Salts, crystal forms, preparation methods and applications of nitrogen-containing heterocyclic derivatives

Abstract

A salt, crystal form, preparation method, and application of a nitrogen-containing heterocyclic derivative are disclosed. Specifically, the salt and crystal form of a compound of general formula (I), a preparation method thereof, and a pharmaceutical composition containing a therapeutically effective amount of the crystal form are disclosed, as well as their use as a KRAS G12C mutation inhibitor in the treatment of diseases or conditions such as leukemia, neuroblastoma, melanoma, breast cancer, lung cancer, and colon cancer, wherein each substituent in general formula (I) is as defined in the specification.

Description

[0001] This application claims priority to Chinese Patent Application No. 2020113542899, filed on November 26, 2020, and Chinese Patent Application No. 2021113892168, filed on November 22, 2021. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to a salt, crystal form, preparation method, and application of a nitrogen-containing heterocyclic derivative. Background Technology

[0003] Rat sarcoma (RAS) is encoded by the proto-oncogenes HRAS, NRAS, and KRAS, and consists of four proteins: HRAS, NRAS, KRAS4A, and KRAS4B. These proteins are GTP (guanosine triphosphate)-binding proteins. RAS is located on the inner surface of the cell membrane, upstream of which is the receptor tyrosine kinase (RTK). Activation of RAS regulates downstream signaling pathways such as PI3K and RAF, thereby controlling cell growth, survival, migration, and differentiation.

[0004] RAS exists in two main states in the body: an inactive state bound to GDP (guanosine diphosphate) and an activated state bound to GTP. Its activity is regulated by two proteins: guanine nucleotide exchange factor (GEF) promotes the release of GDP from the RAS protein, enabling GTP binding and activation of RAS; GTPase activating protein (GAP) activates the GTPase activity of the RAS protein, hydrolyzing the GTP bound to RAS protein into GDP, thus inactivating RAS. Under normal circumstances, the RAS protein is in an inactive state. Mutations alter its conformation, leading to a persistently activated RAS state, and downstream signaling pathways are also continuously activated, resulting in the development of various cancers.

[0005] RAS, the first confirmed oncogene, has the highest mutation rate among oncogenes, accounting for an average of 25% of human cancers. The most common oncogenic mutation in the RAS family is KRAS (85%), while NRAS (12%) and HRAS (3%) are less common. KRAS mutations are prevalent in a range of cancers, including pancreatic cancer (95%), colorectal cancer (52%), and lung cancer (31%). The most common type of KRAS mutation is point mutation, occurring primarily in the G12, G13, and Q61 regions (aa59-76) of the p-loop (aa 10–17), with G12 mutations being the most frequent (83%). In non-small cell lung cancer (NSCLC) and colorectal cancer, KRAS G12C is one of the most common mutations.

[0006] Despite significant clinical need, no drugs directly targeting KRAS have yet been marketed, and chemotherapy is currently the only treatment option for patients with KRAS mutations. The development of KRAS inhibitors faces two main challenges: first, the smooth structure of the RAS protein makes it difficult for small molecules to bind to its surface; second, the RAS GTPase has a high affinity for GTP at the picomolar (pM) level, and high levels of endogenous GTP make it difficult for small molecule drugs to block this binding. Recent studies have found that a mutation at KRAS position 12 (glycine, Gly) to cysteine ​​(Cys) creates a conformational change, forming a new pocket for small molecules to covalently bind, irreversibly locking KRAS G12C in an inactive state bound to GDP. Therefore, KRAS G12C inhibitors hold promise as the first drugs to directly target KRAS.

[0007] Several KRAS G12C inhibitors have entered the clinical research stage, such as AMG510 developed by Amgen, ARS-3248 developed by Wellspring Biosciences, and MTRX849 developed by Mirati. They are all currently in Phase I clinical trials, but none of them have been developed and marketed.

[0008] There are currently no specific targeted drugs for KRAS G12C, resulting in significant clinical demand. KRAS G12C inhibitors, which offer higher selectivity, better activity, and better safety, have the potential to treat a variety of cancers and have a broad market prospect.

[0009] Jiangsu Hansoh Pharmaceutical Group Co., Ltd.'s patent application (application number: PCT / CN2020 / 093285) discloses the structures of a series of pyridazine derivative inhibitors. In subsequent research and development, in order to make the products easy to handle, filter, dry, store, and have long-term stability and high bioavailability, this invention has conducted a comprehensive study on the salts and crystal forms of the above substances, and is committed to obtaining the most suitable crystal form. Summary of the Invention

[0010] All contents relating to patent application PCT / CN2020 / 093285 are incorporated herein by reference.

[0011] The object of this invention is to provide an acid salt of a compound represented by general formula (I):

[0012]

[0013] in:

[0014] R a Each group is independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, and -SR. aa -C(O)R aa -NR aa R bb , haloalkoxy or hydroxyalkyl;

[0015] R1 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, and -SR. aa -C(O)R aa -NR aa R bb , haloalkoxy or hydroxyalkyl;

[0016] R2 is selected from alkyl groups;

[0017] R3 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, and -SR. aa -C(O)R aa -NR aa R bb or hydroxyalkyl;

[0018] R4 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, and -SR. aa -C(O)R aa -NR aa R bb or hydroxyalkyl;

[0019] R5 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, and -SR. aa -C(O)R aa -NR aa R bb or hydroxyalkyl;

[0020] R6 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, and -SR. aa -C(O)R aa -NR aa R bb or hydroxyalkyl;

[0021] R7 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, and -SR. aa -C(O)R aa -NR aa R bb or hydroxyalkyl;

[0022] R aa Selected from deuterium, halogens, alkyl groups, deuterated alkyl groups, or haloalkyl groups;

[0023] R bb Selected from deuterium, halogens, alkyl groups, deuterated alkyl groups, or haloalkyl groups; and

[0024] x is selected from 0, 1, 2 or 3.

[0025] In a preferred embodiment of the present invention, in the acid salt of the compound represented by general formula (I), R a Each group is independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -SR aa -C(O)R aa -NR aa R bb C 1-6 Halogenated alkoxy or C 1-6 Hydroxyalkyl;

[0026] Preferred radicals include hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, -SR aa -C(O)R aa -NRaa R bb C 1-3 Halogenated alkoxy or C 1-3 Hydroxyalkyl;

[0027] More preferably, hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, ethylthio, propylthio, isopropylthio, halomethoxy, haloethoxy, halopropoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, or hydroxyisopropyl;

[0028] R1 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -SR aa -C(O)R aa -NR aa R bb C 1-6 Halogenated alkoxy or C 1-6 Hydroxyalkyl;

[0029] Preferred groups include hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, and C. 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, -SR aa -C(O)R aa -NR aa R bb C 1-3 Halogenated alkoxy or C 1-3 Hydroxyalkyl;

[0030] More preferably, hydrogen, methyl, fluorine, chlorine, amino, hydroxyl, or cyano groups;

[0031] R2 is selected from hydrogen or C. 1-6 alkyl;

[0032] Hydrogen or C is preferred 1-3 alkyl;

[0033] More preferably, hydrogen, methyl, ethyl, propyl, or isopropyl;

[0034] R3 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, and C. 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6Alkoxy, -SR aa -C(O)R aa -NR aa R bb Or C 1-6 Hydroxyalkyl;

[0035] Preferred groups include hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, and C. 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, -SR aa Or C 1-3 Hydroxyalkyl;

[0036] More preferably, hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, -S(CH3), hydroxymethyl, hydroxyethyl, hydroxypropyl, or hydroxyisopropyl;

[0037] R4 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, and C. 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -SR aa -C(O)R aa -NR aa R bb or C 1-6 Hydroxyalkyl;

[0038] Preferred groups include hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, and C. 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, -NR aa R bb or C 1-3 Hydroxyalkyl;

[0039] More preferably, hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, -NH(CH3), -N(CH3)2, hydroxymethyl, hydroxyethyl, hydroxypropyl, or hydroxyisopropyl;

[0040] R5 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-6Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -SR aa -C(O)R aa -NR aa R bb Or C 1-6 Hydroxyalkyl;

[0041] Preferred radicals include hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy or C 1-3 Hydroxyalkyl;

[0042] More preferably hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl or hydroxyisopropyl;

[0043] R6 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -SR aa -C(O)R aa -NR aa R bb Or C 1-6 Hydroxyalkyl;

[0044] Preferred radicals include hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy or C 1-3 Hydroxyalkyl;

[0045] More preferably hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl or hydroxyisopropyl;

[0046] R7 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-6 Alkyl, C1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -SR aa -C(O)R aa -NR aa R bb Or C 1-6 Hydroxyalkyl;

[0047] Preferred radicals include hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, and C. 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy or C 1-3 Hydroxyalkyl;

[0048] More preferably hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl or hydroxyisopropyl;

[0049] R aa Selected from deuterium, halogens, C 1-6 Alkyl, C 1-6 Deuterated alkyl or C 1-6 Halogenated alkyl groups;

[0050] Preferred elements: deuterium, halogens, C 1-3 Alkyl, C 1-3 Deuterated alkyl or C 1-3 Halogenated alkyl groups;

[0051] More preferably, methyl, ethyl, propyl, or isopropyl;

[0052] R bb Selected from deuterium, halogens, C 1-6 Alkyl, C 1-6 Deuterated alkyl or C 1-6 Halogenated alkyl groups;

[0053] Preferred elements: deuterium, halogens, C 1-3 Alkyl, C 1-3 Deuterated alkyl or C 1-3 Halogenated alkyl groups;

[0054] More preferably, methyl, ethyl, propyl, or isopropyl;

[0055] x is selected from 0, 1, 2 or 3; preferably 0, 1 or 2; more preferably 0 or 1.

[0056] In a preferred embodiment of the invention, the acid salt of the compound is shown in general formula (II):

[0057]

[0058] in:

[0059] R a Selected from hydrogen or methyl;

[0060] R1 is selected from hydrogen, fluorine, chlorine, bromine, or methyl;

[0061] R3 is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, methyl, -S(CH3) or trifluoromethyl;

[0062] R4 is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, -N(CH3)2, -NH(CH3) or fluorine;

[0063] R5 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, or isopropyl;

[0064] R6 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, or isopropyl;

[0065] R7 is selected from hydrogen, fluorine, chlorine, bromine, or methyl.

[0066] In a preferred embodiment of the invention, the acid salt of the compound is further shown as general formula (II-A) or (II-B):

[0067]

[0068] In a preferred embodiment of the invention, the compound is an acid salt, wherein the compound is selected from:

[0069]

[0070]

[0071]

[0072] In a more preferred embodiment of the invention, the compound is an acid salt, wherein the compound is selected from:

[0073]

[0074]

[0075] The acid in the acid salt is selected from hydroxyethyl sulfonic acid, sulfuric acid, 1,5-naphthalenedisulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleic acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, pamoic acid, p-toluenesulfonic acid, or fumaric acid, preferably hydroxyethyl sulfonic acid or sulfuric acid.

[0076] In a further preferred embodiment of the present invention, the number of acids is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.

[0077] In a further preferred embodiment of the present invention, the acid salt is a hydrate or anhydrous form, and when the acid salt is a hydrate, the number of water molecules is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.

[0078] In the most preferred embodiment of the present invention, an acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is provided, wherein the acid in the acid salt is selected from hydroxyethylsulfonic acid, sulfuric acid, 1,5-naphthalenedisulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleic acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, pamoic acid, p-toluenesulfonic acid, or fumaric acid, wherein the specific structure of the acid salt of the compound is as follows:

[0079]

[0080]

[0081] In a preferred embodiment of the present invention, the acid salt is in crystalline form; preferably, it is the crystalline form of the acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one.

[0082] The acid salt crystal form of P-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one;

[0083] The acid salt crystal form of P-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one;

[0084] The acid salt crystal form of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one;

[0085] The acid salt crystal form of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one;

[0086] More preferably, the crystal forms are hydroxyethyl sulfonate, sulfate, 1,5-naphthalenedisulfonate, methanesulfonate, hydrobromide, phosphate, benzenesulfonate, oxalate, maleate, adipate, hydrochloride, citrate, malonate, L-malate, pamoate, p-toluenesulfonate, or fumarate.

[0087] In a preferred embodiment of the present invention, the crystal form of the acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is provided.

[0088] In a more preferred embodiment of the present invention, the acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)-one is the crystal form, preferably the hydroxyethyl sulfonate crystal form, sulfate crystal form, 1,5-naphthalene disulfonate crystal form, methanesulfonate crystal form, hydrobromide crystal form, phosphate crystal form, benzenesulfonate crystal form, oxalate crystal form, maleate crystal form, adipate crystal form, hydrochloride crystal form, citrate crystal form, malonate crystal form, L-malate crystal form, pamoate crystal form, p-toluenesulfonate crystal form, or fumarate crystal form.

[0089] In a preferred embodiment of the present invention, the acid salt is crystalline, wherein the number of acids is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.

[0090] In a preferred embodiment of the present invention, hydroxyethyl sulfonate crystal forms I-III and sulfate crystal forms I-IV of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one are provided:

[0091] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form I shows a diffraction peak at 2θ: either at 21.7 ± 0.2°; or at 8.8 ± 0.2°; or at 19.3 ± 0.2°; or at 27.6 ± 0.2°; or at 10.9 ± 0.2°; or at 15.4 ± 0.2°; or at 16.7 ± 0.2°; or at 15.8 ± 0.2°. The diffraction peak is present at ±0.2°; or at 17.5±0.2°; or at 23.8±0.2°; or at 10.2±0.2°; or at 11.8±0.2°; preferably including any 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably including any 6, 7, or 8 of them;

[0092] The X-ray powder diffraction pattern 2θ of hydroxyethyl sulfonate crystal form II has a diffraction peak at 21.7±0.2°; or at 8.8±0.2°; or at 19.3±0.2°; or at 27.6±0.2°; or at 10.9±0.2°; or at 23.8±0.2°; or at 16.7±0.2°; or at 15.4±0.2°; or at 15.8±0.2°; or at 10.0±0.2°; preferably including any 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably including any 6, 7, or 8 of them;

[0093] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form III has a diffraction peak at 2θ of 19.4±0.2°; or at 16.9±0.2°; or at 26.6±0.2°; or at 14.6±0.2°; or at 28.0±0.2°; or at 25.6±0.2°; or at 20.7±0.2°; or at 12.8±0.2°; or at 19.1±0.2°; or at 27.2±0.2°; preferably including any 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably including any 6, 7, or 8 of them;

[0094] The X-ray powder diffraction pattern 2θ of sulfate crystal form I has a diffraction peak at 19.0±0.2°; or at 19.4±0.2°; or at 12.4±0.2°; or at 26.2±0.2°; or at 17.6±0.2°; or at 18.1±0.2°; or at 25.3±0.2°; or at 8.8±0.2°; or at 21.9±0.2°; or at 11.5±0.2°; preferably including any 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably including any 6, 7, or 8 of them;

[0095] The X-ray powder diffraction pattern of sulfate crystal form II has a diffraction peak at 15.5±0.2°; or at 11.1±0.2°; or at 8.9±0.2°; or at 19.3±0.2°; or at 22.3±0.2°; or at 23.6±0.2°; or at 17.4±0.2°; or at 27.3±0.2°; or at 17.0±0.2°; or at 27.9±0.2°; preferably including any 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably including any 6, 7, or 8 of them;

[0096] The X-ray powder diffraction pattern of sulfate crystal form III has a diffraction peak at 2θ at 19.6±0.2°; or at 18.0±0.2°; or at 18.4±0.2°; or at 16.8±0.2°; or at 14.3±0.2°; or at 11.8±0.2°; or at 14.9±0.2°; or at 25.7±0.2°; or at 15.4±0.2°; or at 23.5±0.2°; preferably including any 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably including any 6, 7, or 8 of them;

[0097] The X-ray powder diffraction pattern of sulfate crystal form IV has a diffraction peak at 2θ at 19.4±0.2°; or at 18.9±0.2°; or at 15.5±0.2°; or at 8.8±0.2°; or at 18.1±0.2°; or at 24.9±0.2°; or at 17.4±0.2°; or at 12.3±0.2°; or at 26.1±0.2°; or at 14.5±0.2°; preferably including any 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably including any 6, 7, or 8 of them.

[0098] In a further preferred embodiment of the invention, hydroxyethyl sulfonate crystal forms I-III and sulfate crystal forms I-IV of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one are provided:

[0099] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form I contains at least one or more diffraction peaks located at 2θ of 21.7±0.2°, 8.8±0.2°, and 19.3±0.2°, preferably two, more preferably three; optionally, it may further contain at least one diffraction peak located at 2θ of 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 10.2±0.2°, and 11.8±0.2°, preferably two, three, four, or five; for example,

[0100] 21.7±0.2°, 8.8±0.2°;

[0101] 8.8±0.2°, 27.6±0.2°;

[0102] 21.7±0.2°, 8.8±0.2°, 10.9±0.2°;

[0103] 8.8±0.2°, 19.3±0.2°, 15.4±0.2°;

[0104] 21.7±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°;

[0105] 8.8±0.2°, 19.3±0.2°, 15.4±0.2°, 16.7±0.2°;

[0106] 15.8±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°;

[0107] 11.7±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°;

[0108] 16.7±0.2°, 8.8±0.2°, 19.3±0.2°, 16.7±0.2°, 10.9±0.2°, 15.4±0.2°;

[0109] 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 15.8±0.2°, 10.9±0.2°, 15.4±0.2°;

[0110] 10.9±0.2°, 8.8±0.2°, 10.2±0.2°, 27.6±0.2°, 10.9±0.2°, 15.8±0.2°;

[0111] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form II contains at least one or more diffraction peaks located at 2θ of 21.7±0.2°, 10.0±0.2°, and 8.8±0.2°, preferably two, more preferably three; optionally, it may further contain at least one peak located at 2θ of 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, and 16.7±0.2°, preferably two, three, four, or five; for example,

[0112] 21.7±0.2°, 10.0±0.2°;

[0113] 10.0±0.2°, 8.8±0.2°;

[0114] 21.7±0.2°, 10.0±0.2°, 19.3±0.2°;

[0115] 10.0±0.2°, 8.8±0.2°, 27.6±0.2°;

[0116] 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°;

[0117] 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°;

[0118] 27.6±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 16.7±0.2°, 10.9±0.2°;

[0119] 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 16.7±0.2°, 27.6±0.2°, 10.9±0.2°;

[0120] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form III contains at least one or more diffraction peaks located at 2θ of 19.4±0.2°, 16.9±0.2°, and 26.6±0.2°, preferably two, more preferably three; optionally, it may further contain at least one peak located at 2θ of 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, and 12.8±0.2°, preferably two, three, four, or five; for example,

[0121] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°;

[0122] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 12.8±0.2°, 28.0±0.2°, 25.6±0.2°;

[0123] The X-ray powder diffraction pattern of sulfate crystal form I includes at least one or more diffraction peaks located at 2θ of 19.0±0.2°, 19.4±0.2°, and 12.4±0.2°, preferably two, more preferably three; optionally, it may further include at least one diffraction peak located at 2θ of 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, and 8.8±0.2°, preferably two, three, four, or five; for example,

[0124] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°;

[0125] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 25.3±0.2°;

[0126] The X-ray powder diffraction pattern of sulfate crystal form II includes at least one or more diffraction peaks located at 2θ of 15.5±0.2°, 11.1±0.2°, and 8.9±0.2°, preferably two, more preferably three; optionally, it may further include at least one peak located at 2θ of 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, and 27.3±0.2°, preferably two, three, four, or five; for example,

[0127] 15.5±0.2°, 11.1±0.2°;

[0128] 11.1±0.2°, 8.9±0.2°;

[0129] 15.5±0.2°, 11.1±0.2°, 8.9±0.2°;

[0130] 11.1±0.2°, 8.9±0.2°, 19.3±0.2°;

[0131] 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°;

[0132] 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 27.3±0.2°;

[0133] 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°;

[0134] The X-ray powder diffraction pattern of sulfate crystal form III contains at least one or more diffraction peaks located at 2θ of 19.6±0.2°, 18.0±0.2°, and 18.4±0.2°, preferably two, more preferably three; optionally, it may further contain at least one peak located at 2θ of 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, and 25.7±0.2°, preferably two, three, four, or five; for example,

[0135] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°;

[0136] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 14.9±0.2°;

[0137] The X-ray powder diffraction pattern of sulfate crystal form IV contains at least one or more diffraction peaks located at 2θ of 19.4±0.2°, 18.9±0.2°, and 15.5±0.2°, preferably two, more preferably three; optionally, it may further contain at least one peak located at 2θ of 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, and 12.3±0.2°, preferably two, three, four, or five; for example,

[0138] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°;

[0139] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 12.3±0.2°, 24.9±0.2°.

[0140] In a further preferred embodiment of the invention, hydroxyethyl sulfonate crystal forms I-III and sulfate crystal forms I-IV of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one are provided:

[0141] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form I optionally includes one or more diffraction peaks located at 2θ of 21.7±0.2°, 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.3±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, and 16.7±0.2°; preferably, it includes at least any 2-3 peaks, or 4-5 peaks, or 6-8 peaks; more preferably, it includes any 2, 3, 4, 5, 6, 7, or 8 peaks; for example,

[0142] 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.8±0.2°, 17.5±0.2°;

[0143] 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 17.5±0.2°, 15.8±0.2°, 16.7±0.2°, 17.5±0.2°, 23.8±0.2°;

[0144] 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 17.5±0.2°, 16.7±0.2°, 15.8±0.2°;

[0145] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form II optionally includes one or more diffraction peaks located at 2θ of 10.0±0.2°, 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, and 23.8±0.2°; preferably, it includes at least 2-3, 4-5, or 6-8 peaks; more preferably, it includes any 2, 3, 4, 5, 6, 7, or 8 peaks; for example,

[0146] 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°;

[0147] 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 15.4±0.2°, 15.8±0.2°, 10.0±0.2°;

[0148] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form III optionally includes one or more diffraction peaks located at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, and 20.7±0.2°; preferably, it includes at least 2-3, 4-5, or 6-8 peaks; more preferably, it includes any 2, 3, 4, 5, 6, 7, or 8 peaks; for example,

[0149] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 27.2±0.2°;

[0150] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 27.2±0.2°, 20.7±0.2°, 12.8±0.2°;

[0151] The X-ray powder diffraction pattern of sulfate crystal form I optionally includes one or more diffraction peaks located at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, and 25.3±0.2°; preferably, it includes at least 2-3, 4-5, or 6-8 peaks; more preferably, it includes any 2, 3, 4, 5, 6, 7, or 8 peaks; for example,

[0152] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°;

[0153] The X-ray powder diffraction pattern of sulfate crystal form II optionally includes one or more diffraction peaks located at 2θ of 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, and 17.4±0.2°; preferably, it includes at least 2-3, 4-5, or 6-8 peaks; more preferably, it includes any 2, 3, 4, 5, 6, 7, or 8 peaks; for example,

[0154] 15.5±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°;

[0155] 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°;

[0156] The X-ray powder diffraction pattern of sulfate crystal form III optionally includes one or more diffraction peaks located at 2θ of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, and 14.9±0.2°; preferably, it includes at least 2-3, 4-5, or 6-8 peaks; more preferably, it includes any 2, 3, 4, 5, 6, 7, or 8 peaks; for example,

[0157] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 15.4±0.2°;

[0158] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 23.5±0.2°;

[0159] The X-ray powder diffraction pattern of sulfate crystal form IV optionally includes one or more diffraction peaks located at 2θ of 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, and 17.4±0.2°; preferably, it includes at least 2-3, 4-5, or 6-8 peaks; more preferably, it includes any 2, 3, 4, 5, 6, 7, or 8 peaks; for example,

[0160] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°;

[0161] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 26.1±0.2°;

[0162] In a preferred embodiment of the present invention, hydroxyethyl sulfonate crystal forms I-III and sulfate crystal forms I-IV of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one are provided:

[0163] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form I includes values ​​at 2θ of 21.7±0.2°, 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.1±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 13.3±0.2°, 15.4±0.2°, and 16.7±0.2°. One or more diffraction peaks are selected from the following: 15.8±0.2°, 17.5±0.2°, 23.8±0.2°, 14.7±0.2°, 24.3±0.2°, 27.3±0.2°, 23.4±0.2°, 20.6±0.2°, and 21.2±0.2°. Preferably, four, five, six, eight, or ten diffraction peaks are selected from these peaks. For example,

[0164] 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 20.6±0.2°;

[0165] 19.3±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°;

[0166] 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 20.6±0.2°;

[0167] 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 16.7±0.2°, 23.4±0.2°, 20.6±0.2°;

[0168] 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 16.7±0.2°, 15.8±0.2°, 17.5±0.2°, 24.3±0.2°, 14.7±0.2°, 27.3±0.2°, 23.4±0.2°;

[0169] 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 24.3±0.2°, 23.8±0.2°;

[0170] 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.1±0.2°, 27.6±0.2°, 10.9±0.2°, 13.3±0.2°, 21.2±0.2°, 15.8±0.2°, 17.5±0.2°;

[0171] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form II includes one or more diffraction peaks located at 2θ of 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 17.5±0.2°, 14.7±0.2°, 24.4±0.2°, 27.3±0.2°, and 29.2±0.2°, preferably including any 4, 5, 6, 8, or 10 diffraction peaks; for example,

[0172] 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 29.2±0.2°;

[0173] 8.8±0.2°, 27.6±0.2°, 27.3±0.2°, 29.2±0.2°;

[0174] 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 29.2±0.2°;

[0175] 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 27.3±0.2°, 14.7±0.2°;

[0176] 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 27.3±0.2°, 17.5±0.2°;

[0177] 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 15.8±0.2°, 16.7±0.2°, 15.4±0.2°, 17.5±0.2°;

[0178] 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 17.5±0.2°;

[0179] 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 17.5±0.2°, 27.3±0.2°, 29.2±0.2°;

[0180] The X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form III includes one or more diffraction peaks located at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°, 24.4±0.2°, 15.3±0.2°, 26.2±0.2°, 30.2±0.2°, and 27.4±0.2°, preferably including any 4, 5, 6, 8, or 10 diffraction peaks; for example,

[0181] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°;

[0182] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 28.0±0.2°;

[0183] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 27.4±0.2°;

[0184] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 30.2±0.2°;

[0185] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 27.4±0.2°;

[0186] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 30.2±0.2°;

[0187] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°;

[0188] 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 24.4±0.2°;

[0189] The X-ray powder diffraction pattern of sulfate crystal form I includes one or more diffraction peaks located at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, 21.9±0.2°, and 11.5±0.2°, preferably including any 4, 5, 6, 8, or 10 diffraction peaks; for example,

[0190] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°;

[0191] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 17.6±0.2°;

[0192] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 11.5±0.2°;

[0193] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 21.9±0.2°;

[0194] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°;

[0195] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 21.9±0.2°;

[0196] 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, 21.9±0.2°, 11.5±0.2°;

[0197] The X-ray powder diffraction pattern of sulfate crystal form II includes one or more diffraction peaks located at 2θ of 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°, 15.8±0.2°, 24.2±0.2°, 21.8±0.2°, 10.3±0.2°, and 20.6±0.2°. Preferably, it includes diffraction peaks at any of the selected 4, 5, 6, 8, or 10 peaks. For example,

[0198] 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 21.8±0.2°;

[0199] 15.5±0.2°, 8.9±0.2°, 22.3±0.2°, 10.3±0.2°;

[0200] 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 20.6±0.2°, 27.9±0.2°;

[0201] 15.5±0.2°, 11.1±0.2°, 19.3±0.2°, 22.3±0.2°, 21.8±0.2°, 10.3±0.2°;

[0202] 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 20.6±0.2°, 27.9±0.2°;

[0203] 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 23.6±0.2°, 17.4±0.2°, 10.3±0.2°, 20.6±0.2°;

[0204] 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°, 20.6±0.2°;

[0205] 15.5±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 15.8±0.2°, 10.3±0.2°;

[0206] The X-ray powder diffraction pattern of sulfate crystal form III includes one or more diffraction peaks located at 2θ of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, 15.4±0.2°, 23.5±0.2°, 18.8±0.2°, 24.7±0.2°, 9.5±0.2°, 8.8±0.2°, and 11.1±0.2°, preferably including any 4, 5, 6, 8, or 10 diffraction peaks; for example,

[0207] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°;

[0208] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 14.3±0.2°;

[0209] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.1±0.2°;

[0210] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 8.8±0.2°;

[0211] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 11.1±0.2°;

[0212] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 8.8±0.2°;

[0213] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, 15.4±0.2°, 23.5±0.2°;

[0214] 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, 15.4±0.2°, 18.8±0.2°;

[0215] The X-ray powder diffraction pattern of sulfate crystal form IV includes one or more diffraction peaks located at 2θ of 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, 26.1±0.2°, 14.5±0.2°, 22.2±0.2°, 24.3±0.2°, 21.7±0.2°, and 23.6±0.2°, preferably including any 4, 5, 6, 8, or 10 diffraction peaks; for example,

[0216] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°;

[0217] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 18.1±0.2°;

[0218] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 23.6±0.2°;

[0219] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 21.7±0.2°;

[0220] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 23.6±0.2°;

[0221] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, 26.1±0.2°, 14.5±0.2°;

[0222] 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, 26.1±0.2°, 24.3±0.2°.

[0223] In a further preferred embodiment of the present invention, the hydroxyethyl sulfonate crystal form I of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, using Cu-Kα radiation, shows the characteristic X-ray diffraction peaks expressed in terms of 2θ angle and interplanar spacing d value as shown in Table 1.

[0224] Table 1

[0225]

[0226] The hydroxyethyl sulfonate crystal form I of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one described in Example 13-1 of this invention has an X-ray powder diffraction pattern that is basically as follows: Figure 1 As shown; its DSC spectrum is basically as follows Figure 2 As shown; its TGA spectrum is basically as follows. Figure 3 As shown.

[0227] In a further preferred embodiment of the present invention, the hydroxyethyl sulfonate crystal form II of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, irradiated with Cu-Kα, shows the characteristic X-ray diffraction peaks in terms of 2θ angle and interplanar spacing d as shown in Table 2.

[0228] Table 2

[0229]

[0230]

[0231] The hydroxyethyl sulfonate crystal form II of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one described in Example 13-1 of this invention has an X-ray powder diffraction pattern that is basically as follows: Figure 4 As shown; its DSC spectrum is basically as follows Figure 5 As shown; its TGA spectrum is basically as follows. Figure 6 As shown.

[0232] In a further preferred embodiment of the present invention, the hydroxyethyl sulfonate crystal form III of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, irradiated with Cu-Kα, shows the characteristic X-ray diffraction peaks expressed in terms of 2θ angle and interplanar spacing d as shown in Table 3.

[0233] Table 3

[0234]

[0235]

[0236] The hydroxyethyl sulfonate crystal form III of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one described in Example 13-1 of this invention has an X-ray powder diffraction pattern that is essentially as follows: Figure 7 As shown; its DSC spectrum is basically as follows Figure 8 As shown; its TGA spectrum is basically as follows. Figure 9 As shown.

[0237] In a further preferred embodiment of the present invention, the sulfate crystal form I of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, as shown in Table 4, was obtained by Cu-Kα radiation.

[0238] Table 4

[0239]

[0240] The sulfate crystal form I of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one shown in Example 13-1 of this invention has an X-ray powder diffraction pattern that is basically as follows: Figure 10 As shown.

[0241] In a further preferred embodiment of the present invention, the sulfate crystal form II of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, as shown in Table 5, is obtained by Cu-Kα radiation, and the characteristic X-ray diffraction peaks are expressed in terms of 2θ angle and interplanar spacing d.

[0242] Table 5

[0243]

[0244]

[0245] The sulfate crystal form II of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one shown in Example 13-1 of this invention has an X-ray powder diffraction pattern that is basically as follows: Figure 11 As shown.

[0246] In a further preferred embodiment of the present invention, the sulfate crystal form III of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, as shown in Table 6, is obtained by Cu-Kα radiation, with characteristic X-ray diffraction peaks expressed in terms of 2θ angle and interplanar spacing d.

[0247] Table 6

[0248]

[0249] The sulfate crystal form III of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one shown in Example 13-1 of this invention has an X-ray powder diffraction pattern that is basically as follows: Figure 12 As shown.

[0250] In a further preferred embodiment of the present invention, the sulfate crystal form IV of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, as shown in Table 7, was obtained by Cu-Kα radiation. The characteristic X-ray diffraction peaks, expressed in terms of 2θ angle and interplanar spacing d, are shown in Table 7.

[0251] Table 7

[0252]

[0253] The sulfate crystal form IV of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one described in Example 13-1 of this invention has an X-ray powder diffraction pattern that is basically as follows: Figure 13 As shown.

[0254] In a further preferred embodiment of the present invention, the positions of the top ten diffraction peaks with relative peak intensities in the X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form I are... Figure 1 The 2θ error of the diffraction peak at the corresponding position is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°;

[0255] The positions of the top ten diffraction peaks with relative intensities in the X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form II are as follows: Figure 4 The 2θ error of the diffraction peak at the corresponding position is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°;

[0256] The positions of the top ten diffraction peaks with relative intensities in the X-ray powder diffraction pattern of hydroxyethyl sulfonate crystal form III are as follows: Figure 7 The 2θ error of the diffraction peak at the corresponding position is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°;

[0257] The positions of the top ten diffraction peaks with the highest relative intensities in the X-ray powder diffraction pattern of sulfate crystal form I are... Figure 10 The 2θ error of the diffraction peak at the corresponding position is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°;

[0258] The positions of the top ten diffraction peaks with relative intensities in the X-ray powder diffraction pattern of sulfate crystal form II are... Figure 11 The 2θ error of the diffraction peak at the corresponding position is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°;

[0259] The positions of the top ten diffraction peaks with the highest relative intensities in the X-ray powder diffraction pattern of sulfate crystal form III are... Figure 12 The 2θ error of the diffraction peak at the corresponding position is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°;

[0260] The positions of the top ten diffraction peaks with the highest relative intensities in the X-ray powder diffraction pattern of sulfate crystal form IV are... Figure 13 The 2θ error of the diffraction peak at the corresponding position is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°;

[0261] In a further preferred embodiment of the present invention, the acid salt of the compound is characterized in that the acid salt crystal form is a hydrate or anhydrous form, and when the acid salt crystal form is a hydrate, the number of water molecules is 0.2-3, preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, more preferably 0.5, 1, 2 or 3; furthermore, the water in the hydrate is pipe water or crystal water or a combination of both.

[0262] In a further preferred embodiment of the present invention, the method for preparing the acid salt includes the following steps:

[0263] 1) Weigh an appropriate amount of free alkali and dissolve it in a solvent;

[0264] 2) Add an appropriate amount of acid and stir; the amount of acid is preferably 1.2 equivalents.

[0265] 3) Obtain the salt of the compound by rapid centrifugation or standing.

[0266] The solvent is an organic solvent, preferably at least one of ethanol, 2-methyltetrahydrofuran, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate, or 1,4-dioxane.

[0267] The acids are selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthylcarboxylic acid, acetic acid, ethanesulfonic acid, dichloroacetic acid, trichloroacetic acid, acetoxyxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, 4-aminobenzoic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecyl sulfate, dibenzoyl tartaric acid, and ethane-1,2-disulfonic acid. Ethylenesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethylsulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanate, undecanoic acid, trifluoroacetic acid, benzenesulfonic acid, p-methylbenzenesulfonic acid, or L-malic acid; preferably hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, hydroxyethylsulfonic acid, oxalic acid, or hydrobromic acid.

[0268] In a further preferred embodiment of the present invention, the method for preparing the acid salt of the compound and its crystal form includes the following steps:

[0269] 1) Weigh an appropriate amount of free base and dissolve it in the reaction solvent;

[0270] 2) Add an appropriate amount of acid and stir; the amount of acid is preferably 1.2 equivalents.

[0271] 3) After centrifugation and drying, the crystal form of the acid salt of the compound is obtained;

[0272] The reaction solvent is an organic solvent, preferably at least one of ethanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate, or 1,4-dioxane.

[0273] The acids are selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthylcarboxylic acid, acetic acid, ethanesulfonic acid, dichloroacetic acid, trichloroacetic acid, acetoxyxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, 4-aminobenzoic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecyl sulfate, dibenzoyl tartaric acid, and ethane-1,2-disulfonic acid. Ethylenesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethylsulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanate, undecanoic acid, trifluoroacetic acid, benzenesulfonic acid, p-methylbenzenesulfonic acid, or L-malic acid; preferably hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, hydroxyethylsulfonic acid, oxalic acid, or hydrobromic acid.

[0274] In a further preferred embodiment of the present invention, the method for preparing the acid salt crystal form of the compound includes the following steps:

[0275] 1) Weigh an appropriate amount of the salt of the compound and suspend it in an organic solvent;

[0276] 2) After stirring, centrifuging and drying, the crystal form of the acid salt of the compound is obtained;

[0277] The organic solvent is selected from at least one of ethanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate, or 1,4-dioxane.

[0278] In a further preferred embodiment of the present invention, the method for preparing the acid salt of the compound or its crystal form includes the following steps:

[0279] 1) Weigh an appropriate amount of free base and dissolve it in the reaction solvent;

[0280] 2) Add appropriate amounts of acid and organic solvent and stir until dissolved;

[0281] 3) Optionally, add seed crystals;

[0282] 4) Cool, filter to precipitate solid, wash with solvent, and dry.

[0283] The reaction solvent used in step 1) is an organic solvent, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane.

[0284] The acid in step 2) is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthylcarboxylic acid, acetic acid, ethanesulfonic acid, dichloroacetic acid, trichloroacetic acid, acetoxyxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, 4-aminobenzoic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecyl sulfate, dibenzoyl tartaric acid, ethane-1,2- Disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethylsulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanate, undecanoic acid, trifluoroacetic acid, benzenesulfonic acid, p-methylbenzenesulfonic acid, or L-malic acid; preferably hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, hydroxyethylsulfonic acid, oxalic acid, or hydrobromic acid.

[0285] The organic solvent in step 2) is selected from one or more of alcohols, ethers, ketones or esters, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane.

[0286] The solvent in step 3) is selected from one or more of alcohols, ethers, ketones or esters, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane.

[0287] The present invention also provides a preferred embodiment and relates to a pharmaceutical composition comprising an acid salt or crystal form of a compound of general formula (I) shown in a therapeutically effective dose, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0288] The present invention further relates to the use of any of the acid salts of the compound of general formula (I) shown or its crystal form or the pharmaceutical composition thereof in the preparation of KRAS inhibitor drugs; preferably in the preparation of KRAS G12C mutation inhibitor drugs.

[0289] In some embodiments, the use of pharmaceutically acceptable salts of the compounds of the present invention, crystal forms thereof, or compositions thereof in the treatment of Noonan syndrome, panther syndrome, leukemia, neuroblastoma, melanoma, breast cancer, esophageal cancer, head and neck tumors, gastric cancer, lung cancer, and colon cancer; preferably non-small cell lung cancer, colon cancer, esophageal cancer, and head and neck tumors.

[0290] Detailed description of the invention

[0291] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0292] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 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-Dimethylbutyl, 2-Ethylbutyl, 2-Methylpentyl, 3-Methylpentyl, 4-Methylpentyl, 2,3-Dimethylbutyl, n-Heptyl, 2-Methylhexyl, 3-Methylhexyl, 4-Methylhexyl, 5-Methylhexyl, 2,3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl 3-Ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 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-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester groups. The present invention preferably uses methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuteralkyl, alkoxy-substituted alkyl, and hydroxy-substituted alkyl.

[0293] The term "alkylene" refers to an alkyl group in which one hydrogen atom is further substituted, for example: "methylene" refers to -CH2-, "ethylene" refers to -(CH2)2-, "propylene" refers to -(CH2)3-, "butylene" refers to -(CH2)4-, etc. The term "alkenyl" refers to an alkyl group as defined above, consisting of at least two carbon atoms and at least one carbon-carbon double bond, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc. Alkenyl groups can be substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0294] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups, preferably cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, and cycloheptyl.

[0295] The term "spirocycloalkyl" refers to a polycyclic group consisting of 5 to 20 quintile rings sharing a single carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14 quintiles, more preferably 7 to 10 quintiles. Spirocycloalkyl groups are classified into monospirocycloalkyl, bispirocycloalkyl, or polyspirocycloalkyl groups based on the number of shared spiro atoms between the rings, with monospirocycloalkyl and bispirocycloalkyl groups being preferred. More preferably, it is a 3 / 6, 3 / 5, 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 quintile monospirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups include:

[0296] wait;

[0297] It also includes spirocyclic alkyl groups that share a spiro atom with a heterocyclic alkyl group, and non-limiting examples include:

[0298] wait.

[0299] The term "fused-ring alkyl" refers to a 5- to 20-membered polycyclic carbon group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused-ring alkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused-ring alkyl include:

[0300] wait.

[0301] The term "bridged cycloalkyl" refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly bonded carbon atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0302]

[0303] The cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0304] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 8 ring atoms; most preferably, it contains 3 to 8 ring atoms; even more preferably, it contains a 3-8 membered heterocyclic group containing 1 to 3 nitrogen atoms, optionally substituted with 1 to 2 oxygen atoms, sulfur atoms, or oxo groups, including nitrogen-containing monocyclic heterocyclic groups, nitrogen-containing spirocyclic groups, or nitrogen-containing fused heterocyclic groups.

[0305] Non-limiting examples of monocyclic heterocyclic groups include pyrrolyl, imidazoyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, acrylonitrile, 1,4-diazaheptanyl, pyranyl, etc., preferably pyrrolyl, morpholinyl, piperidinyl, acrylonitrile, 1,4-diazaheptanyl, and piperazinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups; wherein the spirocyclic, fused-ring, and bridged-ring heterocyclic groups involved are optionally connected to other groups by single bonds, or further cyclically linked to other cycloalkyl, heterocyclic, aryl, and heteroaryl groups by any two or more atoms on the ring.

[0306] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 member monocyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m The heteroatom is a carbon atom (where m is an integer from 0 to 2). It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14 fused, more preferably 7 to 10 fused. Spirocyclic groups are classified into monospirocyclic, bispirocyclic, or multispirocyclic groups based on the number of shared spiro atoms between rings, preferably monospirocyclic and bispirocyclic groups. More preferably, they are 3 / 5, 3 / 6, 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 monospirocyclic groups. Non-limiting examples of spirocyclic groups include:

[0307]

[0308] wait.

[0309] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic group, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0310]

[0311]

[0312] wait.

[0313] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic group, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0314]

[0315] wait.

[0316] The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0317] wait.

[0318] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0319] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 12-membered, such as phenyl and naphthyl. More preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, including benzo5- to 10-membered heteroaryl, benzo3- to 8-membered cycloalkyl, and benzo3- to 8-membered heteroalkyl, preferably benzo5- to 6-membered heteroaryl, benzo3- to 6-membered cycloalkyl, and benzo3- to 6-membered heteroalkyl, wherein the heterocyclic group is a heterocyclic group containing 1-3 nitrogen, oxygen, and sulfur atoms; or may further include a ternary nitrogen-containing fused ring containing a benzene ring.

[0320] The ring connected to the parent structure is an aryl ring, and non-limiting examples include:

[0321]

[0322] wait.

[0323] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0324] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12-membered, more preferably 5- or 6-membered, such as imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, oxazolyl, pyrroleyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably triazolyl, thiophene, imidazolyl, pyrazolyl, oxazolyl, pyrimidinyl, or thiazolyl; more preferably pyrazolyl, pyrroleyl, and oxazolyl. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0325]

[0326] wait.

[0327] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0328] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl is defined as described above, preferably alkyl containing 1 to 8 carbon atoms, more preferably alkyl containing 1 to 6 carbon atoms, and most preferably alkyl containing 1 to 3 carbon atoms. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0329] The term "alkathioyl" refers to -S- (alkyl) and -S- (unsubstituted cycloalkyl), wherein the definition of alkyl is as described above. Alkyl groups containing 1 to 8 carbon atoms are preferred, alkyl groups containing 1 to 6 carbon atoms are more preferred, and alkyl groups containing 1 to 3 carbon atoms are most preferred. Non-limiting examples of alkathioyl groups include: methylthioyl, ethylthioyl, propylthioyl, butylthioyl, cyclopropylthioyl, cyclobutylthioyl, cyclopentylthioyl, and cyclohexylthioyl. Alkathioyl groups can be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkathioyl, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkathioyl, heterocycloalkathioyl, carboxyl, or carboxylic acid ester groups.

[0330] "Alkylthio-alkyl" refers to an alkylthio group attached to an alkyl group, where the alkyl and alkylthio groups are as defined above.

[0331] "alkylaminocarbonyl" refers to (alkyl)-NC(O)-, where alkyl is defined as described above.

[0332] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0333] "Haloalkoxy" refers to an alkoxy group that has been substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0334] "Haloalkoxy" refers to an alkylthio group substituted by one or more halogens, wherein the alkylthio group is as defined above.

[0335] "Hydroxyalkyl" refers to an alkyl group that has been replaced by a hydroxyl group, where the alkyl group is as defined above.

[0336] "Alkenyl" refers to an alkenyl group, also known as an olefinic group, preferably an alkyl group containing 2 to 8 carbon atoms, more preferably an alkyl group containing 2 to 6 carbon atoms, and most preferably an alkyl group containing 2 to 3 carbon atoms. The alkenyl group may be further substituted with other related groups, such as: alkyl, alkenyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0337] "Alkyne" refers to (CH≡C-), preferably an alkyl group containing 2 to 8 carbon atoms, more preferably an alkyl group containing 2 to 6 carbon atoms, and most preferably an alkyl group containing 2 to 3 carbon atoms. The alkynyl group may be further substituted with other related groups, such as: alkyl, alkenyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0338] The term "alkenyl carbonyl" refers to -C(O)-(alkenyl), where alkenyl is defined as described above. Non-limiting examples of alkenyl carbonyl include vinyl carbonyl, propenyl carbonyl, and butenyl carbonyl. Alkenyl carbonyl can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester.

[0339] "Hydroxy" refers to the -OH group.

[0340] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0341] "Amino" refers to -NH2.

[0342] “Cyano” refers to -CN.

[0343] "Nitro" refers to -NO2.

[0344] "Carbonyl" refers to -C(O)-.

[0345] "Carboxyl group" refers to -C(O)OH.

[0346] "THF" refers to tetrahydrofuran.

[0347] “EtOAc” refers to ethyl acetate.

[0348] “MeOH” refers to methanol.

[0349] "DMF" refers to N,N-dimethylformamide.

[0350] "DIPEA" refers to diisopropylethylamine.

[0351] "TFA" refers to trifluoroacetic acid.

[0352] “MeCN” refers to Yi Qing.

[0353] “DMA” stands for N,N-dimethylacetamide.

[0354] “Et2O” refers to diethyl ether.

[0355] “DCE” refers to 1,2-dichloroethane.

[0356] "DIPEA" refers to N,N-diisopropylethylamine.

[0357] “NBS” refers to N-bromosuccinimide.

[0358] “NIS” refers to N-iodosuccinimide.

[0359] “Cbz-Cl” refers to benzyl chloroformate.

[0360] “Pd2(dba)3” refers to tris(dibenzylacetone)dipalladium.

[0361] “Dppf” refers to 1,1'-bis(diphenylphosphine)ferrocene.

[0362] “HATU” refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0363] "KHMDS" refers to potassium hexamethyldisilamide.

[0364] "LiHMDS" refers to lithium bis(trimethylsilyl)amine.

[0365] “MeLi” refers to methyl lithium.

[0366] “n-BuLi” refers to n-butyllithium.

[0367] "NaBH(OAc)3" refers to sodium triacetoxyborohydride.

[0368] The different terms such as "X is selected from A, B, or C", "X is selected from A, B, and C", "X is A, B, or C", and "X is A, B, and C" all express the same meaning, that is, X can be any one or more of A, B, and C.

[0369] All hydrogen atoms described in this invention can be replaced by their isotope deuterium, and any hydrogen atom in the compounds of the embodiments of this invention can also be replaced by a deuterium atom.

[0370] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of such event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0371] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0372] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0373] "Medicinal salts" refer to the salts of the compounds of this invention, which are safe and effective when used in mammals and have the appropriate biological activity. Attached Figure Description

[0374] Figure 1 XRPD illustration of crystal form I of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0375] Figure 2 DSC illustration of crystal form I of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0376] Figure 3 TGA illustration of crystal form I of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0377] Figure 4 XRPD illustration of crystal form II of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0378] Figure 5 DSC illustration of crystal form II of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0379] Figure 6 TGA illustration of crystal form II of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0380] Figure 7 XRPD illustration of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate crystal form III.

[0381] Figure 8 DSC illustration of crystal form III of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0382] Figure 9 TGA illustration of crystal form III of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one hydroxyethyl sulfonate.

[0383] Figure 10 XRPD illustration of crystal form I of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one sulfate.

[0384] Figure 11 XRPD illustration of crystal form II of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)-one sulfate.

[0385] Figure 12XRPD illustration of crystal form III of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)-one sulfate.

[0386] Figure 13 XRPD illustration of crystal form IV of P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one sulfate. Detailed Implementation

[0387] The present invention is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the present invention.

[0388] I. Preparation of Compounds

[0389] Example

[0390] The structures of the compounds of this invention were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3) as solvents, and tetramethylsilane (TMS) as the internal standard.

[0391] LC-MS was performed using an Agilent 1200 Infinity Series mass spectrometer. HPLC was performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C). 18 (150×4.6mm chromatographic column).

[0392] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The standard size for TLC is 0.15mm to 0.20mm, while the standard size for separating and purifying products using TLC is 0.4mm to 0.5mm. Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0393] The starting materials used in the embodiments of the present invention are known and commercially available, or can be synthesized using or in accordance with methods known in the art.

[0394] Unless otherwise specified, all reactions in this invention are carried out under continuous magnetic stirring, in a dry nitrogen or argon atmosphere, using a dry solvent, and the reaction temperature is expressed in degrees Celsius.

[0395] Example 1

[0396] 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0397]

[0398] Step 1: Preparation of 4-chloro-2-(prop-1-en-2-yl)pyridine-3-amine

[0399]

[0400] 2,4-Dichloropyridin-3-amine (4.5 g, 27.78 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxoboropentan (5.13 g, 30.56 mmol), potassium carbonate (11.5 g, 83.34 mmol), and Pd(PPh3)4 were added to dioxane (120 mL). After the reaction mixture was thoroughly mixed, it was stirred overnight in an oil bath at 100 °C. The mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography to obtain the target compound 4-chloro-2-(prop-1-en-2-yl)pyridin-3-amine as a colorless oily liquid (4.5 g, 96% yield).

[0401] MS m / z (ESI): 169.1 [M+H] + .

[0402] Step 2: Preparation of 4-(methylthio)-2-(prop-1-en-2-yl)pyridin-3-amine

[0403]

[0404] 4-Chloro-2-(prop-1-en-2-yl)pyridine-3-amine (2 g, 11.9 mmol) and sodium methanethiol (10 mL, 20% aqueous solution) were added to dioxane (3 mL). After the reaction solution was mixed evenly, it was reacted at 100 °C for 2 days. After cooling to room temperature, it was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography to obtain 4-(methylthio)-2-(prop-1-en-2-yl)pyridine-3-amine as a pale yellow liquid (1.7 g, yield 79%).

[0405] MS m / z (ESI): 181.2 [M+H] + .

[0406] Step 3: Preparation of 2-isopropyl-4-(methylthio)pyridine-3-amine

[0407]

[0408] 4-(methylthio)-2-(prop-1-en-2-yl)pyridine-3-amine (2 g, 11.11 mmol) and Pd / C (4 g) were added to methanol (50 mL). After the reaction mixture was homogeneously mixed, it was reacted overnight at room temperature and concentrated under reduced pressure. The crude product was added to a solution of methanol (5 mL), N,N-diisopropylethylamine (0.5 mL), and acrylonitrile (1 mL), and reacted at room temperature for 2 hours. The crude product was concentrated under reduced pressure, and purified by rapid silica gel column chromatography to obtain 2-isopropyl-4-(methylthio)pyridine-3-amine as a colorless liquid (500 mg, yield 25%).

[0409] MS m / z (ESI): 183.2 [M+H] + .

[0410] Step 4: Preparation of 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide

[0411]

[0412] 10 mL of THF was added to 2,6-dichloro-5-fluoronicotinamide (500 mg, 2.44 mmol) and oxalyl chloride (1.32 mL, 2.54 mmol). After the reaction solution was mixed evenly, it was reacted at 60 °C for 3 hours. The reaction temperature was then lowered to room temperature, and triethylamine (680 mg, 6.6 mmol) and 2-isopropyl-4-(methylthio)pyridin-3-amine (400 mg, 2.2 mmol) were added. The reaction was carried out at room temperature for 1 hour, and the mixture was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography to obtain 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide as a white solid (800 mg, yield 87%).

[0413] MS m / z (ESI): 417.1 [M+H] + .

[0414] Step 5: Preparation of 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0415]

[0416] 2,6-Dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (800 mg, 1.92 mmol) was added to THF (20 mL). After the reaction solution was mixed evenly, KHMDS (4.8 mL, 4.8 mmol) was slowly added at 0 °C. The reaction was carried out at room temperature for 1 hour, and the mixture was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography to obtain 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one as a white solid (600 mg, yield 82%).

[0417] MS m / z(ESI): 381.1 [M+H] + .

[0418] Step 6: Preparation of tert-butyl(S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid ester

[0419]

[0420] 7-Chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (300 mg, 0.79 mmol), phosphorus oxychloride (600 mg, 3.95 mmol), and DIPEA (1 g, 7.9 mmol) were added to THF (40 mL). After the reaction solution was thoroughly mixed, it was reacted at 80 °C for 1 hour. The reaction temperature was then lowered to room temperature, and tert-butyl ( S)-3-methylpiperazine-1-carboxylic acid ester (240 mg, 1.19 mmol) was reacted at room temperature for 1 hour, concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography to give tert-butyl(S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid ester as a white solid (400 mg, 90% yield).

[0421] MS m / z(ESI): 563.1 [M+H] + .

[0422] Step 7: Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0423]

[0424] 400 mg (0.71 mmol) of tert-butyl(S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid ester and 2 mL of TFA were added to CH2Cl2 (30 mL). After the reaction solution was mixed evenly, it was reacted at room temperature for 1 hour. The mixture was then concentrated under reduced pressure. CH2Cl2 (20 mL) and DIPEA (0.3 mL) were added to the crude product. The reaction temperature was lowered to 0 °C. Acryloyl chloride (0.1 mL) was slowly added to the reaction solution. The mixture was reacted at room temperature for 1 hour and then concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography to give the compound (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)-one as a yellow solid (200 mg, yield 55%).

[0425] MS m / z (ESI): 517.1 [M+H] + .

[0426] Step 8: Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0427]

[0428] (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (50 mg, 0.1 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (30 mg, 0.2 mmol), Pd(dppf)Cl2 (16 mg, 0.02 mmol), and cesium carbonate (100 mg, 0.3 mmol) were added to dixoane (1.5 mL). After the reaction solution was mixed evenly, it was reacted at 100 °C under microwave heating for 1 hour. The mixture was then concentrated under reduced pressure. CH2Cl2 (20 mL) and DIPEA (0.3 mL) were added to the crude product. The reaction temperature was lowered to 0 °C, and acryloyl chloride (0.1 mL) was slowly added to the reaction solution. The mixture was reacted at room temperature for 1 hour and then concentrated under reduced pressure. The crude product was purified by Pre-HPLC to give 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one as a white solid (14 mg, yield: 24%).

[0429] MS m / z(ESI): 593.1 [M+H] + .

[0430] 1 H NMR(400MHz,MeOD-d4)δ8.40(d,J=5.6Hz,1H),8.22-8.27(m,1H),7.21-7.27(m,2H),6.7 9-6.88(m,1H),6.58-6.66(m,2H),6.28-6.34(m,1H),5.84(d,J=12.0Hz,1H),5.06(s,1H) ,4.43-4.59(m,2H),4.07-4.23(s,1H),3.57-3.85(m,2H),3.20-3.48(m,1H),2.79-2.85 (m,1H),2.41(s,3H),1.47(d,J=4.8Hz,3H),1.20(d,J=6.4Hz,3H),1.06(d,J=6.8Hz,3H).

[0431] Examples 1-1 and 1-2

[0432] (P-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

[0433]

[0434]

[0435] Example 1: Two axial chiral isomers, Example 1-1 and Example 1-2, were obtained by SFC separation. SFC: Chiral preparation conditions:

[0436] instrument SFC-150 (Thar, Waters) Column IC 20*250mm, 10μm (Daicel) Column pressure 100 bar mobile phase <![CDATA[CO2 / Methanol(0.2%Methanol Ammonia)=50 / 50]]> Flow rate 120g / min Detection wavelength UV 214nm Column temperature 35℃

[0437] Example 1-1:

[0438] t R =1.92min

[0439] MS m / z(ESI): 593.1 [M+H] + .

[0440] 1 H NMR(400MHz,MeOD-d4)δ8.40(d,J=5.6Hz,1H),8.22-8.27(m,1H),7.21-7.27(m,2H),6.7 9-6.88(m,1H),6.58-6.66(m,2H),6.28-6.34(m,1H),5.84(d,J=12.0Hz,1H),5.06(s,1H) ,4.43-4.59(m,2H),4.07-4.23(s,1H),3.57-3.85(m,2H),3.20-3.48(m,1H),2.79-2.85 (m,1H),2.41(s,3H),1.47(d,J=4.8Hz,3H),1.20(d,J=6.4Hz,3H),1.06(d,J=6.8Hz,3H).

[0441] Examples 1-2:

[0442] t R =2.43min

[0443] MS m / z(ESI): 593.1 [M+H] + .

[0444] 1 HNMR(400MHz,MeOD-d4)δ8.40(d,J=5.6Hz,1H),8.25(t,J=10.8Hz,1H),7.21-7.27(m,2H),6. 79-6.90(m,1H),6.58-6.66(m,2H),6.28-6.34(m,1H),5.83(dd,J=10.8Hz,2.0Hz,1H),5.05- 5.10(m,1H),4.41-4.57(m,2H),4.07-4.21(m,1H),3.61-3.87(m,2H),3.24-3.36(m,1H),2.7 7-2.83(m,1H),2.41(s,3H),1.46-1.49(m,3H),1.19(d,J=6.8Hz,3H),1.06(d,J=6.8Hz,3H).

[0445] Example 2

[0446] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0447]

[0448] Step 1: Preparation of 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0449]

[0450] At room temperature, N,N-diisopropylethylamine (407 mg, 3.16 mmol) was added to a solution of 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (200 mg, 0.526 mmol) in acetonitrile (10 mL), followed by phosphorus oxychloride (242 mg, 1.58 mmol). The mixture was stirred at 80 °C for 1 hour. After cooling to room temperature, the solution was used directly for the next reaction.

[0451] Step 2: Preparation of tert-butyl(2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylic acid ester

[0452]

[0453] Add N,N-diisopropylethylamine (678 mg, 5.26 mmol) and tert-butyl(2R,5S)-2,5-dimethylpiperazine-1-carboxylic acid ester (224 mg, 1.005 mmol) to the previous reaction solution, and stir at room temperature for 1 hour after the addition is complete. Add water (60 mL), extract with ethyl acetate (40 mL × 3), wash the organic phase with ammonium chloride aqueous solution (40 mL), then with sodium chloride aqueous solution (30 mL), concentrate and purify by column chromatography [eluent: dichloromethane to methanol / dichloromethane from 0% to 2.2%] to give tert-butyl(2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylic acid ester (200 mg, 2-step yield 66%) as a yellow solid.

[0454] MS m / z (ESI): 577.2 [M+H] + 579.2[M+H+2] +

[0455] Step 3: Preparation of 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate

[0456]

[0457] A solution of tert-butyl(2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazin-1-carboxylic acid ester (200 mg, 0.347 mmol) in dichloromethane (6 mL) was added with 1.2 mL of trifluoroacetic acid, and the mixture was stirred at room temperature for 1.5 hours after the addition was complete. The reaction solution was concentrated at low temperature to give 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate (200 mg), which was rapidly used in the next step of the reaction.

[0458] MS m / z (ESI): 477.2 [M+H] + 479.2[M+H+2] + .

[0459] Step 4: Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0460]

[0461] To a solution of 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate (200 mg, 0.347 mmol) in dichloromethane (15 mL), N,N-diisopropylethylamine (447 mg, 3.47 mmol) was added, and acryloyl chloride (63 mg, 0.694 mmol) was added dropwise at 0 °C. The mixture was stirred for 1 hour after the addition was complete. The reaction was quenched with an aqueous solution of ammonium chloride (30 mL), extracted with dichloromethane (30 mL × 3), the dichloromethane layer was washed with a saturated aqueous solution of NaCl (20 mL), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography [eluent: dichloromethane to methanol / dichloromethane from 0% to 2.5%] to give 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (130 mg, 2-step yield 71%) as a yellow solid.

[0462] MS m / z (ESI): 530.2 [M+H] + 532.2[M+H+2] + .

[0463] Step 5: Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0464]

[0465] 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (130 mg, 0.246 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (77 mg, 0.491 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex (40 mg, 0.0491 mmol) and cesium carbonate (240 mg, 0.738 mmol) were added to dioxane (8 mL) and water (1 mL), purged with nitrogen, and microwaved at 100 °C for 1 hour. The reaction solution was concentrated and purified by column chromatography [eluent: dichloromethane to methanol / dichloromethane from 0% to 2.5%] to give 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (90 mg, yield 60%) as a yellow solid.

[0466] MS m / z (ESI): 606.2 [M+H] + .

[0467] 1 H NMR(400MHz,MeOD-d4)δ8.40(d,J=8Hz,1H),8.29-8.18(m,1H),7.30–7.18(m,2H),6.93–6.73 (m,1H),6.70–6.56(m,2H),6.36–6.20(m,1H),5.89–5.75(m,1H),5.15–4.98(m,1H),4.63–4. 22(m,2H),4.11–3.82(m,2H),3.68–3.40(m,1H),2.88–2.65(m,1H),2.40(d,J=4Hz,3H),1.53 –1.43(m,3H),1.36(t,J=8Hz,1H),1.28(t,J=8Hz,2H),1.23–1.16(m,3H),1.10–1.01(m,3H).

[0468] Examples 2-1 and 2-2

[0469] (P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

[0470]

[0471]

[0472] Example 2: Two axial chiral isomers, Example 2-1 and Example 2-2, were obtained by SFC separation. SFC: Chiral preparation conditions:

[0473] instrument SFC-150 (Thar, Waters) Column IC 20*250mm, 10μm (Daicel) Column pressure 100 bar mobile phase <![CDATA[CO2 / Methanol(0.2%Methanol Ammonia)=60 / 40]]> Flow rate 100g / min Detection wavelength UV 214nm Column temperature 35℃

[0474] Example 2-1:

[0475] t R =1.99min

[0476] MS m / z (ESI): 606.2 [M+H] + .

[0477] 1 H NMR(400MHz,MeOD-d4)δ8.40(d,J=8Hz,1H),8.29-8.18(m,1H),7.30–7.18(m,2H),6.93–6.73 (m,1H),6.70–6.56(m,2H),6.36–6.20(m,1H),5.89–5.75(m,1H),5.15–4.98(m,1H),4.63–4. 22(m,2H),4.11–3.82(m,2H),3.68–3.40(m,1H),2.88–2.65(m,1H),2.40(d,J=4Hz,3H),1.53 –1.43(m,3H),1.36(t,J=8Hz,1H),1.28(t,J=8Hz,2H),1.23–1.16(m,3H),1.10–1.01(m,3H).

[0478] Example 2-2:

[0479] t R =2.87min

[0480] MS m / z (ESI): 606.2 [M+H] + .

[0481] 1 H NMR(400MHz,MeOD-d4)δ8.40(d,J=8Hz,1H),8.27-8.18(m,1H),7.30–7.19(m,2H),6.94–6.74(m,1 H),6.70–6.56(m,2H),6.36–6.20(d,J=16Hz,1H),5.90–5.75(m,1H),5.14–4.98(m,1H),4.63–4.22 (m,2H),4.12–3.82(m,2H),3.68–3.41(m,1H),2.87–2.65(m,1H),2.40(d,J=4Hz,3H),1.53–1.42(m ,3H),1.36(t,J=8Hz,1H),1.28(t,J=8Hz,2H),1.23–1.16(d,J=4Hz,3H),1.10–1.01(d,J=4Hz,3H).

[0482] Example 3

[0483] 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one)

[0484]

[0485] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidine-2(1H)-one) is described in Example 1.

[0486] MS m / z (ESI): 609.1 [M+H] + .

[0487] Example 4

[0488] 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylthio)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one)

[0489]

[0490] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylthio)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidine-2(1H)-one) is described in Example 1.

[0491] MS m / z (ESI): 622.8 [M+H] + .

[0492] Example 5

[0493] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0494]

[0495] Step 1: Preparation of N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide

[0496]

[0497] 4-Chloro-3-fluoroaniline (1.45 g, 0.01 mol) was dissolved in THF (150 mL), and Na₂CO₃ (3.18 g, 0.03 mol) was added. Under nitrogen protection, the mixture was cooled to 0 °C, and trifluoroacetic anhydride (4.2 mL, 0.03 mol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 10 hours. The reaction solution was added to water (150 mL). The mixture was extracted three times with ethyl acetate (100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 5:1) to obtain the white solid target product N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide (2.3 g, 95% yield).

[0498] 1 H NMR (400MHz, MeOD-d4) δ7.70 (dd, J=11.1, 2.0Hz, 1H), 7.49–7.40 (m, 2H);

[0499] 19 F NMR(376MHz,MeOD-d4)δ-77.17(s);

[0500] MS m / z(ESI): 242.1 [M+H] + .

[0501] Step 2: Preparation of (6-amino-3-chloro-2-fluorophenyl)boronic acid

[0502]

[0503] N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide (2.3 g, 9.5 mmol) was dissolved in THF (40 mL). Under nitrogen protection, the solution was cooled to -78 °C, and n-BuLi (7.9 mL, 19.0 mmol, 2.4 M) was added dropwise. After the addition was complete, the mixture was stirred at -50 °C for 50 minutes. The reaction solution was then cooled to -78 °C, and triisopropyl borate (2.3 g, 9.5 mmol) (4.8 mL, 20.9 mmol) was added dropwise. After the addition was complete, the mixture was stirred at the same temperature for 20 minutes. The dry ice bath was removed, and the mixture was stirred at room temperature for 2 hours. Then, the reaction solution was cooled to 0 °C, and dilute hydrochloric acid (19 mL, 1 M) was added dropwise. The temperature was raised to 40 °C, and the mixture was stirred for 1 hour. The mixture was extracted three times with ethyl acetate (100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, concentrated to obtain crude product, and purified by column chromatography (PE / EA = 4:1) to obtain the target product (6-amino-3-chloro-2-fluorophenyl)boronic acid (1.1 g, yield 56%) as a gray solid.

[0504] MS m / z(ESI): 190.0 [M+H] + .

[0505] Step 3: Preparation of (2-amino-6-fluorophenyl)boronic acid

[0506]

[0507] (6-Amino-3-chloro-2-fluorophenyl)boronic acid (100 mg, 0.53 mmol) was dissolved in MeOH (20 mL), and Pd / C (20 mg) was added. The mixture was purged with hydrogen three times, and stirred at 15 psi for 2 hours. The reaction was confirmed to be complete by TLC (PE / EA 1:1). The mixture was filtered, and the filtrate was concentrated to obtain the target product (2-amino-6-fluorophenyl)boronic acid (80 mg, 97% yield) as a yellow solid, which was used directly in the next step without purification.

[0508] MS m / z(ESI): 156.0 [M+H] + .

[0509] Step 4: Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0510]

[0511] (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl)boronic acid (23.2 mg, 0.15 mmol), and cesium carbonate (48.87 mg, 0.15 mmol) were dissolved in dioxane / H₂O (1.5 mL / 0.3 mL). The mixture was purged with nitrogen for 1 minute and reacted in a microwave oven at 100 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography (CH2Cl2 / MeOH = 20:1) to obtain the crude product. Then, the crude product was purified by preparative HPLC to obtain the yellow solid target product 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (7.0 mg, yield 24%).

[0512] 1 H NMR(400MHz,MeOD-d4)δ8.46(d,J=5.4Hz,1H),8.25(dd,J=21.2,12.0Hz,1H),7.27(d,J=5.5Hz,1H),7.1 1(dd,J=14.7,8.2Hz,1H),6.84(d,J=14.2Hz,1H),6.49(d,J=8.3Hz,1H),6.41–6.27(m,2H),5.83(dd,J=1 0.6,1.6Hz,1H),4.48(dd,J=52.4,11.6Hz,2H),4.30–3.83(m,2H),3.74(d,J=9.7Hz,2H),3.22(s,1H),2 .98–2.80(m,1H),2.43(d,J=0.7Hz,3H),1.56–1.40(m,3H),1.22(d,J=6.6Hz,3H),1.01(d,J=6.6Hz,3H).

[0513] 19 F NMR(376MHz,MeOD-d4)δ-114.58–-114.95(m),-114.95–-115.34(m),-125.12–-126.48(m).

[0514] MS m / z(ESI): 592.2 [M+H] + .

[0515] Example 6

[0516] 2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-oxy-1,2-dihydropyridin[2,3-d]pyrimidin-7-yl)-3-fluorobenzamide

[0517]

[0518] The preparation of 2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-oxy-1,2-dihydropyridin[2,3-d]pyrimidin-7-yl)-3-fluorobenzamide was carried out in accordance with Example 1.

[0519] MS m / z (ESI): 619.7 [M+H] + .

[0520] Example 7

[0521] 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-(dimethylamino)-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridinyl[2,3-d]pyrimidin-2(1H)-one

[0522]

[0523] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-(dimethylamino)-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridinyl[2,3-d]pyrimidin-2(1H)-one was carried out in accordance with Example 1.

[0524] MS m / z (ESI): 619.7 [M+H] + .

[0525] Example 8

[0526]

[0527] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylamino)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridinyl[2,3-d]pyrimidin-2(1H)-one was carried out in accordance with Example 1.

[0528] MS m / z (ESI): 605.7 [M+H] + .

[0529] Example 9

[0530] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0531]

[0532] (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26.7 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl)boronic acid (28.4 mg, 0.15 mmol), and potassium acetate (15.0 mg, 0.15 mmol) were dissolved in dioxane / H2O (1.5 mL / 0.3 mL). The mixture was purged with nitrogen for 1 minute and reacted in a microwave oven at 100 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography (CH2Cl2 / MeOH = 20:1) to obtain the crude product. Then, the crude product was purified by preparative HPLC to obtain the yellow solid target product 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (3.7 mg, yield 14%).

[0533] MS m / z (ESI): 642.1 [M+H] + .

[0534] 1 H NMR (400MHz, MeOD-d4) δ8.56–8.39(m,2H),7.24(t,J=5.3Hz,1H),7.15(dd,J=15.4,6.9H z,1H),6.84(d,J=9.9Hz,1H),6.53–6.46(m,1H),6.32(d,J=15.9Hz,1H),5.84(d,J=12.2H z,1H),4.68–4.36(m,3H),4.10(dd,J=45.7,31.6Hz,2H),3.76(s,1H),2.94(s,2H),2.42 (d,J=6.2Hz,3H),1.57–1.43(m,3H),1.22(d,J=6.7Hz,3H),1.06(dd,J=42.4,6.7Hz,3H). 19F NMR(376MHz,MeOD)δ-117.04–-117.24(m),-117.24–-117.51(m).

[0535] Examples 9-1 and 9-2

[0536] (P-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

[0537]

[0538] Example 9 yielded two axial chiral isomers, Example 9-1 and Example 9-2, through SFC separation. SFC: Chiral preparation conditions:

[0539] instrument SFC-80 (Thar, Waters) Column IC 20*250mm, 10μm (Daicel) Column pressure 100 bar

[0540] mobile phase <![CDATA[CO2 / Methanol(0.2%Methanol Ammonia)=45 / 55]]> Flow rate 80g / min Detection wavelength UV 214nm Column temperature 35℃

[0541] Example 9-1:

[0542] t R =1.74min

[0543] MS m / z (ESI): 642.1 [M+H] + .

[0544] 1 H NMR (400MHz, MeOD-d4) δ8.56–8.39(m,2H),7.24(t,J=5.3Hz,1H),7.15(dd,J=15.4,6.9H z,1H),6.84(d,J=9.9Hz,1H),6.53–6.46(m,1H),6.32(d,J=15.9Hz,1H),5.84(d,J=12.2H z,1H),4.68–4.36(m,3H),4.10(dd,J=45.7,31.6Hz,2H),3.76(s,1H),2.94(s,2H),2.42 (d,J=6.2Hz,3H),1.57–1.43(m,3H),1.22(d,J=6.7Hz,3H),1.06(dd,J=42.4,6.7Hz,3H).

[0545] 19 F NMR(376MHz,MeOD-d4)δ-117.04–-117.24(m),-117.24–-117.51(m).

[0546] Example 9-2:

[0547] t R =2.49min

[0548] MS m / z (ESI): 642.1 [M+H] + .

[0549] 1 H NMR(400MHz,MeOD-d4)δ8.56–8.39(m,2H),7.27–7.10(m,2H),6.84(dd,J=28.3,17.7Hz ,1H),6.50(d,J=8.8Hz,1H),6.32(d,J=16.9Hz,1H),5.83(d,J=11.7Hz,1H),4.63–4.41( m,2H),4.23–4.02(m,1H),3.79–3.57(m,2H),3.36(s,2H),2.99–2.86(m,1H),2.41(d,J= 7.6Hz, 3H), 1.51 (d, J = 25.9Hz, 3H), 1.21 (d, J = 6.6Hz, 3H), 1.05 (dd, J = 44.8, 6.7Hz, 3H).

[0550] Example 10

[0551] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0552]

[0553] (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl)boronic acid (28.4 mg, 0.15 mmol), and cesium carbonate (48.8 mg, 0.15 mmol) were dissolved in dioxane / H₂O (1.5 mL / 0.3 mL). The mixture was purged with nitrogen for 1 minute and reacted in a microwave oven at 100 °C for 1 hour. After the reaction was completed, the reaction solution was evaporated to dryness and purified by column chromatography (CH2Cl2 / MeOH = 20:1) to obtain the crude product. Then, the crude product was purified by preparative HPLC to obtain the yellow solid target product 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (4.4 mg, yield 14%).

[0554] 1 H NMR(400MHz,MeOD-d4)δ8.47(d,J=5.4Hz,1H),8.38–8.24(m,1H),7.27(d,J=5.4Hz,1H),7.17(t ,J=8.6Hz,1H),6.85(d,J=14.9Hz,1H),6.49(d,J=8.9Hz,1H),6.32(d,J=16.3Hz,1H),5.84(d,J =10.5Hz,1H),4.57(d,J=23.5Hz,2H),4.42(s,1H),4.24–3.89(m,2H),3.73(dd,J=14.4,7.9Hz, 1H),2.92(s,1H),2.43(s,3H),1.54–1.40(m,3H),1.22(d,J=6.7Hz,3H),1.01(d,J=6.6Hz,3H).

[0555] 19 F NMR(376MHz,MeOD-d4)δ-116.46–-116.73(m),-116.87(dd,J=39.0,8.4Hz),-126.18(dd,J=24.9,15.2Hz).

[0556] MS m / z (ESI): 626.1 [M+H] + .

[0557] Example 11

[0558] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2,3-difluoro-6-hydroxyphenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0559]

[0560] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2,3-difluoro-6-hydroxyphenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidine-2(1H)-one is described in Example 2.

[0561] MS m / z(ESI): 611.1 [M+H] + .

[0562] 1 H NMR(400MHz,MeOD-d4)δ8.41(d,J=5.6Hz,1H),8.32-8.25(m,1H),7.25(d,J=5.6Hz,1H),7 .20-7.13(m,1H),6.92-6.82(m,1H),6.62-6.58(m,1H),6.34-6.28(m,1H),5.83(d,J=10.4 Hz,1H),5.14-5.04(m,1H),4.64-4.42(m,2H),4.25-4.07(m,1H),3.89-3.61(m,3H),2.88- 2.77(m,1H),2.42(s,3H),1.52-1.46(m,3H),1.20(d,J=6.4Hz,3H),1.05(d,J=6.4Hz,3H).

[0563] Example 12

[0564] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0565]

[0566] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidine-2(1H)-one is described in Example 2.

[0567] MS m / z(ESI): 611.1 [M+H] + .

[0568] Example 13

[0569] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0570]

[0571] Step 1: Preparation of 2,5,6-trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide

[0572]

[0573] Under N2 protection, 2,5,6-trichloronicotinamide (6.2 g, 27.7 mmol) was dissolved in THF (60 mL), and oxalyl chloride (15.2 mL, 31.5 mmol) (2 M / L dichloromethane solution) was added dropwise at -78 °C. The mixture was stirred at -78 °C for 10 minutes, then at 60 °C for 3 hours. The reaction solution was cooled to 0 °C, and triethylamine (18 mL, 111 mmol) was added dropwise. Then, a THF solution of 2-isopropyl-4-(methylthio)pyridin-3-amine (5 g, 27.7 mmol) was added dropwise. The mixture was stirred at room temperature for 2 hours. Quenching with brine, extraction with water and ethyl acetate (3*100mL), combining the organic layers, drying with anhydrous sodium sulfate, filtering, concentrating to obtain the crude product, and purifying by column chromatography (DCM / MeOH = 100:1 to 70:1) to obtain the target product 2,5,6-trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (8.6g, yield 72%).

[0574] MS m / z(ESI): 433.1 [M+H] + 435.1[M+H+2] + .

[0575] Step 2: Preparation of 6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione

[0576]

[0577] 2,5,6-Trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (10.4 g, 24.1 mmol) was dissolved in anhydrous THF (80 mL), cooled to 0 °C under nitrogen protection, and KHMDS (48 mL, 48.2 mmol) was added dropwise while stirring for 0.5 hours. The mixture was quenched with saturated ammonium chloride solution, and extracted with water and ethyl acetate (3 x 100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by slurrying with ethyl acetate to obtain the target product 6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (8 g, 84% yield).

[0578] MS m / z(ESI): 397.1 [M+H] + 399.1[M+H+2] + .

[0579] Step 3: Preparation of 4,6,7-trichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0580]

[0581] 5.2 g (13.1 mmol) of 6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione was dissolved in 50 mL of ACN, and 23 mL (66 mmol) and 3 mL (19.7 mmol) of POCl3 were added. The mixture was stirred at 80 °C for 0.5 hours. This solution was then used directly in the next reaction.

[0582] MS m / z (ESI): 415.1 [M+H] + 417.1[M+H+2] + .

[0583] Step 4: Preparation of tert-butyl(2R,5S)-4-(6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylic acid ester

[0584]

[0585] In acetonitrile (50 mL) of 4,6,7-trichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one, DIEA (23 mL, 66 mmol) was added, followed by tert-butyl(2R,5S)-2,5-dimethylpiperazine-1-carboxylic acid ester (6.2 g, 26.2 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was quenched with water and extracted with water and ethyl acetate (3 x 100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography (CH2Cl2 / MeOH = 30:1) to obtain the target product tert-butyl(2R,5S)-4-(6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylic acid ester (6.1 g, yield 77%).

[0586] MS m / z(ESI): 593.1 [M+H] + 595.1[M+H+2] + .

[0587] Step 5: Preparation of 6,7-dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0588]

[0589] 6.1 g (10.3 mmol) of tert-butyl(2R,5S)-4-(6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazin-1-carboxylic acid ester was dissolved in dichloromethane (20 mL), and TFA (20 mL) was added. The mixture was stirred at room temperature for 1 hour. The crude product 6,7-dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (6.1 g, 100% yield) was obtained.

[0590] MS m / z(ESI): 493.1 [M+H] + 495.1[M+H+2] + .

[0591] Step 6: Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0592]

[0593] 6,7-Dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (6 g, 12.2 mmol) was dissolved in dichloromethane (30 mL), DIEA (30 mL, 131 mmol) was added, and acryloyl chloride (1.08 mL, 13.13 mmol) was added. The mixture was stirred at room temperature for 1 hour. The solution was quenched with water and extracted with water and ethyl acetate (3 x 100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography (CH2Cl2 / MeOH = 20:1) to obtain the target product 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (1.6 g, yield 22%).

[0594] MS m / z (ESI): 547.1 [M+H] + 549.1[M+H+2] + .

[0595] Step 7: Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0596]

[0597] Under N2 protection, 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (700 mg, 1.3 mmol) and (6-amino-3-chloro-2-fluorophenyl)boronic acid (380 mg, 2.6 mmol) were dissolved in a mixture of 1,4-dioxane and water (6 mL: 0.3 mL), Pd(dppf)Cl2·DCM (100 mg, 0.1 mmol), and KOAc (400 mg, 4 mmol). The mixture was microwaved at 100 °C for 1 hour. The reaction was quenched with water and extracted with water and ethyl acetate (3 x 50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography (CH2Cl2 / MeOH = 200:1 to 80:1) to obtain the target product 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (400 mg, yield 48%).

[0598] MS m / z (ESI): 656.1 [M+H] + 658.1[M+H+2] + .

[0599] 1 H NMR(400MHz, Methanol-d4)δ8.47–8.34(m,2H),7.24-7.20(m,1H),7.10-7.14(m,1H ),6.79-6.68(m,1H),6.42–6.40(d,J=8.0Hz,1H),6.24–6.17(m,1H),5.75-5.71(m,1 H),5.01–4.94(m,2H),4.46-4.40(m,1H),4.26-4.17(m,1H),4.03-3.99(m,1H),3.84 -3.79(m,1H),2.86-2.77(m,1H),2.36(s,3H),1.26-1.19(m,9H),1.14-1.11(m,3H).

[0600] Examples 13-1 and 13-2

[0601] (P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

[0602]

[0603] Example 13: Two axial chiral isomers, Example 13-1 and Example 13-2, were obtained by SFC separation. SFC: Chiral preparation conditions:

[0604] instrument SFC-150 (Thar, Waters) Column IC 20*250mm, 10μm (Daicel) Column pressure 100 bar mobile phase <![CDATA[CO2 / Methanol(0.2%Methanol Ammonia)=40 / 60]]> Flow rate 120g / min Detection wavelength UV 214nm Column temperature 35℃

[0605] Example 13-1:

[0606] t R =1.74min

[0607] MS m / z (ESI): 656.1 [M+H] + 658.1[M+H+2] + .

[0608] 1 H NMR(400MHz,MeOD-d4)δ8.47–8.34(m,2H),7.24-7.20(m,1H),7.10-7.14(m,1H),6 .79-6.68(m,1H),6.42–6.40(d,J=8.0Hz,1H),6.24–6.17(m,1H),5.75-5.71(m,1H) ,5.01–4.94(m,2H),4.46-4.40(m,1H),4.26-4.17(m,1H),4.03-3.99(m,1H),3.84- 3.79(m,1H),2.86-2.77(m,1H),2.36(s,3H),1.26-1.19(m,9H),1.14-1.11(m,3H).

[0609] Example 13-2:

[0610] t R =2.49min

[0611] MS m / z (ESI): 656.1 [M+H] +658.1[M+H+2] + .

[0612] 1 H NMR(400MHz,DMSO-d6)δ8.55–8.38(m,2H),7.25-7.20(m,1H),7.18-7.11( m,1H),6.88-6.76(m,1H),6.51–6.47(d,J=8.0Hz,1H),6.33–6.27(m,1H), 5.84-5.80(m,1H),5.12-5.10(m,2H),4.46-4.23(m,2H),4.15-3.89(m,2H ),3.64-3.50(m,1H),2.89-2.82(m,1H),2.43(s,3H),1.51-0.99(m,12H).

[0613] Example 14

[0614] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0615]

[0616] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0617] MS m / z(ESI): 623.1 [M+H] + 625.1[M+H+2] + .

[0618] 1H NMR(400MHz, Methanol-d4)δ8.47–8.34(m,2H),7.21-7.20(m,2H),6.89-6.77(m,1H),6. 64–6.55(m,2H),6.32–6.26(m,1H),5.84-5.80(m,1H),5.08–5.03(m,2H),4.56-4.49(m,1 H),4.34-4.26(m,1H),4.13-4.04(m,1H),3.92-3.88(m,1H),2.79-2.72(m,1H),2.40(s,3 H),1.55–1.43(m,3H),1.35-1.27(m,3H),1.20-1.17(m,3H),1.08-1.05(t,J=8.0Hz,3H).

[0619] Examples 14-1 and 14-2

[0620] (P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

[0621]

[0622]

[0623] Example 14: Two axial chiral isomers, Example 14-1 and Example 14-2, were obtained by SFC separation. SFC: Chiral preparation conditions:

[0624] instrument SFC-150 (Thar, Waters) Column IC 20*250mm, 10μm (Daicel) Column pressure 100 bar mobile phase <![CDATA[CO2 / Methanol(0.2%Methanol Ammonia)=50 / 50]]> Flow rate 120g / min Detection wavelength UV 214nm Column temperature 35℃

[0625] Example 14-1:

[0626] t R =2.46min

[0627] MS m / z(ESI): 623.1 [M+H] + 625.1[M+H+2] + .

[0628] 1H NMR(400MHz, Methanol-d4)δ8.47–8.34(m,2H),7.21-7.20(m,2H),6.89-6.77(m,1H),6. 64–6.55(m,2H),6.32–6.26(m,1H),5.84-5.80(m,1H),5.08–5.03(m,2H),4.56-4.49(m,1 H),4.34-4.26(m,1H),4.13-4.04(m,1H),3.92-3.88(m,1H),2.79-2.72(m,1H),2.40(s,3 H),1.55–1.43(m,3H),1.35-1.27(m,3H),1.20-1.17(m,3H),1.08-1.05(t,J=8.0Hz,3H).

[0629] Example 14-2:

[0630] t R =3.08min

[0631] MS m / z(ESI): 623.1 [M+H] + 625.1[M+H+2] + .

[0632] 1 H NMR(400MHz, Methanol-d4)δ8.48–8.34(m,2H),7.23-7.21(m,2H),6.90-6.78(m,1H),6. 66–6.58(m,2H),6.33–6.28(m,1H),5.85-5.82(m,1H),5.10–5.06(m,2H),4.58-4.50(m,1 H),4.34-4.27(m,1H),4.13-4.06(m,1H),3.93-3.88(m,1H),2.79-2.71(m,1H),2.41(s,3 H),1.56–1.46(m,3H),1.37-1.29(m,3H),1.21-1.18(m,3H),1.07-1.05(t,J=8.0Hz,3H).

[0633] Example 15

[0634] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0635]

[0636] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)-one is described in Example 2.

[0637] MS m / z (ESI): 595.1 [M+H] + .

[0638] 1 H NMR(400MHz, Methanol-d4)δ8.40–8.32(m,2H),7.51(t,J=7.6Hz,1H),7.22(d,J=5.4Hz, 1H),7.05(t,J=8.4Hz,2H),6.86–6.79(m,1H),6.37–6.26(m,1H),5.84(d,J=10.6Hz,1H) ,5.08(m,2H),4.56-4.46(m,2H),4.21-4.08(m,1H),3.85-3.62(m,2H),2.86-2.82(m,1H ), 2.40 (s, 3H), 1.47 (d, J = 6.6Hz, 3H), 1.21–1.19 (d, J = 6.8Hz, 3H), 1.04 (d, J = 6.8Hz, 3H).

[0639] Example 16

[0640] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0641]

[0642] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)-one is described in Example 2.

[0643] MS m / z (ESI): 576.7 [M+H] +

[0644] Example 17

[0645] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0646]

[0647] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0648] MS m / z (ESI): 690.1 [M+H] + 692.1[M+H+2] + .

[0649] 1 H NMR(400MHz,Methanol-d4)δ8.46–8.34(m,2H),7.25-7.21(m,1H),7.11-7.14( m,1H),6.44–6.42(d,J=8.0Hz,1H),6.23–6.16(m,1H),5.73-5.70(m,1H),5.03– 4.97(m,2H),4.47-4.42(m,1H),4.25-4.16(m,1H),4.06-4.02(m,1H),3.86-3.8 3(m,1H),2.84-2.79(m,1H),2.34(s,3H),1.27-1.19(m,9H),1.16-1.14(m,3H).

[0650] Example 18

[0651] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0652]

[0653] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0654] MS m / z (ESI): 660.1 [M+H] + 662.1[M+H+2] + .

[0655] 1 H NMR(400MHz, Methanol-d4)δ8.58–8.38(m,2H),7.53–7.36(m,1H),7.23–7.15(m,1H) ,6.97–6.79(m,1H),6.22(d,J=16Hz,1H),5.77(d,J=8Hz,1H),5.45–5.40(m,2H),5.0 7–4.82(m,1H),4.50–3.98(m,3H),3.92–3.49(m,2H),3.17–3.02(m,1H),2.93–2.63( m,1H),2.44–2.26(m,3H),1.43–1.27(m,3H),1.08(d,J=4Hz,3H),1.04–0.86(m,3H).

[0656] Example 19

[0657] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0658]

[0659] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0660] MS m / z (ESI): 640.1 [M+H] + 642.1[M+H+2] + .

[0661] 1H NMR(400MHz, Methanol-d4)δ8.57–8.35(m,3H),7.25–7.04(m,2H),6.96–6.79( m,1H),6.29–6.14(m,1H),5.77(d,J=12Hz,1H),5.09–4.82(m,1H),4.76–4.58(m ,2H),4.48–3.98(m,3H),3.94–3.59(m,2H),2.93–2.69(m,1H),2.44–2.29(m,3H ),2.10–1.95(m,3H),1.42–1.26(m,3H),1.08(d,J=4Hz,3H),1.05–0.87(m,3H).

[0662] Example 20

[0663] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0664]

[0665] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0666] MS m / z (ESI): 674.1 [M+H] + 676.1[M+H+2] + .

[0667] 1H NMR(400MHz, Methanol-d4)δ8.61–8.39(m,2H),7.56–7.35(m,1H),7.27–7.14( m,1H),6.96–6.75(m,1H),6.20(d,J=16Hz,1H),5.82–5.71(m,1H),5.53–5.38(m ,2H),4.95–4.69(m,1H),4.57–4.30(m,1H),4.24–4.00(m,2H),3.98–3.79(m,2 H),2.95–2.60(m,1H),2.44–2.25(m,3H),1.40–1.13(m,6H),1.10–0.87(m,6H).

[0668] Example 21

[0669] 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0670]

[0671] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0672] MS m / z (ESI): 674.1 [M+H] + ,

[0673] Example 22

[0674] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0675]

[0676] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0677] MS m / z (ESI): 626.1 [M+H] + 628.1[M+H+2] + .

[0678] 1 H NMR(400MHz, Methanol-d4)δ8.58–8.34(m,2H),7.26–6.99(m,2H),6.95–6.77(m,1 H),6.47–6.27(m,1H),6.26–6.13(m,1H),5.77(d,J=16Hz,1H),5.22(s,2H),5.09–4 .80(m,1H),4.50–3.99(m,3H),3.95–3.53(m,2H),3.20–2.98(m,1H),2.94–2.65(m ,1H),2.42–2.24(m,3H),1.43–1.25(m,3H),1.09(d,J=4Hz,3H),1.04–0.82(m,3H).

[0679] Example 23

[0680] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0681]

[0682] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidine-2(1H)-one was carried out according to Example 13.

[0683] MS m / z(ESI):644.1[M+H]+.

[0684] Example 24

[0685] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0686]

[0687] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0688] MS m / z(ESI):658.1[M+H]+.

[0689] Example 25

[0690] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0691]

[0692] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0693] MS m / z (ESI): 654.1 [M+H] + 656.1[M+H+2] + .

[0694] 1 H NMR(400MHz, Methanol-d4)δ8.58–8.31(m,2H),7.25–7.03(m,2H),6.94–6.73(m,1H),6.19(d,J=16Hz,1H),5.81–5.69(m,1H),4.96–4.59(m,3H),4.5 5–4.38(m,1H),4.29–3.96(m,2H),3.93–3.72(m,2H),3.00–2.60(m,1H),2 .45–2.25(m,3H),2.07–1.94(m,3H),1.43–1.13(m,6H),1.12–0.82(m,6H).

[0695] Example 26

[0696] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0697]

[0698] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0699] MS m / z(ESI): 658.1 [M+H]+,

[0700] Example 27

[0701] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0702]

[0703] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0704] MS m / z(ESI): 644.1 [M+H]+,

[0705] Example 28

[0706] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0707]

[0708] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0709] MS m / z(ESI): 640.2[M+H]+,

[0710] Example 29

[0711] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0712]

[0713] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0714] MS m / z(ESI): 626.1 [M+H]+,

[0715] Example 30

[0716] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0717]

[0718] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0719] MS m / z (ESI): 607.1 [M+H] + 609.1[M+H+2] + .

[0720] 1H NMR(400MHz, Methanol-d4)δ8.57–8.34(m,2H),7.43–7.31(m,1H),7.18(d,J=4Hz,1H),7.15–7 .01(m,2H),6.95–6.78(m,1H),6.28–6.14(m,1H),5.77(d,J=12Hz,1H),5.07–4.86(m,1H),4.4 5–4.25(m,2H),4.22–3.98(m,1H),3.93–3.58(m,2H),3.21–3.02(m,1H),2.87–2.69(m,1H),2. 40–2.27(m,3H),1.98–1.85(m,3H),1.41–1.28(m,3H),1.08(d,J=8Hz,3H),1.02–0.79(m,3H).

[0721] Example 31

[0722] Preparation of 4-((2S,5R)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0723]

[0724] The preparation of 4-((2S,5R)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0725] MS m / z(ESI): 621.2[M+H]+,

[0726] Example 32

[0727] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0728]

[0729] The preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0730] MS m / z (ESI): 627.1 [M+H] + 629.1[M+H+2] + .

[0731] 1 H NMR(400MHz, Methanol-d4)δ8.56–8.30(m,2H),7.58-7.36(m,3H),7.19(s,1H),6.87(s,1H),6.24-6.19(d,J=20.0Hz,1H),5 .79-5.76(d,J=12.0Hz,1H),4.97(s,1H),4.32-4.04(m,3H),3.80-3.49(m,3H),2.72(s,1H),2.35(s,3H),1.34-0.91(m,9H).

[0732] Example 33

[0733] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0734]

[0735] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0736] MS m / z(ESI): 641.6[M+H]+,

[0737] Example 34

[0738] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0739]

[0740] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0741] MS m / z (ESI): 589.1 [M+H] + 591.1[M+H+2] + .

[0742] Example 35

[0743] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0744]

[0745] The preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0746] MS m / z(ESI): 609.6 [M+H]+,

[0747] Example 36

[0748] Preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0749]

[0750] The preparation of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0751] MS m / z(ESI): 661.1[M+H]+,

[0752] Example 37

[0753] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0754]

[0755] The preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0756] MS m / z (ESI): 603.1 [M+H] + 605.1[M+H+2] + .

[0757] Example 38

[0758] Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

[0759]

[0760] The preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0761] MS m / z(ESI): 623.6[M+H]+,

[0762] Example 39

[0763] Preparation of 4-((2S,5R)-(4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0764]

[0765] The preparation of 4-((2S,5R)-(4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0766] MS m / z(ESI): 675.1 [M+H]+,

[0767] Example 40

[0768] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one

[0769]

[0770] Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one is described in Example 13.

[0771] MS m / z (ESI): 676.1 [M+H] + 678.1[M+H+2] + .

[0772] 1 H NMR(400MHz, Methanol-d4)δ8.40–8.32(m,2H),7.51(t,J=7.6Hz,1H),7.22(d,J=5.4Hz, 1H),7.05(t,J=8.4Hz,2H),6.86–6.79(m,1H),6.37–6.26(m,1H),5.84(d,J=10.6Hz,1H) ,5.08(m,2H),4.56-4.46(m,2H),4.21-4.08(m,1H),3.85-3.62(m,2H),2.86-2.82(m,1H ), 2.40 (s, 3H), 1.47 (d, J = 6.6Hz, 3H), 1.21–1.19 (d, J = 6.8Hz, 3H), 1.04 (d, J = 6.8Hz, 3H).

[0773] II. Evaluation of Compound Biological Testing

[0774] The present invention will be further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present invention.

[0775] Test Example 1: Determination of the inhibitory effect of NCI-H358 / Mia PaCa-2 cell proliferation activity

[0776] 1.1 Experimental Objective:

[0777] The inhibitory effects of the compounds in the examples on the proliferation activity of KRAS G12C mutant cell lines NCI-H358 and Mia PaCa-2 were determined.

[0778] 1.2. Experimental Apparatus and Reagents:

[0779] 1.2.1 Instruments:

[0780] Microplate reader (BioTek Synergy H1)

[0781] Pipettes (Eppendorf & Rainin)

[0782] 1.2.2 Reagents:

[0783] NCI-H358 was purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0784] Mia PaCa-2 was purchased from ATCC;

[0785] Cell Titer-Glo cells were purchased from Promega, catalog number G7573;

[0786] RPMI 1640 was purchased from Gibco, part number 22400089;

[0787] DMEM was purchased from Gibco, product number 11995065;

[0788] FBS was purchased from Gibco, item number 10091148;

[0789] PBS was purchased from Gibco, catalog number 10010023;

[0790] The pancreatic enzyme was purchased from Gibco, catalog number 25200056;

[0791] The cell culture plates were purchased from Corning, catalog number 3610.

[0792] 1.3. Experimental Methods:

[0793] When NCI-H358 or Mia PaCa-2 cells reach a suitable confluence, collect the cells and adjust them to a suitable cell concentration using complete culture medium. Spread the cell suspension into 96-well plates (90 μL per well) and incubate overnight at 37°C with 5% CO2. Prepare compound solutions of different concentrations using DMSO and culture medium, and set up a solvent control. Add 10 μL of the compound solution to each well of the 96-well plate and incubate for 72 hours at 37°C with 5% CO2. Then add CellTiter-Glo solution, shake to mix thoroughly, and incubate in the dark for 10 minutes. Read the values ​​using a BioTek Synergy H1 microplate reader.

[0794] 1.4. Experimental Data Processing Methods:

[0795] The inhibition rate was calculated using the emission signal value. The concentration and inhibition rate were then fitted with a nonlinear regression curve using Graphpad Prism software to obtain the IC50. 50 value.

[0796] 1.5. Experimental Results:

[0797] The experimental results are shown in Table 8. The IC50 values ​​of the compounds in the examples on the inhibitory activity of NCI-H358 and Mia PaCa-2 cell proliferation are shown in Table 8. 50 value.

[0798] Table 8

[0799]

[0800]

[0801] Note: "NT" indicates not detected.

[0802] 1.6. Experimental Conclusions:

[0803] According to the data, the compounds in the embodiments of the present invention have a good inhibitory effect on the proliferation of NCI-H358 and Mia PaCa-2 cells.

[0804] Test Example 2: Determination of the ability of the compound of the present invention to enhance the stability (melting temperature) of KRAS G12C protein.

[0805] 2.1. Experimental Objective:

[0806] The ability of the compound to enhance the stability of KRAS G12C protein was tested (the degree of increase in protein melting temperature can characterize the binding ability of the compound to KRAS G12C protein).

[0807] 2.2. Experimental reagents and instruments:

[0808] 2.2.1 Experimental Apparatus:

[0809] The quantitative PCR instrument (Quantstudio 6Flex) was purchased from Life Sciences.

[0810] The pipettes were purchased from Eppendorf or Rainin.

[0811] 2.2.2 Experimental reagents:

[0812] Protein Thermal Shift TM The Dye Kit was purchased from Thermofisher, item number 4461146;

[0813] KRAS G12C protein was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., product number 12259-H07E2;

[0814] HEPES, 1M Buffer Solution, was purchased from Thermofisher, part number 15630080;

[0815] DTT was purchased from Sigma, product number 43816-50mL;

[0816] NaCl was purchased from Sinopharm Chemical Reagent Co., Ltd., product number 10019318.

[0817] 2.3 Experimental Methods:

[0818] This experiment uses the thermal shift method to test the change in melting temperature (Tm) of KRAS G12C protein before and after compound binding, in order to characterize the ability of the compound to improve the stability of KRAS G12C protein.

[0819] The specific experimental procedure is as follows:

[0820] Prepare an experimental buffer solution containing 20 μM HEPES (pH 7.5), 1 mM DTT, 5X SYPRO Orange, and 150 mM NaCl, and add human KRAS G12C protein to a final concentration of 5.37 μM. Aliquot the reaction mixture into 8-tube PCR tubes (19.5 μL per tube), adding 0.5 μL of the test compound or DMSO to each tube, resulting in a total reaction volume of 20 μL and a final compound concentration of 10 μM. Use 2.5% DMSO as a solvent control. After incubating at room temperature in the dark for one hour, place the PCR tubes in a PCR instrument. Open QuantStudio Software v1.3 and use the melt curve function to detect the melting temperature of KRAS G12C protein in different treatment groups (heating from 25℃ to 95℃, 0.03℃ / s).

[0821] 2.4. Experimental Data Processing Methods:

[0822] Import the PCR instrument experimental data file into the thermal shift software to obtain the melting temperature (Tm) of each treatment group, and subtract the Tm of the DMSO solvent control group to obtain the change in melting temperature (ΔTm).

[0823] 2.5. Experimental Results:

[0824] Based on the above methods, the compounds of this invention, in experiments to enhance the stability of KRAS G12C protein, showed the ability to increase the protein melting temperature as shown in Table 9.

[0825] Table 9

[0826] Example number Tm(℃)DMSO Tm (°C) ΔTm (°C) Example 1 48.6 60.2 11.6 Example 2 48.7 57.2 8.5 Example 3 50.6 61.5 10.9 Example 4 49.5 61.2 11.7 Example 5 48.6 64.4 15.8 Example 9 46.8 60.2 13.4 Example 13 47.0 58.0 11.0

[0827] 2.6 Experimental Conclusions:

[0828] The above data show that the compounds in the embodiments of the present invention have good binding ability with KRAS G12C protein.

[0829] Test Example 3: Inhibitory activity of the compound of the present invention against p-ERK in Miapaca-2 cells.

[0830] 3.1. Experimental Objective:

[0831] The inhibitory activity of the compounds in the examples on phosphorylated ERK levels in KRAS G12C mutant cells Mia PaCa-2 was determined.

[0832] 3.2. Experimental Apparatus:

[0833] 3.2.1 Instruments:

[0834] Microplate reader (BioTek Synergy H1);

[0835] Pipettes (Eppendorf & Rainin).

[0836] 3.2.2 Reagents:

[0837] The Phosphorylated ERK1 / 2(T202-Y204)LANCE Ultra Cellular Detection Kit was purchased from PerkinElmer, product number TRF4000M;

[0838] The cell culture plates were purchased from Corning, catalog number 3610;

[0839] White opaque OptiPlate TM The -384 board was purchased from PerkinElmer, part number 6007290.

[0840] 3.3. Experimental Methods:

[0841] When Mia PaCa-2 cells reach a suitable confluence, collect the cells and adjust the cell density to 1×10⁻⁶ using complete culture medium. 6 / mL, the cell suspension was seeded into 96-well plates, 50 μL per well, and incubated overnight at 37°C in a 5% CO2 incubator. Compound solutions of different concentrations were prepared using DMSO and complete culture medium, with a solvent control included. 25 μL of the compound solution was added to each well of the 96-well plate and incubated for 2 hours at 37°C in a 5% CO2 incubator. The supernatant was discarded, and 50 μL of lysis buffer was added to each well. Lysis was performed at room temperature with shaking for 30 minutes. The cells were then centrifuged at 1000 rpm for 1 minute. 15 μL of the supernatant was transferred to 384-well plates, and 5 μL of the detection mixture (0.5 nM Eu-labeled anti-ERK1 / 2 (T202-Y204) antibody and 5 nM Ultra-labeled anti-ERK1 / 2 antibody) were added to each well. The mixture was centrifuged at 1000 rpm for 1 minute to mix thoroughly and incubated overnight at room temperature. BioTek Synergy was then used to analyze the reaction. H1 was used to read the plate, and the signal values ​​at the emission wavelengths of 620 nm and 665 nm were detected using a time-resolved fluorescence procedure.

[0842] 3.4. Experimental Data Processing Methods:

[0843] Calculate the ratio of the signal values at the emission wavelengths of 665 nm and 620 nm, use the ratio to calculate the inhibition rate, and perform non-linear regression curve fitting on the concentration and the inhibition rate using Graphpad Prism software to obtain the IC 50 value.

[0844] 3.5. Experimental results:

[0845] Table 10 IC 50 values

[0846]

[0847] 3.6. Experimental conclusion:

[0848] The above data show that the compound of the embodiment of the present invention has a good inhibitory effect on pERK in Mia PaCa-2 cells.

[0849] Test Example 4, Pharmacokinetics determination in mice

[0850] 4.1. Research purpose:

[0851] Using Balb / c mice as test animals, study the pharmacokinetic behavior of the compound after oral administration in mice (plasma).

[0852] 4.2. Test protocol:

[0853] 4.2.1 Test drug:

[0854] The compound of the embodiment of the present invention, self-made;

[0855] 4.2.2 Test animals:

[0856] Balb / c mice, male, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., animal production license number (SCXK (Shanghai) 2013-0006 N0.311620400001794).

[0857] 4.2.3 Drug preparation:

[0858] Weigh 5 g of hydroxyethyl cellulose (HEC, CMC-Na, viscosity: 800-1200 Cps), dissolve it in 1000 mL of pure water, and add 10 g of Tween80. Mix evenly to form a clear solution.

[0859] Weigh the compound of the embodiment, add it to a 4-mL glass bottle respectively, add 2.4 mL of this solution, and ultrasonic for 10 minutes to obtain a colorless clear solution with a concentration of 1 mg / mL.

[0860] 4.2.4 Administration:

[0861] Balb / c mice, male; fasted overnight, then administered orally at a dose of 10 mg / kg, with a volume of 10 mL / kg.

[0862] 4.2.5 Sample Collection:

[0863] Blood samples were collected before administration and at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h after administration. The blood samples were placed in EDTA-2K tubes and centrifuged at 6000 rpm for 6 min at 4 °C to separate plasma, which was then stored at -80 °C. Patients were fed 4 h after administration. 4.3 Experimental Results:

[0864] The final determination results obtained using the LCMS / MS method are shown in Table 11.

[0865] Table 11: Pharmacokinetic parameters of the compounds in mice

[0866]

[0867] 4.4 Experimental Conclusions:

[0868] The data above show that the compounds in the embodiments of the present invention have good pharmacokinetic parameters in mice.

[0869] Test Example 5: Tumor Inhibition Experiment in MiaPaca 2 Xenograft Model

[0870] 5.1 Experimental Objective:

[0871] Using BALB / c nude mice as test animals, in vivo efficacy experiments were conducted using a human pancreatic cancer cell line MiaPaca 2 xenograft (CDX) model to evaluate the antitumor effects of the test compounds.

[0872] 5.2 Experimental Apparatus and Reagents:

[0873] 5.2.1 Instruments:

[0874] Clean bench (BSC-1300II A2, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory);

[0875] CO2 incubator (Thermo-311, Thermo);

[0876] Centrifuge 5720R, Eppendorf;

[0877] Fully automated cell counter (Countess II, Life Technologies);

[0878] Pipettes (10-20 μL, Eppendorf);

[0879] Microscope (Ts 2, Nikon);

[0880] Vernier calipers (CD-6”AX, Mitutoyo, Japan);

[0881] Cell culture flasks (T25 / T75 / T225, Corning);

[0882] Thermostatic water bath (HWS12, Shanghai Yiheng Science).

[0883] 5.2.2 Reagents:

[0884] DMEM (11995-065, Gibco);

[0885] Fetal bovine serum (FBS) (10091-148, Gibco);

[0886] 0.25% trypsin (25200-056, Gibco);

[0887] Penicillin / streptomycin bispecific antibody (P / S) (SV30010, GE);

[0888] Phosphate-buffered saline (PBS) (10010-023, Gibco);

[0889] Matrigel (356234, Corning);

[0890] Gln(25030-081, Gibco).

[0891] 5.3 Experimental Procedure:

[0892] MiaPaca 2 cells were retrieved from the cell bank, revived, and then cultured in DMEM medium (containing 10% FBS, 1% Glu, and 1% P / S) in a CO2 incubator (37°C, 5% CO2). After the cells reached 80-90% confluence with the bottom of the culture flask, they were passaged and cultured in the CO2 incubator. This process was repeated until the cell count met the in vivo drug efficacy requirements. Cells in the logarithmic growth phase were then collected and counted using an automated cell counter. Based on the count results, the cells were resuspended in PBS and Matrigel (1:1 volume ratio) to prepare a cell suspension (density 8 × 10⁻⁶). 7 ( / mL), place in an ice box for later use.

[0893] The animals used were female BALB / c nude mice, 6-8 weeks old, weighing approximately 18-22 grams. Mice were kept in a specific pathogen-free environment in individual ventilated cages, 5 mice per cage. All cages, bedding, and water were sterilized before use, and all animals had free access to a standard certified commercial laboratory diet. Nude mice were tagged with disposable universal ear tags for rats and mice before the experiment. The injection site was disinfected with 75% medical alcohol before inoculation. Each mouse was subcutaneously inoculated with 0.1 ml (containing 8*10 ml of 8 ... 6 MiaPaca 2 tumor cells (cells). When the average tumor volume reaches 100-200 mm. 3 Dosing was initiated in groups at the specified time. The test compound was administered orally daily via gavage at a dose / frequency of 6 mg / kg QD x 3w. The efficacy of each group at the end of the experiment is shown in Table 5.

[0894] 5.4 Data Processing:

[0895] Measure tumor volume (mm) twice a week using calipers. 3 The calculation formula is: V = 0.5 * D * d * d, where D and d are the long and short diameters of the tumor, respectively. The antitumor efficacy is determined by dividing the average tumor increase volume of the treated animals by the average tumor increase volume of the untreated animals. The tumor inhibition rate is calculated as: TGI(%) = 1 - [(Vt - V0) treated group / (Vt - V0) solvent control group] * 100%. All animals were euthanized after the experiment.

[0896] 5.5 Experimental Results:

[0897] Table 12: Pharmacodynamic parameters of the compounds in xenograft mice

[0898]

[0899] 5.6 Experimental Conclusions:

[0900] The above data show that after 21 days of continuous oral administration, the compound of the present invention significantly inhibited the growth of xenografts in MiaPaca 2 nude mice under the condition of daily oral administration of 6 mg / kg.

[0901] Test Example 6: In vivo pharmacodynamic study on a human lung cancer NCI-H358 cell xenograft tumor model

[0902] 6.1 Experimental Objective:

[0903] To evaluate the in vivo efficacy of the compound in a human lung cancer NCI-H358 cell xenograft model.

[0904] 6.2 Experimental Apparatus and Reagents:

[0905] 6.2.1 Instruments:

[0906] 1) Biosafety cabinet (BSC-1300II A2, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory);

[0907] 2) Clean bench (CJ-2F, Suzhou Fengshi Experimental Animal Equipment Co., Ltd.);

[0908] 3) CO2 incubator (Thermo-311, Thermo);

[0909] 4) Centrifuge (Centrifuge 5720R, Eppendorf);

[0910] 5) Fully automated cell counter (Countess II, Life Technologies);

[0911] 6) Vernier calipers (CD-6”AX, Mitutoyo, Japan);

[0912] 7) Cell culture flasks (T75 / T225, Corning);

[0913] 8) Electronic balance (CPA2202S, Sartorius);

[0914] 9) Electronic balance (BSA2202S-CW, Sartorius);

[0915] 10) Electronic balance (BS124S, Sartorius).

[0916] 6.2.2 Reagents:

[0917] 1) RPMI-1640 medium (22400-089, Gibco);

[0918] 2) DMEM medium (11995-065, Gibco);

[0919] 3) Fetal bovine serum (FBS) (10099-141C, Gibco);

[0920] 4) Phosphate-buffered saline (PBS) (10010-023, Gibco);

[0921] 5) Tween 80 (30189828, Sinopharm Reagent);

[0922] 6) Sodium carboxymethyl cellulose (30036365, Sinopharm Reagent).

[0923] 6.3 Experimental Procedures and Data Processing:

[0924] 6.3.1 Animals:

[0925] BALB / c nude mice, 6-8 weeks old, female, purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.

[0926] 6.3.2 Cell Culture and Cell Suspension Preparation

[0927] 1) Take a MiaPaca-2 cell line from the cell bank and revive the cells in DMEM medium (DMEM + 10% FBS). After revival, place the cells in a cell culture flask (label the flask wall with cell type, date, culturer's name, etc.) and incubate in a CO2 incubator (incubator temperature is 37℃, CO2 concentration is 5%). (The method for reviving NCI-H358 cells is the same as that for MiaPaca-2 cells in test example 5, but the culture medium is changed to RPMI-1640 medium).

[0928] 2) Pass the cells every three to five days, and after passage, continue to culture them in a CO2 incubator. Repeat this process until the cell number meets the requirements for in vivo drug efficacy.

[0929] 3) Collect the cultured MiaPaca-2 cells, count them using an automated cell counter, and resuspend the cells in PBS and Matrigel (1:1 ratio) to prepare a cell suspension (cell density 5 × 10⁻⁶). 7 ( / mL), place in an ice box for later use (NCI-H358 cells do not require Matrigel, resuspend directly in PBS, cell density 1×10⁹ / mL). 8 / mL).

[0930] 6.3.3 Sample preparation:

[0931] 1) Solvent: 0.5% CMC-Na + 1% Tween 80, storage conditions: 4℃.

[0932] Weigh 0.5g of CMC-Na, dissolve it in a certain volume of ddH2O, then add 1.0mL of Tween 80 and stir until well mixed. Finally, bring the volume to 100mL.

[0933] 2) Preparation of the test compound (10 mg / kg):

[0934] Weigh 8.42 mg of AMG510 compound and add 8.260 mL of solvent. Obtain a homogeneous solution by sonication, vortexing, and stirring.

[0935] Weigh 7.81 mg of the compound from Example 13-1, add 7.654 mL of solvent, and obtain a homogeneous solution by sonication, vortexing, and stirring.

[0936] 6.3.3 Cell Seeding

[0937] 1) Tag nude mice with disposable universal ear tags for rats and mice before inoculation;

[0938] 2) When inoculating, mix the cell suspension well, use a 1mL syringe to draw 0.1-1mL of cell suspension, remove air bubbles, and then place the syringe on an ice pack for later use;

[0939] 3) Hold the nude mouse firmly with your left hand, disinfect the right side of the nude mouse's back near the right shoulder with 75% alcohol (inoculation site), and begin inoculation after 30 seconds;

[0940] 4) Inoculate the nude mice sequentially (inoculate each mouse with 0.1 mL of cell suspension);

[0941] 6.3.4 Tumor measurement, grouping, and drug administration in tumor-bearing mice:

[0942] 1) Based on the tumor growth, measure the tumor and calculate its size on the 18th day after inoculation.

[0943] Tumor volume calculation: Tumor volume (mm) 3 = Length (mm) × Width (mm) × Width (mm) / 2

[0944] 2) The mice were randomly assigned to groups based on their weight and tumor size.

[0945] 3) Based on the grouping results, begin administering the test drug (administration route: oral administration; dosage: 10 mg / kg; administration volume: 10 mL / kg; administration frequency: once a day; administration period: 21 days; solvent: 0.5% CMC / 1% Tween 80).

[0946] 4) After starting the test drug, measure and weigh the tumor twice a week.

[0947] 5) Euthanize the animals after the experiment.

[0948] 6) Use software such as Excel to process data.

[0949] 6.4 Data Processing:

[0950] Calculation of the tumor inhibition rate (TGI) of the compound: When there is no tumor regression, TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment)) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%. When there is tumor regression, TGI (%) = [1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / mean tumor volume at the start of treatment] × 100%.

[0951] 6.5 Experimental Results:

[0952] Table 13: Pharmacodynamic parameters of the compounds in xenograft mice

[0953]

[0954] 6.6 Experimental Conclusions:

[0955] The above data show that after 15 days of continuous oral administration, the compound of the present invention significantly inhibited the growth of human lung cancer NCI-H358 cell xenografts in nude mice under the condition of daily oral administration of 10 mg / kg, which was significantly better than the reference data.

[0956] Test Example 7: hERG Potassium Channel Inhibitory Activity Test

[0957] 7.1 Cell Preparation

[0958] 7.1.1 CHO-hERG cells were cultured at 175 cm⁻¹ 2 In the culture flask, when the cell density grows to 60-80%, remove the culture medium, wash once with 7 mL PBS, and then add 3 mL Detachin for digestion.

[0959] 7.1.2 After complete digestion, add 7 mL of culture medium to neutralize, then centrifuge, aspirate the supernatant, and resuspend in 5 mL of culture medium to ensure a cell density of 2–5 × 10⁻⁶ cells / mL. 6 / mL.

[0960] 7.2 Solution Preparation

[0961] Table 14: Composition of intracellular and extracellular fluids

[0962]

[0963] 7.3 Electrophysiological Recording Process

[0964] The single-cell high-impedance sealing and whole-cell pattern formation processes were all automated by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cells were clamped at -80 mV. Before applying a 5-second +40 mV depolarization stimulus, a 50-millisecond -50 mV pre-voltage was applied, followed by repolarization to -50 mV for 5 seconds, and then back to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular fluid was applied for 5 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2.5 minutes. After all concentrations were administered, 3 μM Cisapride, a positive control compound, was administered. At least 3 cells (n≥3) were tested for each concentration.

[0965] 7.4 Compound Preparation

[0966] 7.4.1 Dilute the 20mM compound stock solution with extracellular fluid. Take 5μL of the 20mM compound stock solution and add it to 2495μL of extracellular fluid. Dilute 500 times to 40μM. Then, perform 3-fold serial dilutions in extracellular fluid containing 0.2% DMSO to obtain the final concentration to be tested.

[0967] 7.4.2 The highest test concentration was 40 μM, and the six concentrations were 40, 13.33, 4.44, 1.48, 0.49, and 0.16 μM respectively.

[0968] 7.4.3 The DMSO content in the final test concentration shall not exceed 0.2%, and this concentration of DMSO has no effect on the hERG potassium channel.

[0969] 7.5 Data Analysis

[0970] Experimental data were analyzed using XLFit software.

[0971] 7.6 Quality Control

[0972] Environment: Humidity 20-50%, temperature 22-25℃

[0973] Reagents: All experimental reagents used were purchased from Sigma-Aldrich and had a purity >98%.

[0974] The experimental data in the report must meet the following standards:

[0975] Whole-cell sealing impedance >100MΩ

[0976] Tail current amplitude >400pA

[0977] Pharmacological parameters:

[0978] The inhibitory effect of multiple concentrations of Cisapride on hERG channels was used as a positive control.

[0979] 7.7 Experimental Results:

[0980] Table 15: Suppression results of hERG current at multiple concentrations in the embodiments of the present invention

[0981] Example number hERG(μM) Example 2-1 >30 Example 9-1 >30 Example 13-1 >30 Example 14-1 >30

[0982] 7.8 Experimental Conclusions:

[0983] Drug inhibition of cardiac hERG potassium channels is a major cause of drug-induced QT prolongation syndrome. Experimental results show that the compounds in this invention do not significantly inhibit cardiac hERG potassium channels, thus avoiding cardiotoxic side effects at high doses.

[0984] Test Example 8: Plasma Stability Test Protocol

[0985] 8.1 Experimental Objective

[0986] The purpose of this experiment was to test the stability of the compounds in mouse, rat, dog and human plasma.

[0987] 8.2 Experimental Procedure

[0988] 8.2.1 Solution Preparation

[0989] 1) Plasma preparation

[0990] After collecting whole blood from animals or humans, place it in a test tube containing an anticoagulant, centrifuge at 3500 rpm for 10 minutes, and collect the upper layer of pale yellow plasma.

[0991] 2) 10 μM test compound (m / M / V = C)

[0992] Weigh the compound, prepare the stock solution with DMSO, and prepare the working solution with 100mM phosphate buffer.

[0993] 3) 10μM positive control

[0994] (1) Propantheline (Mr = 449.4 Da)

[0995] Weigh 2.36 mg of bromhexine and dilute it with 1 mL of DMSO to prepare a 10 mM stock solution; transfer 10 μL of the 10 mM stock solution to 1 mL of 100 mM phosphate buffer, with a final concentration of 100 μM.

[0996] (2) Mevinolin (Lovastatin Mr = 404.5 Da)

[0997] Weigh 4.05 mg of lovastatin and dilute it with 1 mL of DMSO to prepare a 10 mM stock solution; transfer 10 μL of the 10 mM stock solution to 1 mL of 100 mM phosphate buffer, with a final concentration of 100 μM.

[0998] 8.2.2 Experimental Procedure:

[0999] 1) Add 285 μL of plasma and 15 μL of 10 μM compound (test compound) sequentially to a 96-well plate and incubate at 37°C.

[1000] 2) Take 40 μL at 0, 15, 30, 60, 90 and 120 min (the sampling points can be finely adjusted) and add 160 μL of acetonitrile stop solution containing internal standard.

[1001] 3) After centrifugation (3500 rpm, 10 min), take 50 μL of supernatant, add 50 μL of DDH2O to dilute, and then inject into LC-MS / MS.

[1002] 8.3 Chromatographic conditions

[1003] Instrument: Shimadzu LC-20AD

[1004] Column: Phenomenex C18 (50*4.6mm, 5μm particle size);

[1005] Mobile phase: A: acetonitrile, B: 0.1% formic acid solution; 0–8 min: 5% A → 95% A, 2.0–2.1 min: 90% A → 5% A; Flow rate: 0.8 mL / min; Run time: 5.0 min; Injection volume: 5 μL.

[1006] 8.4 Mass Spectrometry Conditions:

[1007] Instrument: API 4000 liquid chromatography-mass spectrometry system, AB Corporation, USA;

[1008] The ion source is an electrospray ionization source (ESI);

[1009] Dry gas (N2) temperature 500℃;

[1010] The electrospray voltage is 5500V;

[1011] The detection method is positive ion detection;

[1012] The scanning method was selected as the Mass Reaction Monitoring (MRM) mode;

[1013] The scan time is 0.1s.

[1014] 8.5 Experimental Results:

[1015] Table 16: Plasma stability results of compounds in the examples

[1016]

[1017]

[1018] 8.6 Experimental Conclusions:

[1019] The data above show that the compounds in the embodiments of the present invention have high plasma stability and little species difference.

[1020] Test Example 9: CYP Enzyme Single-Point Inhibition Assay

[1021] 9.1 Experimental Objective

[1022] Using a human liver microsome incubation system, a single-point method was employed to rapidly predict the inhibitory effect of compounds on CYP450 enzyme subtypes.

[1023] 9.2 Experimental Procedure

[1024] 9.2.1 Solution Preparation

[1025] Weigh 4.165 mg of NADPH (reduced nicotinamide adenine dinucleotide phosphate) and add 100 mM phosphate buffer to a final volume of 2 mL. Add 50 μL of 0.25 mg / mL microsomes to 4 mL of 100 mM phosphate buffer and mix well.

[1026] Preparation of reaction solution for the test compound

[1027] Weigh the compound to be tested, dilute it to 10 mM with DMSO, and then dilute it to 100 μM with 100 mM phosphate buffer.

[1028] 9.2.2 Experimental Procedure:

[1029] 1. Add 40 μL of liver microsomes, 10 μL of substrate, and 10 μL of the test compound to a 96-well plate and incubate for 3 min.

[1030] 2. Add 40 μL of NADPH.

[1031] 3. Add 300 μL of acetonitrile stop solution containing internal standard at 20 min.

[1032] 4. Centrifuge and inject the sample.

[1033] 9.3 Experimental Results:

[1034] Table 17: Results of Single-Point Inhibition of CYP Enzyme by Compounds in Examples

[1035]

[1036] Note:

[1037] Strong inhibition: IC 50 <1μM; Moderate inhibition: 1μM <IC 50 <10μM; Weak inhibition: IC50 50 >10μM

[1038] 9.4 Experimental Conclusions:

[1039] The data above show that the compounds in the embodiments of the present invention do not strongly inhibit any CYP enzyme subtypes and have a low risk of DDI.

[1040] Test Example 10: Plasma Protein Binding Rate Experiment

[1041] 10.1 Experimental Objective:

[1042] The purpose of this experimental method is to detect the binding of the compounds in the examples to plasma proteins.

[1043] 10.2 Experimental Instruments and Materials:

[1044] Liquid chromatography-mass spectrometry (LC-MS), centrifuge, vortex mixer, pipette, continuous feeder, 96-well plate, tissue homogenizer (used for tissue sample analysis), 50% methanol aqueous solution with acetonitrile solution added as internal standard, and blank matrix (plasma, urine, or tissue homogenate, etc.).

[1045] 10.3 Experimental Procedure:

[1046] 10.3.1 Preparation of the stock solution of the analyte A

[1047] The compounds from the examples were prepared into a 1 mM solution A using DMSO.

[1048] 10.3.2 Preparation of Plasma Solution B

[1049] Add solution A to the plasma solution to prepare a 5 μM solution B.

[1050] 10.3.3 Processing Flow

[1051] 1) Add 200 μL of solution B to the membrane.

[1052] 2) Add 350 uL of PBS outside the membrane.

[1053] 3) Incubate in a 37℃ water bath for 6 hours.

[1054] 4) The sample is processed, diluted, and then analyzed by mass spectrometry.

[1055] 10.4 Chromatographic conditions:

[1056] Instrument: Shimadzu LC-20AD;

[1057] Column: Phenomenex C18 (50*4.6mm, 5μm particle size);

[1058] Mobile phase: A: acetonitrile, B: 0.1% formic acid solution; 0–0.5 min: 5% A → 90% A, 2.0–2.1 min: 90% A → 5% A; Flow rate: 0.8 mL / min; Run time: 5.0 min; Injection volume: 5 μL.

[1059] 10.5 Mass spectrometry conditions:

[1060] Instrument: API 4000 liquid chromatography-mass spectrometry system, AB Corporation, USA;

[1061] The ion source is an electrospray ionization source (ESI);

[1062] The temperature of the drying gas (N2) is 500 °C;

[1063] The electrospray voltage is 5500 V;

[1064] The detection mode is positive ion detection;

[1065] The scanning mode is the selected reaction monitoring (MRM) mode; the scanning time is 0.1 s.

[1066] 10.6 Experimental results:

[1067] Table 18: Plasma protein binding rate of the compounds in the examples

[1068] serial number people rats mice dog Example 2-1 98.0 90.5 88.4 82.6

[1069] Example 9-1 99.8 94.9 90.1 98.7 Example 13-1 99.7 97.9 93.9 98.7 Example 14-1 96.8 95.4 96.3 92.5

[1070] 10.7 Experimental conclusions:

[1071] The above data show that: the compounds in the examples of the present invention show a high plasma protein binding rate and small species differences.

[1072] Test Example 11, Pharmacokinetic determination in tumor-bearing mice

[1073] 11.1. Research purpose:

[1074] Using MiaPaca 2 tumor-bearing mice as the test animals, study the pharmacokinetic behaviors of Compound Example 13-1 and AMG-510 compound after oral administration at a dose of 6 mg / kg in mice (plasma, tumor tissue and intestine).

[1075] 11.2. Test protocol

[1076] 11.2.1 Test drugs:

[1077] Compound Example 13-1 of the present invention, AMG-510 compound, self-made.

[1078] 11.2.2 Test animals:

[1079] 24 female MiaPaca 2 tumor-bearing mice. 3 mice at each time point (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h). Shanghai Xipu-Bikai Laboratory Animal Co., Ltd., Animal Production License Number (SCXK (Shanghai) 2018-0006.

[1080] 11.2.3 Drug preparation:

[1081] Weigh 5g of hydroxymethyl cellulose, dissolve it in 1000mL of purified water, and add 10g of Tween 80. Mix well to form a clear solution.

[1082] Compound 13-1 from Example 1 was weighed, and AMG-510 was dissolved in the solution. The mixture was shaken well and sonicated for 15 minutes to obtain a homogeneous suspension with a concentration of 0.6 mg / mL.

[1083] 11.2.4 Administration:

[1084] MiaPaca 2 tumor-bearing mice were fasted and administered the drug orally at body weight (no drug was administered to animals at 0h), with a dose of 6 mg / kg and a volume of 10 mL / kg.

[1085] 11.2.5 Sample Collection:

[1086] Mice were euthanized with CO2 before and after drug administration. 0.5 ml of blood was collected from the heart and placed in an EDTA-2K tube. Plasma was separated by centrifugation at 6000 rpm for 6 min at 4°C and stored at -80°C. Tumor tissue was weighed and placed in a 2 mL centrifuge tube and stored at -80°C. Appropriate lengths of duodenal, ileal, and colonic tissue were cut open with scissors, contents removed, and the tissue washed twice with PBS. After blotting with absorbent paper, the tissue was weighed and placed in a 2 mL centrifuge tube and stored at -80°C.

[1087] 11.3 Experimental Results: The final measurement results were obtained using the LCMS / MS method, as shown in Table 11:

[1088] Table 19: Pharmacokinetic parameters of the compounds of the present invention in mice

[1089]

[1090]

[1091] 11.4 Experimental Conclusions:

[1092] At a dose of 6 mg / kg, the ratio of the exposure level of the compound in mouse tumors to its exposure level in blood was higher than that of AMG-510. 1 / 2 And MRT is longer.

[1093] III. Study on the salts and crystal forms of compounds

[1094] As is well known to those skilled in the art, when the compounds of the above embodiments are shown to have good inhibitory effects on the proliferation of NCI-H358 and MiaPaCa-2 cells, their pharmaceutically acceptable salts often have the same pharmacological and efficacy activities. Based on this, the inventors further studied the physicochemical properties of the salt forms and crystal forms of the corresponding compounds. However, the preparation and characterization of the specific salt forms or crystal forms described below do not constitute a limitation on the scope of protection of this invention. Those skilled in the art can use this invention as a basis to obtain more salt forms and crystals of the compounds of this invention through conventional salt-forming or crystallization methods. These salt forms and crystals are all protected by this invention. Specifically:

[1095] 1. Experimental apparatus

[1096] 1.1 Some parameters of physicochemical testing instruments

[1097]

[1098]

[1099] 1.2 Instrument and Liquid Chromatography Analysis Conditions

[1100] 1.2.1 Instruments and Equipment

[1101] Instrument Name model Analytical balance METTLER TOLEDO XA105 Pure water machine Milli-Q Plus, Millipore High Performance Liquid Chromatography Thermo Ultimate 3000

[1102] 1.2.2 Chromatographic conditions

[1103]

[1104] 2. Research on the salt form of compounds

[1105] 2.1 Salt form screening of compounds in Example 13-1

[1106] 2.1.1 Experimental Objective:

[1107] Screening for the salt forms of compounds.

[1108] 2.1.2 Experimental Procedure:

[1109] 1) Instruments and equipment

[1110] name model source Analytical balance XA105 METTLER TOLEDO Ultrasonic cleaner SK5200LHC Shanghai Kedao Ultrasonic Instruments

[1111] pipette Eppendorf (50 mL, 100 μL) Eppendorf

[1112] 2) Operating Procedures

[1113] ① Salt formation through dissolution or suspension

[1114] Weigh 10 mg of the compound, add 200 μL of solvent, stir at room temperature, add different acids, stir overnight, centrifuge and dry or evaporate to obtain the salt of the compound.

[1115]

[1116] ② Salt formation by antisolvent method

[1117] Select a good solvent, weigh the acid, add the good solvent to prepare a stock solution containing the compound at a concentration of 100 mg / mL, add the antisolvent, weigh 100 mg of the compound, add 1 mL of the good solvent, after dissolving completely, filter, take 0.2 mL of the filtrate, add the antisolvent dropwise (stop adding if there is precipitation, the maximum addition is 1.8 mL of antisolvent), stir for a period of time, then quickly centrifuge to remove the filtrate, and obtain the salt of the compound.

[1118]

[1119] 2.1.3 Experimental Results:

[1120] Salt type screening experiments revealed that sulfuric acid, hydroxyethyl sulfonic acid, and 1,5-naphthalenedisulfonic acid are the compounds that can form salts with the free base of the compound.

[1121] As described above, those skilled in the art can obtain more medicinal salts using conventional methods based on the present invention.

[1122] 2.2 Quantitative Analysis of Compound Hydroxyethyl Sulfonate in Example 13-1

[1123] 2.2.1 HPLC quantification of hydroxyethyl sulfonate

[1124] 2.2.1.1 Experimental Objective:

[1125] Determine the number of hydroxyethyl sulfonic acids in the compound hydroxyethyl sulfonate of Example 13-1.

[1126] 2.2.1.2 Experimental Procedure:

[1127] 1) Chromatographic conditions

[1128]

[1129] 2) Operation

[1130] Weigh an appropriate amount of the free base from Example 13-1 and add methanol to prepare a series of linear solutions with a concentration range of 0.05-0.30 mg / mL.

[1131] Weigh an appropriate amount of the hydroxyethyl sulfonate compound from Example 13-1 and prepare a solution containing 0.25 mg / mL of hydroxyethyl sulfonate from Example 13-1 in methanol. Inject the above linear solution and the sample solution separately.

[1132] 2.2.1.3 Experimental Results:

[1133]

[1134]

[1135] The external standard method calculation results show that hydroxyethyl sulfonic acid and free base form a salt in a 1:1 molar ratio.

[1136] 2.2.2 ELSD Quantification of Compound Hydroxyethylsulfonate in Example 13-1

[1137] 2.2.2.1 Experimental Objective:

[1138] Determine the number of hydroxyethyl sulfonic acids in the compound hydroxyethyl sulfonate of Example 13-1.

[1139] 2.2.2.2 Experimental Procedure:

[1140] 1) Chromatographic conditions

[1141] Dilutent MeOH Column ZIC-HILIC (150*4.6mm, 5μm) Mobile phase 75mM ammonium acetate solution (pH 4.80):acetonitrile = 30:70 Injection volume 5μL Flow rate 1.0 mL / min Column Temperature 35℃ ELSD Temperature 40℃

[1142] 2) Operation

[1143] Weigh an appropriate amount of hydroxyethyl sulfonic acid and add methanol to prepare a series of linear solutions containing hydroxyethyl sulfonic acid in the range of 0.5-1 mg / mL.

[1144] Weigh an appropriate amount of the hydroxyethyl sulfonate compound from Example 13-1 and dissolve it in methanol to prepare a solution containing 5.0 mg / mL of the hydroxyethyl sulfonate compound from Example 13-1. Inject the linear solution and the sample solution separately.

[1145] 2.2.2.3 Experimental Results:

[1146]

[1147] The number of hydroxyethyl sulfonic acid molecules in the compound hydroxyethyl sulfonate of Example 13-1 was calculated to be 1.

[1148] 3. Study on the crystal form of compound salts

[1149] 3.1 Crystal form study of the compound in Example 13-1

[1150] 3.1.1 Experimental Objective:

[1151] Screening for salts that form the crystal form of compounds.

[1152] 3.1.2 Experimental Procedure:

[1153] 1) Instruments and equipment

[1154]

[1155] 2) Operating Procedures

[1156] ① Salt crystallization by dissolution or suspension in different solvents

[1157] Weigh 10 mg of the compound from Example 13-1 and add it to different reaction solvents. The final total volume is 200 μL. Stir, add acid, stir for 12 hours, centrifuge and dry, and then measure its XRPD.

[1158]

[1159]

[1160] ② Antisolvent method for salt crystallization

[1161] Select a good solvent, weigh the acid, add the good solvent to prepare a stock solution containing a compound concentration of 100 mg / mL, add the antisolvent, weigh 100 mg of the compound, add 1 mL of the good solvent, after complete dissolution, filter, take 0.2 mL of the filtrate, add the antisolvent dropwise (stop adding if there is precipitation, the maximum addition is 1.8 mL of antisolvent), stir for a period of time, then quickly centrifuge to remove the filtrate, dry the solid and measure its XRPD.

[1162]

[1163] 3.1.3 Experimental Results

[1164] Through experiments studying the crystal forms of the salts of this compound, the crystalline salt forms were found to be hydroxyethyl sulfonate, sulfate, and 1,5-naphthalene disulfonate.

[1165] 3.2 Preparation of the crystal form of the compound in Example 13-1

[1166] 3.2.1 Experimental Objective:

[1167] Preparation of the crystal form of the compound salt in Example 13-1.

[1168] 3.2.2 Experimental Procedure:

[1169] 1) Instruments and equipment

[1170]

[1171] 2) Operating Procedures

[1172] I. Preparation of Hydroxyethyl Sulfonate Crystal Form I

[1173] Weigh 500 mg of the compound from Example 13-1, add 9.08 mL of isopropanol, heat and stir at 50°C, add 0.914 mL of hydroxyethyl sulfonic acid (1.0 M in MeOH), dissolve and precipitate, stir at room temperature for 2 hours, filter, and dry the solid under vacuum at 50°C to obtain hydroxyethyl sulfonate crystal form I. Analysis showed that it possessed the following properties: Figure 1 The XRPD diagram shown is as follows: Figure 2 The DSC diagram shown and as follows Figure 3 The TGA diagram shown.

[1174] Alternatively, compound 100g from Example 13-1 and isopropanol (1200mL) were added to a 3L three-necked flask, heated to 40-45°C, and stirred until dissolved. Then, 2-hydroxyethylsulfonic acid (28.84g) was dispersed in 800mL of ethanol and added dropwise to the reaction system, maintaining the temperature at 39-42°C, for approximately 10 minutes. 500mg of seed crystals were added to the above reaction solution, causing rapid precipitation of the solid. Heating was removed, and the mixture was cooled to 25°C and stirred for 12 hours. The mixture was filtered, and the filter cake was washed with 400mL of isopropanol. The cake was then dried under vacuum at 45°C for 16 hours to obtain 92.57g of a pale yellow solid with a purity of 97.9%, a chiral purity of 99.8%, and a yield of 92%. Analysis showed that it possessed the basic properties of... Figure 1 The XRPD diagram shown is basically as follows: Figure 2 The DSC diagram shown and the basic... Figure 3 The TGA diagram shown.

[1175] II. Preparation of Hydroxyethyl Sulfonate Crystal Form II

[1176] Weigh 10 mg of the compound from Example 13-1, add 0.2 mL of tetrahydrofuran, heat and stir at 50 °C, add 18.3 μL of hydroxyethyl sulfonic acid (1.0 M in MeOH), dissolve and precipitate, stir at room temperature for 2 hours, filter, and dry the solid under vacuum at 50 °C to obtain hydroxyethyl sulfonate crystal form II. Analysis showed that it possessed the following properties: Figure 4 The XRPD diagram shown is as follows: Figure 5 The DSC diagram shown and as follows Figure 6 The TGA diagram shown.

[1177] III. Preparation of Hydroxyethyl Sulfonate Crystal Form III

[1178] Weigh 20 mg of hydroxyethyl sulfonate crystal form I, add 0.2 mL of methanol and 0.45 mL of methyl tert-butyl ether, heat and stir overnight at 50 °C, filter, and dry the solid under vacuum at 50 °C to obtain hydroxyethyl sulfonate crystal form III. Analysis showed that it possessed the following properties: Figure 7 The XRPD diagram shown is as follows: Figure 8 The DSC diagram shown and as follows Figure 9The TGA diagram shown.

[1179] IV. Preparation of Sulfate Crystal Form I

[1180] Weigh 10 mg of the compound from Example 13-1, add 0.2 mL of ethanol, heat and stir at 50 °C, add 18.3 μL of sulfuric acid (1.0 min MeOH), dissolve and precipitate, stir overnight at room temperature, filter, and dry the solid under vacuum at 50 °C to obtain sulfate crystal form I. Analysis showed that it possessed the following properties: Figure 10 The XRPD diagram shown.

[1181] V. Preparation of sulfate crystal form II

[1182] Weigh 100 mg of the compound from Example 13-1, add 1.82 mL of isopropanol, heat and stir at 50 °C, add 183 μL of sulfuric acid (1.0 M in MeOH), dissolve until clear, and precipitate a solid. Stir overnight at room temperature, filter, and dry the solid under vacuum at 50 °C to obtain sulfate crystal form II. Analysis showed that it possessed the following properties: Figure 11 The XRPD diagram shown.

[1183] VI. Preparation of Sulfate Crystal Form III

[1184] Weigh 10 mg of sulfate crystal form I, add 0.2 mL of isopropanol, heat and stir at 50 °C for 5 days, filter, and then vacuum dry the solid at 50 °C to obtain sulfate crystal form III. Analysis showed that it possessed the following properties: Figure 12 The XRPD diagram shown.

[1185] VII. Preparation of Sulfate Crystal Form IV

[1186] 10 mg of sulfate crystal form I was weighed, and 0.2 mL of ethyl acetate was added. The mixture was heated and stirred at 50 °C for 5 days. After filtration, the solid was dried under vacuum at 50 °C to obtain sulfate crystal form III. Analysis showed that it possessed the following properties: Figure 13 The XRPD diagram shown.

[1187] 4. Solid stability test

[1188] 4.1 Stabilization test of hydroxyethyl sulfonate crystal form I in Example 13-1

[1189] 4.1.1 Experimental Objective:

[1190] The study investigated the physicochemical stability of compound crystal forms under high temperature, high humidity, high temperature and high humidity, and light conditions, providing a basis for crystal form screening and storage.

[1191] 4.1.2 Instrument and Liquid Chromatography Analysis Conditions

[1192]

[1193] 4.1.3 Experimental Scheme

[1194] 4.1.3.1 Weigh an appropriate amount of hydroxyethyl sulfonate crystal form I of compound 13-1, and treat it for a certain period of time under light (≥1.2×106 lux·h, 10 days), high humidity (25℃, 75%, 10 days), high humidity (25℃, 90%, 10 days), high temperature (40℃, 30 days), high temperature (60℃, 30 days) and micronized powder conditions, and then measure its XRPD.

[1195] 4.1.4.1 Experimental Results:

[1196]

[1197] 4.1.3.2 Experimental Scheme:

[1198] Weigh an appropriate amount of hydroxyethyl sulfonate crystal form I of compound 13-1, and place it under light (5000±500 lux), high temperature (60℃), high humidity (92.5% RH), and high temperature and high humidity (50℃ & 75% RH) conditions for 10 days respectively. Then add methanol as diluent to prepare a solution containing 0.25 mg / mL of free alkali of compound 13-1. Analyze by HPLC and calculate the changes of related substances according to the peak area normalization method.

[1199] 4.1.4.2 Experimental Results:

[1200]

[1201] The above experimental results show that the hydroxyethyl sulfonate crystal form I of Compound 13-1 is relatively stable under light, high humidity, high temperature and micronized conditions.

[1202] 4.2 Solid stability test of compound sulfate crystal form II in Example 13-1

[1203] 4.2.1 Experimental Objective:

[1204] The study investigated the physicochemical stability of compound crystal forms under high temperature, high humidity, high temperature and high humidity, and light conditions, providing a basis for crystal form screening and storage.

[1205] 4.2.2 Instrument and Liquid Chromatography Analysis Conditions

[1206]

[1207] 4.2.3 Experimental Scheme:

[1208] Weigh an appropriate amount of sulfate crystal form II of compound 13-1 and place it under light (5000±500 lux), high temperature (60℃), high humidity (92.5% RH), and high temperature and high humidity (50℃ & 75% RH) conditions for 10 days. Then add methanol as a diluent to prepare a solution containing 0.25 mg / mL of free alkali from Example 13-1. Analyze the solution by HPLC and calculate the changes in related substances according to the peak area normalization method.

[1209] 4.2.4 Experimental Results:

[1210]

[1211]

[1212] Sulfate crystal form II is relatively stable under light, high humidity, and high temperature and high humidity conditions.

[1213] 5. Solubility experiments in different media

[1214] 5.1 Example 13-1 Solubility experiment of compound in different media

[1215] 5.1.1 Experimental Objective:

[1216] The solubility of hydroxyethyl sulfonate crystal form I and sulfate crystal form II in different pH media, water, artificial gastric juice (FaSSGF), fasting artificial intestinal juice (FaSSIF), and non-fasting artificial intestinal juice (FeSSIF) was investigated to provide a basis for evaluating the druggability of salts.

[1217] 5.1.2 Experimental Procedure:

[1218] Approximately 1 mg of different salt forms of the compound were weighed and suspended in 1 mL of artificial simulated gastric fluid (FaSSGF), fasting artificial simulated intestinal fluid (FaSSIF), non-fasting artificial simulated intestinal fluid (FeSSIF), and pure water for 24 hours. The thermodynamic solubility of the compound at 37 °C was determined by HPLC using the external standard method.

[1219] 5.1.3 Experimental Results: As shown in the table below:

[1220]

[1221] 6. Thermodynamic stability experiment

[1222] 6.1 Polymorphic screening of the hydroxyethyl sulfonate of the compound in Example 13-1

[1223] 6.1.1 Experimental Objective:

[1224] Thermodynamically stable hydroxyethyl sulfonate crystals were obtained through polycrystalline screening.

[1225] 6.1.2 Experimental Procedure:

[1226] Take 10 mg of hydroxyethyl sulfonate crystal form I, add 200 μL of organic solvent, and slurry for 5 days at room temperature and 50 °C. Centrifuge, discard the supernatant, and measure the XRPD of the solid after drying.

[1227] 6.1.3 Experimental results: as shown in the table below:

[1228]

[1229]

[1230] The above results indicate that hydroxyethyl sulfonate crystal form I is the stable crystal form of hydroxyethyl sulfonate.

[1231] 6.2 Polymorphism Screening Experiment of Sulfate of Compound 13-1 in Example 1

[1232] 6.2.1 Experimental Objective:

[1233] Thermodynamically stable sulfate crystal forms were obtained through polycrystalline screening.

[1234] 6.2.2 Experimental Procedure:

[1235] Take 10 mg of sulfate crystal form II, add 200 μL of organic solvent, slurry at 50℃ for 5 days, centrifuge, discard the supernatant, dry the solid and measure its XRPD.

[1236] 6.2.3 Experimental results: as shown in the table below:

[1237] Serial Number solvent sulfates - Initial crystal form Crystal form II 1 ethanol Crystal form II 2 2-Methyltetrahydrofuran Crystal form II 3 2-Butanone Crystal form II 4 Ethyl acetate Crystal form II 5 Toluene Crystal form II 6 Isopropyl acetate Crystal form II 7 tert-Butanol Crystal form II

[1238] The above results indicate that sulfate crystal form II is the stable crystal form of sulfate.

Claims

1. An acid salt of a compound of general formula (II), wherein: R a selected from hydrogen or methyl; R1is selected from hydrogen, fluorine, chlorine, bromine or methyl; R3is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, methyl, -S(CH3) or trifluoromethyl; R4is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, -N(CH3)2or -NH(CH3); R5is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl or isopropyl; R6is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl or isopropyl; R7is selected from hydrogen, fluorine, chlorine, bromine or methyl; the acid of the acid salt is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethanesulfonic acid, dichloroacetic acid, trichloroacetic acid, acetyloxymethane acid, adipic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyl tartaric acid, ethane-1,2-disulfonic acid, formic acid, fumaric acid, galacturonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1,5-naphthalene disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid.

2. The acid salt of the compound according to claim 1, characterized in that, the acid is selected from hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid or hydrobromic acid.

3. The acid salt of the compound according to claim 1, characterized in that, the compound is further of general formula (II-A) or (II-B):

4. The acid salt of the compound according to claim 1, characterized in that, the compound is selected from:

5. The acid salt of the compound according to claim 1, characterized in that, the compound is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2- fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d]pyrimidin-2(1H)-one; P-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)- 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one; P-4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1- (2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one; P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6- hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin- 2(1H)-one; P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-l-yl)-6-fluoro-7-(2-fluoro-6- hydroxyphenyl)-l-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin- 2(lH)-one; the acid in the acid salt is selected from isethionic acid, sulfuric acid, 1,5- naphthalene disulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleic acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, p-toluenesulfonic acid, or fumaric acid.

6. The acid salt of the compound according to claim 5, characterized in that, the acid is selected from isethionic acid or sulfuric acid.

7. The acid salt of the compound according to claim 1, characterized in that, the number of acids is 0.2, 0.5, 1, 1.5, 2, 2.5, or 3.

8. The acid salt of the compound according to claim 7, characterized in that, the number of acids is 0.5, 1, 2, or 3.

9. The acid salt of the compound according to claim 7, characterized in that, the number of acids is 1.

10. The acid salt of the compound according to claim 1, wherein, the acid salt is a hydrate or an anhydrate; when the acid salt is a hydrate, the number of waters is 0.2, 0.5, 1, 1.5, 2, 2.5, or 3.

11. The acid salt of the compound according to claim 1, wherein, the acid salt is an acid salt crystalline form of P-4-((2S,5R)-4-acryloyl-2,5- dimethylpiperazin-l-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-l-(2- isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(lH)-one: which is an isethionic acid salt crystalline form II having an X-ray powder diffraction pattern 2Θ at 21.7 ± 0.2°; or at 8.8 ± 0.2°; or at 19.3 ± 0.2°; or at 27.6 ± 0.2°; or at 10.9 ± 0.2°; or at 23.8 ± 0.2°; or at 16.7 ± 0.2°; or at 15.4 ± 0.2°; or at 15.8 ± 0.2°; or at 10.0 ± 0.2°; comprising any 7 thereof; which is an isethionic acid salt crystalline form II having an X-ray powder diffraction pattern 2Θ at 21.7 ± 0.2°; or at 8.8 ± 0.2°; or at 19.3 ± 0.2°; or at 27.6 ± 0.2°; or at 10.9 ± 0.2°; or at 23.8 ± 0.2°; or at 16.7 ± 0.2°; or at 15.4 ± 0.2°; or at 15.8 ± 0.2°; or at 10.0 ± 0.2°; comprising any 7 thereof; which is hydroxyethylsulfate Form III having an X-ray powder diffraction pattern 2Q at 19.4+0.2°; or at 16.9+0.2°; or at 26.6+0.2°; or at 14.6+0.2°; or at 28.0+0.2°; or at 25.6+0.2°; or at 20.7+0.2°; or at 12.8+0.2°; or at 19.1+0.2°; or at 27.2+0.2°; comprising any 7 thereof; which is sulfate Form I having an X-ray powder diffraction pattern 2Q at 19.0+0.2°; or at 19.4+0.2°; or at 12.4+0.2°; or at 26.2+0.2°; or at 17.6+0.2°; or at 18.1+0.2°; or at 25.3+0.2°; or at 8.8+0.2°; or at 21.9+0.2°; or at 11.5+0.2°; comprising any 7 thereof; which is sulfate Form II having an X-ray powder diffraction pattern 2Q at 15.5+0.2°; or at 11.1+0.2°; or at 8.9+0.2°; or at 19.3+0.2°; or at 22.3+0.2°; or at 23.6+0.2°; or at 17.4+0.2°; or at 27.3+0.2°; or at 17.0+0.2°; or at 27.9+0.2°; comprising any 7 thereof; which is sulfate Form III having an X-ray powder diffraction pattern 2Q at 19.6+0.2°; or at 18.0+0.2°; or at 18.4+0.2°; or at 16.8+0.2°; or at 14.3+0.2°; or at 11.8+0.2°; or at 14.9+0.2°; or at 25.7+0.2°; or at 15.4+0.2°; or at 23.5+0.2°; comprising any 7 thereof; which is Form IV of the sulfate salt having an X-ray powder diffraction pattern with a diffraction peak at 19.4 ± 0.2°; or a diffraction peak at 18.9 ± 0.2°; or a diffraction peak at 15.5 ± 0.2°; or a diffraction peak at 8.8 ± 0.2°; or a diffraction peak at 18.1 ± 0.2°; or a diffraction peak at 24.9 ± 0.2°; or a diffraction peak at 17.4 ± 0.2°; or a diffraction peak at 12.3 ± 0.2°; or a diffraction peak at 26.1 ± 0.2°; or a diffraction peak at 14.5 ± 0.2°; including any 7 thereof.

12. The acid salt of the compound according to claim 11, wherein: the X-ray powder diffraction pattern of the isethionate salt Form I includes diffraction peaks at 2-theta of 21.7 ± 0.2°, 8.8 ± 0.2°, 19.3 ± 0.2°; and can further include 4 diffraction peaks at 2-theta of 27.6 ± 0.2°, 10.9 ± 0.2°, 15.4 ± 0.2°, 16.7 ± 0.2°, 15.8 ± 0.2°, 10.2 ± 0.2°, 11.8 ± 0.2°; the X-ray powder diffraction pattern of the isethionate salt Form II includes diffraction peaks at 2-theta of 21.7 ± 0.2°, 10.0 ± 0.2°, 8.8 ± 0.2°; and can further include 4 diffraction peaks at 2-theta of 19.3 ± 0.2°, 27.6 ± 0.2°, 10.9 ± 0.2°, 23.8 ± 0.2°, 16.7 ± 0.2°; the X-ray powder diffraction pattern of the isethionate salt Form III includes diffraction peaks at 2-theta of 19.4 ± 0.2°, 16.9 ± 0.2°, 26.6 ± 0.2°; and can further include 4 diffraction peaks at 2-theta of 14.6 ± 0.2°, 28.0 ± 0.2°, 25.6 ± 0.2°, 20.7 ± 0.2°, 12.8 ± 0.2°; the X-ray powder diffraction pattern of the sulfate salt Form I includes diffraction peaks at 2-theta of 19.0 ± 0.2°, 19.4 ± 0.2°, 12.4 ± 0.2°; and can further include 4 diffraction peaks at 2-theta of 26.2 ± 0.2°, 17.6 ± 0.2°, 18.1 ± 0.2°, 25.3 ± 0.2°, 8.8 ± 0.2°; the X-ray powder diffraction pattern of the sulfate salt Form II includes diffraction peaks at 2-theta of 15.5 ± 0.2°, 11.1 ± 0.2°, 8.9 ± 0.2°; and can further include 4 diffraction peaks at 2-theta of 19.3 ± 0.2°, 22.3 ± 0.2°, 23.6 ± 0.2°, 17.4 ± 0.2°, 27.3 ± 0.2°; The X-ray powder diffraction pattern of the sulfate Form III comprises diffraction peaks at 2-theta = 19.6±0.2°, 18.0±0.2°, 18.4±0.2°; can further comprise 4 diffraction peaks at 2-theta = 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°; The X-ray powder diffraction pattern of the sulfate Form IV comprises diffraction peaks at 2-theta = 19.4±0.2°, 18.9±0.2°, 15.5±0.2°; can further comprise 4 diffraction peaks at 2-theta = 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°.

13. The acid salt of the compound according to claim 12, wherein: The X-ray powder diffraction pattern of the isethionate Form I optionally further comprises 8 peaks at 2-theta = 21.7±0.2°, 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.3±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 17.5±0.2°, 23.8±0.2°; The X-ray powder diffraction pattern of the isethionate Form II optionally further comprises 8 peaks at 2-theta = 10.0±0.2°, 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9+±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 10.0±0.2°; The X-ray powder diffraction pattern of the isethionate Form III optionally further comprises 8 peaks at 2-theta = 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°; The X-ray powder diffraction pattern of the sulfate Form I optionally further comprises 8 peaks at 2-theta = 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, 21.9±0.2°, 11.5±0.2°; The X-ray powder diffraction pattern of the sulfate Form II optionally further comprises 8 peaks at 2-theta = 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°; The X-ray powder diffraction pattern of the sulfate Form III optionally further comprises 8 of the following peaks at 2-theta of 19.6 ± 0.2°, 18.0 ± 0.2°, 18.4 ± 0.2°, 16.8 ± 0.2°, 14.3 ± 0.2°, 11.8 ± 0.2°, 14.9 ± 0.2°, 25.7 ± 0.2°, 15.4 ± 0.2°, 23.5 ± 0.2°; The X-ray powder diffraction pattern of the sulfate Form IV optionally further comprises 8 of the following peaks at 2-theta of 19.4 ± 0.2°, 18.9 ± 0.2°, 15.5 ± 0.2°, 8.8 ± 0.2°, 18.1 ± 0.2°, 24.9 ± 0.2°, 17.4 ± 0.2°, 12.3 ± 0.2°, 26.1 ± 0.2°, 14.5 ± 0.2°.

14. The acid salt of the compound according to claim 11, wherein: The X-ray powder diffraction pattern of the isethionate Form I comprises 10 of the following peaks at 2-theta of 21.7 ± 0.2°, 8.8 ± 0.2°, 10.2 ± 0.2°, 11.8 ± 0.2°, 13.1 ± 0.2°, 19.3 ± 0.2°, 27.6 ± 0.2°, 10.9 ± 0.2°, 13.3 ± 0.2°, 15.4 ± 0.2°, 16.7 ± 0.2°, 15.8 ± 0.2°, 17.5 ± 0.2°, 23.8 ± 0.2°, 14.7 ± 0.2°, 24.3 ± 0.2°, 27.3 ± 0.2°, 23.4 ± 0.2°, 20.6 ± 0.2°, 21.2 ± 0.2°; The X-ray powder diffraction pattern of the isethionate Form II comprises 10 of the following peaks at 2-theta of 21.7 ± 0.2°, 10.0 ± 0.2°, 8.8 ± 0.2°, 19.3 ± 0.2°, 27.6 ± 0.2°, 10.9 ± 0.2°, 23.8 ± 0.2°, 16.7 ± 0.2°, 15.4 ± 0.2°, 15.8 ± 0.2°, 17.5 ± 0.2°, 14.7 ± 0.2°, 24.4 ± 0.2°, 27.3 ± 0.2°, 29.2 ± 0.2°; The X-ray powder diffraction pattern of the isethionate Form III comprises 10 of the following peaks at 2-theta of 19.4 ± 0.2°, 16.9 ± 0.2°, 26.6 ± 0.2°, 14.6 ± 0.2°, 28.0 ± 0.2°, 25.6 ± 0.2°, 20.7 ± 0.2°, 12.8 ± 0.2°, 19.1 ± 0.2°, 27.2 ± 0.2°, 24.4 ± 0.2°, 15.3 ± 0.2°, 26.2 ± 0.2°, 30.2 ± 0.2°, 27.4 ± 0.2°; The X-ray powder diffraction pattern of the sulfate Form I comprises 10 diffraction peaks at 2-theta of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, 21.9±0.2°, 11.5±0.2°; The X-ray powder diffraction pattern of the sulfate Form II comprises 10 diffraction peaks at 2-theta of 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°, 15.8±0.2°, 24.2±0.2°, 21.8±0.2°, 10.3±0.2°, 20.6±0.2°; The X-ray powder diffraction pattern of the sulfate Form III comprises 10 diffraction peaks at 2-theta of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, 15.4±0.2°, 23.5±0.2°, 18.8±0.2°, 24.7±0.2°, 9.5±0.2°, 8.8±0.2°, 11.1±0.2°; The X-ray powder diffraction pattern of the sulfate Form IV comprises 10 diffraction peaks at 2-theta of 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, 26.1±0.2°, 14.5±0.2°, 22.2±0.2°, 24.3±0.2°, 21.7±0.2°, 23.6±0.2°.

15. The acid salt of the compound according to claim 11, wherein: The X-ray powder diffraction pattern of the isethionate Form I is shown in Figure 1; The X-ray powder diffraction pattern of the isethionate Form II is shown in Figure 4; The X-ray powder diffraction pattern of the isethionate Form III is shown in Figure 7; The X-ray powder diffraction pattern of the sulfate Form I is shown in Figure 10; The X-ray powder diffraction pattern of the sulfate Form II is shown in Figure 11; The X-ray powder diffraction pattern of the sulfate Form III is shown in Figure 12; The X-ray powder diffraction pattern of the sulfate Form IV is shown in Figure 13.

16. The acid salt of the compound according to claim 11, wherein: In the X-ray powder diffraction pattern of the isethionate Form I, the error of the relative peak intensity of the top ten peaks is ±0.2° to ±0.5° in the 2-theta of the corresponding diffraction peak in Figure 1; In the X-ray powder diffraction pattern of the isethionate Form I, the error of the relative peak intensity of the top ten peaks is ±0.2° to ±0.5° in the 2-theta of the corresponding diffraction peak in Figure 1; The X-ray powder diffraction pattern of the hydroxyethanesulfonate salt Form II has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° to ±0.5° from the corresponding position diffraction peak of Figure 4; The X-ray powder diffraction pattern of the hydroxyethanesulfonate salt Form III has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° to ±0.5° from the corresponding position diffraction peak of Figure 7; The X-ray powder diffraction pattern of the sulfate salt Form I has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° to ±0.5° from the corresponding position diffraction peak of Figure 10; The X-ray powder diffraction pattern of the sulfate salt Form II has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° to ±0.5° from the corresponding position diffraction peak of Figure 11; The X-ray powder diffraction pattern of the sulfate salt Form III has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° to ±0.5° from the corresponding position diffraction peak of Figure 12; The X-ray powder diffraction pattern of the sulfate salt Form IV has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° to ±0.5° from the corresponding position diffraction peak of Figure 13.

17. The acid salt of the compound according to claim 16, wherein The X-ray powder diffraction pattern of the said crystal form has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° to ±0.3° from the corresponding position diffraction peak of Figure 1.

18. The acid salt of the compound according to claim 16, wherein, The X-ray powder diffraction pattern of the said crystal form has relative peak intensity of the top ten strongest diffraction peak position with the 2θ error of ±0.2° from the corresponding position diffraction peak of Figure 1.

19. The acid salt of the compound according to claim 11, wherein, The hydroxyethanesulfonate salt Form I has a DSC pattern as shown in Figure 2; The hydroxyethanesulfonate salt Form II has a DSC pattern as shown in Figure 5; The hydroxyethanesulfonate salt Form III has a DSC pattern as shown in Figure 8.

20. The acid salt of the compound according to claim 11, wherein, The acid salt form is a hydrate or an anhydrate, when the acid salt form is a hydrate, the number of water is 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; further, the water in the hydrate is tap water or crystallization water or a combination of both.

21. The acid salt of the compound according to claim 20, wherein When the acid salt form is a hydrate, the number of water is 0.5, 1, 2 or 3.

22. A method for preparing the acid salt of the compound of any one of claims 1-21, comprising the steps of: 1) weighing an appropriate amount of free base, dissolving in a reaction solvent; 2) adding an appropriate amount of acid, stirring; the amount of acid is 1.2 equivalents; 3) obtaining the acid salt of the compound or its crystal form after centrifugal drying; the reaction solvent is selected from at least one of ethanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butyl alcohol, n-butyl alcohol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane; the acid is selected from the group consisting of hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid, or hydrobromic acid.

23. The preparation method according to claim 22, characterized in that, 24. A method of preparing an acid salt of a compound according to any one of claims 1 to 21 comprising the steps of: 1) weighing an appropriate amount of the free base and dissolving in a reaction solvent; 2) adding an appropriate amount of the acid and stirring to dissolve in an organic solvent; 3) optionally, adding seed crystals; 4) cooling, filtering the precipitated solid, washing with a solvent, and drying; the reaction solvent used in step 1) is selected from at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate, or 1,4-dioxane; the acid used in step 2) is selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethanesulfonic acid, dichloroacetic acid, trichloroacetic acid, acetyloxymethane acid, adipic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, formic acid, fumaric acid, galacturonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1,5-naphthalene disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, or L-malic acid. the acid is selected from the group consisting of hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid, or hydrobromic acid.

24. A method of preparing an acid salt of a compound according to any one of claims 1 to 21 comprising the steps of: 1) weighing an appropriate amount of the free base and dissolving in a reaction solvent; 2) adding an appropriate amount of the acid and stirring to dissolve in an organic solvent; 3) optionally, adding seed crystals; 4) cooling, filtering the precipitated solid, washing with a solvent, and drying; the reaction solvent used in step 1) is selected from at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate, or 1,4-dioxane; the acid used in step 2) is selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethanesulfonic acid, dichloroacetic acid, trichloroacetic acid, acetyloxymethane acid, adipic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, formic acid, fumaric acid, galacturonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1,5-naphthalene disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, or L-malic acid. the organic solvent in step 2) is selected from one or more of an alcohol, an ether, a ketone or an ester solvent; the solvent in step 3) is selected from one or more of an alcohol, an ether, a ketone or an ester solvent.

25. The method of claim 24, wherein, the acid in step 2) is selected from hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid or hydrobromic acid; the organic solvent in step 2) is selected from at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl-tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane; the solvent in step 3) is selected from at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl-tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane.

26. A pharmaceutical composition comprising a therapeutically effective amount of an acid salt of a compound according to any one of claims 1-21, and one or more pharmaceutically acceptable carriers, diluents or excipients.

27. Use of an acid salt of a compound according to any one of claims 1-21 or a pharmaceutical composition according to claim 26 for the manufacture of a KRAS inhibitor medicament.

28. The use according to claim 27, characterized in that, the KRAS inhibitor medicament is a KRAS G12C inhibitor medicament.

29. Use of an acid salt of a compound according to any one of claims 1-21 or a pharmaceutical composition according to claim 26 for the manufacture of a medicament for the treatment of Noonan Syndrome, Leopard Syndrome, leukemia, neuroblastoma, melanoma, esophageal cancer, head and neck tumors, breast cancer, lung cancer, pancreatic cancer and colon cancer diseases or conditions.

30. The use according to claim 29, characterized in that, the disease or condition is selected from non-small cell lung cancer, colon cancer, esophageal cancer, pancreatic cancer and head and neck tumors.

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