures, trans-isomer mixtures, and the like.
  • the compound of formula (I) of the present invention also includes tautomers, and tautomers refer to isomeric forms of compounds in equilibrium with each other.
  • tautomers refer to isomeric forms of compounds in equilibrium with each other.
  • the imine-enamine tautomers in the compound structure of the present invention are as follows, and the present invention includes these tautomeric forms isomers and their mixtures,
  • Enantiomers, diastereomers, cis isomers, trans isomers, imine-enamine tautomers, keto-enol tautomers and Mixtures of these isomers are within the protection scope of the present invention.
  • Enantiomers, diastereomers, and cis-trans isomers can be resolved by methods known in the art, such as crystallization and chiral chromatography.
  • the present invention provides methods for preparing compounds of formula (I), which can be synthesized using standard synthetic techniques known to those skilled in the art or using methods known in the art in combination with methods described in the present invention. Solvents, temperatures and other reaction conditions given in this invention can be varied according to the skill in the art. The reactions can be used sequentially to provide compounds of the invention, or they can be used to synthesize fragments which are subsequently added by methods described herein and/or by methods known in the art.
  • the compounds described in the present invention can be synthesized by using appropriate alternative starting materials using methods similar to those described below or the exemplary methods described in the Examples, or relevant publications used by those skilled in the art.
  • Starting materials for the synthesis of the compounds described in this invention can be synthesized or can be obtained from commercial sources.
  • the compounds described in this invention and other related compounds with different substituents can be synthesized using techniques and starting materials known to those skilled in the art.
  • General methods for preparing compounds disclosed herein can be derived from reactions known in the art, and the reactions can be modified with reagents and conditions deemed appropriate by those skilled in the art to introduce various moieties in the molecules provided herein.
  • the main advantage of the present invention is that it provides a series of novel triazinylmethylcycloalkyl carboxylic acid derivatives, which have higher inhibitory activity on P2X3 and higher inhibitory activity on P2X2/3. It is relatively low and has a large safety window, and has the potential to be used in the treatment of diseases related to P2X3 activity or P2X2/3 activity, including but not limited to good analgesic effects. Furthermore, in this class of triazinylmethylcycloalkylcarboxylic acid derivatives, there are obvious activity differences among different stereoisomers.
  • LC-MS Agilent 1290HPLC System/6130/6150MS liquid mass spectrometer (manufacturer: Agilent), column Waters BEH/CHS, 50 ⁇ 2.1mm, 1.7 ⁇ m.
  • Adopt ISCO Combiflash-Rf75 or Rf200 automatic column passing instrument Agela 4g, 12g, 20g, 40g, 80g, 120g disposable silica gel column.
  • thin layer chromatography can be used for monitoring the reaction progress
  • column chromatography can be used for compound purification.
  • the developer system used in column chromatography or TLC can be selected from: dichloromethane and methanol system, n-hexane and ethyl acetate system, petroleum ether and ethyl acetate system and acetone system, etc., the volume ratio of the solvent is based on the polarity of the compound Adjust differently.
  • DCM dichloromethane
  • DCE 1,2-dichloroethane
  • DMF N,N-dimethylformamide
  • DMSO dimethylsulfoxide
  • THF tetrahydrofuran
  • EA ethyl acetate
  • PE petroleum ether
  • n-BuLi n-butyllithium
  • HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • TEA is triethylamine
  • DIEA or DIPEA is N,N-diisopropylethylamine
  • NBS is N-bromosuccinimide
  • NCS N-chlorosuccinimide
  • TBAF tetrabutyl Ammonium fluoride
  • DEAD diethyl azodicarboxylate.
  • the percentage content involved in this article refers to mass percentage for solid-liquid mixing and solid-solid phase mixing, and refers to volume percentage for liquid-liquid mixing.
  • the solvent is water.
  • room temperature means about 20-30°C.
  • hours refers to about 10h-16h.
  • Step 1 Add methyl 2-fluoropyridine-3-carboxylate (557.57mg, 3.59mmol), 4-nitrophenol (500mg, 3.59mmol) and cesium carbonate (1.76g, 5.39mmol) into DMF (10mL) , and the reaction solution was stirred at 80° C. for 16 hours. The reaction solution was filtered and concentrated under reduced pressure. Purification by silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate: 1/0-1/1) gave methyl 2-(4-nitrophenoxy)nicotinate (650 mg). LC-MS m/z (ESI): 275.0 [M+1] + .
  • Step 2 Dissolve methyl 2-(4-nitrophenoxy)nicotinate (470mg, 1.71mmol) in THF (20mL), under the protection of argon, cool to -78°C, and then add lithium hydride dropwise Aluminum in THF (1M, 2.57 mL). The reaction was stirred at -78°C for 1 hour. The reaction was quenched by adding sodium sulfate decahydrate. Filter and concentrate under reduced pressure. Purify with silica gel column chromatography with eluent system (petroleum ether/ethyl acetate: 1/0 ⁇ 0/1) to obtain (2-(4-nitrophenoxy)pyridin-3-yl)methanol (405mg ). MS m/z (ESI): 247.0 [M+1] + .
  • Step 3 Dissolve (2-(4-nitrophenoxy)pyridin-3-yl)methanol (200mg, 812.29 ⁇ mol) in DCM (10mL), cool to 0°C under argon protection, add bis( 2-Methoxyethyl)aminosulfur trifluoride (539.13 mg, 2.44 mmol). The reaction solution was naturally warmed to room temperature and stirred for 5 hours. Saturated sodium bicarbonate solution (30 mL) was added, extracted with DCM (30 mL ⁇ 3), the organic phase was dried, filtered, and concentrated under reduced pressure.
  • Step 4 Mix 3-(fluoromethyl)-2-(4-nitrophenoxy)pyridine (100mg, 402.89 ⁇ mol), iron powder (112.50mg, 2.01mmol), ammonium chloride (107.75mg, 2.01mmol ) was added to methanol (2 mL), THF (2 mL) and water (2 mL). The reaction solution was stirred at 75°C for 2 hours. Filter and concentrate the filtrate under reduced pressure. The resulting residue was purified by silica gel column chromatography with an eluent system (dichloromethane/methanol: 1/0 to 10/1) to give 4-(3-(fluoromethyl)pyridin-2-yloxy)aniline V1 (82 mg). MS m/z (ESI): 219.1 [M+1] + .
  • Step 1 Add 2-fluoronicotinaldehyde (449.65mg, 3.59mmol), 4-nitrophenol (500mg, 3.59mmol) and cesium carbonate (1.76g, 5.39mmol) into DMF (10mL), and the reaction solution was heated at 60°C Stir for 2 hours. The reaction solution was filtered and concentrated under reduced pressure. Purification by silica gel column chromatography with eluent system (petroleum ether/ethyl acetate: 1/0-1/1) gave 2-(4-nitrophenoxy)nicotinaldehyde (450 mg). MS m/z (ESI): 244.9 [M+1] + .
  • Step 2 Dissolve 2-(4-nitrophenoxy)nicotinaldehyde (420mg, 1.72mmol) in DCM (20mL), under argon protection, cool to 0°C, add diethylaminosulfur trifluoride dropwise ( 1.39 g, 8.60 mmol). The reaction solution was allowed to warm up to room temperature naturally, and stirred for 16 hours. 30 mL of saturated sodium bicarbonate solution was added, extracted with DCM (30 mL ⁇ 3), the organic phase was dried, filtered, and concentrated under reduced pressure.
  • Step 1 Dissolve ( ⁇ ) trans-1,2-cyclopropanedicarboxylic acid dimethyl ester (25g, 0.158mol) in methanol (90mL)/water (10mL), cool in an ice bath, add sodium hydroxide ( 6.65g, 0.166mol), stirred overnight at room temperature.
  • Step 2 Dissolve ( ⁇ )trans-2-(methoxycarbonyl)cyclopropane-1-carboxylic acid (22g, 0.153mol) in dry THF (100mL), add borane dropwise under ice cooling THF solution (1M, 183mL, 0.183mol), stirred at room temperature for 2 hours, added water dropwise to the reaction solution, quenched the reaction, after no bubbles were generated, the reaction solution was filtered, the filtrate was concentrated, the residue was diluted with ethyl acetate, washed with water, Wash with saturated sodium chloride solution, dry, and concentrate to obtain ( ⁇ ) trans-2-(hydroxymethyl)cyclopropane-1-carboxylic acid methyl ester (20g), MS m/z (ESI): 131.1 [M+ 1] + .
  • Step 1 Add ( ⁇ )trans-2-(methoxycarbonyl)cyclopropane-1-carboxylic acid (8.5 g) and (S)- ⁇ -methylbenzylamine (7.86 g) to toluene (85 mL) , stirred at 50°C for 1 h, spin-dried, added 85 mL of tertiary methyl ether, stirred, filtered, and dried to obtain 13.5 g of solid. Take 5.0 g of the solid, add 50 mL of acetone to reflux to dissolve, stir for 0.5 h, cool down naturally, stir at room temperature for 2 h, and filter. 1.4 g of white solid were obtained.
  • Step 2 carboxylate (700mg) is dissolved in 5mL water, then add the potassium bisulfate solution of 5mL 10%wt, the aqueous solution is extracted 2 times with methyl tert-butyl ether 10mL, organic phase merges and dries spin-drying, obtains (1S , 2S)-2-(Methoxycarbonyl)cyclopropane-1-carboxylic acid (300 mg).
  • Step 3 Dissolve (1S,2S)-2-(methoxycarbonyl)cyclopropane-1-carboxylic acid (300mg) in 1mL tetrahydrofuran, add borane tetrahydrofuran solution (1mol/L, 10.4mL) dropwise under ice-cooling After dropping, return to normal temperature and stir for 5 hours, add 10 mL of methanol to the reaction solution in an ice bath to quench, spin dry, add 10 mL of water, add methyl tert-butyl ether (10 mL ⁇ 3) for extraction, combine the organic phases, wash with 10 mL of water once, Dry, filter and spin dry to obtain (1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylic acid methyl ester V7 (250mg).
  • Step 1 Add ( ⁇ )methyl cis-2-(hydroxymethyl)cyclopropane-1-carboxylate (52 mg, 403.01 ⁇ mol), 1-(4-chlorobenzyl)-6-(ethylthio) -1,3,5-Triazine-2,4(1H,3H)-dione (100 mg, 335.84 ⁇ mol, 1239691-22-5) and triphenylphosphine (132.13 mg, 503.76 ⁇ mol) were added to DCM (10 mL) , under argon protection, diethyl azodicarboxylate (87.73 mg, 503.76 ⁇ mol, 79.04 ⁇ L) was added dropwise, and the reaction solution was stirred at room temperature for 1 hour.
  • Step 2 Add ( ⁇ )cis-methyl 2-((5-(4-chlorobenzyl)-4-(ethylthio)-2,6-dioxo-5,6-dihydro-1 , 3,5-triazine-1(2H)-))methyl)cyclopropanecarboxylate (50mg, 121.99 ⁇ mol) and 4-(2-pyridyloxy)aniline (22.71mg, 121.99 ⁇ mol) were dissolved in valeric acid (3 mL). The reaction was stirred at 120°C for 5 hours. Saturated sodium bicarbonate solution (30 mL) was added and extracted with DCM (30 mL ⁇ 3).
  • Step 3 Add ( ⁇ )cis-methyl 2-(((-3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-yloxy) Phenylimino)-1,3,5-triazinyl-1-yl)methyl)cyclopropanecarboxylic acid (65 mg, 121.73 ⁇ mol) was added to THF (10 mL), methanol (2 mL) and water (2 mL), and then Add lithium hydroxide monohydrate (10.22 mg, 243.46 ⁇ mol). The reaction solution was stirred at room temperature for 4 hours. The reaction solution was neutralized with 1N hydrochloric acid solution, and concentrated under reduced pressure.
  • Preparative liquid chromatography (preparative column: water-sunfire; system: A: water+0.045% formic acid, B: acetonitrile; wavelength: 254/214nm; gradient: 5%-30% acetonitrile change) purify the resulting residue to obtain ( ⁇ )cis-2-((3-(4-chloro Benzyl)-2,6-dioxo-4-(4-(pyridin-2-yloxy)phenylimino)-1,3,5-triazinyl-1-yl)methyl) ring Propane carboxylic acid H1 (16.21 mg).
  • Step 1 Add ( ⁇ )trans-2-((3-(4-chlorobenzyl)-4-ethylthio-2,6-dioxo-3,6-dihydro-1,3, 5-Triazin-1(2H)-yl)methyl)cyclopropane-1-carboxylic acid methyl ester V6 (1.9g, 4.64mmol) and 4-((5-fluoropyridin-2-yl)oxy)aniline V4 (947 mg, 4.64 mmol) was dissolved in pivalic acid (10 mL). The reaction was stirred at 120°C for 3 hours, neutralized with saturated sodium bicarbonate, extracted with dichloromethane, and dried over anhydrous sodium sulfate.
  • Step 2 Adding ( ⁇ )trans-methyl-2-((3-(4-chlorobenzyl)-4-((4-((5-fluoropyridin-2-yl)oxy)phenyl) Amino)-2,6-dioxo-3,6-dihydro-1,3,5-triazin-1(2H)-yl)methyl)cyclopropane-1-carboxylate (2.00g, 3.62 mmol) was dissolved in methanol (10 mL) and water (10 mL). Then LiOH (433.91 mg, 18.12 mmol) was added.
  • Example 5-1 ( ⁇ ) trans-2-((3-(4-chlorobenzyl)-4-((4-((5-fluoropyridin-2-yl)oxy)phenyl)phenylene Preparation of enantiomers H5-P1 and H5-P2 of amino)-2,6-dioxo-1,3,5-triazinyl-1-yl)methyl)cyclopropane-1-carboxylic acid
  • Example 5-2 (1S,2S)-2-((3-(4-chlorobenzyl)-4-((4-((5-fluoropyridin-2-yl)oxy)phenyl)phenylene Amino)-2,6-dioxo-1,3,5-triazin-1-yl)methyl)cyclopropane-1-carboxylic acid
  • Step 1 Methyl (1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylate V7 (39g, 0.3mol), 1-(4-chlorobenzyl)-6-(ethylthio )-1,3,5-triazine-2,4(1H,3H)-dione (89g, 0.3mol) and triphenylphosphine (102g, 0.39mol) were suspended in dry THF, and DEAD was added dropwise (68g, 0.39mol), the reaction solution gradually became clear and stirred overnight at room temperature.
  • Step 2 (1S,2S)-2-((3-(4-Chlorobenzyl)-4-ethylthio-2,6-dioxo-3,6-dihydro-1,3,5 -triazin-1(2H)-yl)methyl)cyclopropane-1-carboxylic acid methyl ester (120g, 0.293mol) and 4-((5-fluoropyridin-2-yl)oxy)aniline V4 (60g , 0.293mol) was added to the round bottom flask, and then pivalic acid (400g) was added, then the temperature was raised to 130°C and stirred for 5 hours.
  • H5-P2 is (1R, 2R)-2-((3-(4-chlorobenzyl)-4-((4 -((5-fluoropyridin-2-yl)oxy)phenyl)imino)-2,6-dioxo-1,3,5-triazinyl-1-yl)methyl)cyclopropane- 1-Carboxylic acid.
  • Example 8 ( ⁇ ) trans-2-((3-(4-chlorobenzyl)-4-((4-((3-fluoropyridin-2-yl)oxy)phenyl)imino) Preparation of -2,6-dioxo-1,3,5-triazinyl-1-yl)methyl)cyclopropane-1-carboxylic acid H8
  • Test Example 1 FLIPR Assay Screening Compounds for Antagonistic Activity on hP2X 3 /hP2X 2/3 Receptors
  • Cell preparation cells 1321N1/hP2X3 and cells 1321N1/hP2X2/3 (supplier Chempartner) were stably transfected with Versene digestion solution, resuspended and counted with plating medium (DMEM+10% DFBS) after centrifugation, and the cells were adjusted to 3 ⁇ 10 5 cells/mL, spread 50 ⁇ L of cells in each well of a 384-well test plate, and culture in a 37°C incubator with 5% CO 2 for 16-24h.
  • plating medium DMEM+10% DFBS
  • test buffer 1* 1* 1 13.23 ATP hydrolase 0.5U/mL 10 ⁇ L 1U 200 0.07 the the the the Sample volume 14 the the the the the hP2X2/3 Working solution concentration stock solution concentration Dilution factor Required volume (mL) 10*dye stock 0.5* 10* 20 0.7 Carboxybenzsulfonamide 1.25 0.25 200 0.07 test buffer 1* 1* 1 13.23 the the the Sample volume 14
  • Test product In a 384-well polypropylene microwell plate that meets the Echo standard, prepare 180 times the required concentration of the test compound (54mM DMSO stock solution) with DMSO, and add 500nL to the 384-well compound in each well Add 30 ⁇ L of test buffer (1*HBSS+2mM CaCl 2 +20mM HEPES containing 1.26mM Ca 2+ ) to the plate, and shake for 20-40min to mix well.
  • test buffer (1*HBSS+2mM CaCl 2 +20mM HEPES containing 1.26mM Ca 2+
  • Agonist Prepare agonist ( ⁇ , ⁇ -meATP) at 3 times the required concentration with test buffer (both hP2X 3 and hP2X 2/3 cells require a final concentration of 3000nM), add 45 ⁇ L of agonist to each well of 384-well compound board.
  • Dye incubation take out the cell plate, suck off the cell supernatant, add 30uL Dye ( Calcium 4 Assay Kit, diluted in test buffer), incubated for 1h.
  • FLIPR detection add 15 ⁇ L of compound to each well of the cell plate (FLIPR instrument loading), 15 minutes later, add 22.5 ⁇ L of agonist to each well, and detect the fluorescent signal (excitation light wavelength 470nm-495nm, emission wavelength 515nm-575nm).
  • Data processing Take the difference between the signal peak value and the valley value as the basic data, take the data of the highest concentration of the positive drug as the 100% inhibition rate, and the DMSO data as the 0% inhibition rate, and pass (log(inhibitor) vs. response--Variable slope) to fit the inhibitory effect curve of the compound and calculate the IC 50 value.
  • Experimental animals 50 male SD rats, weighing 230-260g, all experimental animals were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. After purchase, food and water were supplied ad libitum, and the feeding temperature was 20-20 25°C, humidity: 40-70%, rearing in separate cages, and marking animals by tail marking method.
  • the experimental design grouping and drug treatment are shown in Table 6.
  • the complete Freund's adjuvant was mixed with normal saline to obtain an emulsion, and rats were subcutaneously injected with a certain amount of the emulsion. Put the animals in a special multi-unit metal mesh cage for pain detection, and let the animals adapt to the environment for 15 minutes. , 1.0, 1.4, 2, 4, 6, 8, 10, 15) pressurize continuously in the hind paw until withdrawal reflex occurs. The testers will conduct a blind test on the experiment, and the experimental results are expressed as "mean ⁇ standard deviation". The data of each group is analyzed by GraphPad Prism 5t test, and P ⁇ 0.05 has a statistical difference. The specific experimental results are shown in Figure 2 and Figure 3.
  • male and female SD rats there are 16 male and female SD rats each, and the grade is SPF grade.
  • Male SD rats weigh about 240g and are 6-9 weeks old; female SD rats weigh about 200g and are 6-9 weeks old. All experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. After purchase, food and water were supplied ad libitum. The feeding temperature was 20-26°C, humidity: 40-70%, and the number of air changes per hour ⁇ 15 times, use 100% fresh air. Automatic lighting, alternating light and dark every 12hrs. Male and female are housed in separate cages, no more than 5 per cage.
  • each group of animals was designed according to the dosage in Table 7, administered once a day for a total of 14 consecutive days.
  • Toxicokinetic blood sampling was carried out in each dosage group before the first and last administration, 30min, 1hr, 4hr, 7hr, 10hr and 24hr after administration, and the toxicokinetic process of each group in SD rats was investigated. The results are as follows: Table 8 shows.
  • the onset dose of compound H5-P1 in the rat pain model is 30 mg/kg, the Cmax at this dose is about 18.4 ⁇ g/mL, and the AUC is about 249hr* ⁇ g/mL.
  • the onset dose of compound D1 in the rat pain pharmacodynamic model is 60 mg/kg, the Cmax at this dose is about 141 ⁇ g/mL, and the AUC is about 1566hr* ⁇ g/mL.
  • the structure of the positive compound D1 in Test Example 3 is as follows:

Abstract

一种取代的三嗪基甲基环烷基羧酸衍生物其结构如式(I)所示。此外还公开了该衍生物的药学上可接受的盐、立体异构体、药物组合物以及其用途。该化合物具有显著的P2X3抑制活性和选择性，具有较高的应用价值。

Description

三嗪基甲基环烷基羧酸衍生物及其药物组合物和用途 技术领域
本发明涉及医药技术领域，特别涉及一种三嗪基甲基环烷基羧酸衍生物、其药学上可接受的盐、立体异构体、药物组合物及其医药上的用途。
背景技术
P2X嘌呤受体是由细胞外腺苷三磷酸(ATP)激活的离子通道家族。嘌呤受体涉及多种生物功能，特别是与疼痛敏感性相关。P2X3受体是这个家族的成员之一，最初从大鼠背根神经节克隆而来(Chen等,Nature,第377卷,第428-431页(1995))。大鼠P2X3和人P2X3两者的核苷酸序列和氨基酸序列现在是已知的(Lewis等,Nature,第377卷,第432-435页(1995)；以及Garcia-Guzman等,BrainRes.Mol.BrainRes.,第47卷,第59-66页(1997))。
据报道，P2X3涉及控制膀胱容量反射的传入通路，因此，抑制P2X3可以治疗储尿和排尿相关的病症，如膀胱过度活动症(Cockayne等,Nature,第407卷,第1011-1015页(2000))。
P2X3还选择性地表达在伤害感受性神经元、小直径感觉神经元(即由疼痛或损伤刺激的神经元)上，这与它在疼痛敏感性中的作用相符。此外，也有报道，阻断P2X3受体在慢性炎症性和神经性病理痛动物模型中具有镇痛作用(Jarvis等,PNAS,99,17179-17184(2002))。因此，降低P2X3水平或活性的方法可用于调节疼痛对象的痛觉。
P2X3还能够与P2X2形成P2X2/3异二聚体，所述P2X2是P2X嘌呤能配体-门控离子通道家族的另一个成员。P2X2/3在感觉神经元的末梢(中枢和外周)上高表达(Chen等,Nature,第377卷,第428-431页(1995))。最新的研究结果还表明P2X2/3主要在膀胱感觉神经元中表达(超过P2X3)，并且有可能在膀胱充盈和伤害性感受的感觉中起作用(Zhong等,Neuroscience,第120卷,第667-675页(2003))。
因此，需要提供一种用于治疗与P2X3和/或P2X2/3相关的各种病症的可能有用的新的P2X3和/或
P2X2/3受体配体，特别是拮抗剂。
发明内容
本发明的目的是提供结构新颖的三嗪基甲基环烷基羧酸衍生物、或其药学上可接受的盐、或其立体异构体、其药物组合物及其作为P2X3拮抗剂的用途，该类化合物对P2X3具有较高的抑制活性，对P2X2/3抑制活性较低，具有显著的抑制选择性。
本发明第一方面提供了式(I)所示化合物、或其药学上可接受的盐、或其立体异构体：
Figure PCTCN2022096597-appb-000001
其中，
r为1、2或3；
W为苯环或5至6元杂芳基环(优选为吡啶环)；所述苯环、5至6元杂芳基环(优选为吡啶环)为未取代的或被1、2、3或4个各自独立地选自下组的取代基取代：氘、卤素(优选为氟、氯)、氰基、羟基、羧基、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、C 2-4烯基、C 2-4炔基、卤代C 1-8烷基(优选为卤代C 1-6烷基，更优选为卤代C 1-3烷基)、氰基取代C 1-8烷基(优选为氰基取代C 1-6烷基，更优选为氰基取代C 1-3烷基)、卤代C 1-8烷氧基(优选为卤代C 1-6烷氧基，更优选为卤代C 1-3烷氧基)、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基和5至6元杂芳基；其中取代基中的所述3至6元杂环烷基、苯基、5至6元杂芳基为未取代的或被1、2或3个各自独立地选自下组的取代基取代：卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基和5至6元杂芳基；
(R a) n表示苯环上的氢被n个R a取代，n为0、1或2；每个R a相同或不同并且各自独立地为氰基、羟基、羧基、卤素(优选为氟或氯)、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、卤素取代C 1-8烷基(优选为卤素取代C 1-6烷基，更优选为卤素取代C 1-3烷基)、氰基取代C 1-8烷基(优选为氰基取代C 1-6烷基，更优选为氰基取代C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)或-C(O)NR a1R b1；
R c为氢、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、C 3-8环烷基(优选为C 3-6环烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、氰基、羟基、羧基或卤素(优选为氟或氯)；
(R b) m表示苯环上的氢被m个R b取代，m为0、1或2；每个R b相同或不同并且各自独立地为氰基、羟基、羧基、卤素(优选为氟或氯)、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、卤代C 1-8烷基(优选为卤代C 1-6烷基，更优选为卤代C 1-3烷基)、氰基取代C 1-8烷基(优选为氰基取代C 1-6烷基，更优选为氰基取代C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)或-C(O)NR a1R b1；
R 0为氢或C 1-6烷基；
R a1、R b1各自独立地为氢或C 1-3烷基；或者R a1、R b1与相连的氮原子共同形成4至6元饱和单杂环；所述4至6元饱和单杂环为未取代的或被1、2或3个各自独立地选自下组的取代基取代：氘、卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NH 2、-C(O)NH(C 1-3烷基)、-C(O)N(C 1-3烷基) 2、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基和3至6元杂环烷基；
R a0、R b0各自独立地为氢、C 1-3烷基或乙酰基；或者R a0、R b0与相连的氮原子共同形成4至6元饱和单杂环；所述4至6元饱和单杂环为未取代的或被1、2或3个各自独立地选自下组的取代基取代：氘、卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NH 2、-C(O)NH(C 1-3烷基)、-C(O)N(C 1-3烷基) 2、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基和3至6元杂环烷基。
在一些实施例中，r为1。
在一些实施例中，W为
Figure PCTCN2022096597-appb-000002
(R d) u表示吡啶环上的氢被u个R d取代，u为0、1或2；每个R d相同或不同并且各自独立地为氘、卤素(优选为氟、氯)、氰基、羟基、羧基、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、C 2-4烯基、C 2-4炔基、卤代C 1-8烷基(优选为卤代C 1-6烷基，更优选为卤代C 1-3烷基)、卤代C 1-8烷氧基(优选为卤代C 1-6烷氧基，更优选为卤代C 1-3烷氧基)、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基、5至6元杂芳基；其中取代基中的所述3至6元杂环烷基、苯基、5至6元杂芳基为未取代的或被1、2或3个各自独立地选自下组的取代基取代：卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基和5至6元杂芳基。
在一些实施例中，u为0或1；R d为氟、氯、氰基、羟基、羧基、C 1-3烷基(优选甲基、乙基、正丙基、异丙基)、C 1-3烷氧基(优选为甲氧基)、卤代C 1-3烷基(优选为一氟甲基、二氟甲基、三氟甲基)、卤代C 1-3烷氧基(优选为一氟甲氧基、二氟甲氧基、三氟甲氧基)、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)C 1-3烷基、-C(O)OC 1-3烷基或-OC(O)C 1-3烷基。
在一些实施例中，R d中含有吸电子基团。
在一些实施例中，R d为吸电子基团。
在一些实施例中，R d为吸电子基团取代的C 1-8烷基；进一步地，R d为卤素取代的C 1-8烷基。
在一些实施例中，u为0或1，R d为卤素(优选为氟)或卤代C 1-3烷基(优选为一氟甲基、二氟甲基、三氟甲基)；进一步地，R d为氟。
在一些实施例中，W为
Figure PCTCN2022096597-appb-000003
Figure PCTCN2022096597-appb-000004
在一些实施例中，W为
Figure PCTCN2022096597-appb-000005
在一些实施例中，m为0或1；R b为氰基、羟基、羧基、氟、氯、C 1-3烷基、C 1-3烷氧基、-C(O)C 1-3烷基、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基或-C(O)NR a1R b1。
在一些实施例中，R b为吸电子基团。
在一些实施例中，m为1，R b为卤素或氰基；进一步地，R b为氯。
在一些实施例中，m为1，R b为对位取代。
在一些实施例中，
Figure PCTCN2022096597-appb-000006
为
Figure PCTCN2022096597-appb-000007
在一些实施例中，n为0或1；R a为氰基、羟基、羧基、氟、氯、C 1-3烷基、C 1-3烷氧基、-C(O)C 1-3烷基、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基或-C(O)NR a1R b1。
在一些实施例中，
Figure PCTCN2022096597-appb-000008
为
Figure PCTCN2022096597-appb-000009
在一些实施例中，n为0。
在一些实施例中，R c为氢、C 1-3烷基、C 3-6环烷基、C 1-3烷氧基、氰基、羟基、羧基、氟、氯、-C(O)NR a1R b1、-C(O)C 1-3烷基或-C(O)OC 1-3烷基。
在一些实施例中，R c为氢。
在一些实施例中，R 0为氢。
在一些实施例中，R 0为C 1-6烷基；进一步地R 0为C 1-4烷基。
在一些实施例中，式(I)结构中的
Figure PCTCN2022096597-appb-000010
为顺式结构。
在一些实施例中，式(I)结构中的
Figure PCTCN2022096597-appb-000011
为反式结构。
在一些实施例中，所述式(I)为式(Ⅱ)所示结构：
Figure PCTCN2022096597-appb-000012
其中R a、R c、R d、n、u如上述所定义。
在一些实施例中，式(II)结构中的
Figure PCTCN2022096597-appb-000013
为顺式结构。
在一些实施例中，式(II)结构中的
Figure PCTCN2022096597-appb-000014
为反式结构。
在一些实施例中，式(I)为式(Ⅱ-1)所示结构：
Figure PCTCN2022096597-appb-000015
其中R a、R c、R d、n、u如上述所定义。
在一些实施例中，式(I)为式(Ⅱ-2)所示结构：
Figure PCTCN2022096597-appb-000016
其中R a、R c、R d、n、u如上述所定义。
在一些实施例中，式(I)为式(Ⅱ-3)所示结构：
Figure PCTCN2022096597-appb-000017
其中R a、R c、R d、n、u如上述所定义。
在一些实施例中，式(I)为式(Ⅱ-4)所示结构：
Figure PCTCN2022096597-appb-000018
其中R a、R c、R d、n、u如上述所定义。
在一些实施例中，式(I)化合物为选自实施例中的具体化合物。
在一些实施例中，式(I)化合物为选自表1中的化合物或其立体异构体：
表1
Figure PCTCN2022096597-appb-000019
在一些实施例中，式(I)化合物为选自表2的化合物：
表2
Figure PCTCN2022096597-appb-000020
在一些实施例中，式(I)化合物为选自表3的化合物：
表3
Figure PCTCN2022096597-appb-000021
本发明第二方面提供了一种药物组合物，包括上述式(I)所示化合物、或其药学上可接受的盐、或其立体异构体；以及药学上可接受的载体。
本发明第三方面提供了上述式(I)所示化合物、或其药学上可接受的盐、或其立体异构体在制备治疗与P2X3活性或与P2X2/3活性相关的疾病的药物中的应用。
本发明第四方面提供了第二方面所述的药物组合物在制备治疗与P2X3活性或与P2X2/3活性相关的疾病的药物中的应用。
本发明第五方面提供了一种治疗与P2X3活性或与P2X2/3活性相关的疾病的方法，包括向患者施用治疗有效量的本发明第一方面所述的化合物、或其药学上可接受的盐、或其立体异构体或本发明第二方面所述的组合物。
在一些实施例中，所述的与P2X3活性或与P2X2/3活性相关的疾病为疼痛、泌尿道病症、胃肠疾病、癌症、免疫相关的疾病、咳嗽、抑郁症、焦虑或应激相关病症。进一步地，所述的与P2X3活性或与P2X2/3活性相关的疾病为P2X3或P2X2/3介导的疼痛、泌尿道病症、胃肠疾病、癌症、免疫相关的疾病、咳嗽、抑郁症、焦虑或应激相关病症。
附图说明
图1为中间体V7的羧酸盐的单晶结构图；
图2显示了给药后2小时不同处理组缩爪阈值的效果；图2中，*、***分别表示待测化合物组与溶媒组相比P<0.05、P<0.001；
图3显示了给药后4小时不同处理组缩爪阈值的效果；图3中，**表示待测化合物组与溶媒组相比P<0.01。
具体实施方式
本发明人经过广泛而深入的研究，意外地发现了这类三嗪基甲基环烷基羧酸衍生物具有显著的P2X3抑制活性和较低的P2X2/3抑制活性，且具有较大的安全窗。因此该系列化合物有望开发成为用于调节P2X3和/或P2X2/3以治疗由P2X3和/或P2X2/3介导(或以其它方式与P2X3和/或P2X2/3相关)的各种疾病的药物，包括但不限于发挥良好的镇痛效果。进一步地，发明人还发现，在该类三嗪基甲基环烷基羧酸衍生物中，不同的立体异构体之间存在较为明显的活性差异。
在此基础上，发明人完成了本发明。
术语定义
为了能够更清楚地理解本发明的技术内容，下面对本发明的术语作进一步说明。
“烷基”指直链和支链的饱和的脂族烃基。“C 1-8烷基”是指具有1至8个碳原子的烷基，优选为C 1-6烷基；更优选为C 1-3烷基；烷基的非限制性的例子包括：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体。
“环烷基”和“环烷基环”可互换使用，均指饱和单环、双环或多环环状烃基，该基团可以与芳基或杂芳基稠合。环烷基环可以任选地被取代。在某些实施方案中，环烷基环含有一个或多个羰基，例如氧代的基团。“C 3-8环烷基”是指具有3至8个碳原子的单环环烷基，环烷基的非限制性实施例包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环丁酮、环戊酮、环戊烷-1,3-二酮等。优选为C 3-6环烷基，包括环丙基、环丁基、环戊基和环己基。
“杂环烷基”和“杂环烷基环”可互换使用，均指包含至少一个选自氮、氧和硫的杂原子的环烷基，该基团可以与芳基或杂芳基稠合。杂环烷基环可以任选地被取代。在某些实施方案中，杂环烷基环含有一个或多个羰基或硫代羰基，例如包含氧代和硫代的基团。“3至6元杂环烷基”是指具有3至6个环原子，其中1、2或3个环原子为选自氮、氧和硫的杂原子的单环环状烃基，优选为3至6元杂环烷基中1或2个环原子为选自氮、氧和硫的杂原子。单环杂环烷基的非限制性实施例包括氮丙环基、环氧乙烷基、氮杂环丁烷基、氧杂环丁烷基、四氢呋喃基、四氢噻吩基、四氢吡咯基、噁唑烷基、二氧戊环基、哌啶基、哌嗪基、吗啉基、二氧六环基、硫代吗啉基、硫代吗啉-1,1-二氧化物、四氢吡喃基、氮杂环丁烷-2-酮基、氧杂环丁烷-2-酮基、二氢呋喃-2(3H)-酮基、吡咯烷-2-酮基、吡咯烷-2,5-二酮基、二氢呋喃-2,5-二酮基、哌啶-2-酮基、四氢-2H-吡喃-2-酮基、哌嗪-2-酮基、吗啉-3-酮基等。
“杂芳基”和“杂芳基环”可互换使用，均指具有环碳原子和环杂原子的单环、双环或多环的4n+2芳族 环体系(例如，具有以环状排列共享的6或10个π电子)的基团，其中每个杂原子独立地选自氮、氧和硫。本发明中，杂芳基还包括其中上述杂芳基环与一个或多个环烷基环、杂环烷基环、环烯基环、杂环烯基环或芳环稠合的环系统。杂芳基环可以任选地被取代。“5至6元杂芳基”是指具有5至6个环原子，其中1、2、3或4个环原子为杂原子的单环杂芳基。非限制性实施例包括噻吩基、呋喃基、噻唑基、异噻唑基、咪唑基、噁唑基、吡咯基、吡唑基、三唑基、1,2,3-三唑基、1,2,4-三唑基、1,2,5-三唑基、1,3,4-三唑基、四唑基、异噁唑基、噁二唑基、1,2,3-噁二唑基、1,2,4-噁二唑基、1,2,5-噁二唑基、1,3,4-恶二唑基、噻二唑基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、四嗪基。“杂原子”是指氮、氧或硫。在含有一个或多个氮原子的杂芳基中，只要化合价允许，连接点可以是碳或氮原子。杂芳基双环系统在一个或两个环中可以包括一个或多个杂原子。
“卤素”指氟、氯、溴或碘。
“卤代”指基团中一个或多个(如1、2、3、4或5个)氢被卤素所取代。
“卤代烷基”指烷基被一个或多个(如1、2、3、4或5个)卤素取代，其中烷基的定义如上所述。优选为卤代C 1-8烷基，更选为卤代C 1-6烷基，更优选为卤代C 1-3烷基。卤代烷基的例子包括(但不限于)一氯甲基、二氯甲基、三氯甲基、一氯乙基、1,2-二氯乙基、三氯乙基、一溴乙基、一氟甲基、二氟甲基、三氟甲基、一氟乙基、二氟乙基、三氟乙基等。
“氘代烷基”指烷基被一个或多个(如1、2、3、4或5个)氘原子取代，其中烷基的定义如上所述。优选为氘代C 1-8烷基，更优选为氘代C 1-6烷基，更优选为氘代C 1-3烷基。氘代烷基的例子包括(但不限于)单氘代甲基、单氘代乙基、二氘代甲基、二氘代乙基、三氘代甲基、三氘代乙基等。
“烷氧基”指-O-烷基，其中烷基的定义如上所述。优选C 1-8烷氧基，更优选C 1-6烷氧基，最优选C 1-3烷氧基。烷氧基的非限制性实施例包含甲氧基、乙氧基、正丙氧基、异丙氧基、丁氧基、叔丁氧基、异丁氧基、戊氧基等。
“环烷基氧基”指-O-环烷基，其中环烷基的定义如上所述。优选C 3-8环烷基氧基，更优选C 3-6环烷基氧基。环烷基氧基的非限制性实施例包含环丙基氧基、环丁基氧基、环戊基氧基、环己基氧基等。
“卤代烷氧基”指烷氧基被一个或多个(如1、2、3、4或5个)卤素取代，其中烷氧基的定义如上所述。优选为卤代C 1-8烷氧基，更优选为卤代C 1-6烷氧基，更优选为卤代C 1-3烷氧基。卤代烷氧基包括(但不限于)三氟甲氧基、三氟乙氧基、一氟甲氧基、一氟乙氧基、二氟甲氧基、二氟乙氧基等。
“氨基”指-NH 2，“氰基”指-CN，“硝基”指-NO 2，“苯甲基”指-CH 2-苯基，“氧代基”指＝O，“羧基”指-C(O)OH，“乙酰基”指-C(O)CH 3，“羟甲基”指-CH 2OH，“羟乙基”指-CH 2CH 2OH或-CHOHCH 3，“羟基”指-OH，“硫醇”指-SH。
“取代的”指基团中的一个或多个氢原子，优选为1～5个氢原子彼此独立地被相应数目的取代基取代，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
本发明中未指出取代基数目时表示任选可取代数目的取代基进行取代。
除非另有定义，本发明所述“各自独立地选自……的取代基”是指当基团上的一个以上的氢被取代基取代时，所述的取代基种类可以相同或不同，所选自的取代基为各自独立的种类。
除非另有定义，本文任一基团可以是取代的或未取代的。上述基团被取代时，取代基优选为1至5个以下基团，独立地选自氘、卤素(优选为氟、氯)、氰基、羟基、羧基、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、C 2-4烯基、C 2-4炔基、卤代C 1-8烷基(优选为卤代C 1-6烷基，更优选为卤代C 1-3烷基)、氰基取代C 1-8烷基(优选为氰基取代C 1-6烷基，更优选为氰基取代C 1-3烷基)、卤代C 1-8烷氧基(优选为卤代C 1-6烷氧基，更优选为卤代C 1-3烷氧基)、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基、5至6元杂芳基；其中取代基中的所述3至6元杂环烷基、苯基、5至6元杂芳基为未取代的或被1、2或3个各自独立地选自卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基、5至6元杂芳基；
R a1、R b1各自独立地为氢或C 1-3烷基；或者R a1、R b1与相连的氮原子共同形成4至6元饱和单杂环；所述4至6元饱和单杂环为未取代的或被1、2或3个各自独立地选自下组的取代基取代：氘、卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NH 2、-C(O)NH(C 1-3烷基)、-C(O)N(C 1-3烷基) 2、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基；
R a0、R b0各自独立地为氢、C 1-3烷基或乙酰基；或者R a0、R b0与相连的氮原子共同形成4至6元饱和单杂环；所述4至6元饱和单杂环为未取代的或被1、2或3个各自独立地选自下组的取代基取代：氘、卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NH 2、-C(O)NH(C 1-3烷基)、-C(O)N(C 1-3烷基) 2、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基。
本发明中，一个方案中出现两个或更多个“优选”时，任意的两个“优选”可以是彼此独立的。
本发明中，当取代基的数量大于1时，任意的两个取代基可以相同或不同。如，可以为两个相同或不同的卤素取代，可以为一个卤素和一个羟基取代。
本文以上所述的各类取代基团其自身也是可以被本文所描述的基团取代。
药物组合物
通常本发明化合物或其药学上可接受的盐、或其立体异构体可以与一种或多种药用载体形成适合的剂型施用。这些剂型适用于口服、直肠给药、局部给药、口内给药以及其他非胃肠道施用(例如，皮下、肌肉、静脉等)。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明的化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。
“药学上可接受的载体”是指无毒、惰性、固态、半固态的物质或液体灌装机、稀释剂、封装材料或辅助制剂或任何类型辅料，其与患者相兼容，最好为哺乳动物，更优选为人，其适合将活性试剂输送到目标靶点而不终止试剂的活性。
本发明的组合物以符合医学实践规范的方式配制，定量和给药。给予化合物的“治疗有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。
“治疗有效量”是指将引起个体的生物学或医学响应，例如降低或抑制酶或蛋白质活性或改善症状、缓解病症、减缓或延迟疾病进程或预防疾病等的本发明化合物的量。
本发明的所述药物组合物或所述药用组合物中含有的本发明化合物或其药学上可接受的盐、或其立体异构体的治疗有效量优选为0.1mg/kg～5g/kg(体重)。
“患者”是指一种动物，优选为哺乳动物，更优选为人。术语“哺乳动物”是指温血脊椎类哺乳动物，包括如猫、狗、兔、熊、狐狸、狼、猴子、鹿、鼠、猪和人类。
“治疗”是指减轻、延缓进展、衰减、预防，或维持现有疾病或病症(例如癌症)。治疗还包括将疾病或病症的一个或多个症状治愈、预防其发展或减轻到某种程度。
所述“药学上可接受的盐”包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其他副作用的与无机酸或有机酸所形成的盐。
“药学上可接受的碱加成盐”，包括但不限于无机碱的盐和有机碱的盐。
本发明的化合物可以含有一个或多个手性中心，并以不同的光学活性形式存在。当化合物含有一个手性中心时，化合物包含对映异构体。当化合物含有多于一个手性中心时，可以存在非对映异构体。本发明包括这两种立体异构体以及这两种立体异构体的混合物，如外消旋体、非对映异构体混合物等。
此外本发明式(I)化合物因存在环烷基结构还可能存在顺反异构体，其中可能存在的式(I)化合物的不对称碳原子可以以(R)构型、(S)构型、(R，R)构型、(S，S)构型、(S，R)构型或(R，S)构型存在。本发明包括顺反异构体及其混合物，例如顺式异构体混合物、反式异构体混合物等。
此外本发明式(I)化合物还包括互变异构体，互变异构体是指相互平衡的化合物异构形式。本领域技术人员容易理解的是，各种官能团和其他结构可能会表现出互变异构形式，例如本发明化合物结构中的亚胺-烯胺互变异构体如下，本发明包括这些互变异构体及其混合物，
Figure PCTCN2022096597-appb-000022
Figure PCTCN2022096597-appb-000023
本发明化合物的对映异构体、非对映异构体、顺式异构体、反式异构体、亚胺-烯胺互变异构体、酮-烯醇互变异构体以及这些异构体的混合物均在本发明的保护范围内。对映异构体、非对映异构体、顺反异构体可以通过本领域已知的方法进行拆分，例如结晶以及手性色谱等方法。
制备方法
本发明提供了式(I)化合物的制备方法，使用本领域技术人员已知的标准合成技术或使用本领域已知的方法与本发明描述的方法组合可以合成式(I)化合物。本发明给出的溶剂、温度和其它反应条件可以根据本领域技术而改变。所述反应可以按顺序使用，以提供本发明的化合物，或者它们可以用于合成片段，所述片段通过本发明所描述的方法和/或本领域已知的方法随后加入。
本发明描述的化合物可以使用与下述类似的方法或实施例中所述的示例性方法，或本领域技术人员所用的相关公开文献，通过使用适当的可选择的起始原料合成化合物。用于合成本发明所描述的化合物的起始原料可以被合成或可以从商业来源获得。本发明描述的化合物和其它相关具有不同取代基的化合物可以使用本领域技术人员已知的技术和原料合成。制备本发明公开的化合物的一般方法可以来自本领域已知的反应，并且该反应可以通过由本领域技术人员所认为适当的试剂和条件修改，以引入本发明提供的分子中的各种部分。
与现有技术相比，本发明的主要优点在于：提供了一系列结构新颖的三嗪基甲基环烷基羧酸衍生物，其对P2X3具有较高的抑制活性，对P2X2/3抑制活性较低，且具有较大的安全窗，具有用于治疗与P2X3活性或与P2X2/3活性相关的疾病的潜能，包括但不限于发挥良好的镇痛效果。进一步地，在该类三嗪基甲基环烷基羧酸衍生物中，不同的立体异构体之间存在较为明显的活性差异。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。除非另行定义，本文所用的术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或同等的方法及材料皆可应用于本发明中。
试剂与仪器
1HNMR：Bruker AVANCE-400核磁仪，内标为四甲基硅烷(TMS)。
LC-MS：Agilent 1290HPLC System/6130/6150MS液质联用质谱仪(生产商：安捷伦)，柱子Waters BEH/CHS，50×2.1mm，1.7μm。
制备高效液相色谱(pre-HPLC)：GX-281(生产商：吉尔森)。
采用ISCO Combiflash-Rf75或Rf200型自动过柱仪，Agela 4g、12g、20g、40g、80g、120g一次性硅胶柱。
已知的起始原料可以采用或按照本领域已知的方法来合成，或可以购自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)和达瑞化学品等公司。
实施例中，反应进程的监测可采用薄层色谱法(TLC)，化合物纯化可采用柱层析。柱层析或TLC所用的展开剂体系可选自：二氯甲烷和甲醇体系、正己烷和乙酸乙酯体系、石油醚和乙酸乙酯体系和丙酮体系等，溶剂的体积比根据化合物的极性不同而进行调节。
如本文所用，DCM为二氯甲烷，DCE为1,2-二氯乙烷，DMF为N,N-二甲基甲酰胺，DMSO为二甲基亚砜，THF为四氢呋喃，EA为乙酸乙酯，PE为石油醚，n-BuLi为正丁基锂，HATU为2-(7-偶氮苯并 三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯，TEA为三乙胺，DIEA或DIPEA为N,N-二异丙基乙胺，NBS为N-溴代丁二酰亚胺，NCS为N-氯代丁二酰亚胺，TBAF为四丁基氟化铵，DEAD为偶氮二甲酸二乙酯。
如本文中涉及的百分比含量，如无特别说明，对于固液混合和固相-固相混合均指质量百分比，对于液相-液相混合指体积百分比。如无特别说明，溶剂均为水。
如本文所用，室温是指约20-30℃。
如本文所用，“过夜”是指约进行10h～16h。
中间体V1的制备
Figure PCTCN2022096597-appb-000024
步骤1：将2-氟吡啶-3-羧酸甲酯(557.57mg,3.59mmol)，4-硝基苯酚(500mg,3.59mmol)和碳酸铯(1.76g,5.39mmol)加入DMF(10mL)中，反应液在80℃搅拌16小时。反应液过滤，减压浓缩。用硅胶柱色谱法以洗脱剂体系(石油醚/乙酸乙酯：1/0～1/1)纯化，得到2-(4-硝基苯氧基)烟酸甲酯(650mg)。LC-MS m/z(ESI):275.0[M+1] +。
步骤2：将2-(4-硝基苯氧基)烟酸甲酯(470mg,1.71mmol)溶于THF(20mL)中，氩气保护下，冷却到-78℃，然后滴加氢化锂铝的THF溶液(1M,2.57mL)。反应液在-78℃继续搅拌1小时。加入十水硫酸钠淬灭反应。过滤，减压浓缩。用硅胶柱色谱法以洗脱剂体系(石油醚/乙酸乙酯：1/0～0/1)纯化，得到(2-(4-硝基苯氧基)吡啶-3-基)甲醇(405mg)。MS m/z(ESI):247.0[M+1] +。
步骤3：将(2-(4-硝基苯氧基)吡啶-3-基)甲醇(200mg,812.29μmol)溶于DCM(10mL)中，氩气保护下冷却到0℃，滴加双(2-甲氧基乙基)氨基三氟化硫(539.13mg,2.44mmol)。反应液自然升温到室温，搅拌5小时。加入饱和碳酸氢钠溶液(30mL)，用DCM(30mL×3)萃取，有机相干燥后，过滤，减压浓缩。用硅胶柱色谱法以洗脱剂体系(石油醚/乙酸乙酯：1/0～3/1)纯化，得到3-(氟甲基)-2-(4-硝基苯氧基)吡啶(177mg)。MS m/z(ESI):249.0[M+1] +。
步骤4：将3-(氟甲基)-2-(4-硝基苯氧基)吡啶(100mg,402.89μmol)，铁粉(112.50mg,2.01mmol)，氯化铵(107.75mg,2.01mmol)加入甲醇(2mL)，THF(2mL)和水(2mL)中。反应液在75℃搅拌2小时。过滤，滤液减压浓缩。用硅胶柱色谱法以洗脱剂体系(二氯甲烷/甲醇：1/0～10/1)纯化所得残余物，得到4-(3-(氟甲基)吡啶-2-基氧基)苯胺V1(82mg)。MS m/z(ESI):219.1[M+1] +。
中间体V2的制备
Figure PCTCN2022096597-appb-000025
步骤1：将2-氟烟醛(449.65mg,3.59mmol)，4-硝基苯酚(500mg,3.59mmol)和碳酸铯(1.76g,5.39mmol)加入DMF(10mL)中，反应液在60℃搅拌2小时。反应液过滤，减压浓缩。用硅胶柱色谱法以洗脱剂体系(石油醚/乙酸乙酯：1/0～1/1)纯化，得到2-(4-硝基苯氧基)烟醛(450mg)。MS m/z(ESI):244.9[M+1] +。
步骤2：将2-(4-硝基苯氧基)烟醛(420mg,1.72mmol)溶于DCM(20mL)，氩气保护，冷却到0℃，滴加二乙胺基三氟化硫(1.39g,8.60mmol)。反应液让其自然升温到室温，搅拌16小时。加入30mL饱和碳酸氢钠溶液，用DCM(30mL×3)萃取，有机相干燥后过滤，减压浓缩。用硅胶柱色谱法以洗脱剂体系(石油醚/乙酸乙酯：1/0～1/1)纯化所得残余物，得到3-(二氟甲基)-2-(4-硝基苯氧基)吡啶V2(405mg)。MS m/z(ESI):267.0[M+1] +。
中间体V3的制备
Figure PCTCN2022096597-appb-000026
将4-氨基-2-氟苯酚(1g,10.30mmol)溶解于DMF(20mL)中，加入碳酸铯(6.71g,20.60mmol)和2- 氟吡啶(1.31g,10.30mmol)，反应液在90℃搅拌12小时。反应液冷却到室温，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系(石油醚/乙酸乙酯：5/0～3/1)纯化，得到3-氟-4-(吡啶-2-氧基)苯胺V3(1.5g)。MS m/z(ESI):205.1[M+1] +。
中间体V4的制备
Figure PCTCN2022096597-appb-000027
将2,5-二氟吡啶(4.4g,38.23mmol)和4-氨基苯酚(4.17g,38.23mmol)溶于DMF(50mL)中，然后加入Cs 2CO 3(13.70g,42.06mmol)，该反应在90℃条件下搅拌16小时，减压旋干后经过柱分离(0～50％EA/PE)得到4-((5-氟吡啶-2-基)氧基)苯胺V4(6g)。MS m/z(ESI):204.6[M+1] +。
中间体V5的制备
Figure PCTCN2022096597-appb-000028
以6-氟吡啶-2-羧酸甲酯为原料，参考中间体V1的制备方法，得到中间体V5。
(±)反式-2-((3-(4-氯苄基)-4-乙基硫基-2,6-二氧代-3,6-二氢-1,3,5-三嗪-1(2H)-基)甲基)环丙烷-1-羧酸甲酯(中间体V6)的制备
Figure PCTCN2022096597-appb-000029
步骤1：将(±)反式-1,2-环丙二羧酸二甲酯(25g，0.158mol)溶解在甲醇(90mL)/水(10mL)中，冰浴冷却，加入氢氧化钠(6.65g，0.166mol)，室温搅拌过夜。反应液浓缩，用水40mL稀释，再用3M盐酸调节PH＝6，乙酸乙酯萃取，合并的有机相用饱和氯化钠溶液洗，硫酸钠干燥，浓缩，得到(±)反式-2-(甲氧基羰基)环丙烷-1-羧酸(22g)，MS m/z(ESI):145.0[M+1] +。
步骤2：将(±)反式-2-(甲氧基羰基)环丙烷-1-羧酸(22g，0.153mol)溶解在干燥的THF(100mL)中，冰浴冷却下，滴加硼烷的四氢呋喃溶液(1M，183mL，0.183mol)，室温搅拌2小时，逐滴向反应液加水，淬灭反应，无气泡生成后，反应液过滤，滤液浓缩，剩余物用乙酸乙酯稀释，水洗，饱和氯化钠溶液洗，干燥，浓缩，得到(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯(20g)，MS m/z(ESI):131.1[M+1] +。
步骤3：将(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯(7g，53.85mmol)，1-(4-氯苄基)-6-(乙硫基)-1,3,5-三嗪-2,4(1H，3H)-二酮(16g，53.85mmol，CAS.NO.1239691-22-5)和三苯基膦(22.6g，86.16mmol)悬浮在干燥的二氯甲烷中，冰浴冷却下，逐滴加入DEAD(15g，86.16mmol)，反应液逐渐澄清，室温搅拌3小时，反应液浓缩，剩余物用90mL乙酸乙酯稀释，搅拌情况下，慢慢加入150mL石油醚，大量固体析出，过滤，滤液浓缩，剩余物用柱层析纯化(EA/PE＝1/1.8)，得到(±)反式-2-((3-(4-氯苄基)-4-乙基硫基-2,6-二氧代-3,6-二氢-1,3,5-三嗪-1(2H)-基)甲基)环丙烷-1-羧酸甲酯V6(25g)，MS m/z(ESI):410.1[M+1] +。
(1S,2S)-2-(羟甲基)环丙烷-1-羧酸甲酯(中间体V7)的制备
Figure PCTCN2022096597-appb-000030
步骤1：将(±)反式-2-(甲氧基羰基)环丙烷-1-羧酸(8.5g)和(S)-α-甲基苄胺(7.86g)加入甲苯(85mL)中，50℃搅拌1h，旋干，加入甲叔醚85mL，搅拌过滤后，烘干得13.5g固体。取固体5.0g，加入50mL丙酮回流溶清，搅拌0.5h，自然降温，再室温搅拌2h，过滤。得1.4g白色固体。将白色固体于21mL丙酮中回流搅拌1h，降至室温，过滤得固体(800mg，ee>99.0％)。取其中100mg固体溶于1mL丙酮中，缓慢挥发，析出无色晶体。通过布鲁克公司D8venture X射线单晶衍射仪对该晶体进行单晶X-ray分析证实其绝对构型为(1S，2S)构型，其结构图如图1所示。因此中间体V7可由羧酸盐的单晶结构推断其绝对构型为(1S，2S)构型。
仪器参数：
Figure PCTCN2022096597-appb-000031
Figure PCTCN2022096597-appb-000032
步骤2：将羧酸盐(700mg)溶于5mL水，再加入5mL 10％wt的硫酸氢钾溶液，水溶液用甲基叔丁基醚10mL萃取2次，有机相合并干燥旋干，得到(1S，2S)-2-(甲氧基羰基)环丙烷-1-羧酸(300mg)。
步骤3：将(1S，2S)-2-(甲氧基羰基)环丙烷-1-羧酸(300mg)溶于1mL四氢呋喃，冰浴下滴加硼烷四氢呋喃溶液(1mol/L，10.4mL)，滴完之后恢复常温搅拌5小时，反应液冰浴下加10mL甲醇淬灭，旋干，加入10mL水，加入甲基叔丁基醚(10mL×3)萃取，合并有机相，10mL水洗一遍，干燥，过滤后旋干，得(1S,2S)-2-(羟甲基)环丙烷-1-羧酸甲酯V7(250mg)。
实施例1：(±)顺式-2-((3-(4-氯苄基)-2,6-二氧代-4-(4-(吡啶-2-基氧基)苯基亚氨基)-1,3,5-三嗪基-1-基)甲基)环丙烷羧酸H1的制备
Figure PCTCN2022096597-appb-000033
步骤1：将(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯(52mg,403.01μmol)，1-(4-氯苄基)-6-(乙硫基)-1,3,5-三嗪-2,4(1H，3H)-二酮(100mg,335.84μmol，1239691-22-5)和三苯基膦(132.13mg,503.76μmol)加入DCM(10mL)中，氩气保护下，滴加偶氮二甲酸二乙酯(87.73mg,503.76μmol,79.04μL)，反应液在室温搅拌1小时。反应液减压旋干，用硅胶柱色谱法以洗脱剂体系(石油醚/乙酸乙酯：1/0～1/1)纯化所得残余物，得到(±)顺式-甲基2-((5-(4-氯苄基)-4-(乙硫基)-2,6-二氧代-5,6-二氢-1,3,5-三嗪-1(2H)-))甲基)环丙烷甲酸酯(125mg)。MS m/z(ESI):410.1[M+H] +。
步骤2：将(±)顺式-甲基2-((5-(4-氯苄基)-4-(乙硫基)-2,6-二氧代-5,6-二氢-1,3,5-三嗪-1(2H)-))甲基)环丙烷甲酸酯(50mg,121.99μmol)和4-(2-吡啶氧基)苯胺(22.71mg,121.99μmol)溶于特戊酸(3mL)中。该反应在120℃条件下搅拌5小时。加入饱和碳酸氢钠溶液(30mL)，用DCM(30mL×3)萃取。有机相干燥，过滤，减压浓缩得到(±)顺式-甲基2-(((-3-(4-氯苄基)-2,6-二氧代-4-(4-(吡啶-2-基氧基)苯基亚氨基)-1,3,5-三嗪基-1-基)甲基)环丙烷甲酸(65mg)。MS m/z(ESI):534.2[M+H] +。
步骤3：将(±)顺式-甲基2-(((-3-(4-氯苄基)-2,6-二氧代-4-(4-(吡啶-2-基氧基)苯基亚氨基)-1,3,5-三嗪基-1-基)甲基)环丙烷甲酸(65mg,121.73μmol)加入THF(10mL)，甲醇(2mL)和水(2mL)中，再加入一水合氢氧化锂(10.22mg,243.46μmol)。反应液在室温搅拌4小时。用1N盐酸溶液中和反应液，减压浓缩。用制备液相色谱(制备柱：water-sunfire；体系：A：水+0.045％甲酸，B：乙腈；波长：254/214nm；梯度：5％-30％乙腈变化)纯化所得残余物，得到(±)顺式-2-((3-(4-氯苄基)-2,6-二氧代-4-(4-(吡啶-2-基氧基)苯基亚氨基)-1,3,5-三嗪基-1-基)甲基)环丙烷羧酸H1(16.21mg)。 1H NMR(400MHz,DMSO-d 6)δ(ppm):12.16(s,1H),9.33(s,1H),8.17–8.11(m,1H),7.84(t,J＝7.0Hz,1H),7.56–7.23(m,6H),7.12-7.02(m,4H),5.28(s,2H),4.07–3.95(m,1H),3.87-3.83(m,1H),1.67-1.61(m,1H),1.50-1.45(m,1H),0.97(t,J＝6.8Hz,2H).MS:520.1[M+H] +。
实施例2：(±)反式-2-((3-(4-氯苄基)-2,6-二氧代-4-(4-(吡啶-2-基氧基)苯基亚氨基)-1,3,5-三嗪基-1-基)甲基)环丙烷羧酸H2的制备
Figure PCTCN2022096597-appb-000034
参考实施例1的制备方法，区别在于用(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯替换化合物(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯，制备得到(±)反式-2-((3-(4-氯苄基)-2,6-二氧代-4-(4-(吡啶-2-基氧基)苯基亚氨基)-1,3,5-三嗪基-1-基)甲基)环丙烷羧酸H2。MS m/z(ESI):520.2[M+1] +； 1H NMR(400MHz,DMSO-d6)δ(ppm):12.03(s,1H),9.37(s,1H),8.16-8.15(m,1H),7.83-7.82(m,1H),7.38-7.37(m,6H), 7.10-7.09(m,3H),7.02-7.01(m,1H),5.28(s,2H),3.79–3.60(m,2H),1.65–1.54(m,2H),0.94-0.92(m,2H)。
实施例3：(±)反式-2-((3-(4-氯苄基)-4-((4-((3-(氟甲基)吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H3的制备
Figure PCTCN2022096597-appb-000035
参考实施例1的制备方法，区别在于用反式-2-(羟甲基)环丙烷-1-羧酸甲酯替换化合物(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯，用中间体V1替换4-(2-吡啶氧基)苯胺，制备得到(±)反式-2-((3-(4-氯苄基)-4-((4-((3-(氟甲基)吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H3。 1H NMR(400MHz,DMSO-d6)δ(ppm):12.13(s,1H),9.38(s,1H),8.14(d,J＝4.7Hz,1H),7.94(d,J＝7.3Hz,1H),7.44-7.34(m,6H),7.21–7.07(m,3H),5.62(s,1H),5.50(s,1H),5.28(s,2H),3.78–3.61(m,2H),1.60-1.57(m,2H),0.99–0.87(m,2H)。MS:552.2[M+H] +。
实施例4：(±)反式-2-((3-(4-氯苄基)-4-((3-((3-氟-4-(吡啶-2-氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H4的制备
Figure PCTCN2022096597-appb-000036
参考实施例1的制备方法，区别在于用(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯替换化合物(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯，用中间体V3替换4-(2-吡啶氧基)苯胺，制备得到(±)反式-2-((3-(4-氯苄基)-4-((3-((3-氟-4-(吡啶-2-氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H4。MS m/z(ESI)＝538.2[M+H] +。 1H NMR(400MHz,DMSO-d6)δ(ppm):12.13(s,1H),9.46(s,1H),8.14–8.06(m,1H),7.85(td,J＝11.9,10.6,5.6Hz,1H),7.51–7.24(m,5H),7.23–6.98(m,3H),6.69(dd,J＝70.9,10.3Hz,1H),5.38–5.02(m,2H),3.87–3.56(m,2H),1.59(t,J＝6.9Hz,2H),0.93(q,J＝7.1,6.3Hz,2H)。
实施例5：(±)反式-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H5的制备
Figure PCTCN2022096597-appb-000037
步骤1：将(±)反式-2-((3-(4-氯苄基)-4-乙基硫基-2,6-二氧代-3,6-二氢-1,3,5-三嗪-1(2H)-基)甲基)环丙烷-1-羧酸甲酯V6(1.9g,4.64mmol)和4-((5-氟吡啶-2-基)氧基)苯胺V4(947mg,4.64mmol)溶于特戊酸(10mL)中。该反应在120℃条件下搅拌3小时，用饱和碳酸氢钠中和，二氯甲烷萃取，无水硫酸钠干燥。减压旋干后经过柱分离(20g，0～5％MeOH/DCM)得到(±)反式-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)氨基)-2,6-二氧代-3,6-二氢-1,3,5-三嗪-1(2H)-基)甲基)环丙烷-1-羧酸甲酯(2g)。MS m/z(ESI):551.4[M+H] +。
步骤2：将(±)反式-甲基-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)氨基)-2,6-二氧代-3,6-二氢-1,3,5-三嗪-1(2H)-基)甲基)环丙烷-1-羧酸酯(2.00g,3.62mmol)溶于甲醇(10mL)和水(10mL)中。然后加入LiOH(433.91mg,18.12mmol)。该反应在室温条件下搅拌16小时，旋干溶剂，用制备液相色谱 (制备柱：21.2×250mm C18柱；体系：10mM HCOOH H 2O；波长：254/214nm；梯度：30％-60％乙腈变化)纯化所得残余物，制备得到(±)反式-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)氨基)-2,6-二氧代-3,6-二氢-1,3,5-三嗪基-1(2H)-基)甲基)环丙烷-1-羧酸H5(1.2g)。MS m/z(ESI):538.1[M+1]。 1HNMR(400MHz,DMSO-d6)δ(ppm):8.14(d,J＝3.1Hz,1H),7.84–7.76(m,1H),7.38(dd,J＝19.6,8.5Hz,4H),7.29–7.00(m,5H),5.23(s,2H),3.67(ddd,J＝20.7,13.7,6.5Hz,2H),1.57(d,J＝3.8Hz,2H),0.97–0.88(m,2H)。
实施例5-1：(±)反式-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸的对映异构体H5-P1和H5-P2的制备
将(±)反式-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸经由手性HPLC柱(系统:Waters UPCC；柱：IG 4.6×100mm 5um溶剂：甲醇；流速：3.0mL/min；)分离，分别得到对映异构体H5-P1(保留时间为：1.960min)和对映异构体H5-P2(保留时间为：2.678min)。
实施例5-2：(1S,2S)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪-1-基)甲基)环丙烷-1-羧酸
Figure PCTCN2022096597-appb-000038
步骤1：将(1S,2S)-2-(羟甲基)环丙烷-1-羧酸甲酯V7(39g，0.3mol)，1-(4-氯苄基)-6-(乙硫基)-1,3,5-三嗪-2,4(1H，3H)-二酮(89g，0.3mol)和三苯基膦(102g，0.39mol)悬浮在干燥的四氢呋喃中，逐滴加入DEAD(68g，0.39mol)，反应液逐渐澄清，室温搅拌过夜。反应液浓缩，剩余物用柱层析纯化(乙酸乙酯/石油醚＝1/2)，得到(1S,2S)-2-((3-(4-氯苄基)-4-乙基硫基-2,6-二氧代-3,6-二氢-1,3,5-三嗪-1(2H)-基)甲基)环丙烷-1-羧酸甲酯(135g)。MS m/z(ESI):410.1[M+1] +。
步骤2：(1S,2S)-2-((3-(4-氯苄基)-4-乙基硫基-2,6-二氧代-3,6-二氢-1,3,5-三嗪-1(2H)-基)甲基)环丙烷-1-羧酸甲酯(120g，0.293mol)和4-((5-氟吡啶-2-基)氧基)苯胺V4(60g，0.293mol)加到圆底烧瓶中，再加入特戊酸(400g)中，然后升温至130℃，搅拌5小时。反应液冷却，用水(800mL)稀释，碳酸钠调节PH＝7-8，二氯甲烷萃取，合并的有机相干燥，过滤，浓缩，剩余物用柱层析纯化(乙酸乙酯/石油醚＝1/3)，得到(1S,2S)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)氨基)-2,6-二氧代--1,3,5-三嗪-1-基)甲基)环丙烷-1-羧酸甲酯(150g)。MS m/z(ESI):552.1[M+1] +。
步骤3：将(1S,2S)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)氨基)-2,6-二氧代-1,3,5-三嗪-1-基)甲基)环丙烷-1-羧酸甲酯(150g，0.272mol)溶解在四氢呋喃/甲醇/水(300/100/100mL)中，加入氢氧化锂(一水合物)(22g，0.544mol)，室温搅拌过夜，反应液过滤，浓缩，剩余物用水稀释，乙酸乙酯萃取两次，水相用3M盐酸溶液调节PH＝5-6左右，再用乙酸乙酯萃取，合并第二次萃取的有机相，饱和氯化钠溶液洗，无水硫酸钠干燥，浓缩，纯化(乙酸乙酯/石油醚＝1/3)得到(1S,2S)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸(100g)。MS m/z(ESI):538.1[M+1] +。 1H NMR(400MHz,DMSO-d6)δ(ppm):8.15(d,J＝3.1Hz,1H),7.82-7.81(m,1H),7.48–7.30(m,6H),7.12-7.10(m,3H),5.36–5.19(m,2H),3.67-3.64(m,2H),1.63–1.54(m,2H),1.00–0.87(m,2H)。
将上述合成得到的(1S,2S)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸与实施例5-1中的对映异构体P1、对映异构体P2通过手性HPLC柱(系统:Waters UPCC；柱：IG 4.6*100mm 5um溶剂：甲醇；流速：3.0mL/min；)进行比对，对映异构体P1与(1S,2S)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸的保留时间一致，因此推定对映异构体P1绝对构型为(1S，2S)构型，即H5-P1为(1S,2S)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸。所以对映异构体P2的绝对构型为(1R，2R)构型， 即H5-P2为(1R,2R)-2-((3-(4-氯苄基)-4-((4-((5-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸。
实施例6：(±)反式-2-((3-(4-氯苄基)-4-((4-((3-二氟甲基)吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H6的制备
Figure PCTCN2022096597-appb-000039
参考实施例1的制备方法，区别在于用(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯替换化合物(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯，用中间体V2替换4-(2-吡啶氧基)苯胺，制备得到(±)反式-2-((3-(4-氯苄基)-4-((4-((3-二氟甲基)吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H6。 1H NMR(400MHz,DMSO-d 6)δ(ppm):12.12(s,1H),9.40(s,1H),8.27(d,J＝4.2Hz,1H),8.08(d,J＝7.3Hz,1H),7.52–6.95(m,10H),5.25(s,2H),3.79–3.57(m,2H),1.59-1.56(m,2H),0.98–0.87(m,2H)。MS:570.2[M+H] +。
实施例7：(±)反式-2-((3-(4-氯苄基)-4-((4-((6-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H7的制备
Figure PCTCN2022096597-appb-000040
参考实施例1的制备方法，区别在于用(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯替换化合物(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯，用4-[(6-氟-2-吡啶)氧基]-苯胺(CAS.NO:62566-16-9)替换4-(2-吡啶氧基)苯胺，制备得到(±)反式-2-((3-(4-氯苄基)-4-((4-((6-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H7。MSm/z(ESI):538.1[M+1] +。 1H NMR(400MHz,DMSO-d6)δ(ppm):12.11(s,1H),9.39(s,1H),8.01-7.97(m,1H),7.41-7.37(m,6H),7.16-7.13(m,2H),6.91-6.85(m,2H),5.28(s,2H),3.73-3.62(m,2H),1.60-1.57(m,2H),0.97-0.90(m,2H)。
实施例8：(±)反式-2-((3-(4-氯苄基)-4-((4-((3-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H8的制备
Figure PCTCN2022096597-appb-000041
参考实施例1的制备方法，区别在于用(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯替换化合物(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯，用4-[(3-氟-2-吡啶)氧基]-苯胺(CAS.NO:1342511-06-1)替换4-(2-吡啶氧基)苯胺，制备得到(±)反式-2-((3-(4-氯苄基)-4-((4-((3-氟吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H8。MSm/z(ESI):538.1[M+1] +。 1H NMR(400MHz,DMSO-d 6)δ(ppm):12.10(s,1H),9.39(s,1H),7.95-7.82(m,2H),7.44-7.36(m,6H),7.20-7.15(m,3H),5.28(s,2H),3.75-3.62(m,2H),1.60-1.57(m,2H),0.95-0.83(m,2H)。
实施例9：(±)反式-2-((3-(4-氯苄基)-4-((4-((6-氟甲基)吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H9的制备
Figure PCTCN2022096597-appb-000042
参考实施例1的制备方法，区别在于用(±)反式-2-(羟甲基)环丙烷-1-羧酸甲酯替换化合物(±)顺式-2-(羟甲基)环丙烷-1-羧酸甲酯，用中间体V5替换4-(2-吡啶氧基)苯胺，制备得到(±)反式-2-((3-(4-氯苄基)-4-((4-((6-氟甲基)吡啶-2-基)氧基)苯基)亚氨基)-2,6-二氧代-1,3,5-三嗪基-1-基)甲基)环丙烷-1-羧酸H9。MS m/z(ESI):552.1[M+1] +。 1H NMR(400MHz,DMSO-d6)δ(ppm):12.14(s,1H),9.37(s,1H),7.91(m,1H),7.49–7.28(m,6H),7.20(d,J＝7.4Hz,1H),7.12-7.11(m,2H),6.99(d,J＝6.5Hz,1H),5.35(s,1H),5.25-5.24(m,2H),3.64-3.63(m,3H),1.58-1.57(m,2H),0.99–0.87(m,2H)。
测试例1：FLIPR测定法筛选化合物对hP2X 3/hP2X 2/3受体的拮抗活性
材料：
Figure PCTCN2022096597-appb-000043
细胞准备：用Versene消化液将细胞1321N1/hP2X3和细胞1321N1/hP2X2/3(供应商Chempartner)稳定转染，离心后用铺板培养基(DMEM+10％DFBS)重悬并计数，调整细胞到3×10 5个细胞/mL，在384孔测试板中每孔铺50μL细胞，置于5％CO 2，37℃培养箱中培养16-24h。
细胞培养培养基配方:
hP2X3 工作液浓度 储备液浓度 稀释倍数 所需体积(mL)
DMEM 1* 1* 1 447.006
FBS 10％ 100％ 10 50
G418二硫酸盐 300μg/ml 50mg/ml 167 2.994012
      配样量 500
         
hP2X2/3 工作液浓度 储备液浓度 稀释倍数 所需体积(mL)
DMEM 1* 1* 1 447.7489
FBS 10％ 100％ 10 50
G418二硫酸盐 150μg/mL 50mg/mL 333 1.501502
潮霉素B 75μg/mL 50mg/mL 667 0.749625
      配样量 500
实验Dye配方:
hP2X3 工作液浓度 储备液浓度 稀释倍数 所需体积(mL)
10*dye stock 0.5* 10* 20 0.7
羧苯磺胺 1.25 0.25 200 0.07
测试缓冲液 1* 1* 1 13.23
ATP水解酶 0.5U/mL 10μL＝1U 200 0.07
      配样量 14
         
hP2X2/3 工作液浓度 储备液浓度 稀释倍数 所需体积(mL)
10*dye stock 0.5* 10* 20 0.7
羧苯磺胺 1.25 0.25 200 0.07
测试缓冲液 1* 1* 1 13.23
      配样量 14
化合物准备：1、供试品：在符合Echo标准的384孔聚丙烯微孔板中，用DMSO配制180倍所需浓度供试化合物(54mM DMSO储备液)，每孔取500nL加到384孔化合物板中，补充30μL测试缓冲液(含1.26mM Ca 2+的1*HBSS+2mM CaCl 2+20mM HEPES)，振摇20-40min以混匀。
2、激动剂：用测试缓冲液配制3倍所需浓度激动剂(α，β-meATP)(hP2X 3和hP2X 2/3细胞均需终浓度3000nM)，每孔加45μL激动剂到384孔化合物板中。
染料孵育：取出细胞板，吸去细胞上清，每孔加入30uL Dye(
Figure PCTCN2022096597-appb-000044
Calcium 4 Assay Kit，测试缓冲液稀释)，孵育1h。
FLIPR检测：向细胞板中每孔加15μL化合物(FLIPR仪器加样)，15分钟后，每孔加22.5μL激动剂，检测荧光信号(激发光波长470nm-495nm，发射波长515nm-575nm)。
数据处理：取信号峰值和谷值的差值作为基础数据，以阳性药最高浓度数据作为100％抑制率，DMSO数据作为0％抑制率，在软件Graphpad prism 6上通过(log(inhibitor)vs.response--Variable slope)拟合化合物的抑制效应曲线并计算IC 50值。
实验均一性标准：每块板做≥12个max值(DMSO作用结果)和≥12个min值(最高浓度阳性药作用结果)，计算Z值，如果Z≥0.5，则认为平行孔均一，数据可信。Z值的计算公式：Z＝1-3*(SDmax+SDmin)/(MEANmax-MEANmin)。测试结果如表4和表5所示。
表4化合物对P2X3的抑制活性
化合物编号 P2X3(IC 50/nM)
H5-P1 23
H5-P2 106
H7 73
H8 148
表5化合物对P2X2/3抑制活性与对P2X3的抑制活性的倍数
化合物编号 选择性倍数
H1 27
H2 151
H5 251
H5-P1 590
H5-P2 176
H7 114
H8 238
由表4和表5可知，本发明实施例化合物对P2X3具有较高的抑制活性，对P2X2/3抑制活性较低，具有较高的抑制选择性。进一步地，由H1与H2之间的比较可知，
Figure PCTCN2022096597-appb-000045
为反式结构的化合物较顺式结构的化合物具有更高的P2X3选择性抑制活性。由H5-P1与H5-P2之间的比较可知，
Figure PCTCN2022096597-appb-000046
为
Figure PCTCN2022096597-appb-000047
构型的化合物较
Figure PCTCN2022096597-appb-000048
构型的化合物具有更高的P2X3选择性抑制活性。
测试例2：大鼠CFA炎症痛模型镇痛作用的评价
实验动物：雄性SD大鼠50只，体重230-260g，实验用动物均采购自北京维通利华实验动物技术有限公司，购买后采用自由采食的方式进行食物和水供应，饲养温度20～25℃，湿度:40～70％，分笼饲养，采用动物尾部标记法进行动物标记。实验设计分组及药物处理如表6所示。
表6实验设计分组及药物处理
Figure PCTCN2022096597-appb-000049
将完全弗氏佐剂与生理盐水等量混合后得到乳剂，大鼠皮下注射一定量的乳剂。将动物置于特制疼痛检测多单元金属网笼内，让动物适应环境15分钟，待动物的梳理、探究活动结束并且适应了检测环境后，开始用一系列校准的von Frey细丝(0.4、0.6、1.0、1.4、2、4、6、8、10、15)在后爪内连续加压，直至出现退缩反射反应。测试人员将对实验进行盲测，实验结果以“均值±标准差”表示，各组数据采用GraphPad Prism 5t检验进行分析，P<0.05具有统计学差异。具体实验结果如图2和图3所示，由图2和图3可以看出，化合物H5-P1在大鼠CFA炎症痛模型中显著抑制机械痛觉超敏，在大鼠CFA炎症痛模型中的起效剂量为30mg/kg，中高剂量(60mg/kg)给药后4h仍具有良好的镇痛效果。
测试例3：毒性实验
实验动物：
雄性和雌性SD大鼠各16只，等级为SPF级。雄性SD大鼠体重约240g，6-9周龄；雌性SD大鼠体重约200g，6-9周龄。实验用动物均采购自浙江维通利华实验动物技术有限公司，购买后采用自由采食的方式进行食物和水供应，饲养温度20～26℃，湿度:40～70％，每小时换气次数≥15次，使用100％的全新风。自动光照，每12hr明暗交替。雌雄分笼饲养，每笼不超过5只。
根据性别及体重进行随机分组，各组动物按表7的剂量设计，每日1次，连续给药共14天。各剂量组分别于首末次给药前、给药后30min、1hr、4hr、7hr、10hr和24hr进行毒代动力学采血，考察各组别在SD大鼠体内的毒代动力学过程，结果如表8所示。
表7实验剂量设计
Figure PCTCN2022096597-appb-000050
安全窗计算依据：
化合物H5-P1在大鼠疼痛模型的起效剂量为30mg/kg，该剂量下Cmax约18.4μg/mL，AUC约为249hr*μg/mL。D1化合物在大鼠疼痛药效模型的起效剂量为60mg/kg，该剂量下Cmax约141μg/mL，AUC约为1566hr*μg/mL。
表8大鼠毒性研究的毒代动力学分析
Figure PCTCN2022096597-appb-000051
试验期间，各组动物体重未见明显异常，动物均未见死亡/濒死。由表8中数据也可以看出，化合物H5-P1具有较高的安全窗。
测试例3中的阳性化合物D1结构如下：
Figure PCTCN2022096597-appb-000052
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (24)

  1. 一种式(I)化合物、或其药学上可接受的盐、或其立体异构体：
    Figure PCTCN2022096597-appb-100001
    其中，
    r为1、2或3；
    W为苯环或5至6元杂芳基环(优选为吡啶环)；所述苯环、5至6元杂芳基环(优选为吡啶环)为未取代的或被1、2、3或4个各自独立地选自下组的取代基取代：氘、卤素(优选为氟、氯)、氰基、羟基、羧基、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、C 2-4烯基、C 2-4炔基、卤代C 1-8烷基(优选为卤代C 1-6烷基，更优选为卤代C 1-3烷基)、氰基取代C 1-8烷基(优选为氰基取代C 1-6烷基，更优选为氰基取代C 1-3烷基)、卤代C 1-8烷氧基(优选为卤代C 1-6烷氧基，更优选为卤代C 1-3烷氧基)、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基和5至6元杂芳基；其中取代基中的所述3至6元杂环烷基、苯基、5至6元杂芳基为未取代的或被1、2或3个各自独立地选自下组的取代基取代：卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基和5至6元杂芳基；
    (R a) n表示苯环上的氢被n个R a取代，n为0、1或2；每个R a相同或不同并且各自独立地为氰基、羟基、羧基、卤素(优选为氟或氯)、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、卤素取代C 1-8烷基(优选为卤素取代C 1-6烷基，更优选为卤素取代C 1-3烷基)、氰基取代C 1-8烷基(优选为氰基取代C 1-6烷基，更优选为氰基取代C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)或-C(O)NR a1R b1；
    R c为氢、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、C 3-8环烷基(优选为C 3-6环烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、氰基、羟基、羧基或卤素(优选为氟或氯)；
    (R b) m表示苯环上的氢被m个R b取代，m为0、1或2；每个R b相同或不同并且各自独立地为氰基、羟基、羧基、卤素(优选为氟或氯)、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、卤代C 1-8烷基(优选为卤代C 1-6烷基，更优选为卤代C 1-3烷基)、氰基取代C 1-8烷基(优选为氰基取代C 1-6烷基，更优选为氰基取代C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)或-C(O)NR a1R b1；
    R 0为氢或C 1-6烷基；
    R a1、R b1各自独立地为氢或C 1-3烷基；或者R a1、R b1与相连的氮原子共同形成4至6元饱和单杂环；所述4至6元饱和单杂环为未取代的或被1、2或3个各自独立地选自下组的取代基取代：氘、卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NH 2、-C(O)NH(C 1-3烷基)、-C(O)N(C 1-3烷基) 2、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基和3至6元杂环烷基；
    R a0、R b0各自独立地为氢、C 1-3烷基或乙酰基；或者R a0、R b0与相连的氮原子共同形成4至6元饱和单杂环；所述4至6元饱和单杂环为未取代的或被1、2或3个各自独立地选自下组的取代基取代：氘、卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NH 2、-C(O)NH(C 1-3烷基)、-C(O)N(C 1-3烷基) 2、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基和3至6元杂环烷基。
  2. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，r为1。
  3. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，W为
    Figure PCTCN2022096597-appb-100002
    (R d) u表示吡啶环上的氢被u个R d取代，u为0、1或2；每个R d相同或不同并且各自独立地为氘、卤素(优选为氟、氯)、氰基、羟基、羧基、C 1-8烷基(优选为C 1-6烷基，更优选为C 1-3烷基)、C 1-8烷氧基(优选为C 1-6烷氧基，更优选为C 1-3烷氧基)、C 2-4烯基、C 2-4炔基、卤代C 1-8烷基(优选为卤代C 1-6烷基，更优选为卤代C 1-3烷基)、卤代C 1-8烷氧基(优选为卤代C 1-6烷氧基，更优选为卤代C 1-3烷氧基)、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)C 1-8烷基(优选为-C(O)C 1-6烷基，更优选为-C(O)C 1-3烷基)、-C(O)OC 1-8烷基(优选为-C(O)OC 1-6烷基，更优选为-C(O)OC 1-3烷基)、-OC(O)C 1-8烷基(优选为-OC(O)C 1-6烷基，更优选为-OC(O)C 1-3烷基)、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基或5至6元杂芳基；其中所述3至6元杂环烷基、苯基、5至6元杂芳基为未取代的或被1、2或3个各自独立地选自下组的取代基取代：卤素、氰基、羟基、羧基、C 1-3烷基、C 1-3烷氧基、C 2-4烯基、C 2-4炔基、卤代C 1-3烷基、卤代C 1-3烷氧基、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)OC 1-3烷基、-OC(O)C 1-3烷基、C 3-6环烷基、C 3-6环烷基氧基、3至6元杂环烷基、苯基和5至6元杂芳基。
  4. 如权利要求3所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，u为0或1；R d为氟、氯、氰基、羟基、羧基、C 1-3烷基(优选甲基、乙基、正丙基、异丙基)、C 1-3烷氧基(优选为甲氧基)、卤代C 1-3烷基(优选为一氟甲基、二氟甲基、三氟甲基)、卤代C 1-3烷氧基(优选为一氟甲氧基、二氟甲氧基、三氟甲氧基)、NR a0R b0、-SO 2C 1-3烷基、-S(O)C 1-3烷基、-C(O)NR a1R b1、-C(O)C 1-3烷基、-C(O)OC 1-3烷基或-OC(O)C 1-3烷基。
  5. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，W为
    Figure PCTCN2022096597-appb-100003
    Figure PCTCN2022096597-appb-100004
  6. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，
    Figure PCTCN2022096597-appb-100005
    为
    Figure PCTCN2022096597-appb-100006
  7. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，
    Figure PCTCN2022096597-appb-100007
    为
    Figure PCTCN2022096597-appb-100008
  8. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，n为0。
  9. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，R c为氢。
  10. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，R 0为氢。
  11. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)结构中的
    Figure PCTCN2022096597-appb-100009
    为顺式结构。
  12. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)结构中的
    Figure PCTCN2022096597-appb-100010
    为反式结构。
  13. 如权利要求3所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，所述式(I)为式(Ⅱ)所示结构：
    Figure PCTCN2022096597-appb-100011
  14. 如权利要求3所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)为式(Ⅱ-1)所示结构：
    Figure PCTCN2022096597-appb-100012
  15. 如权利要求3所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)为式(Ⅱ-2)所示结构：
    Figure PCTCN2022096597-appb-100013
  16. 如权利要求3所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)为式(Ⅱ-3)所示结构：
    Figure PCTCN2022096597-appb-100014
  17. 如权利要求3所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)为式(Ⅱ-4)所示结构：
    Figure PCTCN2022096597-appb-100015
  18. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)化合物为选自以下的化合物或其立体异构体：
    Figure PCTCN2022096597-appb-100016
  19. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)化合物为以下化合物：
    Figure PCTCN2022096597-appb-100017
    Figure PCTCN2022096597-appb-100018
  20. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其中，式(I)化合物为以下化合物：
    Figure PCTCN2022096597-appb-100019
    Figure PCTCN2022096597-appb-100020
  21. 一种药物组合物，包括：
    1)权利要求1-20任一项所述的化合物、或其药学上可接受的盐、或其立体异构体；和
    2)药学上可接受的载体。
  22. 权利要求1-20任一述的化合物、或其药学上可接受的盐、或其立体异构体在制备治疗与P2X3活性或与P2X2/3活性相关的疾病的药物中的应用。
  23. 权利要求21所述的药物组合物在制备治疗与P2X3活性或与P2X2/3活性相关的疾病的药物中的应用。
  24. 根据权利要求22或23所述的应用，其中，所述与P2X3活性或与P2X2/3活性相关的疾病为疼痛、泌尿道病症、胃肠疾病、癌症、免疫相关的疾病、咳嗽、抑郁症、焦虑或应激相关病症。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102395571A (zh) * 2009-02-13 2012-03-28 盐野义制药株式会社 新型三嗪衍生物及含有该三嗪衍生物的药物组合物
CN103153968A (zh) * 2010-08-10 2013-06-12 盐野义制药株式会社 三唑衍生物及含有其的具有镇痛作用的药物组合物
WO2017204316A1 (ja) * 2016-05-27 2017-11-30 塩野義製薬株式会社 ５－オキソ－１，２，４－トリアジン誘導体およびその医薬組成物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102395571A (zh) * 2009-02-13 2012-03-28 盐野义制药株式会社 新型三嗪衍生物及含有该三嗪衍生物的药物组合物
CN103153968A (zh) * 2010-08-10 2013-06-12 盐野义制药株式会社 三唑衍生物及含有其的具有镇痛作用的药物组合物
WO2017204316A1 (ja) * 2016-05-27 2017-11-30 塩野義製薬株式会社 ５－オキソ－１，２，４－トリアジン誘導体およびその医薬組成物

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
COCKAYNE ET AL., NATURE, vol. 407, 2000, pages 1011 - 1015
GARCIA-GUZMAN ET AL., BRAINRES. MOL. BRAINRES., vol. 47, 1997, pages 59 - 66
JARVIS ET AL., PNAS, vol. 99, 2002, pages 17179 - 17184
LEWIS ET AL., NATURE, vol. 377, 1995, pages 432 - 435
no. 1342511-06-1
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
ZHONG ET AL., NEUROSCIENCE, vol. 120, 2003, pages 667 - 675

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