WO2022161462A1 - Dérivé de morpholine, composition pharmaceutique et leur utilisation - Google Patents

Dérivé de morpholine, composition pharmaceutique et leur utilisation Download PDF

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WO2022161462A1
WO2022161462A1 PCT/CN2022/074593 CN2022074593W WO2022161462A1 WO 2022161462 A1 WO2022161462 A1 WO 2022161462A1 CN 2022074593 W CN2022074593 W CN 2022074593W WO 2022161462 A1 WO2022161462 A1 WO 2022161462A1
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alkyl
compound
substituted
pharmaceutically acceptable
cycloalkyl
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PCT/CN2022/074593
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English (en)
Chinese (zh)
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胡斌
关慧平
杨文�
朱经峰
石晓永
张凯
卢荣昌
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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 CN202280008061.5A priority Critical patent/CN116635389A/zh
Publication of WO2022161462A1 publication Critical patent/WO2022161462A1/fr

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    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • 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
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/30—1,4-Oxazines; Hydrogenated 1,4-oxazines not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • 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
    • C07D471/02—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 two hetero rings
    • C07D471/04—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
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention relates to the technical field of medicine, in particular to a morpholine derivative, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a pharmaceutical composition and a use thereof.
  • P2X purinoceptors are a family of ion channels activated by extracellular adenosine triphosphate (ATP). Purinoceptors are involved in a variety of biological functions, particularly in relation to pain sensitivity.
  • the P2X3 receptor is a member of this family and 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. Brain Res., Vol. 47, pp. 59-66 (1997)).
  • P2X3 has been reported to be involved in afferent pathways that control the bladder volume reflex. Thus, inhibition of P2X3 may 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, small diameter sensory neurons (ie, neurons stimulated by pain or injury), which correlates with its role in pain sensitivity.
  • nociceptive, small diameter sensory neurons ie, neurons stimulated by pain or injury
  • blockade of P2X3 receptors has also been reported to have analgesic effects in animal models of chronic inflammatory and neuropathic pain (Jarvis et al., PNAS, 99, 17179-17184 (2002)).
  • methods of reducing P2X3 levels or activity would be useful in modulating pain perception in subjects with pain.
  • P2X3 is also capable of forming P2X2/3 heterodimers with P2X2, another member of the P2X family of purinergic ligand-gated ion channels.
  • P2X2/3 is highly expressed on the terminals (central and peripheral) of sensory neurons (Chen et al., Nature, Vol. 377, pp. 428-431 (1995)). The latest findings also suggest that P2X2/3 is predominantly expressed in bladder sensory neurons (over P2X3) and has a potential role in bladder filling and nociceptive sensations (Zhong et al., Neuroscience, Vol. 120, No. 667- 675 pages (2003)).
  • the object of the present invention is to provide structurally novel morpholine derivatives, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, pharmaceutical compositions thereof, and uses thereof as P2X3 antagonists, which not only have effects on P2X3 Higher inhibitory activity, lower P2X2/3 inhibitory activity, significant inhibitory selectivity, and more importantly, this class of compounds also has favorable pharmacokinetic parameters, such as lower clearance and higher The exposure in vivo is more conducive to the development of effective and safe drugs.
  • the first aspect of the present invention provides a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:
  • Q is -C(O)NHCH 3
  • R 1 is hydrogen, C 1-6 alkyl (preferably C 1-3 alkyl, more preferably methyl) or halogen (preferably fluorine or chlorine);
  • R 2 is hydrogen or halogen (preferably fluorine);
  • R 3 , R 4 are as follows:
  • R 3 is hydrogen;
  • R 4 is -C(O)R 4a or a 3- to 6-membered heterocycloalkyl group that is unsubstituted or separated by 1, 2 or 3 Substituents independently selected from the group consisting of C 1-3 alkyl (preferably methyl), hydroxy, carboxyl, cyano, halogen (preferably fluoro), C 1-3 alkoxy, haloC 1 -3 alkyl, halogenated C 1-3 alkoxy, -NR a0 R b0 , -SO 2 C 1-3 alkyl, -C(O)NR a0 R b0 , -C(O)C 1-3 Alkyl, -C(O)OC 1-3 alkyl, -OC(O)C 1-3 alkyl, C 3-6 cycloalkyl, C 3-6 cycloalkyloxy, 3- to 6-membered hetero cycloalkyl;
  • R 4a is C 1-6 alkyl (preferably C 1-3 alkyl), C 3-8 cycloalkyl (preferably C 3-6 cycloalkyl, more preferably cyclopropyl), 3- to 6-membered Heterocycloalkyl; the C 1-6 alkyl (preferably C 1-3 alkyl) is replaced by 1 or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more multiple) substituted with substituents independently selected from the group consisting of deuterium, hydroxyl, carboxyl, cyano, halogen (preferably fluorine), C 1-3 alkoxy, halo C 1-3 alkyl, halo C 1-3 alkoxy, -NR a0 R b0 , -SO 2 C 1-3 alkyl, -C(O)NR a0 R b0 , -C(O)C 1-3 alkyl, -C(O) OC 1-3 alkyl, -OC(O)C 1-3 alkyl, C 3-6 cyclo
  • R 3 and R 4 are connected together to form a 3- to 6-membered heterocycloalkyl ring, a 5- to 6-membered heterocycloalkenyl ring, and a 5- to 6-membered heteroaryl ring together with the connected carbon and nitrogen atoms; wherein the 3- to 6-membered heterocycloalkyl ring has 1, 2 or 3 nitrogen atoms and 0, 1 or 2 oxygen atoms as ring atoms; wherein the 5- to 6-membered heterocycloalkenyl ring has 2, 3 or 4 nitrogen atoms and 0, 1 or 2 oxygen atoms as ring atoms; the 5- to 6-membered heteroaryl ring has 2, 3 or 4 nitrogen atoms and 0 or 1 oxygen atom as ring atoms; the 3- to 6-membered heterocycloalkyl rings, 5- to 6-membered heterocycloalkenyl rings, and 5- to 6-membered heteroaryl rings are unsubstituted or by 1, 2 or 3 substituents independently selected from
  • Z 1 , Z 2 , Z 3 , and Z 4 represent ring atoms, each independently being C or N (preferably Z 1 is C, Z 2 is C or N, Z 3 is C or N, and Z 4 is N);
  • Z 5 is CH 2 or O
  • Z 6 and Z 7 are each independently O, S or NR a0 ;
  • R 5 n represents a ring
  • the hydrogen on is replaced by n R 5 , n is 0, 1, 2, 3 or 4, each R 5 is the same or different, each independently is C 1-3 alkyl, hydroxyl, carboxyl, cyano, halogen ( Preferably it is fluorine or chlorine), C 1-3 alkoxy, halogenated C 1-3 alkyl, halogenated C 1-3 alkoxy, -NR a0 R b0 , -SO 2 C 1-3 alkyl, -C(O)NR a0 R b0 , -C(O)C 1-3 alkyl, -C(O)OC 1-3 alkyl, -OC(O)C 1-3 alkyl, C 3-6 cycloalkyl, C 3-6 cycloalkyloxy, or 3- to 6-membered heterocycloalkyl; and
  • R a0 and R b0 are each independently hydrogen or C 1-3 alkyl.
  • one of R 1 and R 2 is C 1-3 alkyl, and the other is hydrogen; further, one of R 1 and R 2 is methyl, and the other is hydrogen; further, R 1 is methyl and R 2 is hydrogen.
  • one of R 1 and R 2 is halogen and the other is hydrogen; further, one of R 1 and R 2 is chlorine and the other is hydrogen; still further, R 1 is chlorine and R 2 for hydrogen.
  • one of R 1 and R 2 is halogen, and the other is C 1-3 alkyl; further, one of R 1 and R 2 is fluorine, and the other is methyl; further, R 1 is methyl and R 2 is fluorine.
  • R 4a is C 1-3 alkyl, C 3-6 cycloalkyl (preferably cyclopropyl), or 3- to 6-membered heterocycloalkyl, wherein said C 1-3 alkyl substituted with 1 or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of deuterium, hydroxy, halogen (preferably fluorine); the C 3-6 cycloalkyl (preferably cyclopropyl) and 3- to 6-membered heterocycloalkyl are unsubstituted or replaced by 1 or more (preferably 1, 2, 3, 4, 5 , 6, 7, 8, 9 or more) are substituted with substituents independently selected from the group consisting of deuterium, C1-3 alkyl (preferably methyl), hydroxy, halogen (preferably fluoro).
  • R 4a is deuterated C 1-6 alkyl; further, R 4a is deuterated C 1-3 alkyl.
  • R 4a is deuterated C 1-3 alkyl, further, selected from the group consisting of: mono-deuterated methyl, mono-deuterated ethyl, di-deuterated methyl, di-deuterated ethyl, tri-deuterated Substituted methyl, trideuteroethyl.
  • R 4a is hydroxy substituted C 1-6 alkyl; further, R 4a is hydroxy substituted C 1-3 alkyl; still further, R 4a is hydroxy substituted methyl.
  • R 4a is C 3-6 cycloalkyl; further, R 4a is cyclopropyl. In some embodiments, R 4a is halogen substituted C 3-6 cycloalkyl or hydroxy substituted C 3-6 cycloalkyl; further, R 4a is fluoro cyclopropyl or hydroxy substituted cyclopropyl.
  • R 4a is C 1-3 alkyl, C 3-6 cycloalkyl (preferably cyclopropyl), or 3- to 6-membered heterocycloalkyl, wherein the 3- to 6-membered heterocycloalkyl Cycloalkyl groups contain 1, 2 or 3 oxygen atoms as ring atoms; the C 1-3 alkyl group is surrounded by 1 or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituted with substituents independently selected from the group consisting of hydroxy, halogen (preferably fluorine); the C 3-6 cycloalkyl, 3- to 6-membered heterocycloalkyl are unsubstituted or substituted by 1 One or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of hydroxy, C 1-3 alkyl, halogen (preferably fluorine).
  • the 3- to 6-membered heterocycloalkyl group is selected from the group consisting of: oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, dioxolanyl, and dioxane .
  • the 3- to 6-membered heterocycloalkyl is selected from the following groups:
  • R 3 , R 4 are linked together to form a 3- to 6-membered heterocycloalkyl ring, a 5- to 6-membered heterocycloalkenyl ring, or a 5- to 6-membered heteroaryl ring together with the attached carbon and nitrogen atoms base ring; wherein the 3- to 6-membered heterocycloalkyl ring is The 5- to 6-membered heterocycloalkenyl ring is The 5- to 6-membered heteroaryl ring is wherein the 3- to 6-membered heterocycloalkyl ring, 5- to 6-membered heterocycloalkenyl ring, 5- to 6-membered heteroaryl ring is unsubstituted or is independently selected from the group consisting of 1, 2 or 3 Substituent substitution: C 1-3 alkyl, hydroxyl, carboxyl, cyano, halogen, C 1-3 alkoxy, halogenated C 1-3 alkyl, halogenated C 1-3 alkoxy, -NR
  • R 3 and R 4 are linked together to form the following structure together with the linked carbon and nitrogen atoms:
  • the compound represented by the formula (I) has the structure represented by the formula (II):
  • R 1 is methyl; R 2 is hydrogen.
  • R 3 and R 4 are connected together, and the 3- to 6-membered heterocycloalkyl ring formed together with the connected carbon and nitrogen atoms is
  • the 5- to 6-membered heterocycloalkenyl ring is
  • the 5- to 6-membered heteroaryl ring is wherein the 3- to 6-membered heterocycloalkyl ring, the 5- to 6-membered heterocycloalkenyl ring, and the 5- to 6-membered heteroaryl ring are unsubstituted or by 1, 2 or 3 substituents independently selected from the group consisting of Substituted: C 1-3 alkyl, hydroxyl, carboxyl, cyano, halogen, C 1-3 alkoxy, halogenated C 1-3 alkyl, halogenated C 1-3 alkoxy, -NR a0 R b0 , -SO 2 C 1-3 alkyl, -C(O)NR a0 R b0 , -C(O)C
  • R 3 and R 4 are linked together to form the following structure together with the linked carbon and nitrogen atoms:
  • the compound represented by the formula (I) has the structure represented by the formula (III):
  • R 1 is C 1-6 alkyl (preferably C 1-3 alkyl, more preferably methyl);
  • R 2 is hydrogen or halogen (preferably fluorine or chlorine).
  • R 1 is methyl; R 2 is hydrogen or fluorine.
  • Z 5 is CH 2 .
  • n 0.
  • R 5 is hydroxyl, carboxyl, -COOCH 3 or -CONH 2 .
  • R 4a is deuterated C 1-6 alkyl, or deuterated C 1-3 alkyl, or mono-deuterated methyl, mono-deuterated ethyl group, di-deuteromethyl, di-deuteroethyl, tri-deuteromethyl, tri-deuteroethyl, or tri-deuteromethyl (CD 3 ).
  • R 4a is trideuteromethyl (CD 3 ).
  • R 4a is C 3-8 cycloalkyl (preferably C 3-6 cycloalkyl, more preferably cyclopropyl), or by 1, 2 or C 1-6 alkyl substituted with 3 hydroxy groups (preferably C 1-3 alkyl substituted with 1 hydroxy).
  • R 4a is cyclopropyl, hydroxy-substituted methyl, and hydroxy-substituted isopropyl.
  • the compound represented by the formula (I) has the structure represented by the formula (IV):
  • R 2 is H or fluorine.
  • Z 6 is NH, and Z 7 is S; or Z 6 is S, and Z 7 is NH.
  • Z 6 is CH 2 and Z 7 is S; or Z 6 is S and Z 7 is CH 2 .
  • Z 6 is CH 2
  • Z 7 is S.
  • Z 6 is S
  • Z 7 is CH 2 .
  • R 4a is C 1-3 alkyl, C 3-6 cycloalkyl (preferably cyclopropyl), or 3- to 6-membered heterocycloalkyl, wherein the C 1-3 alkyl group is substituted with 1 or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of : deuterium, hydroxyl, halogen (preferably fluorine); the C 3-6 cycloalkyl (preferably cyclopropyl), 3- to 6-membered heterocycloalkyl are unsubstituted or substituted by one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) substituents independently selected from the group consisting of deuterium, C1-3 alkyl (preferably methyl), hydroxy , halogen (preferably fluorine).
  • R 4a is selected from: C 3-8 cycloalkyl substituted with 1, 2 or 3 hydroxy groups (preferably C 3-6 cycloalkyl substituted with 1 hydroxy group) alkyl, more preferably cyclopropyl substituted with 1 hydroxy), C 3-8 cycloalkyl substituted with 1, 2 or 3 halogens (preferably C 3-6 cycloalkyl substituted with 1 fluorine) , more preferably cyclopropyl substituted with 1 fluorine), and C 1-6 alkyl substituted with 1, 2 or 3 hydroxy (preferably C 1-3 alkyl substituted with 1 hydroxy).
  • R 4a is selected from the group consisting of: hydroxy-substituted cyclopropyl, fluoro-cyclopropyl, hydroxy-substituted methyl, and hydroxy-substituted ethyl.
  • the compound of formula (I) is any of the following compounds:
  • 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 medicament for treating diseases related to P2X3 activity or P2X2/3 activity application.
  • the fourth aspect of the present invention provides the use of the pharmaceutical composition described in the second aspect in the preparation of a medicament for the treatment of diseases related to P2X3 activity or P2X2/3 activity.
  • the disease associated with P2X3 activity or P2X2/3 activity is pain, urinary tract disorder, gastrointestinal disease, cancer, immune-related disease, cough, depression, anxiety, or stress-related disorder .
  • a fifth aspect of the present invention provides a method for treating a disease associated with P2X3 activity or P2X2/3 activity, comprising administering to a patient a therapeutically effective amount of the compound of the first aspect of the present invention, or a pharmaceutically acceptable form thereof A salt, or a stereoisomer thereof or the pharmaceutical composition of the second aspect of the present invention.
  • this kind of morpholine derivatives not only has significant P2X3 inhibitory activity and low P2X2/3 inhibitory activity, but also has excellent in vivo pharmacokinetic parameters, especially relatively Low clearance and high absorption. Therefore, this series of compounds is expected to be developed into drugs for modulating P2X3 and/or P2X2/3 to treat various diseases mediated by (or otherwise associated with) P2X3 and/or P2X2/3 . On this basis, the inventors have completed the present invention.
  • Alkyl refers to straight and branched chain saturated aliphatic hydrocarbon groups.
  • C 1-6 alkyl refers to an alkyl group having 1 to 6 carbon atoms, preferably C 1-3 alkyl; non-limiting examples of alkyl include: methyl, ethyl, n-propyl , isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-di Methylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethyl propyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-
  • Cycloalkyl and “cycloalkyl ring” are used interchangeably and both refer to a saturated monocyclic, bicyclic or polycyclic cyclic hydrocarbon group which may be fused to an aryl or heteroaryl group. Cycloalkyl rings can be optionally substituted. In certain embodiments, the cycloalkyl ring contains one or more carbonyl groups, eg, oxo groups.
  • C 3-8 cycloalkyl refers to a monocyclic cycloalkyl having 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 group may be combined with an aryl or heteroaryl group fused. Heterocycloalkyl rings can be optionally substituted. In certain embodiments, the heterocycloalkyl ring contains one or more carbonyl or thiocarbonyl groups, eg, oxo- and thio-containing groups.
  • 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 1 or 2 ring atoms in the 6-membered heterocycloalkyl are heteroatoms selected from nitrogen, oxygen and sulfur.
  • Non-limiting examples of monocyclic heterocycloalkyl groups include aziridine, oxiranyl, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyrrolyl , oxazolidinyl, dioxolanyl, piperidinyl, piperazinyl, morpholinyl, dioxane, thiomorpholinyl, thiomorpholine-1,1-dioxide, tetra Hydropyranyl, azetidine-2-one, oxetane-2-one, dihydrofuran-2(3H)-one, pyrrolidin-2-one, pyrrolidine- 2,5-dione, dihydrofuran-2,5-dione, piperidin-2-one, tetrahydro-2H-pyran-2-one, piperazin-2-one, Lino-3-one group, etc.
  • Heterocycloalkenyl and “heterocycloalkenyl ring” are used interchangeably to refer to a heterocycloalkyl group containing one or more carbon-carbon double bonds or carbon-nitrogen double bonds within the ring, but are not intended to include, for example, Heteroaryl moiety as defined herein. This group can be fused to an aryl or heteroaryl group. Heterocycloalkenyl rings can be optionally substituted. In certain embodiments, the heterocycloalkenyl ring contains one or more carbonyl or thiocarbonyl groups, eg, oxo- and thio-containing groups.
  • 5- to 6-membered heterocycloalkenyl ring refers to a heterocycloalkenyl ring having 5 to 6 ring atoms, wherein 1, 2 or 3 ring atoms are heteroatoms selected from nitrogen, oxygen and sulfur.
  • heterocycloalkenyl rings include 4,5-dihydro-1H-imidazole rings, 1,4,5,6-tetrahydropyrimidine rings, 3,4,7,8-tetrahydro-2H- 1,4,6-oxadiazosine ring, 1,6-dihydropyrimidine ring, 4,5,6,7-tetrahydro-1H-1,3-diazepine ring, 2,5,6, 7-Tetrahydro-1,3,5-oxadiazepine.
  • Heteroaryl and “heteroaryl ring” are used interchangeably and both refer to a monocyclic, bicyclic, or polycyclic 4n+2 aromatic ring system (eg, having a ring carbon atom and a ring heteroatom) Arrangement of shared 6 or 10 pi electrons) groups wherein 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, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl 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 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, tetraziny
  • 8- to 10-membered heteroaryl refers to a bicyclic heteroaryl group having 8 to 10 ring atoms, of which 1, 2, 3 or 4 are heteroatoms, non-limiting examples include indolyl, iso Indolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzoisofuryl, benzimidazolyl, benzoxazolyl, benziso oxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indenyl, purinyl, pyrido[3,2-d]pyrimidinyl, pyrido [2,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, 1,8-naphthyridinyl, 1,
  • Heteroatom refers to nitrogen, oxygen or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as valence allows. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Halogen refers to fluorine, chlorine, bromine or iodine.
  • Halo refers to the replacement of one or more (eg, 1, 2, 3, 4 or 5) hydrogens in a group with a halogen.
  • Haloalkyl means an alkyl group substituted with 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 and the like.
  • Deuterated alkyl means an alkyl group substituted with one or more (eg, 1, 2, 3, 4, or 5) deuterium atoms, wherein alkyl is as defined above. Preferably it is deuterated C 1-8 alkyl, more preferably deuterated C 1-6 alkyl, more preferably deuterated C 1-3 alkyl. Examples of deuterated alkyl groups include, but are not limited to, mono-deuteromethyl, mono-deuteroethyl, di-deuteromethyl, di-deuteroethyl, tri-deuteromethyl, tri-deuteroethyl, and the like.
  • Alkoxy refers to -O-alkyl, wherein alkyl is as defined above. Preferred is C 1-8 alkoxy, more preferred is C 1-6 alkoxy, and most preferred is C 1-3 alkoxy.
  • alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, isobutoxy, pentoxy, and the like.
  • Cycloalkyloxy refers to -O-cycloalkyl, wherein cycloalkyl is as defined above. C 3-8 cycloalkyloxy is preferred, and C 3-6 cycloalkyloxy is more preferred. Non-limiting examples of cycloalkyloxy include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • Haloalkoxy means an alkoxy group substituted with one or more (eg, 1, 2, 3, 4, or 5) halogens, wherein alkoxy 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 refers to NH2
  • cyano refers to CN
  • nitro refers to NO2
  • benzyl refers to -CH2 - phenyl
  • oxo O
  • carboxy refers to -C (O)OH
  • acetyl refers to -C(O)CH 3
  • hydroxymethyl refers to -CH 2 OH
  • hydroxyethyl refers to -CH 2 CH 2 OH or -CHOHCH 3
  • hydroxyl refers to -OH
  • thiol refers to SH.
  • Substituted means that one or more hydrogen atoms in a group, preferably 1 to 5 hydrogen atoms, independently of each other, are substituted by a corresponding number of substituents, more preferably 1 to 3 hydrogen atoms are independently of each other replaced by a corresponding number of substituents Substituents are substituted. It goes without saying that the substituents are only in their possible chemical positions, and the person skilled in the art can determine (either experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (eg, olefinic) bonds.
  • substituted substituents independently selected from " in the present invention means that when more than one hydrogen on a group is substituted 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 groups independently selected from the following groups: cyano, halogen (preferably fluorine or chlorine), 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), halogenated C 1-8 alkyl (preferably halogenated C 1-6 alkyl, more preferably halogenated C 1-3 alkyl), C 3-8 cycloalkyl (preferably C 3-6 cycloalkyl), halogenated C 1-8 alkoxy (preferably halogenated C 1-6 alkoxy, more preferably halogenated C 1-3 alkoxy), C 1-8 alkyl substituted amino, halogenated C 1-8 alkyl substituted amino, acetyl, hydroxyl, methylol base, hydroxyethyl, carb
  • a compound of the present invention may be administered in a suitable dosage form with one or more pharmaceutically acceptable carriers.
  • These dosage forms are suitable for oral, rectal, topical, intraoral, 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 dosage forms can be prepared from the active compound and one or more carriers or excipients by conventional methods of pharmacy.
  • 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 may be in solution or suspension with the carriers described above.
  • “Pharmaceutically acceptable carrier” means a non-toxic, inert, solid, semi-solid substance or liquid filling machine, diluent, encapsulating material or auxiliary preparation or any type of auxiliary material, 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 standard medical practice.
  • a "therapeutically effective amount" of a compound to be administered will be determined by such factors 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.
  • a “therapeutically effective amount” refers to an amount of a compound of the invention that will elicit a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or ameliorating symptoms, alleviating a disorder, slowing or delaying disease progression, or preventing disease, and the like.
  • the pharmaceutical composition of the present invention or a therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof, contained in the pharmaceutical composition of the present invention It is preferably 0.1 mg to 5 g/kg (body weight).
  • Patient means 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.
  • Treatment refers to alleviating, delaying the progression, attenuating, preventing, or maintaining an existing disease or disorder (eg, cancer). Treatment also includes curing, preventing the development or alleviating to some extent one or more symptoms of a disease or disorder.
  • compositions include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salts” refers to salts with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects.
  • Inorganic acid salts include but are not limited to hydrochloride, hydrobromide, sulfate, phosphate, etc.
  • organic acid salts include but are not limited to formate, acetate, propionate, glycolate, gluconate , Lactate, Oxalate, Maleate, Succinate, Fumarate, Tartrate, Citrate, Glutamate, Aspartate, Benzoate, Mesylate , p-toluenesulfonate and salicylate, etc. These salts can be prepared by methods known in the art.
  • “Pharmaceutically acceptable base addition salts” include, but are not limited to, salts of inorganic bases, such as sodium, potassium, calcium, and magnesium salts, and the like, and include, but are not limited to, salts of organic bases, such as ammonium, tris Ethylamine salts, lysine salts, arginine salts, etc. These salts can be prepared by methods known in the art.
  • the compounds of the present invention may contain one or more chiral centers and exist in various optically active forms.
  • a compound contains one chiral center, the compound contains enantiomers.
  • the present invention includes both isomers and mixtures of isomers, such as racemic mixtures. Enantiomers can be resolved by methods known in the art, such as crystallization and chiral chromatography. When a compound contains more than one chiral center, diastereomers may exist.
  • the present invention includes resolved optically pure specific isomers as well as mixtures of diastereomers. Diastereomers can be resolved by methods known in the art, such as crystallization and preparative chromatography.
  • the present invention provides methods for the preparation of 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 the methods described herein.
  • the solvents, temperatures and other reaction conditions given in the present 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 that are subsequently added by the methods described herein and/or by methods known in the art.
  • the compounds described herein can be synthesized using methods analogous to those described below or exemplified in the Examples, or relevant publications available to those skilled in the art, using appropriate alternative starting materials.
  • the starting materials used to synthesize the compounds described herein may be synthetic or may be obtained from commercial sources.
  • the compounds described herein and other related compounds having various substituents can be synthesized using techniques and starting materials known to those skilled in the art.
  • the general methods for preparing the compounds disclosed herein can be derived from reactions known in the art, and the reactions can be modified to introduce various moieties in the molecules provided herein by reagents and conditions deemed appropriate by those skilled in the art.
  • the main advantage of the present invention is that it provides a series of morpholine derivatives with novel structures, which have high inhibitory activity on P2X3 and low inhibitory activity on P2X2/3, and are useful for treating and Potential for P2X3 activity or diseases associated with P2X2/3 activity.
  • LC-MS Agilent 1290 HPLC System/6130/6150 MS Liquid Mass Spectrometer (manufacturer: Agilent), column Waters BEH/CHS, 50 ⁇ 2.1 mm, 1.7 ⁇ m.
  • ISCO Combiflash-Rf75 or Rf200 automatic column passing instrument Agela 4g, 12g, 20g, 40g, 80g, 120g disposable silica gel column.
  • Known starting materials can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc) and Darui Chemicals, etc. company.
  • the progress of the reaction can be monitored by thin-layer chromatography (TLC), and the compound can be purified by column chromatography.
  • TLC thin-layer chromatography
  • the developing solvent 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-butyl lithium
  • HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • TEA is triethylamine
  • DIEA or DIPEA is N,N-diisopropylethylamine
  • NBS is N-bromosuccinimide
  • NCS N-chlorosuccinimide
  • TBAF tetrabutyl Ammonium fluoride.
  • room temperature refers to about 20-30°C.
  • Step 1 Dissolve NaOH (14.42g, 360.56mmol) in water (300mL), then drop to 0°C, slowly add Br 2 (19.45g, 121.69mmol) dropwise to the reaction system and keep the reaction at 0°C for half an hour , and then ((benzyloxy)carbonyl)-L-asparagine (30 g, 112.68 mmol) was added to the reaction system in batches, and the reaction was heated to 55° C. for 3 hours.
  • reaction solution was lowered to room temperature, extracted and washed twice with EA, and then the aqueous phase was adjusted to pH 1 with 6M HCl, a solid was precipitated, filtered, and the filter cake was spin-dried to obtain V1-1 (24.6 g).
  • Step 2 Dissolve V1-1 (6.3 g, 23.84 mmol) in MeOH (50 mL). The reaction solution was cooled to -20°C, then SOCl 2 (2.13 g, 17.88 mmol, 1.30 mL) was added to the reaction solution. The reaction solution was stirred at -20°C for 2 hours and at 25°C for 16 hours. After the reaction was complete, the solvent was spin-dried, water and EtOAc were added, separated, extracted twice with EtOAc, and dried over Na 2 SO 4 . Spin dry EtOAc to give V1-2 (6 g). MS m/z (ESI): 279.0 [M+1] + .
  • Step 3 Dissolve V1-2 (4 g, 14.38 mmol) in DMF (40 mL), then add Cs2CO3 ( 7.03 g, 21.56 mmol) and iodomethane (4.08 g, 28.75 mmol). The reaction was stirred at room temperature for 16 hours. After the reaction was completed, water was added, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. After filtration, the solvent was spin-dried to give V1-3 (3.4 g). MS m/z (ESI): 293.0 [M+1] + .
  • Step 4 Dissolve V1-3 (3.4 g, 11.63 mmol) in methanol (40 mL), then add 10% Pd/C (1.23 g, 1.16 mmol, 10% purity). The reaction was stirred at room temperature under hydrogen for 2 hours and monitored by LC-MS and filtered after completion. After filtration, V1-4 (1.5 g) was obtained. MS m/z (ESI): 159.1 [M+1] + .
  • Step 5 Dissolve V1-4 in DMF (20 mL), then add 3 -bromoprop-1-ene (2.29 g, 18.97 mmol) and Cs2CO3 (4.64 g, 14.23 mmol). The reaction was stirred at room temperature for 16 hours and monitored by LC-MS. After the reaction was completed, water was added to obtain a solid, which was extracted with EtOAc and dried over anhydrous sodium sulfate. V1-5 (1.3 g) was obtained after spin-drying the solvent. MS m/z (ESI): 199.1 [M+1] + .
  • Step 6 Dissolve V1-5 (1.3 g, 6.56 mmol) in ethanol (15 mL), then add NaBH4 (248.10 mg, 6.56 mmol). The reaction was stirred at room temperature for 2 hours and monitored by LC-MS. After the reaction was completed, acetic acid was added for quenching, sodium carbonate was neutralized, filtered, and the filtrate was spin-dried to obtain V1-6 (1.1 g). MS m/z (ESI): 171.1 [M+1] + .
  • Step 7 Dissolve V1-6 (1.1 g, 6.46 mmol) in DCM (15 mL), then add p-toluenesulfonyl chloride (2.46 g, 12.93 mmol) and TEA (1.96 g, 19.39 mmol, 2.70 mL). The reaction was stirred at room temperature for 16 hours and monitored by LC-MS when the reaction was complete. After spin-drying under reduced pressure, column separation (20 g, 0-80% EA/PE) was performed to obtain V1-7 (410 mg). MS m/z (ESI): 325.1 [M+1] + .
  • Step 8 AD-mix-beta (CAS NO: 148618-32-0) (9.85 g, 12.64 mmol) was added to a mixed solvent of water (150 mL) and tert-butanol (150 mL), and stirred at room temperature to obtain a yellow clear solution. After cooling to 0°C, V1-7 (410 mg, 1.26 mmol) was added. The reaction solution was stirred at 0°C for 24 hours, monitored by LC-MS, and the reaction was completed. After adding 35 g of sodium sulfite, the temperature was raised to room temperature and stirred for 30 minutes. Saturated sodium chloride solution (150 mL) was added and extracted with DCM (250 mL ⁇ 5).
  • Step 9 V1-8 (170 mg, 474.32 ⁇ mol) was dissolved in DMF (5 mL), then imidazole (129.16 mg, 1.90 mmol) and TBSCl (142.98 mg, 948.64 ⁇ mol) were added. The reaction solution was stirred at room temperature for 4 hours, monitored by LC-MS, and the reaction was completed. Saturated brine and ethyl acetate were added, the ethyl acetate was washed twice with brine, dried over anhydrous sodium sulfate, and the solvent was spin-dried. Purification by silica gel column chromatography with eluent system (dichloromethane/methanol: 1/0 to 10/1) gave V1-9 (190 mg). MS m/z (ESI): 473.2 [M+1] + .
  • Step 10 Dissolve V1-9 (190 mg, 401.97 ⁇ mol) in THF, cool to 0° C. and add NaH (32.16 mg, 803.94 ⁇ mol, 60% purity). The reaction solution was stirred at 0° C. for 4 hours, monitored by LC-MS, and the reaction was completed. Quenched by addition of saturated ammonium chloride solution and extracted with DCM. The organic phase was dried and concentrated under reduced pressure to give V1-10 (105 mg). MS m/z (ESI): 301.1 [M+1] + .
  • Step 11 Dissolve V1-10 (160 mg, 532.50 ⁇ mol) in THF (5 mL). Then TBAF (1M, 639.00 ⁇ L) was added, the reaction solution was stirred at room temperature for 2 hours, monitored by LC-MS, the reaction was completed, and the solvent was spin-dried. The resulting residue was purified by silica gel column chromatography with an eluent system (dichloromethane/methanol: 1/0 to 10/1) to give V1-11 (95 mg). MS m/z (ESI): 187.1 [M+1] + .
  • Step 12 V1-11 (95 mg, 510.18 ⁇ mol) was added to DCM (2 mL). Dess-Martin oxidant (259.67 mg, 612.22 ⁇ mol) was added after cooling to 0°C. The reaction solution was stirred at room temperature for 2 hours, monitored by LC-MS, and the reaction was completed. After adding sodium sulfite, the temperature was raised to room temperature and stirred for 5 minutes. Saturated sodium chloride solution was added and extracted with DCM. The organic phase was dried and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with an eluent system (dichloromethane/methanol: 1/0 to 10/1) to give V1 (90 mg). MS m/z(ESI): 203.1[M+1+18] + .
  • Step 1 A solution of triphosgene (19.07 g, 64.28 mmol) in THF (100 mL) was added to (2S)-methyl 2-amino-3-hydroxypropionate (10 g, 64.28 mmol, HCl) in THF (200 mL) , heated to 80°C and stirred for 4 hours. The solvent was spin-dried under reduced pressure and purified by column chromatography (petroleum ether containing 80-90% ethyl acetate as mobile phase) to obtain V2-1 (8 g). MS m/z (ESI): 146.1 [M+1] + .
  • Step 2 V2-1 (6 g, 41.35 mmol) and cesium carbonate (26.96 g, 82.69 mmol) were dissolved in DMF (25 mL), then propene bromide (10.00 g, 82.69 mmol) was added. The reaction was stirred at room temperature for 12 hours, and LCMS showed the reaction was complete. Filtered, and the filtrate was spin-dried under reduced pressure. Purification by column chromatography (petroleum ether containing 90% tetrahydrofuran as mobile phase) gave V2-2 (5 g). MS m/z (ESI): 186.0 [M+1] + .
  • Step 3 Dissolve V2-2 (4 g, 21.60 mmol) in EtOH ( 5 mL), then add NaBH4 (4.09 g, 108.00 mmol). The reaction was stirred at room temperature for 1 hour. The completion of the reaction was monitored by LC-MS. The reaction was quenched with acetic acid, filtered, and the filtrate was spin-dried, dissolved in DCM, solid potassium carbonate was added, filtered, and the filtrate was directly spin-dried to obtain V2-3 (2.5 g). MS m/z (ESI): 158.1 [M+1] + .
  • Step 4 V2-3 (2 g, 12.73 mmol), p-toluenesulfonyl chloride (2.91 g, 15.27 mmol) were dissolved in DCM (30 mL) and TEA (2.57 g, 25.45 mmol, 3.54 mL) was added. The reaction was stirred at room temperature for 6 hours. The completion of the reaction was monitored by LC-MS.
  • Step 5 AD-MIX-BETA (55.29 g, 70.98 mmol) was dissolved in water (100 mL) solvent, cooled to 0°C, and V2-4 (1.7 g, 5.46 mmol) solution was added to it. The reaction was stirred at 0°C for 24 hours. The completion of the reaction was monitored by LC-MS. Add 55 g of sodium sulfite and stir for half an hour.
  • Step 6 V2-5 (1.4 g, 4.05 mmol) and imidazole (1.10 g, 16.21 mmol) were dissolved in DMF (15 mL), cooled to 0 °C and TBSCl (1.22 g, 8.11 mmol) was added. The reaction was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LC-MS. Water was added to quench the reaction and extracted with ethyl acetate. The organic phase was dried, concentrated under reduced pressure, and purified by column chromatography (petroleum ether containing 80-100% ethyl acetate) to obtain product V2-6 (1.4 g). MS m/z (ESI): 460.1 [M+1] + .
  • Step 7 Dissolve V2-6 in THF (5 mL), cool to 0°C, and add NaH (365.48 mg, 9.14 mmol, 60% purity). The reaction was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LC-MS. After the reaction was completed, the reaction was quenched with saturated brine, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was spin-dried under reduced pressure. Purification by column chromatography (dichloromethane containing 20% methanol as mobile phase) gave V2-7 (0.6 g), MS m/z (ESI): 288.1 [M+1] + .
  • Step 8 V2-7 (0.6 g, 2.09 mmol) was dissolved in THF (6 mL) and TBAF (654.96 mg, 2.50 mmol) was added to it. The reaction was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LC-MS. Water was added to quench the reaction, and the solvent was spin-dried under reduced pressure. Purification by column chromatography (petroleum ether containing 90% tetrahydrofuran as mobile phase) gave V2-8 (320 mg), MS m/z (ESI): 174.1 [M+1] + .
  • Step 9 V2-8 (300 mg, 1.73 mmol) was dissolved in acetonitrile (10 mL) and IBX (727.68 mg, 2.60 mmol) was added to it. The reaction was stirred at 100°C for 2 hours. The completion of the reaction was monitored by LC-MS. Cooled to room temperature, filtered, and the filter cake was washed with DCM. The filtrate was spin-dried under reduced pressure to obtain V2 (230 mg). MS m/z (ESI): 172.0 [M+1] + .
  • Step 1 The starting material (R)-3-aminopropane-1,2-diol (9.1 g, 0.1 mol) was dissolved in DCM (500 ml), cooled to -20°C, and TEA (12.1 g, 0.12 mol) was added , the DCM solution of chloroacetyl chloride (12.43 g, 0.12 mol) was slowly added dropwise into it, then the temperature was naturally raised to room temperature, stirred overnight, filtered, the filtrate was washed with water, dried, concentrated, and the remaining yellow solid was treated with methyl tert-butyl ether. After washing, the remaining solid was dried in vacuo to give V3-1 (16 g) MS m/z (ESI): 168.0 [M+1] + .
  • Step 4 Dissolve V3-3 (2.35 g, 10 mmol) in DCM (80 ml), add trimethyloxonium tetrafluoroborate (1.78 g, 12 mmol), stir at room temperature for 6 hours, then add methyl carbazate (1.08 g, 12 mmol) was added, stirred at room temperature overnight, and the reaction solution was concentrated to obtain V3-4 (5 g). MS m/z (ESI): 308.1 [M+1] + .
  • Step 5 Dissolve V3-4 (2g) in DMF (20ml), heat to 170°C under microwave conditions, react for 1 hour, cool to room temperature, and concentrate the reaction solution to obtain a red oil.
  • Step 1 Referring to the method of step 4 of intermediate V3, the difference is that acetyl hydrazide is used instead of methyl carbazate to prepare V4-1.
  • Step 2 Referring to the method of step 5 of intermediate V3, V4 can be prepared. MS m/z (ESI): 274.1 [M+1] + .
  • V5 can be prepared.
  • V6 can be prepared.
  • V7 can be prepared. MS m/z (ESI): 316.1 [M+1] + .
  • Step 1 N-Chloroacetamide (20 g, 145.39 mmol) and triphenylphosphine (38.13 g, 145.39 mmol) were dissolved in acetonitrile (200 mL), and then reacted at 90°C for 12 hours. The reaction solution was spin-dried, then dissolved in DCM, washed with 2N aqueous potassium hydroxide solution, extracted with DCM, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to obtain compound V8-1 (45 g). MS m/z (ESI): 364.1 [M+1] + .
  • Step 2 Compound V8-1 (18 g, 83.63 mmol) and N-methoxy-N-methyl-2-(triphenylphosphino)acetamide (30.39 g, 83.63 mmol) were added to DCM (200 mL) The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate: 1/0-1/1) to obtain compound V8-2 (27 g), MS m/z (ESI): 245.1[M+1] + .
  • Step 3 Compound V8-2 (30 g, 99.88 mmol) was dissolved in methanol (300 mL), and Pd/C (10%) (30 g, 99.88 mmol) was added. The reaction solution was stirred under the protection of hydrogen for 12 hours, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound V8-3 (30 g). MS m/z (ESI): 247.1 [M-55] ⁇ .
  • Step 4 1-Bromo-3,5-difluorobenzene (15.32 g, 79.37 mmol) was dissolved in THF (100 mL), cooled to -78°C under nitrogen protection, and LDA (2M, 49.61 mL) was added dropwise. The reaction solution was stirred at -78°C for 1 hour. Then a solution of compound V8-3 (20 g, 66.15 mmol) in tetrahydrofuran (20 mL) was added dropwise. After the reaction solution was stirred at -78°C for 1 hour, the temperature was raised to 25°C and stirring was continued for 3 hours.
  • Step 5 Compound V8-4 (20 g, 46.05 mmol) was dissolved in THF (200 mL), cooled to 0° C., then NaH (3.53 g, 88.24 mmol, 60% purity) was added, and the reaction solution was stirred at room temperature for half an hour. Then, tert-butyldimethylsilyl chloride (13.88 g, 92.11 mmol) was added, and the reaction solution was stirred for 1 hour.
  • Step 6 Compound V8-5 (25 g, 45.58 mmol) was added to water (100 mL) and THF (100 mL), cooled to 0°C, then N-bromosuccinimide (8.11 g, 45.58 mmol) was added . The reaction solution was stirred at 0°C for 1 hour. Water (100 mL) was added, extracted with ethyl acetate (100 mL ⁇ 3), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was spin-dried. ether/ethyl acetate: 1/0 to 4/1) to obtain compound V8 (19 g). MS m/z (ESI): 413.9 [M+1] + .
  • Step 1 Compound V10 (2.4 g, 4.79 mmol) was dissolved in water (2 mL) and methanol (20 mL), lithium hydroxide (343.81 mg, 14.36 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Add 6N hydrochloric acid solution to adjust the pH to 1, add dichloromethane for extraction, dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound V9-1 (2.2g), MS m/z (ESI): 488.1 [M+1] + .
  • Step 2 Compound V9-1 (2.2 g, 4.51 mmol) was dissolved in DMF (21.85 mL), methylamine hydrochloride (609.40 mg, 9.03 mmol), HATU (2.55 g, 6.77 mmol) and triethylamine ( 2.28 g, 22.56 mmol, 3.15 mL), stirred at room temperature overnight, and concentrated under reduced pressure. Isolation by column chromatography (0-30%, DCM/MeOH) gave V9-2 (2.1 g), MS m/z (ESI): 501.1 [M+1] + .
  • Step 3 Dissolve V9-2 (2.1 g, 4.20 mmol) in methanol (20 mL), add HCL (4.0 M, 3.15 mL), and stir at room temperature for 2 hours. Concentrate under reduced pressure, add saturated sodium bicarbonate solution to adjust pH to 8, and extract with dichloromethane/methanol (10/1). The organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain V9 (2 g), MS m/z (ESI): 401.1 [M+1] + .
  • Step 1 Dissolve 4-methyl-2-nitroaniline (1 g, 6.57 mmol) in THF (20 mL), add sodium hydrogen (788.61 mg, 19.72 mmol, 60% purity) at 0 °C, stir at 0 °C for half an hour , Boc 2 O (2.15 g, 9.86 mmol) was added, and the mixture was stirred at room temperature for half an hour. Water was added to quench the reaction, ethyl acetate was added for extraction, and the organic phase was concentrated under reduced pressure to obtain compound V11-1 (1.6 g). MS m/z (ESI): 153.1 (M+H-100).
  • Step 2 Compound V11-1 (1.6 g, 6.34 mmol) was dissolved in methanol (20 mL), 10% Pd/C (770.33 mg, 6.34 mmol) was added, and the reaction was carried out at room temperature under hydrogen for 2 hours. Filtration and concentration under reduced pressure gave compound V11 (1.4 g). MS m/z (ESI): 167.1 (M+H-56).
  • Step 1 3,5-Difluoro-4-formyl-benzoic acid (1 g, 5.37 mmol) and methylamine hydrochloride (362.78 mg, 5.37 mmol) were dissolved in DMF (12.75 mL), then HATU ( 3.04 g, 8.06 mmol) and TEA (1.63 g, 16.12 mmol, 2.25 mL) were added. The reaction was stirred at room temperature overnight. After the completion of the reaction, the solvent was spin-dried under reduced pressure and subjected to column separation (40 g, 0-60% EA/PE) to obtain compound V12-1 (650 mg). MS m/z (ESI): 218.0 (M+H+18).
  • Step 2 Compound V12-1 (5.5 g, 27.62 mmol) was dissolved in DMF (6 mL), potassium peroxysulfate (25.44 g, 41.43 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow oil, which was separated by column (80 g, 0-30% MeOH/DCM) to obtain compound V12-2 (2.1 g). MS m/z(ESI): 216.1[M+1].
  • Step 3 Compound V11 (300 mg, 1.35 mmol) was dissolved in acetonitrile (20 mL), compound V12-2 (290.38 mg, 1.35 mmol) and N,N,N',N'-tetramethylchloromethylaminohexafluoro were added Phosphate (1.14 g, 4.05 mmol), stirred at room temperature for half an hour. N-methylimidazole (224.34 mg, 2.70 mmol) was added and stirred at room temperature overnight. It was concentrated under reduced pressure, and the oil was separated by column (20 g, 0-90% EA/PE) to give compound V12-3 (420 mg). MS m/z (ESI): 320.1 (M+H-100).
  • Step 4 Compound V12-3 (410 mg, 977.54 ⁇ mol) was dissolved in methanol (10 mL), hydrochloric acid methanol (10 mL) was added, the reaction solution was stirred for 4 hours, concentrated under reduced pressure, and saturated sodium bicarbonate solution was added. Compound V12 (180 mg) was obtained. Yield 57.67%. MS m/z (ESI): 320.1 [M+1].
  • Step 1 Combine 4-bromo-2,6-difluorobenzaldehyde (9.38 g, 42.43 mmol), 5-fluoro-4-methylpyridin-2-amine (4.46 g, 35.36 mmol), (2S)-2 - Ethynylmorpholine-4-carboxylate tert-butyl ester (8.96 g, 42.43 mmol), copper(II) trifluoromethanesulfonate (3.84 g, 10.61 mmol), N,N-dimethylacetamide (924.17 g mg, 10.61 mmol) and cuprous chloride (1.05 g, 10.61 mmol) were dissolved in Xylene (100 mL).
  • Step 2 Compound V13-1 (2.3 g, 4.26 mmol) was dissolved in methanol (10 mL) and HCl/dioxane (10 mL). The reaction was stirred at room temperature for 4 hours. The reaction was monitored by LC-MS. After the reaction was completed, the solvent was spin-dried to obtain compound V13-2 (1.7 g). MS m/z (ESI): 440.0 [M+1].
  • Step 3 Compound V13-2 (1.7 g, 3.86 mmol) was dissolved in DCM (19.33 mL) followed by DIEA (998.09 mg, 7.72 mmol, 1.35 mL). The reaction solution was cooled to 0°C, and then 2,2,2-trideuteroacetyl chloride (472.15 mg, 5.79 mmol) was added. The reaction was stirred at room temperature for 0.5 hours. After the reaction was complete, water and DCM were added, and the aqueous phase was extracted twice with DCM and dried over anhydrous sodium sulfate. After spin drying under reduced pressure, compound V13 (1.8 g) was obtained. MS m/z (ESI): 485.0 [M+1].
  • Step 1 Intermediate V8 (147.47 mg) was dissolved in ethanol (1 mL). The reaction was stirred in the air at 120° C., the solvent evaporated in about half an hour, ethanol (1 mL ⁇ 8) was added repeatedly, and the mixture was stirred for 8 hours. LCMS showed that the reaction was complete. The solvent was spin-dried under reduced pressure and purified by column chromatography (petroleum ether containing 50-60% ethyl acetate as mobile phase) to give V18-1 (60 mg). MS m/z (ESI): 540.0 [M+1] + .
  • Step 2 V18-1 (59.73 mg) and pyrrolidin-2-one (18.81 mg) were dissolved in dioxane (10 mL), followed by Pd2dba3 ( 10.12 mg), Xantphos (12.79 mg), Cesium carbonate (72.07 mg) was added. The reaction was stirred at 100°C for 8 hours under argon. LCMS showed the reaction was complete. After the reaction was completed, the solvent was spin-dried under reduced pressure, and purified by column chromatography (dichloromethane containing 10-15% methanol as mobile phase) to obtain V18-2 (40 mg). MS m/z (ESI): 545.2 [M+1] + .
  • Step 3 Dissolve V18-2 (40 mg, 73.45 ⁇ mol) in dioxane (2.85 mL), then add hydrochloric acid gas (dioxane) (4 M, 146.91 ⁇ L). The reaction was stirred at room temperature for 1 hour. The end of the reaction was monitored by LC-MS. The solvent was spun dry under reduced pressure. The solvent was spin-dried under reduced pressure to give V18 (35 mg), MS m/z (ESI): 445.1 [M+1] + .
  • Step 1 Dissolve 2-chloro-5-fluoro-4-methylpyridine (4 g, 27.48 mmol) and tert-butyl carbamate (4.83 g, 41.22 mmol) in toluene (40 mL), add cesium carbonate (13.43 g) , 41.22mmol), tris(dibenzylideneacetone)dipalladium (2.52g, 2.75mmol) and 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (1.59g, 2.75mmol) . The reaction solution was stirred at 100°C for 12 hours under nitrogen protection.
  • Step 2 V25-1 (5 g, 22.10 mmol) was added to DCM (50 mL), then trifluoroacetic acid (8.53 g, 74.79 mmol, 5.56 mL) was added dropwise to the reaction solution, and the mixture was stirred at room temperature for 12 hours.
  • the reaction solution was quenched with 1N aqueous sodium hydroxide solution (50 mL), then extracted three times with DCM (50 mL), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was spin-dried to obtain V25-2 (2.5 g).
  • Step 3 V25-2 (630.22 mg, 5.00 mmol) and methyl 3,5-difluoro-4-formylbenzoate (1 g, 5.00 mmol) were added to ethanol (20 mL), and the reaction was stirred at 80 °C for 12 hours .
  • reaction solution was spin-dried, dissolved in toluene (20 mL), tert-butyl (S)-2-ethynylmorpholine-4-carboxylate (1.06 g, 5.00 mmol), copper trifluoromethanesulfonate ( II) (542.14mg, 1.50mmol), cuprous chloride (148.40mg, 1.50mmol) and N,N-dimethylacetamide (130.59mg, 1.50mmol), the reaction solution was stirred at 85°C for 12 hours under nitrogen protection .
  • reaction solution was concentrated under reduced pressure, and purified by silica gel column chromatography with an eluent system (dichloromethane/methanol: 1/0 to 10/1) to obtain V25-3 (1 g).
  • Step 4 V25-3 (0.3 g, 577.47 ⁇ mol) was dissolved in DCM (30 mL) and 4M hydrochloric acid in 1,4-dioxane (1.44 mL) was added. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was directly spin-dried to obtain V25-4 (220 mg). MS m/z (ESI): 420.1 [M+1] + .
  • Step 5 V25-4 (0.5 g, 1.19 mmol) was added to DCM (20 mL), followed by TEA (241.28 mg, 2.38 mmol, 332.56 ⁇ L) and methyl chloroformate (168.99 mg, 1.79 mmol), the reaction solution was room temperature React for 2 hours. Water (20 mL) was added and extracted with DCM (20 mL ⁇ 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated 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 V25-5 (400 mg). MS m/z (ESI): 478.1 [M+1] + .
  • Step 6 V25-5 (0.4 g, 837.82 ⁇ mol) was dissolved in ethanol (20 mL), hydrazine hydrate (524.27 mg, 8.38 mmol) was added, and the reaction solution was stirred at 90° C. for 4 hours. The reaction solution was directly spin-dried to obtain V25-6 (400 mg). MS m/z (ESI): 478.1 [M+1] + .
  • Step 7 Dissolve V25-6 (0.4 g, 837.81 ⁇ mol) in DCM (20 mL), add TEA (169.56 mg, 1.68 mmol, 233.71 ⁇ L) and di-tert-butyl dicarbonate (274.27 mg, 1.26 mmol), room temperature Stir overnight.
  • the reaction solution was concentrated under reduced pressure, and V25-7 (400 mg) was obtained by silica gel column chromatography using an eluent system (petroleum ether/ethyl acetate: 2/1 to 1/1). MS m/z (ESI): 578.2 [M+1] + .
  • Step 8 V25-7 (200 mg, 346.29 ⁇ mol) and Lawson’s reagent (210.10 mg, 519.43 ⁇ mol) were added to 1,4 dioxane (4 mL) and stirred in a microwave reactor at 140° C. for 40 minutes. After cooling to room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with an eluent system (dichloromethane/methanol: 10/1 to 5/1) to give V25-8 (150 mg). MS m/z (ESI): 594.2 [M+1] + .
  • Step 9 V25-8 (150 mg, 252.69 ⁇ mol) was dissolved in DCM (6 mL) and trifluoroacetic acid (2 mL) was added dropwise. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, diluted with DCM (20 mL), washed with saturated aqueous sodium bicarbonate solution (20 mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to obtain V25-9 (100 mg ). MS m/z (ESI): 494.1 [M+1] + .
  • Step 10 V25-9 (100 mg, 243.16 ⁇ mol) and N,N′-carbonyldiimidazole (39.43 mg, 243.16 ⁇ mol) were dissolved in tetrahydrofuran (20 mL), and the reaction solution was stirred at room temperature for 1 hour. It was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with an eluent system (dichloromethane/methanol: 10/1 to 5/1) to give V25-10 (57 mg). MS m/z (ESI): 520.1 [M+1] + .
  • Step 11 V25-10 (57 mg, 109.72 ⁇ mol) was added to 4N hydrochloric acid in 1,4-dioxane solution (2 mL) and concentrated hydrochloric acid (2 mL), the reaction solution was heated to 110°C and stirred overnight. The reaction solution was concentrated under reduced pressure to obtain V25 (50 mg). MS m/z (ESI): 462.1 [M+1] + .
  • Step 1 Intermediate V8 (450 mg, 876.91 ⁇ mol) and 4-chloropyridin-2-amine (112.73 mg, 876.91 ⁇ mol) were placed in a microwave tube and ethanol (0.5 mL) was added. The reaction was stirred at 120°C for 4 hours (open reaction, keeping very little solvent). The solvent was spun dry under reduced pressure. The resulting residue was purified by silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate: 1/0 to 2/1) to give V28-1 (142 mg). LC-MS m/z (ESI): 542.1 [M+1] + .
  • Step 2 V28-1 (120 mg, 0.221 mmol) and zinc cyanide (18.17 mg, 154.75 ⁇ mol) were placed in a microwave tube, DMF (5 mL) was added, followed by bis(tri-tert-butylphosphine)palladium(0) (11.30 mg, 22.11 ⁇ mol) was added. The reaction was stirred at 90°C for 30 minutes in a microwave reactor. After the reaction was completed, it was cooled to room temperature, and the solvent was spin-dried under reduced pressure to obtain V28-2 (108 mg). MS m/z (ESI): 489.2 [M+1] + .
  • Step 3 V28-2 (108 mg, 220.90 ⁇ mol) and hydroxylamine hydrochloride (23.03 mg, 331.35 ⁇ mol) were added to ethanol (10 mL) followed by DIPEA (57.10 mg, 441.80 ⁇ mol). The reaction solution was stirred at 80°C for 2 hours. The reaction solution was directly concentrated under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography with a chromatography system (dichloromethane/methanol: 100/7) to obtain V28-3 (60 mg). MS m/z (ESI): 522.2 [M+1] + .
  • Step 4 V28-3 (60 mg, 114.96 ⁇ mol) and N,N′-thiocarbonyldiimidazole (30.73 mg, 172.43 ⁇ mol) were dissolved in tetrahydrofuran (10 mL). The reaction solution was stirred at room temperature for 2 hours. The reaction solution was added with 20 mL of water, and extracted with ethyl acetate (30 mL ⁇ 3). The organic phase was dried, filtered, and concentrated under reduced pressure to give V28-4 (72 mg). MS m/z (ESI): 632.2 [M+1] + .
  • Step 5 Dissolve V28-4 (72 mg, 126.41 ⁇ mol) in tetrahydrofuran (20 mL) and cool to 0 °C. Boron trifluoride ether (89.71 mg, 632.04 ⁇ mol) was added. The reaction solution was stirred at room temperature for 16 hours. Concentration under reduced pressure gave V28-5 (63 mg). MS m/z (ESI): 564.1 [M+1] + .
  • Step 6 V28-5 (63 mg, 111.70 ⁇ mol) was dissolved in methanol (2.86 mL) and hydrochloric acid in 1,4-dioxane (4M, 1 mL) was added. The reaction solution was stirred at room temperature for 1 hour. It was concentrated under reduced pressure, and the pH was adjusted to neutrality by adding 7N ammonia in methanol. The resulting residue was purified by preparative thin layer chromatography with a chromatography system (dichloromethane/methanol/ammonia in methanol: 100/10/2) to give V28 (50 mg). MS m/z (ESI): 464.1 [M+1] + .
  • Example 2 refer to the method of Example 1, except that 2-hydroxyacetic acid was used instead of 2-hydroxy-2-methylpropionic acid to prepare compound Z-2 (12 mg).
  • Example 3 can refer to the method of Example 1, except that 2-hydroxypropionic acid is used instead of 2-hydroxy-2-methylpropionic acid to prepare compound Z-3 (12 mg).
  • MS m/z(ESI): 473.2[M+1] + ; 1H NMR (400MHz, CDCl3) 8.17(br s, 1H), 7.43(br s, 3H), 7.13(br s, 1H), 6.72(br s, 1H) s,1H),5.32(d,J 8Hz,1H),4.39-4.19(m,2H),3.91-3.77(m,1H),3.57(br s,1H),3.44-3.36(m,2H) , 3.16-3.00 (m, 5H), 2.81-2.74 (m, 1H), 2.48-2.42 (m, 4H), 1.27-1.23 (m, 3H).
  • Example 5 can refer to the method of Example 1, except that 2-hydroxy-2-methylpropionic acid is replaced by oxetane-2-carboxylic acid to prepare compound Z-5 (25 mg).
  • Example 6 refer to the method of Example 1, except that tetrahydrofuran-2-carboxylic acid was used instead of 2-hydroxy-2-methylpropionic acid to prepare compound Z-6 (25 mg).
  • Example 6 can refer to the method of Example 1, except that tetrahydro-2H-pyran-2-carboxylic acid is used instead of 2-hydroxy-2-methylpropionic acid to prepare compound Z-7 (20 mg).
  • Step 1 Compound V12 (156.02 mg, 488.62 ⁇ mol) and compound V1 (90 mg, 488.62 ⁇ mol) were added to methanol (10 mL) and stirred at room temperature for 0.5 hours. Then NaBH3CN (92.11 mg, 1.47 mmol) was added. The reaction solution was stirred at room temperature for 16 hours and monitored by LC-MS. After the reaction was completed, it was concentrated under reduced pressure to obtain a yellow oil. The resulting residue was purified by silica gel column chromatography with an eluent system (dichloromethane/methanol: 1/0 to 10/1) to give 8-1 (120 mg). MS m/z(ESI): 488.2[M+1] + .
  • Step 2 Compound 8-1 (120 mg, 246.15 ⁇ mol) was added to 1.2-dichloroethane (5 mL). Anhydrous magnesium sulfate (100 mg) and TFA (28.07 mg, 246.15 ⁇ mol) were then added. The reaction solution was microwaved at 120°C for 1 hour, and the situation was monitored by LC-MS. After the reaction, the filtrate was filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by preparative liquid chromatography (preparative column: 21.2X250mm C18 column; system: 10mM NH4HCO3H2O; wavelength: 254/214nm; gradient: 30%-60% acetonitrile change) , to give Z-8 (5.04 mg).
  • Example 9 refer to the method of Example 8, except that Compound V2 was used instead of Compound V1 to prepare Compound Z-9 (20.86 mg).
  • Step 1 Dissolve intermediate V3 (0.26g, 0.9mmol) in a mixed solvent of tetrahydrofuran/methanol/water (10/1/1) (24ml), add lithium hydroxide (0.19g, 4.5mmol), stir at room temperature for 2 After hours, the reaction solution was concentrated to obtain 10-1 (0.4 g). MS m/z(ESI): 186.1[M+1] +
  • Step 3 Compound 10-2 (0.25 g, 1 mmol) was dissolved in DMF (15 ml), sodium azide (0.14 g, 2 mmol) was added, the temperature was raised to 80° C. and stirred overnight. After cooling to room temperature, the reaction solution was diluted with ethyl acetate, washed with water, washed with saturated sodium chloride solution, dried and concentrated to obtain 10-3 (0.2 g). MS m/z(ESI): 211.1[M+1]+
  • Step 4 Compound 10-3 (0.2 g) was dissolved in ethanol (20 ml), 10% wet palladium on carbon (80 mg) was added, hydrogen was replaced three times, stirred at room temperature for 20 hours, filtered, and the filtrate was concentrated to obtain 10-4 ( 0.17g). MS m/z(ESI): 185.2[M+1]
  • Step 6 Suspend compound 10-5 (0.11 g, 0.34 mmol) in absolute ethanol (20 ml), add 10% wet palladium on carbon (60 mg), replace with hydrogen three times, stir at room temperature for 1 hour, filter, and concentrate to obtain 10 -6 (0.07g).
  • Step 8 Compound 10-7 (76 mg, 0.156 mmol) was dissolved in 1,2-dichloroethane (8 ml), trifluoroacetic acid (1 ml) and anhydrous magnesium sulfate solid (76 mg) were added, and heated under microwave conditions to 130°C, reacted for 40 minutes, cooled to room temperature, filtered and concentrated.
  • the product was purified by preparative HPLC (preparative column: 21.2X250mm C18 column; system: 10 mM NH4HCO3H2O ; wavelength: 254/214 nm; gradient: 30%--60% acetonitrile change) to give Z-10 (19 mg) .
  • Example 11 can refer to the method of Example 10, except that compound V4 is used instead of compound V3, and compound Z-11 (24 mg) can be prepared.
  • MS m/z (ESI): 453.2 [M+1] + ; 1 H NMR (400 MHz, DMSO-d 6 ) ⁇ 8.75-8.72 (m, 1H), 7.75-7.62 (m, 3H), 7.53-7.50 (m,1H), 7.20-7.16(m,1H), 4.80-4.76(m,1H), 4.63-4.47(m,2H), 4.25-4.14(m,2H), 4.10-4.00(m,1H) ,3.66-3.62(m,1H),2.80(d,J 4.5Hz,3H),2.43(s,3H),2.30(s,3H).
  • Example 12 can refer to the method of Example 10, the difference is that compound V5 is used instead of compound V3, and compound Z-12 (32 mg) can be prepared.
  • Example 13 can refer to the method of Example 10, except that compound V6 is used instead of compound V3, and compound Z-13 (34 mg) can be prepared.
  • MS m/z (ESI): 537.2 [M+1] + ; 1 H NMR (400 MHz, DMSO-d 6 ) ⁇ 8.74-8.72 (m, 1H), 7.70-7.68 (m, 3H), 7.50 (s ,1H),7.21-7.16(m,1H),4.59-4.42(m,4H),4.32-4.30(m,1H),4.17-4.14(m,1H),4.07-4.02(m,1H),3.84 -3.81(m, 1H), 3.23-3.20(m, 1H), 2.80(d, J 4.5Hz, 3H), 2.43(s, 3H).
  • Example 14 can refer to the method of Example 10, except that compound V7 is used instead of compound V3, and compound Z-14 (27.19 mg) can be prepared.
  • Example 17 The method of Example 16 can be referred to, except that 2-hydroxy-2-methylpropionic acid is used instead of glycolic acid to prepare compound Z-17 (16 mg).
  • Example 30 can refer to the method of Example 29, the difference is that (2S)-5-oxopyrrolidine-2-carboxylic acid methyl ester is used instead of (2R)-5-oxopyrrolidine-2-carboxylic acid methyl ester, which can be prepared Compound Z-30 (18 mg) was obtained.
  • Example 34 can refer to the method of Example 31, the difference is that (4S)-4-hydroxypyrrolidone-2-one is used instead of (4R)-4-hydroxypyrrolidone-2-one to prepare compound Z-34 (23mg) .
  • reaction solution was concentrated under reduced pressure, and the obtained residue was purified by preparative liquid chromatography (preparative column: 21.2X250mm C18 column; system: 10mM NH4HCO3H2O; wavelength: 254/214nm; gradient: 30%-60% acetonitrile change) to obtain compound Z- 35 (8 mg).
  • Example 36 can refer to the method of Example 29, the difference is that oxazolidin-2-one is used instead of (2R)-5-oxopyrrolidine-2-carboxylate methyl ester to prepare compound Z-36 (13.32 mg ).
  • Example 37 can refer to the method of Example 19, except that acetyl chloride is used instead of deuterated acetyl chloride to prepare compound Z-37.
  • Example 38 can refer to the method of Example 29, except that methyl chloroformate is used instead of deuterated acetyl chloride to prepare compound Z-38.
  • Test Example 1 FLIPR assay to screen compounds for antagonistic activity at 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 in plating medium (DMEM+10% DFBS) after centrifugation, and cells were adjusted to 3 *10 5 cells/mL, plated 50 ⁇ L cells per well in a 384-well test plate, placed in 5% CO 2 , and cultured in a 37°C incubator for 16-24 hours.
  • plating medium DMEM+10% DFBS
  • hP2X3 Working fluid concentration stock concentration Dilution factor Required volume (ml) DMEM 1* 1* 1 447.006 FBS 10% 100% 10 50 G418 Disulfate 300 ⁇ g/ml 50mg/ml 167 2.994012
  • Sample size 500 hP2X2/3 Working fluid concentration stock concentration Dilution factor Required volume (ml) DMEM 1* 1* 1 447.7489 FBS 10% 100% 10 50 G418 Disulfate 150 ⁇ g/ml 50mg/ml 333 1.501502 Hygromycin B 75 ⁇ g/ml 50mg/ml 667 0.749625 Sample size 500
  • Test substance in a 384-well polypropylene microplate conforming to the Echo standard, prepare 180 times the required concentration of the test compound (54mM DMSO stock solution) with DMSO, and add 500nL per well to the 384-well compound The plate was supplemented with 30 ⁇ L of assay buffer (1*HBSS + 2 mM CaCl 2 + 20 mM HEPES containing 1.26 mM Ca 2+ ) and shaken for 20-40 min to mix.
  • assay buffer (1*HBSS + 2 mM CaCl 2 + 20 mM HEPES containing 1.26 mM Ca 2+
  • Agonist Prepare 3 times the required concentration of agonist ( ⁇ , ⁇ -meATP) in test buffer (the final concentration of 3000nM for both hP2X 3 and hP2X 2/3 cells), add 45 ⁇ L of agonist to each well of 384 wells of compound in the board.
  • Dye incubation remove the cell plate, aspirate the cell supernatant, add 30uL Dye ( Calcium 4Assay Kit, diluted in assay buffer), incubated for 1 h.
  • FLIPR detection add 15uL of compound to each well of the cell plate (loaded by FLIPR instrument), and after 15 minutes, add 22.5 ⁇ L of agonist to each well to detect the fluorescence signal (excitation wavelength 470nm-495nm, emission wavelength 515nm-575nm).
  • Data processing take the difference between the signal peak and the trough as the basic data, the highest concentration of the positive drug as the 100% inhibition rate, and the DMSO data as the 0% inhibition rate, on the software Graphpad prism 6 to pass (log(inhibitor) vs. response--Variable slope) to fit inhibitory effect curves of compounds and calculate IC50 values.
  • Test Example 2 In vivo pharmacokinetic test in rats
  • the LC/MS/MS method was used to determine the drug concentration in the plasma at different times after intravenous injection and intragastric administration of the compound of the present invention in rats, to study the pharmacokinetic behavior of the compound of the present invention in rats, and to evaluate its pharmacokinetics. academic characteristics.
  • Test animals healthy adult male SD rats (body weight 200-300g, 6 rats, rats in the intravenous injection group drank water and diet freely, the gavage group fasted all night, and freely drank water and diet 4h after administration), administered by Vital Provided by River Laboratory Animal Co.LTD.
  • SD rats were administered by tail vein (1 mg/kg, 5% DMSO in 0.9% saline) and intragastric administration (5 mg/kg, 5% DMSO in 0.9% saline).
  • Blood sample collection select animals that meet the experimental requirements before administration, weigh and mark. Before collecting blood samples, bind the rats, and each dosed rat will collect blood at a predetermined blood collection time point (intravenous administration: 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 hours after administration, respectively). , a total of 8 time points; intragastric administration: blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 hours after administration, a total of 8 time points), and about 200 ⁇ L of blood was collected through the orbit. The blood was transferred to a 1.5 mL test tube pre-added with K 2 EDTA, centrifuged for 6 min (8000 rpm, 4° C.), and the plasma was taken out. The whole process was completed within 15 min after blood collection. All samples need to be stored in a -20°C freezer until sample analysis. The drug concentration was determined by LC/MS/MS method.
  • Table 2 shows the pharmacokinetic properties parameters of some example compounds of the present invention in rats under the same dose of intravenous administration:
  • Table 3 shows the parameters of pharmacokinetic properties in rats of some example compounds of the present invention under the same dosage of gavage administration:

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Abstract

L'invention concerne un dérivé de morpholine substitué ayant une structure telle que représentée dans la formule (I). L'invention concerne en outre un sel pharmaceutiquement acceptable, un stéréoisomère, une composition pharmaceutique et l'utilisation du dérivé.
PCT/CN2022/074593 2021-01-29 2022-01-28 Dérivé de morpholine, composition pharmaceutique et leur utilisation WO2022161462A1 (fr)

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Citations (9)

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