WO2017016463A1 - Egfr抑制剂及其药学上可接受的盐和多晶型物及其应用 - Google Patents

Egfr抑制剂及其药学上可接受的盐和多晶型物及其应用 Download PDF

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WO2017016463A1
WO2017016463A1 PCT/CN2016/091591 CN2016091591W WO2017016463A1 WO 2017016463 A1 WO2017016463 A1 WO 2017016463A1 CN 2016091591 W CN2016091591 W CN 2016091591W WO 2017016463 A1 WO2017016463 A1 WO 2017016463A1
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compound
formula
crystal
pharmaceutically acceptable
another preferred
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PCT/CN2016/091591
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French (fr)
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兰炯
江涛涛
李道飞
刘星
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上海海雁医药科技有限公司
扬子江药业集团有限公司
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Priority to CN201680027448.XA priority Critical patent/CN107531678B/zh
Priority to EP16829828.9A priority patent/EP3327014A4/en
Priority to US15/746,966 priority patent/US10561646B2/en
Publication of WO2017016463A1 publication Critical patent/WO2017016463A1/zh

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Definitions

  • the invention belongs to the technical field of medicine, in particular to an EGFR inhibitor and a pharmaceutically acceptable salt and polymorph thereof, and the use thereof, the inhibitor is N-(2-(4-(2) Methylamino)piperidin-1-yl)-5-(5-fluoro-4-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)-4-methoxybenzene Base) acrylamide.
  • Lung cancer is the world's highest incidence of cancer. It ranks first among all cancers in China. It is also the cancer with the highest morbidity and mortality in China.
  • EGFR mutations of which L858R and Exon 19 deletion mutations account for more than 90%, and such patients are more sensitive to EGFR inhibitors.
  • the first-generation EGFR inhibitors such as erlotinib have been marketed, and gefitinib is effective in such patients, which can reduce tumors in more than 60% of patients and significantly prolong the progression-free survival of patients.
  • the second-generation irreversible pan-EGFR inhibitor (Afatinib (BIBW2992)) currently on the market is significantly better than the first-generation EGFR inhibitor in patients with EGFR-mutant lung cancer.
  • the second-generation inhibitor also has a strong wild-type EGFR inhibitory activity, and the inhibitory activity against wild-type EGFR is significantly higher than that of the resistant T790M mutation.
  • the patient's rash and other toxic side effects are serious and the curative effect on drug-resistant patients is poor.
  • a small number of first-generation EGFR inhibitor-resistant patients respond to these drugs.
  • the present invention has developed various salt forms and crystal forms of inhibitors of EGFR mutants, which contribute to further drug development.
  • the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sulfate, phosphate, acetate, L-lactate, maleate, fumarate. , succinate, L-malate, adipate, L-tartrate, hippurate, citrate, mucate, glycolate, D-glucuronate, benzoate, Citrate, nicotinate, ethanedisulfonate, oxalate, methanesulfonate, besylate, 2-hydroxyethanesulfonate and hydrobromide.
  • the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, phosphate, maleate, L-malate, adipate, sulfate, fumaric acid Salt, succinate, L-tartrate, citrate, methanesulfonate, benzoate, and besylate.
  • the compound of formula X or a pharmaceutically acceptable salt thereof is in the form of an anhydrous form, a hydrated form or a solvate.
  • the solvate comprises a methanol solvate, an ethyl acetate solvate.
  • the compound of the formula X or a pharmaceutically acceptable salt thereof is a crystal.
  • the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, phosphate, maleate, L-malate, adipate, sulfate, fumarate , succinate, L-tartrate, citrate, methanesulfonate, benzoate, and besylate.
  • the pharmaceutically acceptable salt is a hydrochloride salt, wherein the molar ratio of hydrochloric acid to the compound of the formula X is (0.8 - 2.1): 1, preferably (0.9 - 1.1): 1.
  • the pharmaceutically acceptable salt is a phosphate wherein the molar ratio of phosphoric acid to the compound of formula X is 1: (0.9-2.1), preferably 1: (1.1-2.1).
  • the pharmaceutically acceptable salt is a maleate salt, wherein the molar ratio of maleic acid to the compound of formula X is (0.8-1.2): 1, preferably (0.9-1.1) :1, better 1:1.
  • the pharmaceutically acceptable salt is L-malate, wherein the molar ratio of L-malic acid to the compound of formula X is (0.5-1.4): 1, preferably (0.7- 1.3): 1.
  • the pharmaceutically acceptable salt is adipate, wherein the molar ratio of adipic acid to the compound of formula X is (0.8-1.2): 1, preferably (0.9-1.1) :1.
  • the pharmaceutically acceptable salt is a fumarate wherein the molar ratio of fumaric acid to the compound of formula X is (0.5-1.2):1, preferably (0.5-1). ):1.
  • the pharmaceutically acceptable salt is a citrate salt, wherein the molar ratio of citric acid to the compound of formula X The ratio is (0.5-1.2): 1, preferably (0.5-1): 1.
  • the pharmaceutically acceptable salt is a mucic acid salt, wherein the molar ratio of the mucic acid to the compound of the formula X is (0.5-1.2): 1, preferably (0.5-1): 1.
  • the pharmaceutically acceptable salt is a succinate salt, wherein the molar ratio of succinic acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is L-tartrate, wherein the molar ratio of L-tartaric acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is nicotinate, wherein the molar ratio of nicotinic acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is a horse urate wherein the molar ratio of hippuric acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is a glycolate wherein the molar ratio of glycolic acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is a benzoate wherein the molar ratio of benzoic acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is gentisate, wherein the molar ratio of gentisic acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is ethanedisulfonate, wherein the molar ratio of ethanedisulfonic acid to the compound of formula X is (0.9-1.1):1, preferably 1: 1.
  • the pharmaceutically acceptable salt is an oxalate wherein the molar ratio of oxalic acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is a methanesulfonate wherein the molar ratio of methanesulfonic acid to the compound of formula X is (0.9-1.1):1, preferably 1:1.
  • the pharmaceutically acceptable salt is a besylate salt, wherein the molar ratio of the benzenesulfonic acid to the compound of the formula X is (0.9-1.1): 1, preferably 1:1.
  • the pharmaceutically acceptable salt is 2-hydroxyethanesulfonate, wherein the molar ratio of 2-hydroxyethanesulfonic acid to the compound of formula X is (0.9-1.1):1, preferably. The ground is 1:1.
  • the pharmaceutically acceptable salt is D-glucuronide, wherein the molar ratio of D-glucuronic acid to the compound of formula X is (0.9-1.1): 1, preferably 1:1.
  • the crystal is a type A crystal of the hydrochloride salt of the compound of formula X, ie, crystal form A, and its X-ray powder diffraction pattern has a diffraction angle 2 ⁇ (°) value of the lower group A1: 8.47 ⁇ 0.10. , 20.32 ⁇ 0.10, 23.31 ⁇ 0.10 (highest peak) and 25.98 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form A further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group A2: 15.54 ⁇ 0.10, 16.97 ⁇ 0.10, 17.66. ⁇ 0.10, 17.90 ⁇ 0.10, 21.54 ⁇ 0.10, 22.19 ⁇ 0.10, 23.37 ⁇ 0.10, 24.81 ⁇ 0.10.
  • the X-ray powder diffraction pattern of the Form A further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group A3: 9.44 ⁇ 0.10, 11.78 ⁇ 0.10, 13.29. ⁇ 0.10, 14.91 ⁇ 0.10, 15.23 ⁇ 0.10, 18.20 ⁇ 0.10, 18.68 ⁇ 0.10, 20.05 ⁇ 0.10, 21.07 ⁇ 0.10, 23.87 ⁇ 0.10, 24.19 ⁇ 0.10, 25.30 ⁇ 0.10, 25.61 ⁇ 0.10, 26.45 ⁇ 0.10, 27.42 ⁇ 0.10 28.49 ⁇ 0.10, 29.96 ⁇ 0.10, 32.00 ⁇ 0.10, 34.01 ⁇ 0.10, 35.25 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form A comprises 6 or more or all selected from the group A1, A2, and A3 (eg, 6, 7, 8, 9, 10, 2 ⁇ (°) values of 11, 12, 13, 14, 15 etc.).
  • the X-ray powder diffraction pattern of Form A has the values shown in Table A2 below:
  • the X-ray powder diffraction pattern of Form A is substantially as characterized in Figure 1.
  • the molar ratio of hydrochloric acid to the compound of the formula X is (0.8 - 2.1): 1, preferably (0.9 - 1.1): 1, more preferably 0.9: 1.
  • the crystalline form A further has one or more characteristics selected from the group consisting of:
  • the initial temperature is 259 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially characterized by Figure 2C;
  • thermogravimetric analysis maps are substantially characterized as shown in Figure 2C;
  • the crystalline form A has a melting point of from 259 ° C to 267 ° C, preferably from 261 ° C to 265 ° C.
  • the crystalline form A is in the form of a hydrate.
  • the crystal is a B-type crystal of the compound phosphate of the formula X, that is, the crystal form B, and the X-ray powder diffraction pattern has a diffraction angle 2 ⁇ (°) value of the lower group B1: 11.94 ⁇ 0.10, 19.92 ⁇ 0.10 (highest peak), 22.27 ⁇ 0.10, 23.93 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form B further comprises a diffraction angle 2 ⁇ (°) value of the lower group B2: 7.97 ⁇ 0.10, 9.50 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form B further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group B3: 4.01 ⁇ 0.10, 10.71 ⁇ 0.10, 12.11. ⁇ 0.10, 12.56 ⁇ 0.10, 12.89 ⁇ 0.10, 13.94 ⁇ 0.10, 14.47 ⁇ 0.10, 15.16 ⁇ 0.10, 15.52 ⁇ 0.10, 16.54 ⁇ 0.10, 17.00 ⁇ 0.10, 17.42 ⁇ 0.10, 17.99 ⁇ 0.10, 18.38 ⁇ 0.10, 18.91 ⁇ 0.10 , 20.81 ⁇ 0.10, 20.96 ⁇ 0.10, 21.29 ⁇ 0.10, 21.65 ⁇ 0.10, 21.99 ⁇ 0.10, 22.58 ⁇ 0.10, 23.02 ⁇ 0.10, 23.45 ⁇ 0.10, 24.31 ⁇ 0.10, 24.53 ⁇ 0.10, 24.76 ⁇ 0.10, 25.01 ⁇ 0.10, 27.29 ⁇ 0.10, 28.98 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form B comprises 6 or more or all selected from Groups B1, B2, and B3 (eg, 6, 7, 8, 9, 10, 2 ⁇ (°) values of 11, 12, 13, 14, 15 etc.).
  • the X-ray powder diffraction pattern of Form B has the values shown in Table B2 below:
  • the X-ray powder diffraction pattern of Form B is substantially characterized as in Figure 3.
  • the molar ratio of phosphoric acid to the compound of the formula X is 1: (0.9 - 2.1), preferably 1: (1.1 - 2.1), more preferably 1: 1.1.
  • the crystalline form B has one or more characteristics selected from the group consisting of:
  • the initial temperature is 229.7 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially characterized by Figure 4C;
  • thermogravimetric analysis map is substantially characterized by Figure 4C;
  • the crystalline form B has a melting point of from 229 ° C to 239 ° C, preferably from 231 ° C to 237 ° C.
  • the crystalline form B is in the form of a hydrate.
  • the crystal is a C-1 type crystal of the compound of the formula X maleate, that is, the crystal form C-1, and the X-ray powder diffraction pattern thereof has the diffraction angle of the lower group C-1-1. 2 ⁇ (°) values: 8.73 ⁇ 0.10, 13.37 ⁇ 0.10, 18.08 ⁇ 0.10, and 25.55 ⁇ 0.10 (highest peak).
  • the X-ray powder diffraction pattern of the crystalline form C-1 further comprises two or more diffraction angles 2 ⁇ (°) selected from the group C-1-2: 9.10 ⁇ 0.10. , 14.14 ⁇ 0.10, 15.15 ⁇ 0.10, 17.20 ⁇ 0.10, 17.42 ⁇ 0.10, 18.61 ⁇ 0.10, 18.95 ⁇ 0.10, 19.15 ⁇ 0.10, 19.74 ⁇ 0.10, 20.59 ⁇ 0.10, 21.07 ⁇ 0.10, 21.49 ⁇ 0.10, 22.09 ⁇ 0.10, 22.58 ⁇ 0.10, 22.97 ⁇ 0.10, 23.32 ⁇ 0.10, 24.52 ⁇ 0.10, 24.86 ⁇ 0.10, 26.41 ⁇ 0.10, 26.77 ⁇ 0.10, 27.99 ⁇ 0.10, 28.80 ⁇ 0.10, 37.10 ⁇ 0.10.
  • the X-ray powder diffraction pattern of the crystalline form C-1 further comprises two or more diffraction angles 2 ⁇ (°) selected from the group C-1-3: 10.12 ⁇ 0.10. 11.56 ⁇ 0.10, 23.85 ⁇ 0.10, 27.21 ⁇ 0.10, 27.55 ⁇ 0.10, 27.85 ⁇ 0.10, 29.54 ⁇ 0.10, 30.27 ⁇ 0.10, 30.76 ⁇ 0.10, 32.93 ⁇ 0.10, 33.47 ⁇ 0.10, 34.20 ⁇ 0.10, 35.03 ⁇ 0.10, 35.34 ⁇ 0.10, 36.04 ⁇ 0.10, 36.47 ⁇ 0.10, 37.56 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form C-1 comprises 6 or more selected from the group consisting of C-1-1, C-1-2, and C-1-3.
  • the value of 2 ⁇ (°) of all or all eg 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 etc.).
  • the X-ray powder diffraction pattern of Form C-1 has the values shown in Table C-1-2 below:
  • the X-ray powder diffraction pattern of Form C-1 is substantially characterized as in Figure 5.
  • the molar ratio of maleic acid to the compound of formula X in the crystalline form C-1 is (0.8-1.2):1, preferably (0.9-1.1):1, more preferably 1:1.
  • the crystalline form C-1 further has one or more characteristics selected from the group consisting of:
  • the onset temperature is 211.6 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially as characterized in Figure 6A;
  • thermogravimetric analysis map is substantially characterized as shown in Figure 6A;
  • the crystalline form C-1 has a melting point of from 211 ° C to 218 ° C, preferably from 212 ° C to 216 ° C.
  • the crystalline form C-1 is in the anhydrous form.
  • the crystal is a C-2 type crystal of the compound of the formula X maleate, that is, the crystal form C-2, and the X-ray powder diffraction pattern thereof has the diffraction angle of the lower group C-2-1. 2 ⁇ (°) values: 7.48 ⁇ 0.10 (highest peak), 8.60 ⁇ 0.10, 20.63 ⁇ 0.10 and 23.27 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form C-2 further comprises a diffraction angle 2 ⁇ (°) value of the lower group C-2-2: 17.21 ⁇ 0.10, 18.71 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form C-2 further comprises two or more diffraction angles 2 ⁇ (°) selected from the group C-2-3: 3.77 ⁇ 0.10. 11.75 ⁇ 0.10, 12.97 ⁇ 0.10, 15.36 ⁇ 0.10, 15.70 ⁇ 0.10, 18.03 ⁇ 0.10, 18.36 ⁇ 0.10, 20.13 ⁇ 0.10, 25.03 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form C-2 comprises 6 or more selected from the group consisting of C-2-1, C-2-2, and C-2-3.
  • the value of 2 ⁇ (°) of all or all eg 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 etc.).
  • the X-ray powder diffraction pattern of Form C-2 has the values shown in Table C-2-2 below:
  • the X-ray powder diffraction pattern of Form C-2 is substantially characterized as in Figure 7.
  • the molar ratio of maleic acid to the compound of formula X in the crystalline form C-2 is (0.8-1.2):1, preferably (0.9-1.1):1, more preferably 1:1.
  • the crystalline form C-2 further has one or more characteristics selected from the group consisting of:
  • the initial temperature is 208.4 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially characterized as shown in FIG. 8;
  • the crystalline form C-2 has a melting point of from 208 ° C to 216 ° C, preferably from 210 ° C to 214 ° C.
  • the crystalline form C-2 is in the anhydrous form.
  • the crystal is a D-type crystal of the compound L-malate of the formula X, that is, the crystal form D, and the X-ray powder diffraction pattern has the diffraction angle 2 ⁇ (°) of the lower group D1: 7.47 ⁇ 0.10, 18.75 ⁇ 0.10, 22.69 ⁇ 0.10 (highest peak) and 24.39 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form D further comprises two or more diffraction angles 2 ⁇ (°) selected from the group D2: 17.79 ⁇ 0.10, 18.32 ⁇ 0.10, 20.81 ⁇ 0.10.
  • the X-ray powder diffraction pattern of the crystal form D further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group D3: 11.23 ⁇ 0.10, 11.68 ⁇ 0.10, 12.72. ⁇ 0.10, 13.58 ⁇ 0.10, 14.67 ⁇ 0.10, 15.24 ⁇ 0.10, 20.09 ⁇ 0.10, 20.58 ⁇ 0.10, 21.15 ⁇ 0.10, 21.88 ⁇ 0.10, 22.20 ⁇ 0.10, 23.34 ⁇ 0.10, 24.81 ⁇ 0.10, 25.60 ⁇ 0.10, 26.33 ⁇ 0.10 , 26.68 ⁇ 0.10, 32.07 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form D comprises 6 or more or all selected from Groups D1, D2, and D3 (eg, 6, 7, 8, 9, 10, 2 ⁇ (°) values of 11, 12, 13, 14, 15 etc.).
  • the X-ray powder diffraction pattern of Form D has the values shown in Table D2 below:
  • the X-ray powder diffraction pattern of Form D is substantially characterized as in Figure 9.
  • the molar ratio of L-malic acid to the compound of the formula X is (0.5-1.4): 1, preferably (0.7-1.3): 1, more preferably About 1.3:1.
  • the crystalline form D further has one or more characteristics selected from the group consisting of:
  • the onset temperature is 201.4 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially as characterized in Figure 10A;
  • thermogravimetric analysis map is substantially characterized as shown in Figure 10A;
  • the crystalline form D has a melting point of from 201 ° C to 209 ° C, preferably from 202 ° C to 207 ° C.
  • the crystalline form D is in the anhydrous form.
  • the crystal is an E-type crystal of the compound adipate of formula X, ie, Form E, and the X-ray powder diffraction pattern has a diffraction angle of the lower group E1. 2 ⁇ (°) value: 7.43 ⁇ 0.10, 18.45 ⁇ 0.10, 21.64 ⁇ 0.10 (highest peak) and 24.22 ⁇ 0.10.
  • the X-ray powder diffraction pattern of the Form E further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group E2: 15.66 ⁇ 0.10, 18.83 ⁇ 0.10, 21.16. ⁇ 0.10, 21.39 ⁇ 0.10, 24.38 ⁇ 0.10.
  • the X-ray powder diffraction pattern of the Form E further comprises two or more diffraction angles 2 ⁇ (°) selected from the group E3: 11.10 ⁇ 0.10, 11.43 ⁇ 0.10, 12.11. ⁇ 0.10, 13.78 ⁇ 0.10, 15.07 ⁇ 0.10, 15.25 ⁇ 0.10, 17.32 ⁇ 0.10, 20.07 ⁇ 0.10, 20.62 ⁇ 0.10, 20.96 ⁇ 0.10, 23.72 ⁇ 0.10, 25.41 ⁇ 0.10, 25.85 ⁇ 0.10, 26.42 ⁇ 0.10, 27.60 ⁇ 0.10 .
  • the X-ray powder diffraction pattern of Form E comprises 6 or more or all selected from the group E1, E2, and E3 (eg, 6, 7, 8, 9, 10, 2 ⁇ (°) values of 11, 12, 13, 14, 15 etc.).
  • the X-ray powder diffraction pattern of Form E has the values shown in Table E2 below:
  • the X-ray powder diffraction pattern of Form E is substantially characterized as in Figure 11.
  • the molar ratio of the adipic acid to the compound of the formula X is (0.8-1.2): 1, preferably. Ground (0.9-1.1): 1, more preferably about 1.1:1.
  • Form E further has one or more characteristics selected from the group consisting of:
  • the initial temperature is 203.2 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially as characterized in Figure 12A;
  • thermogravimetric analysis map is substantially characterized as shown in Figure 12A;
  • the crystalline form E has a melting point of from 203 ° C to 208 ° C, preferably from 204 ° C to 206 ° C.
  • the crystalline form E is in the anhydrous form.
  • the crystal is Form I of the compound of Formula X, and its X-ray powder diffraction pattern has a diffraction angle 2 ⁇ (°) of Group I-1: 8.74 ⁇ 0.10, 9.80 ⁇ 0.10, 15.63 ⁇ 0.10, and 21.38 ⁇ 0.10 (the highest peak).
  • the X-ray powder diffraction pattern of Form I further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group I-2: 15.45 ⁇ 0.10, 17.50 ⁇ 0.10. 17.81 ⁇ 0.10, 20.56 ⁇ 0.10, 20.89 ⁇ 0.10, 21.51 ⁇ 0.10, 21.91 ⁇ 0.10, 24.14 ⁇ 0.10, 25.99 ⁇ 0.10, 28.26 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form I further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group I-3: 10.68 ⁇ 0.10, 12.94 ⁇ 0.10. 14.47 ⁇ 0.10, 16.39 ⁇ 0.10, 16.76 ⁇ 0.10, 18.69 ⁇ 0.10, 19.59 ⁇ 0.10, 24.84 ⁇ 0.10, 27.41 ⁇ 0.10, 31.13 ⁇ 0.10, 31.89 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form I comprises 6 or more or all selected from Groups I-1, I-2, and I-3 (eg, 6, 7, 2 ⁇ (°) values of 8, 9, 10, 11, 12, 13, 14, 15 and so on.
  • the X-ray powder diffraction pattern of Form I has the values shown in Table I2 below:
  • the X-ray powder diffraction pattern of Form I is substantially characterized as in Figure 13.
  • Form I further has one or more characteristics selected from the group consisting of:
  • the initial temperature is 159.6 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially characterized as shown in FIG. 14;
  • thermogravimetric analysis maps are substantially characterized as shown in Figure 14;
  • the crystalline form I has a melting point of from 159 ° C to 165 ° C, preferably from 160 ° C to 163 ° C.
  • the crystalline form I is in anhydrous form.
  • the crystal is Form II of the compound of Formula X, and its X-ray powder diffraction pattern has a diffraction angle 2 ⁇ (°) of Group II-1: 9.47 ⁇ 0.10 (highest peak), 17.34 ⁇ 0.10, 18.87 ⁇ 0.10, and 23.89 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form II further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group II-2: 6.61 ⁇ 0.10, 12.06 ⁇ 0.10. 16.96 ⁇ 0.10, 19.19 ⁇ 0.10, 19.84 ⁇ 0.10, 20.66 ⁇ 0.10, 20.96 ⁇ 0.10, 24.22 ⁇ 0.10, 28.93 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form II comprises 6 or more or all selected from Groups II-1, II-2 (eg, 6, 7, 8, 9, 2 ⁇ (°) values of 10, 11, 12, 13, 14, 15, etc.).
  • the X-ray powder diffraction pattern of Form II has the values shown in Table II2 below:
  • the X-ray powder diffraction pattern of Form II is substantially characterized as in Figure 15.
  • Form II further has one or more characteristics selected from the group consisting of:
  • the initial temperature is 159.9 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially characterized as shown in FIG. 16;
  • the crystalline form II has a melting point of from 159 ° C to 164 ° C, preferably from 160 ° C to 163 ° C.
  • the Form II is in the form of a hydrate, preferably in the form of a dihydrate.
  • the crystal is a crystalline form III of the compound of the formula X
  • the X-ray powder diffraction pattern has a diffraction angle 2 ⁇ (°) of the group III-1: 9.72 ⁇ 0.10, 18.41 ⁇ 0.10, 23.89 ⁇ 0.10 (the highest peak) and 28.02 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form III further comprises two or more diffraction angles 2 ⁇ (°) selected from the group III-2: 11.78 ⁇ 0.10, 16.34 ⁇ 0.10. 16.57 ⁇ 0.10, 17.54 ⁇ 0.10, 18.19 ⁇ 0.10, 20.06 ⁇ 0.10, 21.33 ⁇ 0.10, 23.68 ⁇ 0.10, 25.05 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form III further comprises two or more diffraction angles 2 ⁇ (°) selected from the group III-3: 6.68 ⁇ 0.10, 8.25 ⁇ 0.10. , 13.19 ⁇ 0.10, 13.92 ⁇ 0.10, 15.54 ⁇ 0.10, 19.58 ⁇ 0.10, 21.66 ⁇ 0.10, 22.39 ⁇ 0.10, 22.96 ⁇ 0.10, 24.26 ⁇ 0.10, 26.34 ⁇ 0.10, 27.36 ⁇ 0.10, 29.06 ⁇ 0.10, 29.41 ⁇ 0.10, 31.00 ⁇ 0.10, 34.62 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form III comprises 6 or more or all selected from Groups III-1, III-2, and III-3 (eg, 6, 7, 2 ⁇ (°) values of 8, 9, 10, 11, 12, 13, 14, 15 and so on.
  • the X-ray powder diffraction pattern of Form III has the values shown in Table III2 below:
  • the X-ray powder diffraction pattern of Form III is substantially characterized as in Figure 17.
  • Form III further has one or more characteristics selected from the group consisting of:
  • the initial temperature is 159.9 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially as characterized in Figure 18A;
  • the crystalline form III of (iii) has a melting point of from 159 ° C to 164 ° C, preferably from 160 ° C to 162 ° C.
  • the crystalline form III is in the form of a solvate, preferably a methanol solvate.
  • the crystal is Form IV of the compound of Formula X, and its X-ray powder diffraction pattern has a diffraction angle 2 ⁇ (°) of Group IV-1: 7.69 ⁇ 0.10 (highest peak), 18.90 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form IV further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group IV-2: 5.38 ⁇ 0.10, 9.43 ⁇ 0.10. 13.31 ⁇ 0.10, 18.06 ⁇ 0.10, 23.19 ⁇ 0.10, 24.18 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form IV comprises 4 or more or all selected from Groups IV-1 and IV-2 (eg, 4, 5, 6, 7, 8) 2 ⁇ (°) values of 9, 10, 11, 12, 13, etc.).
  • the X-ray powder diffraction pattern of Form IV has the values shown in Table IV2 below:
  • the X-ray powder diffraction pattern of Form IV is substantially characterized as in Figure 19.
  • Form IV further has one or more characteristics selected from the group consisting of:
  • the initial temperature is 159.5 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially characterized as shown in FIG. 20;
  • thermogravimetric analysis map is substantially characterized as shown in FIG. 20;
  • the crystalline form IV has a melting point of from 159 ° C to 164 ° C, preferably from 160 ° C to 162 ° C.
  • the crystalline form IV is in the form of a hydrate.
  • the crystal is a crystalline form V of the compound of the formula X
  • the X-ray powder diffraction pattern has a diffraction angle 2 ⁇ (°) of the group V-1: 8.28 ⁇ 0.10, 8.89 ⁇ 0.10 (the highest peak) ), 9.44 ⁇ 0.10, 17.76 ⁇ 0.10.
  • the X-ray powder diffraction pattern of the Form V further comprises two or more diffraction angles 2 ⁇ (°) values selected from the group V-2: 14.01 ⁇ 0.10, 15.39 ⁇ 0.10. 16.55 ⁇ 0.10, 18.46 ⁇ 0.10, 18.91 ⁇ 0.10, 21.71 ⁇ 0.10, 22.91 ⁇ 0.10, 23.24 ⁇ 0.10, 24.87 ⁇ 0.10.
  • the X-ray powder diffraction pattern of Form V comprises 6 or more or all selected from Groups V-1 and V-2 (eg, 6, 7, 8, 9, 2 ⁇ (°) values of 10, 11, 12, 13, 14, 15, etc.).
  • the X-ray powder diffraction pattern of Form V has the values shown in Table V2 below:
  • the X-ray powder diffraction pattern of Form V is substantially as characterized in FIG.
  • the crystalline form V also has one or more characteristics selected from the group consisting of:
  • the initial temperature is 150.7 ⁇ 2 ° C; preferably, the differential scanning calorimetry analysis pattern is substantially as characterized in Figure 22A;
  • the crystalline form V has a melting point of from 149 ° C to 157 ° C, preferably from 151 ° C to 155 ° C.
  • the Form V is in the form of a solvate, preferably in the form of an ethyl acetate solvate.
  • the compound of the formula X formed by the step (1) or (2) or a pharmaceutically acceptable salt thereof is subjected to crystallization treatment to obtain a crystal.
  • the inert solvent is selected from the group consisting of 1,4-dioxane, dimethyl sulfoxide, and DMF.
  • the method further comprises the steps of:
  • the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, or a mixture thereof, preferably For ethanol.
  • the concentration of the compound of the formula X in the organic solvent in the step 1-1) is from 0.1 to 0.5 g/mL.
  • the method comprises any of the following sub-methods (A)-(E) and (I)-(V):
  • the crystal is a type A crystal of the hydrochloride salt of the compound of the formula X, that is, the crystal form A, and the step (3) comprises: crystallization treatment of the compound of the formula X in the presence of hydrochloric acid in an organic solvent, Thereby forming a crystal form A;
  • the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, or a mixture thereof.
  • the organic solvent is acetonitrile.
  • the concentration of hydrochloric acid in the organic solvent is 0.1 to 0.5 g/mL.
  • the molar ratio of hydrochloric acid to the compound of the formula X is (1 to 2): 1, preferably (1 to 1.2): 1.
  • the crystallization treatment is suspension stirring.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 72 hours, preferably from 10 to 50 hours.
  • the crystal is a type B crystal of the compound phosphate of the formula X, that is, the crystal form B, and in the step (3), the crystal of the compound of the formula X is crystallized in the presence of phosphoric acid in an organic solvent, thereby Forming a crystal form B;
  • the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, or a mixture thereof.
  • the organic solvent is acetonitrile.
  • the concentration of phosphoric acid in the organic solvent is 0.1 to 0.5 g/mL.
  • the molar ratio of phosphoric acid to the compound of the formula X is (1 to 2): 1, preferably (1 to 1.2): 1.
  • the crystallization treatment is suspension stirring.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 72 hours, preferably from 10 to 50 hours.
  • the crystal is a C-1 type crystal of the compound of the formula X maleate, ie, Form C-1, and in the step (3) includes: in an organic solvent, in maleic acid In the presence of the compound of formula X, it is subjected to a crystallization treatment to form crystal form C-1.
  • the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, or A mixture thereof, preferably, the organic solvent is acetonitrile.
  • the charge ratio (m:v) of maleic acid to the organic solvent is 5 to 12 mg/mL.
  • the molar ratio of maleic acid to the compound of the formula X is (1 to 2): 1, preferably (1 to 1.2): 1.
  • the crystallization treatment is suspension stirring.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 72 hours, preferably from 10 to 50 hours.
  • the crystal is a C-1 type crystal of the maleic acid salt of the compound of the formula X, that is, the crystal form C-1, and includes in the step (3):
  • the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, and propylene glycol, and Preferably, the organic solvent is methanol, ethanol, acetone, acetonitrile.
  • the molar ratio of maleic acid to the compound of the formula X is (1 to 3): 1, preferably (1 to 1.5): 1.
  • the concentration of the compound of the formula X in an organic solvent is 0.1 to 1 g/mL, preferably 0.2 to 0.6 g/mL.
  • the cooling time is from 1 to 10 hours.
  • the crystal is a D-form crystal of the compound L-malate of the formula X, ie, Form D, and includes in step (3): in the presence of L-malic acid in an organic solvent, the formula X The compound is subjected to a crystallization treatment to form a crystal form D;
  • the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, or a mixture thereof.
  • the organic solvent is acetonitrile.
  • the charge ratio (m:v) of the L-malic acid to the organic solvent is 5 to 12 mg/mL.
  • the molar ratio of the L-malic acid to the compound of the formula X is (1 to 2): 1, preferably (1 to 1.2): 1.
  • the crystallization treatment is suspension stirring.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 72 hours, preferably from 10 to 50 hours.
  • the crystal is an E-type crystal of the compound adipate of the formula X, ie, Form E, and in the step (3), the compound of the formula X is carried out in an organic solvent in the presence of adipic acid. Crystallizing treatment to form crystal form E;
  • the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, or a mixture thereof.
  • the organic solvent is acetonitrile.
  • the feed ratio (m:v) of the adipic acid to the organic solvent is 5 to 12 mg/mL.
  • the molar ratio of adipic acid to the compound of the formula X is (1 to 2): 1, preferably (1 to 1.2): 1.
  • the crystallization treatment is suspension stirring.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 72 hours, preferably from 10 to 50 hours.
  • step (3) comprises: crystallizing the compound of formula X in a solvent to form crystal form I;
  • the solvent is selected from the group consisting of water, methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, ethyl acetate, Methyl isobutyl ketone, isopropyl acetate, 2-methyltetrahydrofuran, dichloromethane, methyl tert-butyl ether, dimethyl sulfoxide, toluene, N,N-dimethylacetamide, N-A Pyrrolidone, or a mixture thereof.
  • the solvent is selected from the group consisting of methanol, isopropanol, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, 2-methyltetrahydrofuran, Acetonitrile, dichloromethane/methyl tert-butyl ether (V:V: 1:1 to 1:4), dimethyl sulfoxide/toluene (V:V: 1:1 to 1:4), N, N - dimethylacetamide / isopropyl acetate (V: V is 1:1 - 1: 4), N-methylpyrrolidone / methyl tert-butyl ether (V: V is 1:1 - 1: 4) , water / acetone.
  • the crystallization treatment is suspension stirring or slow evaporation.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 10 days, preferably from 4 to 8 days.
  • the crystal is a crystalline form II of the compound of the formula X, and in the step (3) comprises: crystallizing the compound of the formula X in a solvent to form a crystal form II;
  • the solvent is selected from the group consisting of water, methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, ethyl acetate, Methyl isobutyl ketone, isopropyl acetate, 2-methyltetrahydrofuran, dichloromethane, methyl tert-butyl ether, dimethyl sulfoxide, toluene, N,N-dimethylacetamide, N-A Pyrrolidone, or a mixture thereof.
  • the solvent is selected from the group consisting of methanol, isopropanol, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, 2-methyltetrahydrofuran, Acetonitrile, dichloromethane/methyl tert-butyl ether (V:V: 1:1 to 1:4), dimethyl sulfoxide/toluene (V:V: 1:1 to 1:4), N, N -Dimethylacetamide/isopropyl acetate (V:V: 1:1 to 1:4), N-methylpyrrolidone/ Methyl tert-butyl ether (V: V is 1:1 to 1:4), water/acetone.
  • the crystallization treatment method is a suspension stirring or an anti-solvent addition method.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 10 days, preferably from 4 to 8 days.
  • step (3) the crystal is a crystalline form III of the compound of formula X, and in step (3) comprises: crystallizing the compound of formula X in a solvent to form crystal form III;
  • the solvent is selected from the group consisting of water, methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, ethyl acetate, Methyl isobutyl ketone, isopropyl acetate, 2-methyltetrahydrofuran, dichloromethane, methyl tert-butyl ether, dimethyl sulfoxide, toluene, N,N-dimethylacetamide, N-A Pyrrolidone, or a mixture thereof.
  • the solvent is selected from the group consisting of methanol, isopropanol, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, 2-methyltetrahydrofuran, Acetonitrile, dichloromethane/methyl tert-butyl ether (V:V: 1:1 to 1:4), dimethyl sulfoxide/toluene (V:V: 1:1 to 1:4), N, N - dimethylacetamide / isopropyl acetate (V: V is 1:1 - 1: 4), N-methylpyrrolidone / methyl tert-butyl ether (V: V is 1:1 - 1: 4) , water / acetone.
  • the crystallization treatment is suspension stirring.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 5 days, preferably from 2 to 4 days.
  • step (3) comprises: crystallizing the compound of formula X in an organic solvent to form crystalline form IV;
  • the solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, ethyl acetate, 1, 4-dioxane, 2-methyltetrahydrofuran, or a mixture thereof.
  • the crystallization treatment is slow volatilization.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 10 days, preferably from 4 to 8 days.
  • step (3) comprises: crystallizing the compound of the formula X in an organic solvent to form a crystalline form V;
  • the solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, ethyl acetate, 1, 4-dioxane, 2-methyltetrahydrofuran, or a mixture thereof.
  • the crystallization treatment is a slow volatilization or an anti-solvent addition.
  • the crystallization treatment temperature is 5 to 30 ° C, preferably 10 to 20 ° C.
  • the crystallization treatment time is from 1 to 10 days, preferably from 4 to 6 days.
  • the crystal is a crystal of a compound of formula X, and
  • the solvent F is selected from the group consisting of water, methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, and tetra Hydrofuran, propylene glycol, ethyl acetate, methyl isobutyl ketone, isopropyl acetate, 2-methyltetrahydrofuran, dichloromethane, methyl tert-butyl ether, dimethyl sulfoxide, toluene, N, N- Dimethylacetamide, N-methylpyrrolidone, or a mixture thereof.
  • the solvent F is selected from the group consisting of methanol, isopropanol, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, 2-methyltetrahydrofuran, acetonitrile, dichloromethane.
  • V:V is 1:1 to 1:4
  • dimethyl sulfoxide/toluene V:V is 1:1 to 1:4
  • N,N-dimethyl Amide / isopropyl acetate V: V is 1:1 - 1: 4
  • N-methylpyrrolidone / methyl tert-butyl ether V: V is 1:1 - 1: 4
  • water / acetone
  • the solvent H is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol, ethyl acetate, 1,4-dioxane, 2-methyltetrahydrofuran. , or a mixture thereof.
  • the crystallization comprises suspension agitation, concentration crystallization, supersaturation crystallization, evaporation crystallization, volatilization, or a combination thereof.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition further comprises one or more additional drugs for treating and/or preventing a tumor.
  • the other drug is one or more selected from the group consisting of gefitinib, erlotinib, ectinib, lapatinib, XL647, NVP-DEE-788 , DRRY-334543, EKB-569, BIBW2992, HKI272, BMS-690514, CI-1033, vandetanib, PF00299804, WZ4002, cetuximab, trastuzumab, panitumumab, matto Chemuzumab, nimotuzumab, zarumimumab, pertuzumab, MDX-214, CDX-110, IMC-11F8, ZemDb, Her2 vaccine PX 1041, HSP90 inhibitor, CNF2024, Spirulina , aspiramycin, IPI-504, SNX-5422, NVP-DUY922, or a combination thereof.
  • a compound of formula X according to any one of the first aspects of the invention, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a pharmaceutical composition according to the third aspect, for the preparation of drugs that modulate EGFR tyrosine kinase or treat EGFR-related diseases.
  • the EGFR-related disease is selected from the group consisting of cancer, diabetes, immune system diseases, neurodegenerative diseases, cardiovascular diseases, diseases with acquired resistance during treatment with EGFR modulators.
  • the acquired resistance disease is caused by a T790 mutation encoded by EGFR exon 20 or by a T790 mutation comprising the EGFR exon 20 coding.
  • Figure 1 shows an XRPD pattern of Form A.
  • FIG. 2 shows an HPLC chart of Form A (FIG. 2A), an IC chart of Form A (FIG. 2B), and a TGA/DSC chart of Form A (FIG. 2C).
  • Figure 3 shows an XRPD pattern of Form B.
  • FIG. 4 shows an HPLC chart of Form B (FIG. 4A), an IC chart of Form B (FIG. 4B), and a TGA/DSC chart of Form B (FIG. 4C).
  • Figure 5 shows an XRPD pattern of Form C-1.
  • Figure 6 shows the TGA/DSC pattern (Figure 6A) of Form C-1 and the 1 H NMR spectrum of Form C-1 ( Figure 6B).
  • Figure 7 shows an XRPD pattern of Form C-2.
  • Figure 8 shows a TGA/DSC chart of Form C-2.
  • Figure 9 shows an XRPD pattern of Form D.
  • Figure 10 shows the TGA/DSC pattern of Form D ( Figure 10A) and the 1 H NMR spectrum of Form D ( Figure 10B).
  • Figure 11 shows an XRPD pattern of Form E.
  • Figure 12 shows the TGA/DSC pattern (Figure 12A) of Form E and the 1 H NMR spectrum of Form E ( Figure 12B).
  • Figure 13 shows an XRPD pattern of Form I.
  • Figure 14 shows a TGA/DSC pattern of Form I.
  • Figure 15 shows an XRPD pattern of Form II.
  • Figure 16 shows a TGA/DSC pattern of Form II.
  • Figure 17 shows an XRPD pattern of Form III.
  • Figure 18 shows the TGA/DSC pattern of Form III ( Figure 18A) and the 1 H NMR spectrum of Form III ( Figure 18B).
  • Figure 19 shows an XRPD pattern of Form IV.
  • Figure 20 shows a TGA/DSC chart of Form IV.
  • Figure 21 shows an XRPD pattern of Form V.
  • Figure 22 shows a TGA/DSC pattern of Form V (Figure 22A) and a 1 H NMR spectrum of Form V ( Figure 22B).
  • Figure 23 shows a comparison of XRPD of Form C-1 before and after standing at 80 ° C for 24 hours.
  • Figure 24 shows a HPLC comparison of Form C-1 before and after standing at 80 ° C for 24 hours.
  • Figure 25 shows the DVS pattern of Form B.
  • Figure 26 shows a comparison of XRPD of Form B before and after DVS testing.
  • Figure 27 shows the DVS pattern of Form C-1.
  • Figure 28 shows the DVS pattern of Form E.
  • Figure 29 shows the DVS pattern of Form II.
  • Figure 30 shows an XRPD overlay of crystalline C-1 and Form C-2 suspension competition.
  • Figure 31 shows the DVS pattern of Form I.
  • Figure 32 shows a comparison of XRPD before and after the Form I DVS test.
  • Figure 33 shows a comparison of XRPD before and after the Form II DVS test.
  • Figure 34 shows a comparison of XRPD before and after the Crystalline I and II suspension competition tests.
  • Figure 35 shows a comparison of XRPD before and after the Crystalline II and IV suspension competition tests.
  • the inventors After long-term and intensive research, the inventors have discovered a series of polymorphs of the free base polymorphs, salts and salts of the compound of formula X.
  • In vitro enzyme and cell growth inhibition experiments showed that the free base of the compound of formula X showed strong inhibitory activity against EGFR T790M mutant enzyme and cells, but showed weak inhibitory activity against EGFR wild-type enzyme and cells, and showed a better inhibition. Low cytotoxicity, therefore the compound of formula X can be used as an ideal third-generation EGFR mutant selective inhibitor.
  • compound of the invention includes a compound of formula X of the invention, a pharmaceutically acceptable salt of a compound of formula X of the invention, and a polymorph of the invention.
  • a compound of the formula X or "a compound of the formula X” may be used interchangeably, and unless otherwise specified, generally means a free base form.
  • the compound of the formula X is N-(2-(4-(dimethylamino)piperidin-1-yl)-5-(5-fluoro-4-(1-methyl-1H-pyrazole) 4-yl)pyrimidin-2-ylamino)-4-methoxyphenyl)acrylamide, which has better selective inhibitory activity and lower cytotoxicity against T790M mutant EGFR, as well as favorable metabolic characteristics.
  • free base sample or “free base” means the free base of the compound of formula X prepared in Example 1.
  • the pharmaceutically acceptable salt is preferably selected from the group consisting of hydrochloride, sulfate, phosphate, acetate, L-lactate, maleate, fumarate, amber.
  • Acid salt L-malate, adipate, L-tartrate, hippurate, citrate, mucate, glycolate, glucuronate, benzoate, gentisate Nicotinate, ethanedisulfonate, oxalate, methanesulfonate, besylate, hydroxyethanesulfonate and hydrobromide.
  • the solid does not exist in an amorphous form or in a crystalline form.
  • the molecules are positioned within the three-dimensional lattice lattice.
  • polymorphism When a compound crystallizes out of a solution or slurry, it can crystallize in different spatial lattices (this property is called "polymorphism"), forming crystals with different crystalline forms, and these various crystalline forms are It is called "polymorph”.
  • Different polymorphs of a given substance may differ from one another in one or more physical properties such as solubility and dissolution rate, true specific gravity, crystalline form, bulk mode, flowability, and/or solid state stability.
  • the solubility limit of the compound of interest can be exceeded by operating the solution to complete production-scale crystallization. This can be done in a number of ways, for example by dissolving the compound at relatively high temperatures and then cooling the solution below the saturation limit. Alternatively, the volume of liquid can be reduced by boiling, atmospheric evaporation, vacuum drying, or by other methods. The solubility of the compound of interest can be lowered by adding an antisolvent or a solvent having a low solubility in the compound or a mixture of such a solvent. Another alternative is to adjust the pH to reduce solubility. For a detailed description of crystallization, see Crystallization, Third Edition, J W Mullens, Butterworth-Heineman Ltd., 1993, ISBN 0750611294.
  • the term "suspended agitation" means that a solution of a compound of formula X and the corresponding acid or corresponding acid is combined in a suitable solvent to form a turbid liquid, or a compound of formula X is mixed with a suitable solvent to form a turbid liquid, followed by stirring.
  • a suitable solvent can be water or an organic solvent.
  • slowly volatile refers to a process in which a solution of a compound of formula X or a solution of a compound of formula X and the corresponding acid is slowly volatilized at a temperature to yield a crystal.
  • anti-solvent addition refers to a process in which a solution of a compound of formula X is added to another suitable solvent to precipitate crystals.
  • salt formation is desired to occur simultaneously with crystallization, if the salt is less soluble than the starting material in the reaction medium, the addition of a suitable acid or base can result in direct crystallization of the desired salt. Similarly, in the final desired form of the medium having less solubility than the reactants, the completion of the synthesis reaction allows the final product to crystallize directly.
  • optimization of crystallization can include seeding the crystal in a desired form with the crystal as a seed.
  • many crystallization methods use a combination of the above strategies.
  • One embodiment is to dissolve the compound of interest in a solvent at elevated temperatures, followed by controlled addition of an appropriate volume of anti-solvent to bring the system just below the level of saturation. At this point, seed crystals of the desired form can be added (and the integrity of the seed crystals maintained) and the system cooled to complete crystallization.
  • room temperature generally refers to 4-30 ° C, preferably 20 ⁇ 5 ° C.
  • crystal of the invention As used herein, “crystal of the invention”, “crystal form of the invention”, “polymorph of the invention” and the like are used interchangeably, including polymorphs of the compounds of formula X of the invention and the invention A polymorph of a pharmaceutically acceptable salt of a compound of formula X.
  • polymorph of the compound of the formula X and “polymorph of the compound of the formula X” are used interchangeably.
  • polymorph of the invention includes a free base of a compound of formula X or a pharmaceutically acceptable salt of a compound of formula X (eg, hydrochloride, phosphate, maleate, L-malate, Adipate, sulfate, fumarate, succinate, L-tartrate, citrate, methanesulfonate, benzoate, and besylate), or various solvents of the compound of formula X Polymorphs of the complex, including the same salts (eg, hydrochloride, phosphate, maleate, L-malate, adipate, sulfate, fumarate, succinate, Different polymorphs of L-tartrate, citrate, methanesulfonate, benzoate, and besylate) or solvates.
  • a pharmaceutically acceptable salt of a compound of formula X eg, hydrochloride, phosphate, maleate, L-malate, Adipate, sulfate, fumarate, succinate, L
  • Preferred polymorphs of the invention include, but are not limited to:
  • the polymorph of the compound of formula X or a pharmaceutically acceptable salt can be characterized by known methods or apparatus, for example, in a variety of ways and apparatus as follows.
  • Methods for determining X-ray powder diffraction of crystalline forms are known in the art.
  • an X-ray powder diffractometer is used to acquire a spectrum using a copper radiation target at a scanning speed of 2° per minute.
  • the polymorph of the compound of the formula X of the present invention or a pharmaceutically acceptable salt thereof has a specific crystal form and has a specific characteristic peak in an X-ray powder diffraction (XRPD) pattern.
  • XRPD X-ray powder diffraction
  • DSC differential calorimetric scanning analysis
  • a compound of formula X of the present invention can be administered in a suitable dosage form with one or more pharmaceutically acceptable carriers.
  • These dosage forms are suitable for oral, rectal, topical, intraoral, and other parenteral administration (eg, subcutaneous, intramuscular, intravenous, etc.).
  • dosage forms suitable for oral administration include capsules, tablets, granules, and syrups and the like.
  • the compound of the present invention contained in these preparations may be a solid powder or granule; a solution or suspension in an aqueous or non-aqueous liquid; a water-in-oil or oil-in-water emulsion or the like.
  • the above dosage forms can be prepared from the active compound with one or more carriers or excipients via conventional pharmaceutical methods.
  • the above carriers need to be compatible with the active compound or other excipients.
  • commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like.
  • Carriers for liquid preparations include water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like.
  • the active compound can form a solution or suspension with the above carriers.
  • compositions of the present invention are formulated, quantified, and administered in a manner consistent with medical practice.
  • the "effective amount" of a compound administered is determined by the particular condition being treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • the present invention provides the use of the compound of the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, for the preparation of a medicament for regulating EGFR tyrosine kinase activity or treating an EGFR-related disease.
  • the EGFR-related disease is cancer, diabetes, immune system disease, neurodegenerative disease or heart Vascular disease.
  • the cancer is non-small cell lung cancer, head and neck cancer, breast cancer, kidney cancer, pancreatic cancer, cervical cancer, esophageal cancer, pancreatic cancer, prostate cancer, bladder cancer, colorectal cancer, ovarian cancer, stomach cancer, brain.
  • Malignant tumors include glioblastoma and the like, or any combination thereof.
  • the non-small cell lung cancer is caused by a mutation in EGFR, including a sensitive mutation (such as a L858R mutation or exon 19 deletion) and a drug resistance mutation (such as an EGFR T790M mutation).
  • a sensitive mutation such as a L858R mutation or exon 19 deletion
  • a drug resistance mutation such as an EGFR T790M mutation
  • the compound, or a pharmaceutically acceptable salt thereof, or a prodrug thereof can also be used in the manufacture of a medicament for the treatment of a disease in which EGFR is abnormally expressed or a disease having acquired resistance during treatment with an EGFR modulator.
  • the acquired resistance is caused by a T790 mutation encoded by EGFR exon 20 or by a T790 mutation comprising a EGFR exon 20 encoding, such as T790M.
  • an EGFR modulator refers to a small molecule tyrosine kinase inhibitor that targets EGFR, such as gefitinib, erlotinib, ectinib, lapatinib or afatinib.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, and a selected from the group consisting of One or more other drugs: gefitinib, erlotinib, ectinib, lapatinib, XL647, NVP-AEE-788, ARRY-334543, EKB-569, BIBW2992, HKI272, BMS- 690514, CI-1033, vandetanib, PF00299804, WZ4002, cetuximab, trastuzumab, panituzumab, matuzumab, nimotuzumab, zarulimumab , Pertuzumab, MDX-214, CDX-110, IMC-11F8, Zemab, Her2 vaccine PX 1041, HSP90 inhibitor, CNF2024, Tan spir
  • terapéuticaally effective amount refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • the therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, contained in the pharmaceutical composition of the present invention or the pharmaceutical composition is preferably 0.1 mg to 5 g. /kg (weight).
  • the pharmaceutical composition of the present invention can be used for the treatment of EGFR-related diseases such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, or diseases having acquired resistance during treatment with EGFR modulators.
  • EGFR-related diseases such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, or diseases having acquired resistance during treatment with EGFR modulators.
  • the acquired drug-resistant disease is caused by a T790 mutation encoded by EGFR exon 20 or by a T790 mutation encoded by EGFR exon 20.
  • the EGFR exon 20 encodes a T790 of T790M.
  • the compounds of the present invention or pharmaceutically acceptable salts thereof can be used in combination with other drugs in certain diseases to achieve the desired therapeutic effect.
  • An example of a joint application is to treat advanced NSCLC.
  • the polymorph of the compound of formula X or a pharmaceutically acceptable salt thereof has excellent physicochemical stability and outstanding related pharmacological activity and is an ideal third-generation EGFR mutant selective inhibitor.
  • LC-MS liquid chromatography-mass spectrometry
  • ISCO Combiflash-Rf75 or Rf200 automatic column analyzer Use ISCO Combiflash-Rf75 or Rf200 automatic column analyzer, Agela 4g, 12g, 20g, 40g, 80g, 120g disposable silica gel column.
  • Known starting materials can be synthesized by or according to methods known in the art, or can be used by companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc and Dary Chemicals. Buy at.
  • the reactions were all carried out under a nitrogen or argon atmosphere.
  • the solution means an aqueous solution.
  • DMF means dimethylformamide
  • DMSO means dimethyl sulfoxide
  • THF means tetrahydrofuran
  • DIEA means N,N-diisopropylethylamine
  • EA means ethyl acetate
  • PE means petroleum ether.
  • BINAP stands for (2R,3S)-2,2'-bisdiphenylphosphino-1,1'-binaphthyl
  • NBS stands for N-bromosuccinimide
  • NCS stands for N-chlorosuccinimide
  • Pd 2 (dba) 3 represents tris(dibenzylideneacetone)dipalladium
  • Pd(dppf)Cl 2 represents [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.
  • room temperature refers to about 25 °C.
  • the powder X-ray diffraction pattern was obtained by a method known in the art using a PANalytacal Empyrean X-ray powder diffraction analyzer.
  • the instrument test conditions are as shown in Table i below:
  • each crystal form has a diffraction peak having the highest peak height as a base peak, and its relative intensity is defined as 100%, and as a peak of I 0 (for example, a peak of the crystal form I having a 2 ⁇ (°) value of 21.38 is a base peak.
  • the peak of the 2 ⁇ (°) value of the crystal form II is 9.47
  • the peak of the crystal form III having a 2 ⁇ (°) value of 23.89 is the base peak
  • the peak of the crystal form IV having the 2 ⁇ (°) value of 7.69 is the base.
  • the peak of the crystal form V has a 2 ⁇ (°) value of 8.89 as a base peak
  • the peak of the crystal form A having a 2 ⁇ (°) value of 23.31 is a base peak
  • the peak of the crystal form B having a 2 ⁇ (°) value of 19.92 is The base peak
  • the peak of the crystal form C-1 having a 2 ⁇ (°) value of 25.55 is the base peak
  • the peak of the crystal form C-2 having a 2 ⁇ (°) value of 7.48 is the base peak
  • the 2 ⁇ (°) value of the crystal form D is
  • the peak of 22.69 is the base peak
  • the peak of the crystal form E has a 2 ⁇ (°) value of 21.64 as the base peak
  • the other peaks have the ratio of the peak height to the peak height of the base peak as the relative intensity I/I 0 .
  • the division of peak relative intensity is defined as shown in Table ii below:
  • the salt of the present invention or its crystal form determines the acid-base molar ratio by HPLC/IC or 1 H NMR.
  • HPLC High performance liquid chromatography
  • TGA and DSC spectra were acquired on a TA Q500/5000 thermogravimetric analyzer and a TA Q200/2000 differential scanning calorimeter, respectively.
  • the instrument test conditions are shown in Table iii below:
  • Dynamic moisture adsorption (DVS) curve acquired on DVS Intrinsic of SMS (Surface Measurement Systems). The relative humidity at 25 ° C was corrected with the deliquescent point of LiCl, Mg(NO 3 ) 2 and KCl. The instrument test conditions are shown in the following table iv:
  • Water content Tested using a Metrohm 870 Karl Fischer moisture analyzer, using a titration test solution for commercial Sigma-aldrich R-Composite 5 (34805-1L-R, Batch #SZBD3330V) using analytically pure MeOH as solvent. Calibration was performed with high purity water before moisture measurement.
  • Step a Compound 1a1 (10.6 g, 58 mmol) was placed in a 500 mL reaction flask, and dissolved in a mixed solution of THF/water (100 mL / 60 mL). Ammonium chloride (15.5 g, 292 mmol) and reduced iron powder (26 g, 467 mmol) were successively added thereto with stirring at room temperature, and then the reaction system was heated to 65 ° C and stirring was continued for 3 h. The progress of the reaction was checked by TLC. After the reaction was completed, excess iron powder was removed by filtration, and the filter cake was rinsed three times with EA.
  • Step b Compound 1a2 (8.0 g, 43 mmol) was placed in a 500 mL reaction flask, and concentrated sulfuric acid (100 mL) was added to dissolve the substrate. Concentrated nitric acid (6.15 mL, 48 mmol) was slowly added dropwise with stirring at minus 20 ° C, and stirred for 5 min. The progress of the reaction was detected by TLC, and after the reaction was completed, it was poured into ice water. The ice bath was kept at minus 20 ° C, and a sodium hydroxide/water solution (150 mL / 300 mL) was slowly added to the reaction system to adjust the pH to 8-9. The reaction mixture was extracted with EA/water system three times.
  • Step 1 Compound 2a1 (500 g, 2.51 mol) and dimethylamine hydrochloride (244 g, 3.01 mol) were added to methanol (2.2 L), triethylamine (508 g, 5.03 mol) and 10% palladium carbon ( 50 g), and then the mixture was reacted with hydrogen (15 kg, 40 ° C) for 24 hours, and the reaction of the crude material was confirmed by LCMS, filtered, and the filtrate was evaporated to dryness to give the crude compound 2a2 (542 g). MS m/z (ESI): 228 [M-H].
  • Step 2 Compound 2a2 (542 g, 2.38 mol) was dissolved in methanol (1.5 L), and concentrated hydrochloric acid (1 L) was added slowly and portionwise. After the addition was completed, the mixture was reacted at 50 ° C for 2 hours, and evaporated to dryness under reduced pressure. The crude solid was taken from EtOAc (EtOAc) (EtOAc) 1 H NMR (400 MHz, DMSO) ⁇ 3.47 (m, 3H), 2.97 (m, 2H), 2.78 (s, 6H), 2.22 (m, 2H), 1.87 (m, 2H).
  • Step 2 Compound 3a2 (11.46 g, 40 mmol) was dissolved in 60 mL of N,N-dimethylacetamide, and compound 2a (9.6 g, 48 mmol), N,N-diisopropylethylamine (7.74 g, 60 mmol), heated to 90 ° C and stirred for 6 h. TLC was used to detect the progress of the reaction. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into ice water, extracted with ethyl acetate, washed with brine, dried over anhydrous Na 2 SO 4 13.43g, 85%). Used directly in the next step. MS m/z (ESI): 495 [M+H] + .
  • Step 3 Compound 3a3 (12.8 g, 32.6 mmol) was dissolved in 200 mL of methanol, and 1.0 g of 10% Pd/C was added. After replacing the air with hydrogen, the hydrogen gas was hydrogenated at room temperature, and the reaction was stirred for 1 h. TLC was used to detect the progress of the reaction. After the reaction was completed, the filter cake was suction filtered through a celite funnel, and the filtrate was washed with a small amount of methanol, and the filtrate was concentrated to give the title compound 3a4 (11.54 g, 97%). Used directly in the next step. MS m/z (ESI): 355 [M+H] + .
  • Step 4 Compound 3a4 (10.95 g, 30 mmol) and triethylamine (6.12 g, 60 mmol) were dissolved in dichloromethane (200 mL), cooled to 0 ° C, acryloyl chloride (3.24 g, 36 mmol). Stir for 3 h. TLC was used to detect the progress of the reaction. After the reaction was completed, the mixture was washed successively with saturated aqueous sodium hydrogen carbonate and brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated to give compound 3a5 (10.3 g, 82%). Used directly in the next step. MS m/z (ESI): 495 [M+H] + .
  • Step 1 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole under a nitrogen atmosphere (1.1 g, 5.25 mmol), a mixture of compound I-1 (664 mg, 4 mmol), Pd(dppf)Cl 2 (109.7 mg, 0.15 mmol) and sodium carbonate solution (5 mL, 2M) in acetonitrile (30 mL) Stir at °C for 6 h.
  • Step 2 To a compound 4a (159 mg, 0.5 mmol), compound I-2 (106 mg, 0.5 mmol), Pd 2 (dba) 3 (45.75 mg, 0.05 mmol) and Xantphos (28.4 mg, 0.05 mmol) of 1,4 To a solution of dioxane (4 ml), cesium carbonate (487.5 mg, 0.5 mmol) was added. After the reaction mixture was stirred at 100 ° C for 3 h, EA (50 ml ⁇ 2) and water (10 ml) were added to the reaction mixture. After washing with water (10 ml ⁇ 3), it was dried over Na 2 SO 4 and concentrated to give a crude product.
  • cesium carbonate 487.5 mg, 0.5 mmol
  • Comparative Compounds 2 and 3 were prepared using the corresponding boron ester and 5,methoxy substituted or unsubstituted 2,4-dichloropyrimidine, respectively, in a similar manner to the compound of formula X, as shown in Table v.
  • the starting sample of the free base of the compound of formula X (5.50 g, 11.1 mmol) was dissolved in ethanol (16.5 mL), and the temperature was raised to 70 ° C, and maleic acid (1.55 g, 13.3 mmol) was added with stirring, and the temperature of the reaction liquid was controlled at 70.
  • Stir at °C for 30min precipitate a large amount of solids, stop heating, cool down to 5 °C with stirring, cool down for 2 hours, stir for 1 hour, filter, filter cake with ethanol, blast dry at 50 °C for 2h, 80 After drying at 0.45 C for 6 h, 6.05 g of a yellow crystalline powder was obtained, yield: 89.1%.
  • the X-ray diffraction pattern of the obtained crystal powder is shown in Fig. 5, and the TGA/DSC spectrum is characterized as shown in Fig. 6A; the 1 H NMR spectrum is shown in Fig. 6B, the acid-base molar ratio is 1:1, and the melting point is 212 ° C - 216 ° C .
  • This crystal form is defined in this application as Form C-1.
  • the TGA/DSC pattern of Form II is shown in Figure 16.
  • the TGA chart shows that the sample loses about 7.2% after heating to about 80 °C.
  • the DSC chart shows a comparison before melting at 158 °C - 162 °C (starting temperature). Wide endothermic peak (peak temperature range 98 °C-102 °C), Karl Fischer test (moisture test) results show that Form II contains 7.2% moisture, and the sample TGA characterizes the knot
  • the weight loss before decomposition in Fig. 16 is similar.
  • the crystal form II was heated to 140 ° C and then cooled to room temperature.
  • the XRPD pattern showed that the crystal form II was cooled and cooled to form an amorphous form I.
  • Form II is present in the form of a hydrate, more preferably in the form of a dihydrate. (The theoretical weight loss of the free base dihydrate is 6.8%).
  • the TGA/DSC pattern of Form III is shown in Figure 18A.
  • the TGA plot shows that the sample loses about 6.4% before heating to about 130 °C.
  • the DSC plot shows how much before 158 °C - 162 °C (starting temperature) melts.
  • An endothermic peak, after heating Form III to 130 ° C and then cooling to room temperature, the XRPD pattern shows that Form III is converted to an amorphous form I.
  • the 1 H NMR result of Form III (Fig. 18B) showed a methanol content of 4.9%. Thus Form III is present as a methanolate.
  • the TGA/DSC pattern of Form IV is shown in Figure 20.
  • the TGA plot shows that the sample loses about 5.8% before heating to about 150 ° C.
  • the DSC plot shows a 158 ° C -162 ° C (starting temperature) before melting.
  • Endothermic peak peak temperature range is 105 ° C - 109 °C
  • the XRPD pattern shows that Form IV is heated to 130 ° C and then cooled to room temperature and then converted to Form I, which is also present as a hydrate.
  • the TGA/DSC pattern of Form V is shown in Figure 22A, which shows that the sample has a weight loss of about 0.7% between heating to about 130 ° C to 160 ° C, and the sample is detected by 1 H NMR ( Figure 22B).
  • the EtOAc content is consistent.
  • the DSC chart shows that there is no significant endothermic or exothermic peak before melting at 149 ° C - 153 ° C (starting temperature), and the XRPD pattern shows that Form V can be converted into a crystalline form after stirring in isopropanol at room temperature for about 24 hours.
  • I 1 H NMR results of Form V ( Figure 22B) showed that Form V was present as ethyl acetate solvate.
  • a corresponding solid acid having a molar ratio of 1:1 to 10 mg of free base was weighed into a reaction flask, and a solution of 0.2 mol/L of a corresponding solvent of hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid was weighed. Then, 10 mg/mL of the clear liquid of the compound X compound free base in four corresponding solvents (isopropyl alcohol, acetonitrile, ethyl acetate, acetone: water (19:1, v/v)) was separately prepared and separately weighed. 1.0 mL of the clear liquid was placed in a reaction flask containing the corresponding acid, and magnetically stirred at a certain temperature at a speed of about 1000 rpm.
  • a solution B of a corresponding acid (the molar ratio of the acid to the compound of the formula X is 1.05:1) is added, and after reacting for a certain period of time at a certain reaction temperature, the reaction solution is filtered and washed with a solvent. The filter cake was dried under vacuum to obtain the corresponding salt.
  • Table 2 The original feed and reaction conditions of the reaction are shown in Table 2 below.
  • Form C-1 was allowed to stand at 80 ° C for 24 hours and samples were collected for XRPD and HPLC characterization. According to Fig. 23 and Fig. 24, the crystal form and the HPLC purity of the crystal form C-1 were not changed before and after the test, indicating that the crystal form C-1 had good physical and chemical stability.
  • the dynamic solubility of the salt form of the compound of formula X and the starting sample of the free base in water and three biosolvents: simulated gastric juice (SGF), simulated fasting intestinal fluid (FaSSIF), simulated feeding state intestinal fluid (FeSSIF) were performed at room temperature. test. In the test, 20 mg of solid and 2 ml of the solvent were mixed in a 4 ml centrifuge tube, and the tube was sealed and fixed on a rotating plate rotating at 25 rpm.
  • N/A pH data was not collected due to less supernatant remaining after separation of solids.
  • Form B, Form C-1, Form D and Form E have good solubility in three biosolvents and water, and the solubility of the salt form in water and FaSSIF compared to the free base starting sample. Significantly improved (>8 mg/ml).
  • the DVS test results of Form B are shown in Figure 25.
  • the sample weight loss rate increases when the humidity is lowered to 20% RH at 25 ° C.
  • the XRPD comparison of the samples before and after the DVS test is shown in Figure 26, indicating that Form B is before and after the DVS test.
  • the crystal form is unchanged.
  • Solvent Solubility S (mg/ml) Solvent Solubility S (mg/ml) Methanol 7.0 ⁇ S ⁇ 10.5 1,4-dioxane S ⁇ 1.0 Ethanol S ⁇ 1.1 Acetonitrile 1.4 ⁇ S ⁇ 1.5 Isopropanol S ⁇ 0.9 Dichloromethane 4.6 ⁇ S ⁇ 5.8 acetone S ⁇ 1.1 acetic acid 3.8 ⁇ S ⁇ 4.8 Methyl isobutyl ketone S ⁇ 0.9 Hexane S ⁇ 1.0 Ethyl acetate S ⁇ 0.9 Toluene S ⁇ 1.1 Isopropyl acetate S ⁇ 1.0 Dimethyl sulfoxide S>22.0 Methyl tert-butyl ether S ⁇ 1.0 N,N-dimethylacetamide S>20.0 Tetrahydrofuran S ⁇ 1.2 N-methylpyrrolidone S>23.0 2-methyltetrahydrofuran S ⁇ 0.9 water 10.0 ⁇ S ⁇ 20.0
  • the DVS test was performed on the Form II sample under the constant temperature of 25 ° C.
  • the DVS result (Fig. 29) showed that the sample had no weight loss when the humidity was higher than 10% RH, indicating that the Form II had better stability.
  • the reagents used in the following z'-lyte test methods were purchased from Invitrogen.
  • the inhibitory effect of the test substance on the activity of the double mutant EGFR kinase (EGFR T790M/L858R kinase) (Invitrogen, PV4879) and wild type EGFR kinase (EGFR WT) (Invitrogen, PV3872) was determined by the z'-lyte method.
  • the working concentrations of the components in the 10 ⁇ L T790M/L858R kinase reaction system were: 25 ⁇ M ATP, 0.08 (or 0.1) ng/ ⁇ L EGFR T790M/L858R kinase, 2 ⁇ M Tyr04 substrate (Invitrogen, PV3193, the same below).
  • the concentration of DMSO after addition of the compound prepared in the above examples of the present invention was 2 vol%.
  • the working concentrations of the components in the 10 ⁇ L EGFR WT kinase reaction system were: 10 ⁇ M ATP, 0.8 ng/ ⁇ L EGFR WT kinase, and 2 ⁇ M Tyr04 substrate.
  • the concentration of DMSO after adding the test substance was 2 vol%.
  • the 10 mM stock solution of the test compound was dissolved at room temperature and diluted with a water gradient of 4 vol% DMSO to a final concentration of 10-0.005 ⁇ M.
  • 2.5 ⁇ L of the test substance solution and 5 ⁇ L of a mixture of EGFR T790M/L858R kinase (or EGFR WT kinase) diluted with the reaction buffer and Tyr04 substrate were added to each well, and 2.5 ⁇ L of ATP was added to initiate the reaction.
  • the C1 well was replaced with ATP with a reaction buffer, no drug was added to the C2 well, and the phosphorylated substrate was added to the C3 well as described.
  • the inhibition rate calculation method (refer to the specification of Invitrogen, PV3193) is as follows:
  • Phosphorylation rate (1-((ER ⁇ C3 520nm - C3 450nm ) / ((C1 450nm - C3 450nm ) + ER ⁇ (C3 520nm - C1 520nm )))) ⁇ 100%
  • Inhibition rate (IR) (1 - (phosphorylation rate of test compound) / (phosphorylation rate of C2)) ⁇ 100%
  • the half-inhibitory concentration IC 50 was calculated using XLFIT 5.0 software (IDBS, UK).
  • the free base of the compound of the formula X of the present invention exhibits a strong inhibitory activity against the EGFR mutant enzyme (T790M/L858R), while the inhibitory activity against the EGFR wild-type enzyme (T790M WT) is weak, and Compared to the positive control BIBW2992 (Afatinib), the compounds of the present invention have significant selective inhibitory activity against EGFR mutant enzymes.
  • the selectivity was 8.5 times higher than that of Comparative Compound 1 (the specific structure is shown below, and can be found in WO2013014448A1).
  • Example 28 MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) method for detecting cell inhibitory activity
  • MTT test method steps are carried out using methods well known to those skilled in the art, and the reagents used in the methods are commercially available.
  • the medium was removed and 1 mL of 0.25% trypsin/EDTA (Gibco, 25200-056) was added. After washing once, 1.5 mL of trypsin/EDTA was added to digest the adherent cells until the cells were separated, and then 3.5 mL of the medium was added to terminate the digestion.
  • the digested cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 1300 rpm for 3 min, the supernatant was discarded, and the cells were suspended with fresh medium. The cells were then counted and the cells were diluted to the following concentrations: A431 and H1975 cells per mL 2.78 million, NIH3T3 per mL 333,000. The cells were seeded in a 96-well plate (BD 3072), 90 ⁇ L per well, and cultured overnight.
  • BD 3072 96-well plate
  • the A431 cell culture medium is: DMEM (Hyclone SH30243.01B) containing 10% FBS (Gibco, 10099-141);
  • NIH3T3 cell culture medium DMEM (Hyclone SH30243.01B) containing 10% FBS (Gibco, 10099-141);
  • H1975 cell culture medium RPMI-1640 (Hyclone SH30809.01B) containing 10% FBS (Gibco, 10099-141);
  • the cells were placed in an incubator. After 72 hours of culture, 10 ⁇ L of 5 mg/ml MTT (Sigma, M5655) solution was added to each well, and then the 96-well plate was incubated in a 37 ° C 5% CO 2 incubator for 4 hours.
  • the plate was again centrifuged at 2000 rpm for 5 min. After removing the supernatant, 150 ⁇ L of DMSO was added to each well, and the plate was shaken in a shaker until all crystal violet was dissolved (about 10-20 min). Finally, the absorbance at 492 nm was measured using a microplate reader, and the IC 50 was calculated using XLFIT 5.0 software (IDBS, UK).
  • the free base showed strong inhibitory activity against EGFR mutant cells (H1975 cells), and showed weak inhibitory activity against EGFR wild-type cells (A431 cells), compared with the positive control BIBW2992.
  • the compounds of the present invention have significant selective inhibitory activity against EGFR mutant cell growth.
  • the reagents, solution preparation methods, and cell treatment and lysate preparation steps and ELISA detection steps in the following methods were carried out in accordance with the instructions of R&D DYC3570, R&D DYC1095E and R&D DYC1095BE.
  • Cell lysis buffer 1% (w/v) NP-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% (v/v) glycerol, 1 mM NaVO 3 , 2 mM EDTA.
  • Cell lysate cell lysis buffer + 10 ⁇ g / mL Aprotinin (Sigma) + 10 ⁇ g / mL Leupeptin (Sigma), now available.
  • 1x PBS buffer NaCl: 0.137 M, KCl: 0.0027 M, Na 2 PO 4 -12H 2 O: 0.01 M, KH 2 PO 4 : 0.0015 M, pH 7.4.
  • Wash Buffer PBS buffer containing 0.05% (v/v) Tween-20.
  • Antibody dilutions were detected: 20 mM Tris, 137 mM NaCl, 0.05% (v/v) Tween-20, 0.1% (w/v) BSA, pH 7.2-7.4.
  • Blocking solution PBS buffer containing 1% (w/v) BSA.
  • ELISA kits R&D DYC3570, R&D DYC1095E and R&D DYC1095BE.
  • H1975 cells purchased from the Cell Culture Bank of the Chinese Academy of Sciences
  • H1975 cells were seeded into 96-well plates at a density of 1 ⁇ 10 4 /well, 90 ⁇ l per well (10% (V/V) FBS, RPMI1640
  • the medium was incubated overnight at 37 ° C, 5% (V/V) CO 2 .
  • the R&D capture antibody ((DYC1095BE or DYC1095E)) was diluted with PBS 1:180, and the diluted antibody 100 ⁇ L/well was added to an ELISA reaction plate (Corning costar 42592), and shaken overnight at 25 ° C;
  • the detection antibody is diluted with the detection antibody dilution solution in the prescribed ratio of the kit, and 100 ⁇ L is added to each well, and the mixture is incubated at 25 ° C for 1 hour in the dark;
  • Inhibition rate (%) 100% ⁇ (OD cell- OD drug treatment ) / (OD cell - OD blank )
  • A431 cells purchased from the Cell Culture Bank of the Chinese Academy of Sciences
  • DMEM medium containing 10% FBS per well at 37 ° C. Incubate overnight with 5% CO 2 .
  • the A431 cell culture medium was changed to 90 ⁇ l of serum-free DMEM medium, and cultivation was continued overnight.
  • R&D capture antibody (DYC3570E) was diluted with PBS 1:180, diluted antibody 100 ⁇ L/well was added to an ELISA reaction plate (Corning costar 42592), and shaken overnight at 25 ° C;
  • the detection antibody is diluted with the detection antibody dilution solution in the prescribed ratio of the kit, and 100 ⁇ L is added to each well, and the mixture is incubated at 25 ° C for 1 hour in the dark;
  • Inhibition rate (%) 100% ⁇ (OD EGF - OD drug ) / (OD EGF - OD cells )
  • the compound of the formula X of the present invention has a significant selective inhibitory activity against a cell-level target as compared with the positive control BIBW2992 and the comparative compound 1.
  • the pyrazole ring was replaced by a pyridine ring or a quinoline ring, the selective inhibitory activity against H1975 cell activity and cell-level target was significantly reduced, and even no selection was made. Selective inhibitory activity.
  • the drug concentration in plasma at different times after administration of the compound of the example by intragastric administration was determined by LC/MS/MS method.
  • the pharmacokinetic behavior of the compound of the present invention in rats was investigated, and its pharmacokinetic characteristics were evaluated.
  • Test animals healthy adult male SD rats (body weight 200-225 g, 21 rats, fasted), provided by Slark;
  • Blood sample collection First, select the animals that meet the experimental requirements before the administration, and weigh the markers. Before the blood sample is collected, the rats are bound, and each rat is administered at a predetermined blood collection time point (administered by gavage: 0.083, 0.25, 0.5, 1, 2, 4 after administration, respectively, before administration). , 8, 24h blood collection, a total of 9 time points), blood collection through the tail vein, or blood collection by the heart (final blood collection) about 150 ⁇ L. Blood was collected through the eyelids, or blood was collected by the heart (final blood collection) by about 150 ⁇ L. The blood was transferred to a 1.5 mL tube pre-added to K 2 EDTA.
  • the collected blood samples were placed on wet ice, centrifuged for 5 min (2000 g, 4 ° C), and the plasma was taken out. The whole process was completed within 15 min after blood collection. All samples need to be stored in a -70 ° C refrigerator until sample analysis.
  • the drug concentration was determined by LC/MS/MS method.
  • the pharmacokinetic properties of the compounds of some embodiments of the present invention in the same dosage and mode of administration are shown in Table 11 below:
  • the salt of the present invention has good absorption of the drug, has obvious pharmacological absorption effect, and exhibits good bioavailability.
  • the compound of the formula X adipate, starch is mixed and sieved in a conventional manner, and then uniformly mixed with the other components described above, and directly compressed.
  • the crystal form C-1 and the starch are mixed and sieved, and then uniformly mixed with the other components described above, and directly compressed.
  • Capsules of Form II were prepared from the components shown in Table 14 below:
  • the crystal form II and the starch are mixed and sieved, and then uniformly mixed with the other components described above, and filled into ordinary gelatin capsules.
  • a suspension competition test at room temperature and 50 ° C was set in the aqueous system.
  • a free base suspension of acetone/H 2 O (aw ⁇ 0.6) was prepared at room temperature and 50 ° C, and after equilibrating at the corresponding temperature for about 1 hour, it was filtered to obtain a near-saturated solution at the corresponding temperature.
  • Equal masses of Form II and IV samples (about 6 mg each) were added to 0.8 ml of a saturated solution to form a suspension, which was magnetically stirred ( ⁇ 800 rpm) at room temperature and 50 ° C for about 5 minutes. After sampling, test XRPD.

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Abstract

提供了一种EGFR抑制剂及其药学上可接受的盐和多晶型物及其应用。具体地，提供了N-(2-(4-(二甲基氨基)哌啶-1-基)-5-(5-氟-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺的多晶型物、药学上可接受的盐或前药。此外，还提供了含该抑制剂的药物组合物及其应用。

Description

EGFR抑制剂及其药学上可接受的盐和多晶型物及其应用 技术领域
本发明属于医药技术领域，具体地说，本发明涉及一种EGFR抑制剂及其药学上可接受的盐和多晶型物及其应用，该抑制剂为N-(2-(4-(二甲基氨基)哌啶-1-基)-5-(5-氟-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺。
背景技术
肺癌是全球发病率最高的癌症，在中国肺癌发病率位居所有癌症中第一位，也是中国发病率和死亡率最高的癌症，在中国肺癌病人中，30％病人具有EGFR突变，其中L858R和外显子19缺失突变占90％以上，这类病人对EGFR抑制剂更为敏感。现有已上市第一代EGFR抑制剂例如厄洛替尼，吉非替尼对这类病人效果良好，能够使其中60％以上病人肿瘤缩小，明显延长病人无进展生存期。但绝大多数病人在6-12个月获得耐药，第一代EGFR抑制剂将不再起效，而这类病人目前处于无药可用状态。临床发现对第一代EGFR抑制剂产生耐药的病人中有50％检测到EGFR T790M突变。在T790M突变细胞系H1975中第一代EGFR抑制剂，吉非替尼和厄洛替尼，均大于3uM，基本没有活性。
目前开发上市的第二代不可逆pan-EGFR抑制剂(Afatinib(BIBW2992))对EGFR突变肺癌病人疗效显著好于第一代EGFR抑制剂。但第二代抑制剂同时也具有很强的野生型EGFR抑制活性，对野生型EGFR的抑制活性显著高于耐药T790M突变，病人皮疹等毒副作用严重且对耐药病人疗效较差，仅有小部分第一代EGFR抑制剂耐药病人对这类药物产生应答。
为了提高对耐药EGFR T790M突变抑制活性的同时降低对野生型EGFR的抑制活性，开发活性更高、选择性更好、毒性更低的第三代EGFR突变体选择性抑制剂具有重要的意义。本发明在前述工作的基础上开发了EGFR突变体的抑制剂的多种盐型和晶型，有助于进一步的药物开发。
发明内容
本发明的目的在于提供一种可有效抑制EGFR突变体的抑制剂、及其药学上可接受的盐和多晶型物及其应用。
在本发明的第一方面，提供了一种结构如式X所示的化合物、或其药学上可接受的盐或其前药:
Figure PCTCN2016091591-appb-000001
在另一优选例中，所述的药学上可接受的盐选自下组：盐酸盐、硫酸盐、磷酸盐、乙酸盐、L-乳酸盐、马来酸盐、富马酸盐、琥珀酸盐、L-苹果酸盐、己二酸盐、L-酒石酸盐、马尿酸盐、柠檬酸盐、粘酸盐、乙醇酸盐、D-葡萄糖醛酸盐、苯甲酸盐、龙胆酸盐、烟酸盐、乙二磺酸盐、草酸盐、甲磺酸盐、苯磺酸盐、2-羟基乙磺酸盐和氢溴酸盐。
在另一优选例中，所述的药学上可接受的盐选自下组：盐酸盐、磷酸盐、马来酸盐、L-苹果酸盐、己二酸盐、硫酸盐、富马酸盐、琥珀酸盐、L-酒石酸盐、柠檬酸盐、甲磺酸盐、苯甲酸盐、和苯磺酸盐。
在另一优选例中，所述的式X化合物或其药学上可接受盐为无水形式、水合物形式或溶剂合物形式。
在另一优选例中，所述的溶剂合物包括甲醇溶剂合物、乙酸乙酯溶剂合物。
在另一优选例中，所述的式X化合物或其药学上可接受盐为晶体。
在另一优选例中，所述的药学上可接受盐选自下组：盐酸盐、磷酸盐、马来酸盐、L-苹果酸盐、己二酸盐、硫酸盐、富马酸盐、琥珀酸盐、L-酒石酸盐、柠檬酸盐、甲磺酸盐、苯甲酸盐、和苯磺酸盐。
在另一优选例中，所述的药学上可接受盐为盐酸盐，其中，盐酸与式X化合物的摩尔比为(0.8-2.1)：1，较佳地(0.9-1.1)：1。
在另一优选例中，所述的药学上可接受盐为磷酸盐，其中，磷酸与式X化合物的摩尔比为1:(0.9-2.1)，较佳地1:(1.1-2.1)。
在另一优选例中，所述的药学上可接受盐为马来酸盐，其中，马来酸与式X化合物的摩尔比为(0.8-1.2)：1，较佳地(0.9-1.1)：1，更佳地1：1。
在另一优选例中，所述的药学上可接受盐为L-苹果酸盐，其中，L-苹果酸与式X化合物的摩尔比为(0.5-1.4)：1，较佳地(0.7-1.3)：1。
在另一优选例中，所述的药学上可接受盐为己二酸盐，其中，己二酸与式X化合物的摩尔比为(0.8-1.2)：1，较佳地(0.9-1.1)：1。
在另一优选例中，所述的药学上可接受盐为富马酸盐，其中，富马酸与式X化合物的摩尔比为(0.5-1.2)：1，较佳地为(0.5-1)：1。
在另一优选例中，所述的药学上可接受盐为柠檬酸盐，其中，柠檬酸与式X化合物的摩尔 比为(0.5-1.2)：1，较佳地为(0.5-1)：1。
在另一优选例中，所述的药学上可接受盐为粘酸盐，其中，粘酸与式X化合物的摩尔比为(0.5-1.2)：1，较佳地为(0.5-1)：1。
在另一优选例中，所述的药学上可接受盐为琥珀酸盐，其中，琥珀酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为L-酒石酸盐，其中，L-酒石酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为烟酸盐，其中，烟酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为马尿酸盐，其中，马尿酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为乙醇酸盐，其中，乙醇酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为苯甲酸盐，其中，苯甲酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为龙胆酸盐，其中，龙胆酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为乙二磺酸盐，其中，乙二磺酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为草酸盐，其中，草酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为甲磺酸盐，其中，甲磺酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为苯磺酸盐，其中，苯磺酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为2-羟基乙磺酸盐，其中，2-羟基乙磺酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的药学上可接受盐为D-葡萄糖醛酸盐，其中，D-葡萄糖醛酸与式X化合物的摩尔比为(0.9-1.1)：1，较佳地为1：1。
在另一优选例中，所述的晶体为式X化合物盐酸盐的A型结晶，即晶型A，其X射线粉末衍射图具有下组A1的衍射角2θ(°)值：8.47±0.10、20.32±0.10、23.31±0.10(最高峰)和25.98±0.10。
在另一优选例中，所述的晶型A的X射线粉末衍射图还包含2个或2个以上选自下组A2的衍射角2θ(°)值：15.54±0.10、16.97±0.10、17.66±0.10、17.90±0.10、21.54±0.10、22.19±0.10、23.37±0.10、24.81±0.10。
在另一优选例中，所述的晶型A的X射线粉末衍射图还包含2个或2个以上选自下组A3的衍射角2θ(°)值：9.44±0.10、11.78±0.10、13.29±0.10、14.91±0.10、15.23±0.10、18.20±0.10、18.68±0.10、20.05±0.10、21.07±0.10、23.87±0.10、24.19±0.10、25.30±0.10、25.61±0.10、26.45±0.10、27.42±0.10、28.49±0.10、29.96±0.10、32.00±0.10、34.01±0.10、35.25±0.10。
在另一优选例中，所述的晶型A的X射线粉末衍射图包含选自组A1、A2和A3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型A的X射线粉末衍射图具有下表A2所示值：
表A2
Figure PCTCN2016091591-appb-000002
在另一优选例中，所述的晶型A的X射线粉末衍射图谱基本如图1所表征。
在另一优选例中，所述的晶型A中，盐酸与式X化合物的摩尔比为(0.8-2.1)：1，较佳地(0.9-1.1)：1，更佳地0.9:1。
在另一优选例中，所述的晶型A还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为259±2℃；较佳地，其差示扫描量热法分析图谱基本如图2C所表征；
(ii)热重分析图谱基本如图2C所表征；
(iii)所述的晶型A的熔点为259℃-267℃，较佳地为261℃-265℃。
在另一优选例中，所述的晶型A为水合物形式。
在另一优选例中，所述的晶体为式X化合物磷酸盐的B型结晶，即晶型B，其X射线粉末衍射图具有下组B1的衍射角2θ(°)值：11.94±0.10、19.92±0.10(最高峰)、22.27±0.10、23.93±0.10。
在另一优选例中，所述的晶型B的X射线粉末衍射图还包含下组B2的衍射角2θ(°)值：7.97±0.10、9.50±0.10。
在另一优选例中，所述的晶型B的X射线粉末衍射图还包含2个或2个以上选自下组B3的衍射角2θ(°)值：4.01±0.10、10.71±0.10、12.11±0.10、12.56±0.10、12.89±0.10、13.94±0.10、14.47±0.10、15.16±0.10、15.52±0.10、16.54±0.10、17.00±0.10、17.42±0.10、17.99±0.10、18.38±0.10、18.91±0.10、20.81±0.10、20.96±0.10、21.29±0.10、21.65±0.10、21.99±0.10、22.58±0.10、23.02±0.10、23.45±0.10、24.31±0.10、24.53±0.10、24.76±0.10、25.01±0.10、27.29±0.10、28.98±0.10。
在另一优选例中，所述的晶型B的X射线粉末衍射图包含选自组B1、B2和B3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型B的X射线粉末衍射图具有下表B2所示值：
表B2
Figure PCTCN2016091591-appb-000003
Figure PCTCN2016091591-appb-000004
在另一优选例中，所述的晶型B的X射线粉末衍射图谱基本如图3所表征。
在另一优选例中，所述的晶型B中，磷酸与式X化合物的摩尔比为1:(0.9-2.1)，较佳地1:(1.1-2.1)，更佳地1:1.1。
在另一优选例中，所述的晶型B具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为229.7±2℃；较佳地，差示扫描量热法分析图谱基本如图4C所表征；
(ii)其热重分析图谱基本如图4C所表征；
(iii)所述的晶型B的熔点为229℃-239℃，较佳地为231℃-237℃。
在另一优选例中，所述的晶型B为水合物形式。
在另一优选例中，所述的晶体为式X化合物马来酸盐的C-1型结晶，即晶型C-1，其X射线粉末衍射图具有下组C-1-1的衍射角2θ(°)值：8.73±0.10、13.37±0.10、18.08±0.10和25.55±0.10(最高峰)。
在另一优选例中，所述的晶型C-1的X射线粉末衍射图还包含2个或2个以上选自下组C-1-2的衍射角2θ(°)值：9.10±0.10、14.14±0.10、15.15±0.10、17.20±0.10、17.42±0.10、18.61±0.10、18.95±0.10、19.15±0.10、19.74±0.10、20.59±0.10、21.07±0.10、21.49±0.10、22.09±0.10、22.58±0.10、22.97±0.10、23.32±0.10、24.52±0.10、24.86±0.10、26.41±0.10、26.77±0.10、27.99±0.10、28.80±0.10、37.10±0.10。
在另一优选例中，所述的晶型C-1的X射线粉末衍射图还包含2个或2个以上选自下组C-1-3的衍射角2θ(°)值：10.12±0.10、11.56±0.10、23.85±0.10、27.21±0.10、27.55±0.10、27.85±0.10、29.54±0.10、30.27±0.10、30.76±0.10、32.93±0.10、33.47±0.10、34.20±0.10、35.03±0.10、35.34±0.10、36.04±0.10、36.47±0.10、37.56±0.10。
在另一优选例中，所述的晶型C-1的X射线粉末衍射图包含选自组C-1-1、C-1-2和C-1-3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型C-1的X射线粉末衍射图具有下表C-1-2所示的值：
表C-1-2
Figure PCTCN2016091591-appb-000005
Figure PCTCN2016091591-appb-000006
在另一优选例中，所述的晶型C-1的X射线粉末衍射图谱基本如图5所表征。
在另一优选例中，所述的晶型C-1中，马来酸与式X化合物的摩尔比为(0.8-1.2)：1，较佳地(0.9-1.1)：1，更佳地1：1。
在另一优选例中，所述的晶型C-1还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为211.6±2℃；较佳地，其差示扫描量热法分析图谱基本如图6A所表征；
(ii)热重分析图谱基本如图6A所表征；
(iii)所述的晶型C-1的熔点为211℃-218℃，较佳地为212℃-216℃。
在另一优选例中，所述的晶型C-1为无水形式。
在另一优选例中，所述的晶体为式X化合物马来酸盐的C-2型结晶，即晶型C-2，其X射线粉末衍射图具有下组C-2-1的衍射角2θ(°)值：7.48±0.10(最高峰)、8.60±0.10、20.63±0.10和23.27±0.10。
在另一优选例中，所述的晶型C-2的X射线粉末衍射图还包含下组C-2-2的衍射角2θ(°)值：17.21±0.10、18.71±0.10。
在另一优选例中，所述的晶型C-2的X射线粉末衍射图还包含2个或2个以上选自下组C-2-3的衍射角2θ(°)值：3.77±0.10、11.75±0.10、12.97±0.10、15.36±0.10、15.70±0.10、18.03±0.10、18.36±0.10、20.13±0.10、25.03±0.10。
在另一优选例中，所述的晶型C-2的X射线粉末衍射图包含选自组C-2-1、C-2-2和C-2-3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型C-2的X射线粉末衍射图具有下表C-2-2所示的值：
表C-2-2
Figure PCTCN2016091591-appb-000007
在另一优选例中，所述的晶型C-2的X射线粉末衍射图谱基本如图7所表征。
在另一优选例中，所述的晶型C-2中，马来酸与式X化合物的摩尔比为(0.8-1.2)：1，较佳地(0.9-1.1)：1，更佳地1：1。
在另一优选例中，所述的晶型C-2还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为208.4±2℃；较佳地，其差示扫描量热法分析图谱基本如图8所表征；
(ii)热重分析图谱基本如图8所表征；
(iii)所述的晶型C-2的熔点为208℃-216℃，较佳地为210℃-214℃。
在另一优选例中，所述的晶型C-2为无水形式。
在另一优选例中，所述的晶体为式X化合物L-苹果酸盐的D型结晶，即晶型D，其X射线粉末衍射图具有下组D1的衍射角2θ(°)值：7.47±0.10、18.75±0.10、22.69±0.10(最高峰)和24.39±0.10。
在另一优选例中，所述的晶型D的X射线粉末衍射图还包含2个或2个以上选自下组D2的衍射角2θ(°)值：17.79±0.10、18.32±0.10、20.81±0.10。
在另一优选例中，所述的晶型D的X射线粉末衍射图还包含2个或2个以上选自下组D3的衍射角2θ(°)值：11.23±0.10、11.68±0.10、12.72±0.10、13.58±0.10、14.67±0.10、15.24±0.10、20.09±0.10、20.58±0.10、21.15±0.10、21.88±0.10、22.20±0.10、23.34±0.10、24.81±0.10、25.60±0.10、26.33±0.10、26.68±0.10、32.07±0.10。
在另一优选例中，所述的晶型D的X射线粉末衍射图包含选自组D1、D2和D3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型D的X射线粉末衍射图具有下表D2所示的值：
表D2
Figure PCTCN2016091591-appb-000008
在另一优选例中，所述的晶型D的X射线粉末衍射图谱基本如图9所表征。
在另一优选例中，所述的晶型D中，L-苹果酸与式X化合物的摩尔比为(0.5-1.4)：1，较佳地(0.7-1.3)：1，更佳地为约1.3:1。
在另一优选例中，所述的晶型D还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为201.4±2℃；较佳地，其差示扫描量热法分析图谱基本如图10A所表征；
(ii)热重分析图谱基本如图10A所表征；
(iii)所述的晶型D的熔点为201℃-209℃，较佳地为202℃-207℃。
在另一优选例中，所述的晶型D为无水形式。
在另一优选例中，所述的晶体为式X化合物己二酸盐的E型结晶，即晶型E，其X射线粉末衍射图具有下组E1的衍射角2θ(°)值：7.43±0.10、18.45±0.10、21.64±0.10(最高峰)和24.22±0.10。
在另一优选例中，所述的晶型E的X射线粉末衍射图还包含2个或2个以上选自下组E2的衍射角2θ(°)值：15.66±0.10、18.83±0.10、21.16±0.10、21.39±0.10、24.38±0.10。
在另一优选例中，所述的晶型E的X射线粉末衍射图还包含2个或2个以上选自下组E3的衍射角2θ(°)值：11.10±0.10、11.43±0.10、12.11±0.10、13.78±0.10、15.07±0.10、15.25±0.10、17.32±0.10、20.07±0.10、20.62±0.10、20.96±0.10、23.72±0.10、25.41±0.10、25.85±0.10、26.42±0.10、27.60±0.10。
在另一优选例中，所述的晶型E的X射线粉末衍射图包含选自组E1、E2和E3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型E的X射线粉末衍射图具有下表E2所示的值：
表E2
Figure PCTCN2016091591-appb-000009
在另一优选例中，所述的晶型E的X射线粉末衍射图谱基本如图11所表征。
在另一优选例中，所述的晶型E中，己二酸与式X化合物的摩尔比为(0.8-1.2)：1，较佳 地(0.9-1.1)：1，更佳地为约1.1:1。
在另一优选例中，所述的晶型E还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为203.2±2℃；较佳地，其差示扫描量热法分析图谱基本如图12A所表征；
(ii)热重分析图谱基本如图12A所表征；
(iii)所述的晶型E的熔点为203℃-208℃，较佳地为204℃-206℃。
在另一优选例中，所述的晶型E为无水形式。
在另一优选例中，所述的晶体为式X化合物的晶型Ⅰ，其X射线粉末衍射图具有组I-1的衍射角2θ(°)值：8.74±0.10、9.80±0.10、15.63±0.10、和21.38±0.10(最高峰)。
在另一优选例中，所述的晶型Ⅰ的X射线粉末衍射图还包含2个或2个以上选自下组I-2的衍射角2θ(°)值：15.45±0.10、17.50±0.10、17.81±0.10、20.56±0.10、20.89±0.10、21.51±0.10、21.91±0.10、24.14±0.10、25.99±0.10、28.26±0.10。
在另一优选例中，所述的晶型Ⅰ的X射线粉末衍射图还包含2个或2个以上选自下组I-3的衍射角2θ(°)值：10.68±0.10、12.94±0.10、14.47±0.10、16.39±0.10、16.76±0.10、18.69±0.10、19.59±0.10、24.84±0.10、27.41±0.10、31.13±0.10、31.89±0.10。
在另一优选例中，所述的晶型Ⅰ的X射线粉末衍射图包含选自组I-1、I-2和I-3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型I的X射线粉末衍射图具有下表I2所示的值：
表I2
Figure PCTCN2016091591-appb-000010
Figure PCTCN2016091591-appb-000011
在另一优选例中，所述的晶型I的X射线粉末衍射图谱基本如图13所表征。
在另一优选例中，所述的晶型I还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为159.6±2℃；较佳地，其差示扫描量热法分析图谱基本如图14所表征；
(ii)热重分析图谱基本如图14所表征；
(iii)所述的晶型I的熔点为159℃-165℃，较佳地为160℃-163℃。
在另一优选例中，所述的晶型I为无水形式。
在另一优选例中，所述的晶体为式X化合物的晶型II，其X射线粉末衍射图具有组II-1的衍射角2θ(°)值：9.47±0.10(最高峰)、17.34±0.10、18.87±0.10、和23.89±0.10。
在另一优选例中，所述的晶型II的X射线粉末衍射图还包含2个或2个以上选自下组II-2的衍射角2θ(°)值：6.61±0.10、12.06±0.10、16.96±0.10、19.19±0.10、19.84±0.10、20.66±0.10、20.96±0.10、24.22±0.10、28.93±0.10。
在另一优选例中，所述的晶型II的X射线粉末衍射图包含选自组II-1、II-2中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型II的X射线粉末衍射图具有下表II2所示的值：
表II2
Figure PCTCN2016091591-appb-000012
在另一优选例中，所述的晶型II的X射线粉末衍射图谱基本如图15所表征。
在另一优选例中，所述的晶型II还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为159.9±2℃；较佳地，其差示扫描量热法分析图谱基本如图16所表征；
(ii)热重分析图谱基本如图16所表征；
(iii)所述的晶型II的熔点为159℃-164℃，较佳地为160℃-163℃。
在另一优选例中，所述的晶型II为水合物形式，较佳地为二水合物形式。
在另一优选例中，所述的晶体为式X化合物的晶型Ⅲ，其X射线粉末衍射图具有组III-1的衍射角2θ(°)值：9.72±0.10、18.41±0.10、23.89±0.10(最高峰)和28.02±0.10。
在另一优选例中，所述的晶型Ⅲ的X射线粉末衍射图还包含2个或2个以上选自下组III-2的衍射角2θ(°)值：11.78±0.10、16.34±0.10、16.57±0.10、17.54±0.10、18.19±0.10、20.06±0.10、21.33±0.10、23.68±0.10、25.05±0.10。
在另一优选例中，所述的晶型Ⅲ的X射线粉末衍射图还包含2个或2个以上选自下组III-3的衍射角2θ(°)值：6.68±0.10、8.25±0.10、13.19±0.10、13.92±0.10、15.54±0.10、19.58±0.10、21.66±0.10、22.39±0.10、22.96±0.10、24.26±0.10、26.34±0.10、27.36±0.10、29.06±0.10、29.41±0.10、31.00±0.10、34.62±0.10。
在另一优选例中，所述的晶型Ⅲ的X射线粉末衍射图包含选自组III-1、III-2和III-3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型Ⅲ的X射线粉末衍射图具有下表Ⅲ2所示的值：
表Ⅲ2
Figure PCTCN2016091591-appb-000013
Figure PCTCN2016091591-appb-000014
在另一优选例中，所述的晶型Ⅲ的X射线粉末衍射图谱基本如图17所表征。
在另一优选例中，所述的晶型Ⅲ还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为159.9±2℃；较佳地，其差示扫描量热法分析图谱基本如图18A所表征；
(ii)热重分析图谱基本如图18A所表征；
(iii)所述的晶型Ⅲ的熔点为159℃-164℃，较佳地为160℃-162℃。
在另一优选例中，所述的晶型Ⅲ为溶剂合物形式，较佳地为甲醇溶剂合物。
在另一优选例中，所述的晶体为式X化合物的晶型Ⅳ，其X射线粉末衍射图具有组IV-1的衍射角2θ(°)值：7.69±0.10(最高峰)、18.90±0.10。
在另一优选例中，所述的晶型Ⅳ的X射线粉末衍射图还包含2个或2个以上选自下组IV-2的衍射角2θ(°)值：5.38±0.10、9.43±0.10、13.31±0.10、18.06±0.10、23.19±0.10、24.18±0.10。
在另一优选例中，所述的晶型Ⅳ的X射线粉末衍射图包含选自组IV-1和IV-2中4个或更多个或全部(如4、5、6、7、8、9、10、11、12、13等)的2θ(°)值。
在另一优选例中，所述的晶型Ⅳ的X射线粉末衍射图具有下表Ⅳ2所示的值：
表Ⅳ2
Figure PCTCN2016091591-appb-000015
在另一优选例中，所述的晶型Ⅳ的X射线粉末衍射图谱基本如图19所表征。
在另一优选例中，所述的晶型Ⅳ还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为159.5±2℃；较佳地，其差示扫描量热法分析图谱基本如图20所表征；
(ii)热重分析图谱基本如图20所表征；
(iii)所述的晶型IV的熔点为159℃-164℃，较佳地为160℃-162℃。
在另一优选例中，所述的晶型Ⅳ为水合物形式。
在另一优选例中，所述的晶体为式X化合物的晶型V，其X射线粉末衍射图具有组V-1的衍射角2θ(°)值：8.28±0.10、8.89±0.10(最高峰)、9.44±0.10、17.76±0.10。
在另一优选例中，所述的晶型V的X射线粉末衍射图还包含2个或2个以上选自下组V-2的衍射角2θ(°)值：14.01±0.10、15.39±0.10、16.55±0.10、18.46±0.10、18.91±0.10、21.71±0.10、22.91±0.10、23.24±0.10、24.87±0.10。
在另一优选例中，所述的晶型V的X射线粉末衍射图包含选自组V-1和V-2中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值。
在另一优选例中，所述的晶型V的X射线粉末衍射图具有下表V2所示的值：
表V2
Figure PCTCN2016091591-appb-000016
在另一优选例中，所述的晶型V的X射线粉末衍射图谱基本如图21所表征。
在另一优选例中，所述的晶型V还具有选自下组的一个或多个特征：
(i)差示扫描量热法分析图谱中，起始温度为150.7±2℃；较佳地，其差示扫描量热法分析图谱基本如图22A所表征；
(ii)热重分析图谱基本如图22A所表征；
(iii)所述的晶型V的熔点为149℃-157℃，较佳地为151℃-155℃。
在另一优选例中，所述的晶型V为溶剂合物形式，较佳地为乙酸乙酯溶剂合物形式。
在本发明的第二方面，提供了一种制备本发明第一方面所述的式X化合物或其药学上可接受盐的方法，包括步骤：
(1)在惰性溶剂中，将化合物I-2与化合物3a进行反应，从而形成式X化合物；和
Figure PCTCN2016091591-appb-000017
(2)任选地将式X化合物与酸进行成盐反应，从而形成药学上可接受的盐；
(3)任选地将步骤(1)或(2)所形成的式X化合物或其药学上可接受的盐进行结晶处理，从而获得晶体。
在另一优选例中，步骤(1)中，所述的惰性溶剂选自下组：1,4-二氧六环、二甲亚砜、DMF。
在另一优选例中，在步骤(1)中，还包括步骤：
1-1)将形成的式X化合物加入有机溶剂中，加热回流溶解形成溶液；
1-2)向所述溶液中加入水，并使得有机溶剂和水的体积比为(1～2):1(较佳地(1.2～1.8):1)；
1-3)冷却，析出固体，并过滤，水洗得式X化合物。
在另一优选例中，步骤1-1)中，所述有机溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇，或其混合物，优选为乙醇。
在另一优选例中，步骤1-1)中，式X化合物在有机溶剂中的浓度为0.1～0.5g/mL。
在另一优选例中，所述方法包括以下子方法(A)-(E)以及(I)-(V)中任一子方法：
(A)所述晶体为式X化合物盐酸盐的A型结晶，即晶型A，并且在步骤(3)中包括：在有机溶剂中，在盐酸存在下，对式X化合物进行结晶处理，从而形成晶型A；
在另一优选例中，子方法(A)中，所述有机溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇，或其混合物，较佳地，所述有机溶剂为乙腈。
在另一优选例中，子方法(A)中，盐酸在所述有机溶剂中的浓度为0.1～0.5g/mL。
在另一优选例中，子方法(A)中，盐酸与式X化合物的摩尔比为(1～2):1，较佳地为(1～1.2):1。
在另一优选例中，子方法(A)中，结晶处理方式为悬浮搅拌。
在另一优选例中，子方法(A)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(A)中，结晶处理时间为1-72小时，较佳地为10-50小时。
(B)所述晶体为式X化合物磷酸盐的B型结晶，即晶型B，并且在步骤(3)中包括：在有机溶剂中，在磷酸存在下，对式X化合物进行结晶处理，从而形成晶型B；
在另一优选例中，子方法(B)中，所述有机溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇，或其混合物，较佳地，所述有机溶剂为乙腈。
在另一优选例中，子方法(B)中，磷酸在所述有机溶剂中的浓度为0.1～0.5g/mL。
在另一优选例中，子方法(B)中，磷酸与式X化合物的摩尔比为(1～2):1，较佳地为(1～1.2):1。
在另一优选例中，子方法(B)中，结晶处理方式为悬浮搅拌。
在另一优选例中，子方法(B)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(B)中，结晶处理时间为1-72小时，较佳地为10-50小时。
(C-1-1)所述晶体为式X化合物马来酸盐的C-1型结晶，即晶型C-1，并且在步骤(3)中包括：在有机溶剂中，在马来酸存在下，对式X化合物进行结晶处理，从而形成晶型C-1。
在另一优选例中，子方法(C-1-1)中，所述有机溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇，或其混合物，较佳地，所述有机溶剂为乙腈。
在另一优选例中，子方法(C-1-1)中，马来酸与所述有机溶剂的投料比(m:v)为5～12mg/mL。
在另一优选例中，子方法(C-1-1)中，马来酸与式X化合物的摩尔比为(1～2):1，较佳地为(1～1.2):1。
在另一优选例中，子方法(C-1-1)中，结晶处理方式为悬浮搅拌。
在另一优选例中，子方法(C-1-1)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(C-1-1)中，结晶处理时间为1-72小时，较佳地为10-50小时。
(C-1-2)所述晶体为式X化合物马来酸盐的C-1型结晶，即晶型C-1，并且在步骤(3)中包括：
向式X化合物的回流状态的有机溶剂中加入马来酸，搅拌冷却，从而形成晶型C-1；
在另一优选例中，子方法(C-1-2)中，所述有机溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃和丙二醇，及其混合物，较佳地，所述有机溶剂为甲醇、乙醇、丙酮、乙腈。
在另一优选例中，子方法(C-1-2)中，马来酸与式X化合物的摩尔比为(1～3):1，较佳地为(1～1.5):1。
在另一优选例中，子方法(C-1-2)中，式X化合物在有机溶剂中的浓度为0.1～1g/mL，优选0.2～0.6g/mL。
在另一优选例中，子方法(C-1-2)中，冷却时间为1～10小时。
在另一优选例中，子方法(C-1-2)中，冷却至5～15℃。
(D)所述晶体为式X化合物L-苹果酸盐的D型结晶，即晶型D，并且在步骤(3)中包括：在有机溶剂中，在L-苹果酸存在下，对式X化合物进行结晶处理，从而形成晶型D；
在另一优选例中，子方法(D)中，所述有机溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇，或其混合物，较佳地，所述有机溶剂为乙腈。
在另一优选例中，子方法(D)中，L-苹果酸与所述有机溶剂的投料比(m:v)为5～12mg/mL。
在另一优选例中，子方法(D)中，L-苹果酸与式X化合物的摩尔比为(1～2):1，较佳地为(1～1.2):1。
在另一优选例中，子方法(D)中，结晶处理方式为悬浮搅拌。
在另一优选例中，子方法(D)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(D)中，结晶处理时间为1-72小时，较佳地为10-50小时。
(E)所述晶体为式X化合物己二酸盐的E型结晶，即晶型E，并且在步骤(3)中包括：在有机溶剂中，在己二酸存在下，对式X化合物进行结晶处理，从而形成晶型E；
在另一优选例中，子方法(E)中，所述有机溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇，或其混合物，较佳地，所述有机溶剂为乙腈。
在另一优选例中，子方法(E)中，己二酸与所述有机溶剂的投料比(m:v)为5～12mg/mL。
在另一优选例中，子方法(E)中，己二酸与式X化合物的摩尔比为(1～2):1，较佳地为(1～1.2):1。
在另一优选例中，子方法(E)中，结晶处理方式为悬浮搅拌。
在另一优选例中，子方法(E)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(E)中，结晶处理时间为1-72小时，较佳地为10-50小时。
(I)所述晶体为式X化合物的晶型I，并且在步骤(3)中包括：在溶剂中，对式X化合物进行结晶处理，从而形成晶型I；
在另一优选例中，子方法(I)中，所述溶剂选自下组：水、甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇、乙酸乙酯、甲基异丁基酮、乙酸异丙酯、2-甲基四氢呋喃、二氯甲烷、甲基叔丁基醚、二甲基亚砜、甲苯、N,N-二甲基乙酰胺、N-甲基吡咯烷酮，或其混合。
在另一优选例中，子方法(I)中，所述溶剂选自下组：甲醇、异丙醇、甲基异丁基酮、乙酸乙酯、乙酸异丙酯、2-甲基四氢呋喃、乙腈、二氯甲烷/甲基叔丁基醚(V:V为1:1-1:4)、二甲基亚砜/甲苯(V:V为1:1-1:4)、N,N-二甲基乙酰胺/乙酸异丙酯(V:V为1:1-1:4)、N-甲基吡咯烷酮/甲基叔丁基醚(V:V为1:1-1:4)、水/丙酮。
在另一优选例中，子方法(I)中，结晶处理方式为悬浮搅拌或缓慢挥发。
在另一优选例中，子方法(I)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(I)中，结晶处理时间为1-10天，较佳地为4-8天。
(II)所述晶体为式X化合物的晶型II，并且在步骤(3)中包括：在溶剂中，对式X化合物进行结晶处理，从而形成晶型II；
在另一优选例中，子方法(II)中，所述溶剂选自下组：水、甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇、乙酸乙酯、甲基异丁基酮、乙酸异丙酯、2-甲基四氢呋喃、二氯甲烷、甲基叔丁基醚、二甲基亚砜、甲苯、N,N-二甲基乙酰胺、N-甲基吡咯烷酮，或其混合。
在另一优选例中，子方法(II)中，所述溶剂选自下组：甲醇、异丙醇、甲基异丁基酮、乙酸乙酯、乙酸异丙酯、2-甲基四氢呋喃、乙腈、二氯甲烷/甲基叔丁基醚(V:V为1:1-1:4)、二甲基亚砜/甲苯(V:V为1:1-1:4)、N,N-二甲基乙酰胺/乙酸异丙酯(V:V为1:1-1:4)、N-甲基吡咯烷酮/ 甲基叔丁基醚(V:V为1:1-1:4)、水/丙酮。
在另一优选例中，子方法(II)中，结晶处理方式为悬浮搅拌或反溶剂添加方式。
在另一优选例中，子方法(II)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(II)中，结晶处理时间为1-10天，较佳地为4-8天。
(Ⅲ)所述晶体为式X化合物的晶型Ⅲ，并且在步骤(3)中包括：在溶剂中，对式X化合物进行结晶处理，从而形成晶型Ⅲ；
在另一优选例中，子方法(Ⅲ)中，所述溶剂选自下组：水、甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇、乙酸乙酯、甲基异丁基酮、乙酸异丙酯、2-甲基四氢呋喃、二氯甲烷、甲基叔丁基醚、二甲基亚砜、甲苯、N,N-二甲基乙酰胺、N-甲基吡咯烷酮，或其混合。
在另一优选例中，子方法(Ⅲ)中，所述溶剂选自下组：甲醇、异丙醇、甲基异丁基酮、乙酸乙酯、乙酸异丙酯、2-甲基四氢呋喃、乙腈、二氯甲烷/甲基叔丁基醚(V:V为1:1-1:4)、二甲基亚砜/甲苯(V:V为1:1-1:4)、N,N-二甲基乙酰胺/乙酸异丙酯(V:V为1:1-1:4)、N-甲基吡咯烷酮/甲基叔丁基醚(V:V为1:1-1:4)、水/丙酮。
在另一优选例中，子方法(Ⅲ)中，结晶处理方式为悬浮搅拌。
在另一优选例中，子方法(Ⅲ)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(Ⅲ)中，结晶处理时间为1-5天，较佳地为2-4天。
(IV)所述晶体为式X化合物的晶型IV，并且在步骤(3)中包括：在有机溶剂中，对式X化合物进行结晶处理，从而形成晶型IV；
在另一优选例中，子方法(IV)中，所述溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇、乙酸乙酯、1,4-二氧六环、2-甲基四氢呋喃，或其混合物。
在另一优选例中，子方法(IV)中，结晶处理方式为缓慢挥发。
在另一优选例中，子方法(IV)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(IV)中，结晶处理时间为1-10天，较佳地为4-8天。
(V)所述晶体为式X化合物的晶型V，并且在步骤(3)中包括：在有机溶剂中，对式X化合物进行结晶处理，从而形成晶型V；
在另一优选例中，子方法(V)中，所述溶剂选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇、乙酸乙酯、1,4-二氧六环、2-甲基四氢呋喃，或其混合物。在另一优选例中，子方法(V)中，结晶处理方式为缓慢挥发或反溶剂添加。
在另一优选例中，子方法(V)中，结晶处理温度为5-30℃，较佳地为10-20℃。
在另一优选例中，子方法(V)中，结晶处理时间为1-10天，较佳地为4-6天。
在另一优选例中，所述晶体为式X化合物的结晶，并且
1)在溶剂F中，对式X化合物进行析晶，从而获得晶型I、II、或Ⅲ；或
2)在溶剂H中，对式X化合物进行析晶，从而获得晶型IV或V；
其中，所述的溶剂F选自下组：水、甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四 氢呋喃、丙二醇、乙酸乙酯、甲基异丁基酮、乙酸异丙酯、2-甲基四氢呋喃、二氯甲烷、甲基叔丁基醚、二甲基亚砜、甲苯、N,N-二甲基乙酰胺、N-甲基吡咯烷酮，或其混合。
在另一优选例中，所述的溶剂F选自下组：甲醇、异丙醇、甲基异丁基酮、乙酸乙酯、乙酸异丙酯、2-甲基四氢呋喃、乙腈、二氯甲烷/甲基叔丁基醚(V:V为1:1-1:4)、二甲基亚砜/甲苯(V:V为1:1-1:4)、N,N-二甲基乙酰胺/乙酸异丙酯(V:V为1:1-1:4)、N-甲基吡咯烷酮/甲基叔丁基醚(V:V为1:1-1:4)、水/丙酮。
所述的溶剂H选自下组：甲醇、乙醇、丙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃、丙二醇、乙酸乙酯、1,4-二氧六环、2-甲基四氢呋喃，或其混合物。
在另一优选例中，所述的析晶包括悬浮搅拌析晶、浓缩析晶、过饱和析晶、蒸发析晶、挥发析晶、或其组合。
在本发明的第三方面，提供了一种药物组合物，所述药物组合物包括：
(a)本发明第一方面中任一所述的式X化合物、其药学上可接受盐、或其前药；以及(b)药学可接受的载体。
在另一优选例中，所述药物组合物还包含一种或多种用于治疗和/或预防肿瘤的其它药物。
在另一优选例中，所述其它药物为选自下组的一种或多种：吉非替尼、厄洛替尼、埃克替尼、拉帕替尼、XL647、NVP-DEE-788、DRRY-334543、EKB-569、BIBW2992、HKI272、BMS-690514、CI-1033、凡德他尼、PF00299804、WZ4002、西妥昔单抗、曲妥珠单抗、帕尼突单抗、马妥珠单抗、尼妥珠单抗、扎鲁木单抗、帕妥珠单抗、MDX-214、CDX-110、IMC-11F8、ZemDb、Her2疫苗PX 1041、HSP90抑制剂、CNF2024、坦螺旋霉素、阿螺旋霉素、IPI-504、SNX-5422、NVP-DUY922、或其组合。
在本发明的第四方面，提供了本发明第一方面中任一所述的式X化合物、其药学上可接受盐、或其前药，或第三方面所述药物组合物的用途，用于制备调控EGFR酪氨酸激酶或者治疗EGFR相关疾病的药物。
在另一优选例中，所述的EGFR相关疾病选自下组：癌症、糖尿病、免疫系统疾病、神经退行性疾病、心血管疾病、使用EGFR调节剂治疗期间具有获得性耐药性的疾病。
在另一优选例中，所述获得性耐药性的疾病是由EGFR外显子20编码的T790突变所引起的或者是包含EGFR外显子20编码的T790突变所引起的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了晶型A的XRPD图。
图2显示了晶型A的HPLC图(图2A)、晶型A的IC图(图2B)和晶型A的TGA/DSC图(图2C)。
图3显示了晶型B的XRPD图。
图4显示了晶型B的HPLC图(图4A)、晶型B的IC图(图4B)和晶型B的TGA/DSC图(图4C)。
图5显示了晶型C-1的XRPD图。
图6显示了晶型C-1的TGA/DSC图(图6A)和晶型C-1的1HNMR谱图(图6B)。
图7显示了晶型C-2的XRPD图。
图8显示了晶型C-2的TGA/DSC图。
图9显示了晶型D的XRPD图。
图10显示了晶型D的TGA/DSC图(图10A)和晶型D的1HNMR谱图(图10B)。
图11显示了晶型E的XRPD图。
图12显示了晶型E的TGA/DSC图(图12A)和晶型E的1HNMR谱图(图12B)。
图13显示了晶型I的XRPD图。
图14显示了晶型I的TGA/DSC图。
图15显示了晶型II的XRPD图。
图16显示了晶型II的TGA/DSC图。
图17显示了晶型Ⅲ的XRPD图。
图18显示了晶型Ⅲ的TGA/DSC图(图18A)和晶型Ⅲ的1HNMR谱图(图18B)。
图19显示了晶型Ⅳ的XRPD图。
图20显示了晶型Ⅳ的TGA/DSC图。
图21显示了晶型Ⅴ的XRPD图。
图22显示了晶型Ⅴ的TGA/DSC图(图22A)和晶型Ⅴ的1HNMR谱图(图22B)。
图23显示了晶型C-1在80℃下放置24小时前后的XRPD对比图。
图24显示了晶型C-1在80℃下放置24小时前后的HPLC对比图。
图25显示了晶型B的DVS图。
图26显示了晶型B在DVS测试前后的XRPD对比图。
图27显示了晶型C-1的DVS图。
图28显示了晶型E的DVS图。
图29显示了晶型II的DVS图。
图30显示了晶型C-1和晶型C-2混悬竞争的XRPD叠图。
图31显示了晶型I的DVS图。
图32显示了晶型I DVS测试前后的XRPD对比图。
图33显示了晶型II DVS测试前后的XRPD对比图。
图34显示了晶型I和II混悬竞争试验前后的XRPD对比图。
图35显示了晶型II和IV混悬竞争试验前后的XRPD对比图。
具体实施方式
发明人经过长期而深入的研究，发现了一系列式X化合物的游离碱多晶型物、盐以及盐的多晶型物。从体外酶、细胞生长抑制实验显示，式X化合物游离碱对EGFR T790M突变型酶、细胞表现出较强的抑制活性，而对EGFR野生型酶、细胞表现出较弱抑制活性，并且表现出较低的细胞毒性，因此式X化合物可作为理想的第三代EGFR突变体选择性抑制剂。研究还发现，式X化合物的一系列游离碱多晶型物、其盐以及盐的多晶型物不仅具有较好的物理化学稳定性，还具有较好的体内、体外相关药理活性，因此具有进一步开发成为药物的可能。
术语
如本文所用，术语“本发明化合物”包括本发明的式X化合物，本发明的式X化合物药学上可接受的盐以及本发明的多晶型物。
式X化合物
本发明中，“式X化合物”或“式X所示的化合物”可以互换使用，除非特别说明，一般都是指游离碱形式。
在本发明中，式X化合物为N-(2-(4-(二甲基氨基)哌啶-1-基)-5-(5-氟-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺，其对T790M突变的EGFR有较好的选择抑制活性和较低的细胞毒性，以及有利的代谢特征。
本发明中，“游离碱样品”或“游离碱”是指实施例1制备的式X化合物游离碱。
式X化合物药学上可接受的盐
在本发明中，所述的药学上可接受的盐优选自下组：盐酸盐、硫酸盐、磷酸盐、乙酸盐、L-乳酸盐、马来酸盐、富马酸盐、琥珀酸盐、L-苹果酸盐、己二酸盐、L-酒石酸盐、马尿酸盐、柠檬酸盐、粘酸盐、乙醇酸盐、葡萄糖醛酸盐、苯甲酸盐、龙胆酸盐、烟酸盐、乙二磺酸盐、草酸盐、甲磺酸盐、苯磺酸盐、羟基乙磺酸盐和氢溴酸盐。
多晶型物
固体不是以无定形的形式就是以结晶的形式存在。在结晶形式的情况下，分子定位于三维晶格格位内。当化合物从溶液或浆液中结晶出来时，它可以不同的空间点阵排列结晶(这种性质被称作“多晶型现象”)，形成具有不同的结晶形式的晶体，这各种结晶形式被称作“多晶型物”。给定物质的不同多晶型物可在一个或多个物理属性方面(如溶解度和溶解速率、真比重、晶形、堆积方式、流动性和/或固态稳定性)彼此不同。
结晶
可以通过操作溶液，使得感兴趣化合物的溶解度极限被超过，从而完成生产规模的结晶。这可以通过多种方法来完成，例如，在相对高的温度下溶解化合物，然后冷却溶液至饱和极限以下。或者通过沸腾、常压蒸发、真空干燥或通过其它的一些方法来减小液体体积。可通过加入抗溶剂或化合物在其中具有低的溶解度的溶剂或这样的溶剂的混合物，来降低感兴趣化合物的溶解度。另一种可选方法是调节pH值以降低溶解度。有关结晶方面的详细描述请参见Crystallization,第三版,J W Mullens,Butterworth-Heineman Ltd.，1993,ISBN 0750611294。
如本文所用，术语“悬浮搅拌”是指将式X化合物和相应的酸或相应酸的溶液在合适的溶剂中混合形成浑浊液，或者将式X化合物与合适的溶剂混合形成浑浊液后搅拌得到晶体的一种方法。合适的溶剂可以为水或有机溶剂。
如本文所用，术语“缓慢挥发”是指将式X化合物的溶液或含式X化合物和相应酸的溶液置于一定温度下缓慢挥发掉溶剂，得到晶体的一种方法。
如本文所用，术语“反溶剂添加”是指向式X化合物的一种溶液中加入另一种合适溶剂后析出得到晶体的一种方法。
假如期望盐的形成与结晶同时发生，如果盐在反应介质中比原料溶解度小，那么加入适当的酸或碱可导致所需盐的直接结晶。同样，在最终想要的形式比反应物溶解度小的介质中，合成反应的完成可使最终产物直接结晶。
结晶的优化可包括用所需形式的晶体作为晶种接种于结晶介质中。另外，许多结晶方法使用上述策略的组合。一个实施例是在高温下将感兴趣的化合物溶解在溶剂中，随后通过受控方式加入适当体积的抗溶剂，以使体系正好在饱和水平之下。此时，可加入所需形式的晶种(并保持晶种的完整性)，将体系冷却以完成结晶。
如本文所用，术语“室温”一般指4-30℃，较佳地指20±5℃。
本发明的多晶型物
如本发明所用，“本发明的晶体”、“本发明的晶型”、“本发明的多晶型物”等可互换使用，包括本发明的式X化合物的多晶型物和本发明的式X化合物药学上可接受的盐的多晶型物。
本发明中，“式X化合物的多晶型物”和“式X化合物游离碱的多晶型物”等可互换使用。
如本文所用，术语“本发明的多晶型物”包括式X化合物游离碱或式X化合物药学上可接受的盐(如盐酸盐、磷酸盐、马来酸盐、L-苹果酸盐、己二酸盐、硫酸盐、富马酸盐、琥珀酸盐、L-酒石酸盐、柠檬酸盐、甲磺酸盐、苯甲酸盐、和苯磺酸盐)，或式X化合物各种溶剂合物的多晶型物，还包括相同的盐(如盐酸盐、磷酸盐、马来酸盐、L-苹果酸盐、己二酸盐、硫酸盐、富马酸盐、琥珀酸盐、L-酒石酸盐、柠檬酸盐、甲磺酸盐、苯甲酸盐、和苯磺酸盐)或溶剂合物的不同多晶型物。
优选的本发明多晶型物包括(但并不限于)：
(i)晶型A、B、C(包括C-1、C-2)、D、E(盐的晶型)；
(ii)晶型I、II、III、IV、V(式X化合物的晶型)。
在本发明中，某些晶型可以互相转化，因此本发明还提供了部分晶型互相转化的方法。
多晶型物的鉴定和性质
可采用已知的方法或仪器对式X化合物或药学上可接受盐的多晶型物进行表征，例如，采用如下多种方式和仪器。
X射线粉末衍射
测定晶型的X射线粉末衍射的方法在本领域中是已知的。例如使X射线粉末衍射仪，以2°每分钟的扫描速度，采用铜辐射靶获取图谱。
本发明的式X化合物或其药学上可接受的盐的多晶型物，具有特定的晶型形态，在X-射线粉末衍射(XRPD)图中具有特定的特征峰。
示差扫描量热分析
又称“差示量热扫描分析”(DSC)，是在加热过程中，测量被测物质与参比物之间的能量差与温度之间关系的一种技术。DSC图谱上的峰位置、形状和峰数目与物质的性质有关，故可以定性地用来鉴定物质。本领域常用该方法来检测物质的相变温度、玻璃化转变温度、反应热等多种参数。
式X化合物的药物组合物及其应用
通常，本发明式X化合物或其药学可接受的盐可以与一种或多种药用载体形成适合的剂型施用。这些剂型适用于口服、直肠给药、局部给药、口内给药以及其他非胃肠道施用(例如，皮下、肌肉、静脉等)。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明的化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。
本发明的组合物以符合医学实践规范的方式配制，定量和给药。给予化合物的“有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。
本发明提供了本发明第一方面所述的化合物，或其药学上可接受的盐、或其前药可用于制备调控EGFR酪氨酸激酶活性或治疗EGFR相关疾病的药物中的应用。
作为优选，所述EGFR相关疾病为癌症，糖尿病，免疫系统疾病，神经退行性疾病或心 血管疾病。
作为优选，所述癌症为非小细胞肺癌、头颈癌、乳腺癌、肾癌、胰腺癌、子宫颈癌、食道癌、胰腺癌、前列腺癌、膀胱癌、结肠直肠癌、卵巢癌、胃癌、脑恶性肿瘤包括成胶质细胞瘤等，或它们的任何组合。
作为优选，所述非小细胞肺癌是由EGFR突变引起的，包括敏感型突变(如L858R突变或外显子19缺失)和耐药性突变(如EGFR T790M突变)。
作为优选，所述的化合物，或其药学上可接受的盐、或其前药还可用于制备治疗EGFR异常表达的疾病或在使用EGFR调节剂治疗期间具有获得性耐药性的疾病的药物中的应用。
作为优选，所述获得性耐药性是由EGFR外显子20编码的T790突变引起的或者是包含EGFR外显子20编码的T790突变所引起的耐药性，如T790M。
在本发明中，EGFR调节剂是指靶向EGFR的小分子酪氨酸激酶抑制剂，如吉非替尼、厄洛替尼、埃克替尼、拉帕替尼或阿法替尼。
本发明还提供了一种药用组合物，其包括治疗有效量的本发明第一方面所述的化合物，或其药学上可接受的盐、或其前药，以及选自下组药物中的一种或多种其它药物：吉非替尼、厄洛替尼、埃克替尼、拉帕替尼、XL647、NVP-AEE-788、ARRY-334543、EKB-569、BIBW2992、HKI272、BMS-690514、CI-1033、凡德他尼、PF00299804、WZ4002、西妥昔单抗、曲妥珠单抗、帕尼突单抗、马妥珠单抗、尼妥珠单抗、扎鲁木单抗、帕妥珠单抗、MDX-214、CDX-110、IMC-11F8、Zemab、Her2疫苗PX 1041、HSP90抑制剂、CNF2024、坦螺旋霉素、阿螺旋霉素、IPI-504、SNX-5422、NVP-AUY922、或其组合。除本发明的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药以外，上述药用组合物中的其它药物均为本领域技术人员熟知的抗肿瘤药物。
如本文所用，“治疗有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
本发明的药物组合物或所述药用组合物中含有的本发明化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药的治疗有效量优选为0.1mg-5g/kg(体重)。
本发明的药用组合物可用于治疗EGFR相关疾病，如癌症、糖尿病、免疫系统疾病、神经退行性疾病或心血管疾病，或使用EGFR调节剂治疗期间具有获得性耐药性的疾病。
所述获得性耐药性的疾病是由EGFR外显子20编码的T790突变所引起的，或者是包含EGFR外显子20编码的T790突变所引起的。
在另一优选例中，所述的EGFR外显子20编码的T790为T790M。
本发明所述的化合物或其药学上可接受的盐在某些疾病中可以与其它药物联合应用，以达到预期的治疗效果。一个联合应用的例子是用来治疗晚期NSCLC。例如，将治疗有效量的本发明式X所示化合物与mTOR抑制剂联用(例如雷帕霉素)；或与Met抑制剂(包括Met抗体MetMAb和Met小分子抑制剂PF02341066)联用；或与IGF1R抑制剂联用(例如OSI-906)；或与热休克蛋白抑制剂联用等。
本发明的主要优点在于：
式X化合物或其药学上可接受的盐的多晶型物具有优异的物理化学稳定性和突出的相关药理活性，是理想的第三代EGFR突变体选择性抑制剂。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
试剂与仪器
化合物的结构和纯度通过核磁共振(1HNMR)和/或液质联用质谱(LC-MS)来确定。1HNMR：BrukerAVANCE-400核磁仪，内标为四甲基硅烷(TMS)。LC-MS：Agilent 1200HPLC System/6140MS液质联用质谱仪(购自安捷伦)，柱子WatersX-Bridge,150×4.6mm,3.5μm。制备高效液相色谱(pre-HPLC)：用Waters PHW007，柱子XBridge C18,4.6*150mm,3.5um。
采用ISCO Combiflash-Rf75或Rf200型自动过柱仪，Agela 4g、12g、20g、40g、80g、120g一次性硅胶柱。
已知的起始原料可以采用或按照本领域已知的方法来合成，或可以于ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)和达瑞化学品等公司处购买。
实施例中无特殊说明，反应均在氮气或氩气氛下进行。实施例中无特殊说明，溶液是指水溶液。
如本文所用，DMF表示二甲基甲酰胺，DMSO表示二甲基亚砜，THF表示四氢呋喃，DIEA表示N,N-二异丙基乙胺，EA表示乙酸乙酯，PE表示石油醚。BINAP表示(2R,3S)-2,2’-双二苯膦基-1,1’-联萘，NBS表示N-溴代丁二酰亚胺，NCS表示N-氯代丁二酰亚胺，Pd2(dba)3表示三(二亚苄基丙酮)二钯，Pd(dppf)Cl2表示[1,1’-双(二苯基磷)二茂铁]二氯化钯。
如本文所用，室温指的是约25℃。
通用方法
粉末X衍射图谱是通过本领域的已知方法，使用PANalytacal Empyrean X射线粉末衍射分析仪获得。仪器测试条件如下表i所示：
表i
Figure PCTCN2016091591-appb-000018
Figure PCTCN2016091591-appb-000019
在粉末X衍射图中，各峰的位置由2θ(°)确定。可以理解，不同的仪器和/或条件可导致产生的数据会略有不同，各峰的位置和相对强度会有变化。峰的强度划分仅仅反映了各位置上峰的近似大小。在本发明中，各晶型均以其峰高最高的衍射峰作为基峰，定义其相对强度为100％，作为I0(如晶型Ⅰ的2θ(°)值为21.38的峰为基峰，晶型II的2θ(°)值为9.47的峰为基峰，晶型Ⅲ的2θ(°)值为23.89的峰为基峰，晶型Ⅳ的2θ(°)值为7.69的峰为基峰，晶型Ⅴ的2θ(°)值为8.89的峰为基峰，晶型A的2θ(°)值为23.31的峰为基峰，晶型B的2θ(°)值为19.92的峰为基峰，晶型C-1的2θ(°)值为25.55的峰为基峰，晶型C-2的2θ(°)值为7.48的峰为基峰，晶型D的2θ(°)值为22.69的峰为基峰，晶型E的2θ(°)值为21.64的峰为基峰)，其它各峰以其峰高与基峰峰高的比值作为其相对强度I/I0，各峰相对强度的划分定义如下表ii所示：
表ii
相对强度I/I0(％) 定义
50～100 VS(很强)
20～50 S(强)
5～20 M(中等)
1～5 W(弱)
本发明的盐或其晶型通过HPLC/IC或1H NMR确定酸碱摩尔比。
高效液相色谱(HPLC)在Agilent 1100/1260HPLC上采集。
TGA和DSC图谱：TGA和DSC图谱分别在TA Q500/5000热重分析仪和TA Q200/2000差示扫描量热仪上采集。仪器测试条件如下表iii所示：
表iii
参数 TGA DSC
方法 线性升温 线性升温
样品盘 铂金盘，敞开 铝盘，压盖
温度范围 室温-设定温度 25℃-设定温度
扫描速率(℃/分钟) 10 10
保护气体 氮气 氮气
动态水分吸附(DVS)曲线：在SMS(Surface Measurement Systems)的DVS Intrinsic上采集。在25℃时的相对湿度用LiCl，Mg(NO3)2和KCl的潮解点校正。仪器测试条件如下表iv所示：
表iv
Figure PCTCN2016091591-appb-000020
含水量：使用万通870卡氏水分测定仪测试，使用滴定试液规格为市售Sigma-aldrich的
Figure PCTCN2016091591-appb-000021
R-Composite5(34805-1L-R,Batch#SZBD3330V)，使用分析纯的MeOH作溶剂。水分测定前用高纯水进行校正。
可以理解的是，使用与上述仪器作用相同的其他类型的仪器或使用不同与本发明中使用的测试条件时，可能会得到另外的数值，因此，所引用的数值不应视为绝对的数值。
由于仪器的误差或操作人员的区别，本领域技术人员能理解，以上用于表征晶体的物理性质的参数可能有微小的差别，所以上述的参数仅用于辅助表征本发明提供的多晶型物，而不能视为是对本发明的多晶型物的限制。
中间体的制备
化合物1a的制备
Figure PCTCN2016091591-appb-000022
步骤a:将化合物1a1(10.6g,58mmol)置于500mL反应瓶中，加入THF/水(100mL/60mL)混合溶液溶解。在室温下，边搅拌边依次加入氯化铵(15.5g,292mmol)和还原铁粉(26g,467mmol)，随后将反应体系加热至65℃并持续搅拌3h。TLC检测反应进度，待反应完成后，过滤除去多余铁粉，滤饼用EA淋洗三次。滤液用EA/水体系萃取三次，分离出有机层，经水洗，饱和食盐水洗涤，无水Na2SO4干燥后，减压浓缩得到化合物1a2(8.0g)，直接用于下一步反应。产率：93％；纯度：90％；MS m/z(ESI):142.0[M+H]+。
步骤b:将化合物1a2(8.0g,43mmol)置于500mL反应瓶中，匀速搅拌下加入浓硫酸(100 mL)使底物溶解。在零下20℃下，边搅拌边缓慢滴加浓硝酸(6.15mL,48mmol)，并保温搅拌5min。TLC检测反应进度，待反应完成后，倒入冰水中。保持零下20℃冰浴，向反应体系中缓慢加入氢氧化钠/水溶液(150mL/300mL)，调节pH值至8-9。然后反应液用EA/水体系萃取三次，分离出有机层，经水洗，饱和食盐水洗涤，无水Na2SO4干燥后，减压浓缩得到化合物1a(8.7g)，直接用于下一步反应。产率：80％；纯度：100％；MS m/z(ESI)：187.0[M+H]+；1H NMR(400MHz,DMSO-d6):δ7.34(d,J＝7.8Hz,1H),7.04(d,J＝13.4Hz,1H),5.25(brs,2H),3.90(s,3H)。
中间体2a的制备
Figure PCTCN2016091591-appb-000023
步骤1：将化合物2a1(500g,2.51mol)和二甲胺盐酸盐(244g,3.01mol)加入到甲醇(2.2L)中，加入三乙胺(508g,5.03mol)和10％钯碳(50g)，然后混合体系在氢气(15kg，40℃)反应24小时，LCMS检测原料反应完全，过滤，滤液减压蒸干，得到油状粗产物化合物2a2(542g)，不用提纯可以直接投入下一步。MS m/z(ESI)：228[M-H]。
步骤2：将化合物2a2(542g,2.38mol)溶解于甲醇(1.5L)中，缓慢并分批加入浓盐酸(1L),加完，混合体系在50℃反应2小时，减压蒸干，得到固体粗产物，将其在甲醇(2L)中打浆1小时，过滤得到白色的固体2a(389g，80％)。1H NMR(400MHz,DMSO)δ3.47(m,3H),2.97(m,2H),2.70(s,6H),2.22(m,2H),1.87(m,2H)。
中间体3a的制备
Figure PCTCN2016091591-appb-000024
步骤1：将化合物1a(11.16g，60mmol)溶于150mL二氯甲烷，加入二碳酸二叔丁酯(15.60g，72mmol)、三乙胺(12.24g，120mmol)和4-二甲氨基吡啶(0.74g，6mmol)室温下搅拌反应18h。TLC检测反应进度，待反应完成后，减压浓缩反应液，柱层析[PE:EA＝80:20]分离纯化得到目标产物化合物3a2(12.56g，73％)。MS m/z(ESI):285[M-H]+。
步骤2：将化合物3a2(11.46g，40mmol)溶于60mL N,N-二甲基乙酰胺，加入化合物2a(9.6g， 48mmol)，N,N-二异丙基乙基胺(7.74g，60mmol)，加热至90℃搅拌反应6h。TLC检测反应进度，待反应完成后，将反应液冷却至室温，倒入冰水中，用乙酸乙酯萃取，饱和食盐水洗涤，无水Na2SO4干燥，减压浓缩得目标产物化合物3a3(13.43g，85％)。直接用于下一步反应。MS m/z(ESI):395[M+H]+。
步骤3：将化合物3a3(12.8g，32.6mmol)溶于200mL甲醇，加入1.0g 10％Pd/C。以氢气置换空气后，于室温下以氢气球加氢，搅拌反应1h。TLC检测反应进度，待反应完成后，经砂芯漏斗抽滤，以少量甲醇洗涤滤饼，将滤液浓缩得到目标产物化合物3a4(11.54g，97％)。直接用于下一步反应。MS m/z(ESI):365[M+H]+。
步骤4：将化合物3a4(10.95g，30mmol)和三乙胺(6.12g，60mmol)溶于200mL二氯甲烷，冷却至0℃，加入丙烯酰氯(3.24g，36mmol)，在氮气保护下，室温搅拌3h。TLC检测反应进度，待反应完成后，以饱和碳酸氢钠水溶液、饱和食盐水顺次洗涤，无水Na2SO4干燥，抽滤，经减压浓缩得到化合物3a5(10.3g，82％)。直接用于下一步反应。MS m/z(ESI):419[M+H]+。
步骤5：将化合物3a5(10g，24mmol)溶解在100mL二氯甲烷中，冷却到0℃，加入20mL三氟乙酸，在氮气保护下室温搅拌18h。TLC检测反应进度，待反应完成后，减压浓缩反应液。将残留物以300mL二氯甲烷溶解，用饱和碳酸氢钠水溶液、饱和食盐水顺次洗涤，无水Na2SO4干燥，抽滤，减压浓缩得到粗品。经柱层析[DCM:MeOH＝10:1]纯化得到目标产物化合物3a(3.54g，46.5％)。MS m/z(ESI):319[M+H]+。
实施例1式X化合物的制备
Figure PCTCN2016091591-appb-000025
步骤1：在氮气气氛下，将1-甲基-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吡唑(1.1g，5.25mmol)、化合物Ⅰ-1(664mg，4mmol)、Pd(dppf)Cl2(109.7mg，0.15mmol)和碳酸钠溶液(5mL，2M)的混合物的乙腈(30mL)溶液在85℃下搅拌6h。待反应完成后，加水淬灭，向反应混合物中加入EA(150ml)分层后，水相用EA(50mL×2)萃取两次，合并的有机相用Na2SO4干燥后浓缩，得到的粗产品经Combi-flash柱层析[PE:EA＝5:1-2:1]分离纯化得0.73g目标产物Ⅰ-2。MS m/z(ESI)：213[M+H]+。
步骤2：向化合物3a(159mg，0.5mmol)、化合物Ⅰ-2(106mg，0.5mmol)、Pd2(dba)3(45.75mg，0.05mmol)和Xantphos(28.4mg，0.05mmol)的1,4-二氧六环(4ml)溶液中加入碳酸铯(487.5mg，0.5mmol)，反应化合物在100℃下搅拌3h后，向反应混合物中加入EA(50ml×2)和水(10ml)， 有机相用水(10ml×3)洗涤后经Na2SO4干燥，浓缩得到粗产品，将所得粗产品经制备液相分离纯化得式X化合物(22mg，9％)。
MS m/z(ESI):495.0[M+H]+；1HNMR(400MHz，DMSO-d6)δ9.05(1s,1H),9.01(1s,1H),8.80(s,1H),8.48(d,J＝4Hz,1H),8.18(s,1H),7.89(s,1H),6.87(s,1H),6.73(m,1H),6.32(m,1H),5.78(t,1H),3.97(s,3H),3.88(s,3H),3.01(d,J＝12Hz,2H),2.66(t,2H),2.19(s,6H),2.16(m,1H),1.84(m,2H),1.71(m,2H)。
对比例
对比化合物2和3分别以相应的硼酯和5位甲氧基取代或未取代的2,4-二氯嘧啶为原料，参照式X化合物的类似方法制备，如表v所示。
表v
Figure PCTCN2016091591-appb-000026
实施例2式X化合物游离碱起始样品的制备
将55.8g式X化合物加入到230ml乙醇中，回流搅拌溶解，回流下加入160ml水，反应液变浑浊，再加入10ml乙醇使反应液变澄清后停止加热，反应液缓慢冷却至室温，体系析出大量黄色固体，过滤，用水洗涤，真空干燥得黄色粉末52.8g，收率94.7％。
实施例3式X化合物晶型A的制备
将盐酸的乙腈(0.2mol/L，3.3ml)溶液和乙腈(2.0mL)加入到装有300mg式X化合物游离碱起始样品的反应瓶中，形成浑浊液，15℃下磁力搅拌约43小时，转速约1000转/分。真空抽滤，分离出固体。50℃真空干燥约4小时后收集得到黄色结晶性粉末245mg，收率：81.7％。所得结晶的粉末X衍射图如图1所示(2θ角已标出)，结晶的HPLC和IC图分别如图2A和2B中所示，酸碱摩尔比为0.9:1，TGA/DSC谱图如图2C所表征；熔点为261℃-265℃。在本申请中定义该晶型为晶型A。
实施例4式X化合物晶型B的制备
将磷酸的乙腈(0.2mol/L，3.3ml)溶液和乙腈(1.0mL)加入到装有300mg式X化合物游离碱起始样品的反应瓶中，形成浑浊液，15℃下磁力搅拌约43小时，转速约1000转/分。真空 抽滤，分离出固体。50℃真空干燥约4小时后收集得到黄色结晶性粉末280mg，收率：93.4％。所得结晶的粉末X衍射图如图3所示，结晶的HPLC和IC图分别如图4A和4B中所示，酸碱摩尔比为0.9:1，TGA/DSC谱图如图4C所表征；熔点为231℃-237℃。在本申请中定义该晶型为晶型B。
实施例5式X化合物晶型C-1的制备
将70.2mg马来酸和乙腈(10.0mL)加入到装有300mg式X化合物游离碱起始样品的反应瓶中，形成浑浊液，15℃下磁力搅拌约28小时，转速约1000转/分。真空抽滤，分离出固体。50℃真空干燥约24小时后收集得到黄色结晶性粉末286.2mg，收率：95.4％。所得结晶的粉末X衍射图如图5所示，TGA/DSC谱图如图6A所表征；1HNMR谱图如图6B所示，酸碱摩尔比为1:1，熔点为212℃-216℃。在本申请中定义该晶型为晶型C-1。
实施例6式X化合物晶型C-1的制备
将式X化合物游离碱起始样品(5.50g，11.1mmol)溶于乙醇(16.5mL)中，升温至70℃，搅拌下加入马来酸(1.55g，13.3mmol)，反应液温度控制在70℃搅拌30min，析出大量固体，停止加热，搅拌下自然降温冷却至5℃，冷却时间为2小时，冷却后再搅拌1h，过滤，滤饼用乙醇淋洗，50℃下鼓风干燥2h，80℃鼓风干燥6h，得黄色结晶性粉末6.05g，收率89.1％。所得结晶的粉末X衍射图如图5所示，TGA/DSC谱图如图6A所表征；1HNMR谱图如图6B所示，酸碱摩尔比为1:1，熔点为212℃-216℃。在本申请中定义该晶型为晶型C-1。
实施例7式X化合物晶型C-2的制备
将1.5ml的DCM加入到装有10.1mg晶型C-1的反应瓶中，溶解后15℃下用封口膜将瓶口密封，用针头刺3-4个小孔后放置在通风橱中自然缓慢挥发，10天后样品挥干，得黄色结晶性粉末9mg，收率89.1％。所得结晶的粉末X衍射图如图7所示，酸碱摩尔比为1:1，TGA/DSC谱图如图8所表征；熔点为210℃-214℃。在本申请中定义该晶型为晶型C-2。
实施例8式X化合物晶型D的制备
将81.3mg L-苹果酸和乙腈(8.0mL)加入到装有300mg式X化合物游离碱起始样品的反应瓶中，形成浑浊液，15℃下磁力搅拌约28小时，转速约1000转/分。真空抽滤，分离出固体。50℃真空干燥约24小时后收集得到黄色结晶性粉末253mg，收率：84.4％。所得结晶的粉末X衍射图如图9所示，1HNMR谱图如图10B所示，酸碱摩尔比为1.3:1，TGA/DSC谱图如图10A所表征；熔点为202℃-207℃。在本申请中定义该晶型为晶型D。
实施例9式X化合物晶型E的制备
将88.5mg己二酸和乙腈(8.0mL)加入到装有300mg式X化合物游离碱起始样品的反应瓶 中，形成浑浊液，15℃下磁力搅拌约28小时，转速约1000转/分。真空抽滤，分离出固体。50℃真空干燥约24小时后收集得到黄色结晶性粉末278mg，收率：92.8％。所得结晶的粉末X衍射图如图11所示，1HNMR谱图如图12B所示，酸碱摩尔比为1.1:1，TGA/DSC谱图如图12A所表征；熔点为204℃-206℃。在本申请中定义该晶型为晶型E。
实施例10式X化合物游离碱晶型Ⅰ的制备
将异丙醇(0.5mL)加入到装有15mg式X化合物游离碱起始样品的反应瓶中，形成浑浊液，15℃下磁力搅拌约5天，转速约1000转/分。离心分离出固体。(离心机转速：10000转/分；时间：3分钟)。15℃下放置在通风橱内晾干，收集得到黄色结晶性粉末10mg，收率：66.7％。所得结晶的粉末X衍射图如图13所示，TGA/DSC谱图如图14所表征；熔点为160℃-163℃。在本申请中定义该晶型为晶型Ⅰ。
实施例11式X化合物游离碱晶型Ⅰ的制备
将1,4-二氧六环(1.5mL)加入到装有10mg式X化合物游离碱起始样品的反应瓶中，形成澄清液，在15℃下用封口膜封住反应瓶口，用针头刺3-4个小孔后放置在通风橱中自然缓慢挥发。约7天后样品挥干，收集得到黄色结晶性粉末8.7mg，收率：87％。所得结晶的粉末X衍射图如图13所示，TGA/DSC谱图如图14所表征；熔点为160℃-163℃。在本申请中定义该晶型为晶型Ⅰ。
实施例12式X化合物游离碱晶型II的制备
将丙酮(0.4mL)加入到装有10.1mg式X化合物游离碱起始样品的反应瓶中，得澄清溶液，15℃下边磁力搅拌边向瓶中逐滴添加反溶剂H2O，加至1.0mL后出现浑浊，离心分离出固体。(离心机转速：10000转/分；时间：3分钟)。15℃下放置在通风橱内晾干，收集得到黄色结晶性粉末8mg，收率：79.2％。所得结晶的粉末X衍射图如图15所示，TGA/DSC谱图如图16所表征；熔点为160℃-163℃。在本申请中定义该晶型为晶型II。
实施例13式X化合物游离碱晶型II的制备
将乙酸乙酯(0.5mL)加入到装有15mg式X化合物游离碱起始样品的反应瓶中，形成浑浊液，15℃下磁力搅拌约5天，转速约1000转/分。离心分离出固体。(离心机转速：10000转/分；时间：3分钟)。15℃下放置在通风橱内晾干，收集得到黄色结晶性粉末13mg，收率：86.7％。所得结晶的粉末X衍射图如图15所示，熔点为160℃-163℃。在本申请中定义该晶型为晶型II。
晶型II的TGA/DSC图如图16所示，TGA图中显示样品在加热到约80℃失重约为7.2％，DSC图显示在158℃-162℃(起始温度)熔化前有一个较宽的吸热峰(峰值温度范围为98℃-102℃)，Karl Fischer测试(水分测试)结果显示，晶型II中含有7.2％的水分，与该样品TGA表征结 果(图16)中分解前的失重相似。将晶型II加热到140℃后冷却至室温，XRPD图显示，晶型II加热冷却后转变成无水晶型I。因此晶型II是以水合物形式存在，更佳地以二水合物形式存在。(游离碱二水合物的理论失重为6.8％)。
实施例14式X化合物游离碱晶型Ⅲ的制备
将甲醇(0.5mL)加入到装有40mg式X化合物游离碱起始样品的反应瓶中，形成浑浊液，15℃下磁力搅拌约47小时，转速约1000转/分。离心分离出固体。(离心机转速：10000转/分；时间：3分钟)。15℃下放置在通风橱内晾干，收集得到黄色结晶性粉末36mg，收率：90％。所得结晶的粉末X衍射图如图17所示，熔点为160℃-162℃。在本申请中定义该晶型为晶型Ⅲ。
实施例15式X化合物游离碱晶型Ⅲ的制备
将10mg式X化合物游离碱起始样品装入3.0-mL反应瓶中，将甲醇(4mL)加入20-mL玻璃瓶中，将装有样品的3.0-mL小瓶敞口置于20-mL大瓶中，瓶盖盖紧后放在通风橱内静置。约7天后取出3.0-mL小瓶，收集得到黄色结晶性粉末7.8mg，收率：78％。所得结晶的粉末X衍射图如图17所示，熔点为160℃-162℃。在本申请中定义该晶型为晶型Ⅲ。
晶型Ⅲ的TGA/DSC图如图18A所示，TGA图中显示样品在加热到约130℃前失重约为6.4％，DSC图显示在158℃-162℃(起始温度)熔化前有多个吸热峰，将晶型Ⅲ加热到130℃再冷却至室温后，XRPD图显示，晶型Ⅲ转变成无水晶型I。晶型Ⅲ的1H NMR结果(图18B)显示，甲醇含量为4.9％。因此晶型Ⅲ是以甲醇合物形式存在。
实施例16式X化合物游离碱晶型Ⅳ的制备
将丙酮(1.5mL)加入到装有10.4mg式X化合物游离碱起始样品的反应瓶中，形成澄清液，在15℃下用封口膜封住反应瓶口，用针头刺3-4个小孔后放置在通风橱中自然缓慢挥发。约6天后样品挥干，收集得到黄色结晶性粉末9.3mg，收率：89.4％。所得结晶的粉末X衍射图如图19所示，熔点为160℃-162℃。在本申请中定义该晶型为晶型Ⅳ。
实施例17式X化合物游离碱晶型Ⅳ的制备
将10mg式X化合物游离碱起始样品装入3.0-mL反应瓶中，将四氢呋喃(0.5mL)加入反应瓶中，得到澄清溶液。再量取4.0mL反溶剂正己烷加至20-mL玻璃瓶中。将装有样品的3.0-mL小瓶敞口置于20-mL大瓶中，瓶盖盖紧后放在通风橱内静置。约4天后3.0-mL小瓶中析出固体，取出小瓶，在空气中晾干后收集得到黄色结晶性粉末8.7mg，收率：87％。所得结晶的粉末X衍射图如图19所示，熔点为160℃-162℃。在本申请中定义该晶型为晶型Ⅳ。
晶型Ⅳ的TGA/DSC图如图20所示，TGA图中显示样品在加热到约150℃前失重约为5.8％，DSC图显示在158℃-162℃(起始温度)熔化前有一个吸热峰(峰值温度范围为105℃-109 ℃)，XRPD图显示，将晶型Ⅳ加热至130℃再冷却至室温后转变成晶型I，晶型Ⅳ也是以水合物形式存在。
实施例18式X化合物游离碱晶型Ⅴ的制备
将乙酸乙酯(5mL)加入到装有100mg式X化合物游离碱起始样品的反应瓶中，得澄清溶液，将该澄清液用0.22μm的尼龙滤头过滤至新的5.0-mL玻璃小瓶中。在21℃下用封口膜将小瓶封口，用针头刺3-4个小孔后放置在通风橱中自然挥发，约6天后样品挥干，收集得到黄色结晶性粉末93mg，收率：93％。所得结晶的粉末X衍射图如图21所示，熔点为151℃-155℃。在本申请中定义该晶型为晶型Ⅴ。
实施例19式X化合物游离碱晶型Ⅴ的制备
将10mg式X化合物游离碱起始样品装入3.0-mL反应瓶中，将丙酮(0.5mL)加入反应瓶中，得到澄清溶液。再量取4.0mL反溶剂正己烷加至20-mL玻璃瓶中。将装有样品的3.0-mL小瓶敞口置于20-mL大瓶中，瓶盖盖紧后放在通风橱内静置。约4天后3.0-mL小瓶中析出固体，取出小瓶，在空气中晾干后收集收集得到黄色结晶性粉末8.4mg，收率：84％。所得结晶的粉末X衍射图如图21所示，熔点为151℃-155℃。在本申请中定义该晶型为晶型Ⅴ。
晶型Ⅴ的TGA/DSC图如图22A所示，TGA图中显示样品在加热到约130℃到160℃之间有约为0.7％的失重，与该样品由1HNMR(图22B)检测到的EtOAc含量一致。DSC图显示在149℃-153℃(起始温度)熔化前没有明显的吸热或放热峰，XRPD图显示，晶型V在室温下异丙醇中搅拌约24小时后可转变成晶型I，晶型Ⅴ的1H NMR结果(图22B)显示晶型Ⅴ以乙酸乙酯溶剂合物形式存在。
实施例20式X化合物各类盐的制备方法
称取与10mg游离碱摩尔比为1:1的相应的固体酸至反应瓶中，盐酸、硫酸、磷酸和氢溴酸配制成0.2mol/L相应溶剂的溶液按体积量取。再分别配制10mg/mL的式X化合物游离碱在四种相应溶剂(异丙醇,乙腈,乙酸乙酯，丙酮：水(19:1，v/v))中的澄清液，并分别量取1.0mL的澄清液至装有相应的酸的反应瓶中，在一定温度下磁力搅拌，转速约1000转/分。反应液析出固体后，离心分离，50℃真空干燥约1小时后收集固体，得到相应酸的盐。其中，L-乳酸和苯磺酸在使用丙酮和水的混合溶剂时，在搅拌前在澄清液中加入适量正己烷溶剂。粘酸在四种溶剂下得到的是盐与酸的混合物。各类酸相应的反应温度和时间如下表1所示：
表1
Figure PCTCN2016091591-appb-000027
Figure PCTCN2016091591-appb-000028
N/A：未得到固体。
表1(续)
Figure PCTCN2016091591-appb-000029
Figure PCTCN2016091591-appb-000030
实施例21式X化合物各类盐的制备方法
向含300mg式X化合物的A溶液中加入相应酸(酸与式X化合物的摩尔比为1.05:1)的B溶液，在一定的反应温度下，反应一段时间后，过滤反应液，用溶剂洗涤滤饼，真空干燥滤饼后得到相应的盐。反应原来的投料和反应条件如下表2所示。
表2
Figure PCTCN2016091591-appb-000031
Figure PCTCN2016091591-appb-000032
各个酸的物理性质如下表3所示：
表3
盐 固体形态 熔点(℃)
富马酸盐 淡黄色固体 214.3-215.4
L-苹果酸盐 淡黄色固体 188.4-191.7
L-酒石酸盐 黄色固体 191.3-193.1
柠檬酸盐 淡黄色固体 168.4-172.0
乙醇酸盐 淡黄色固体 NA
苯甲酸盐 黄色固体 181.0-182.7
烟酸盐 淡黄色固体 NA
草酸盐 黄色固体 227.4-228.9
苯磺酸盐 淡黄色固体 213.0-214.0
乙酸盐 淡黄色固体 139.5-144.2
马来酸盐 淡黄色固体 201.5-202.3
琥珀酸盐 淡黄色固体 183.9-184.8
己二酸盐 淡黄色固体 195.2-196.2
马尿酸盐 淡黄色固体 NA
粘酸盐 淡黄色固体 NA
D-葡萄糖醛酸盐 黄色固体 NA
龙胆酸盐 淡黄色固体 227.9-228.9
乙二磺酸盐 黄色固体 NA
甲磺酸盐 黄色固体 NA
2-羟基乙磺酸盐 黄色固体 NA
实施例22固态稳定性实验
分别称取适量样品在25℃/60％RH和40℃/75％RH条件下敞口放置，同时另将一组样品放在5℃条件下密封保存作为对照。一周后用XRPD、HPLC和TGA对所有样品进行表征，分别检测晶型、纯度和失重变化。测试结果如下表4所示：
表4
Figure PCTCN2016091591-appb-000033
*：将一组样品放在5℃下保存作为评估对照。相对纯度：与对照组比较
式X化合物上述几种盐的晶型、游离碱起始样品和HPLC纯度均没有发生明显变化，表明式X化合物上述几种盐的晶型都具有较好的物理和化学稳定性。TGA测试结果显示，上述几种盐的晶型在25℃/60％RH和40℃/75％RH保存一周后失重变化较小，进一步验证了式X化合物上述几种盐晶型较好的物理稳定性。
将晶型C-1在80℃下放置24小时，收集样品进行XRPD和HPLC表征。根据图23和图24，晶型C-1在测试前后晶型和HPLC纯度均没有发生变化，表明晶型C-1具有较好的物理和化学稳定性。
实施例23动态溶解度实验
室温下对式X化合物盐晶型和游离碱起始样品在水中和三种生物溶媒：模拟胃液(SGF)、模拟禁食状态肠液(FaSSIF)、模拟喂食状态肠液(FeSSIF)的动态溶解度进行了测试。试验中，分别将20毫克固体与2毫升溶媒在4毫升离心管中混合，再将离心管密封固定在转速为25转/分钟的旋转盘上。一小时后，观察到晶型B、晶型C-1、晶型D和晶型E样品在四种溶媒中完全溶清，分别取样测试了准确的浓度和pH值；当平衡24小时后，清样中均未观察到固体析出，取清液测试HPLC纯度。由结果可知，分散24小时后，所有样品均无降解，亦无析出现象。结果如表5所示。
表5
Figure PCTCN2016091591-appb-000034
*：固体分散1小时后测的溶解度(毫克/毫升)和pH。
**：固体分散24小时后观察的现象。
N/A：由于分离固体后剩余上清液较少，pH数据未采集。
晶型B、晶型C-1、晶型D和晶型E在三种生物溶媒和水中均具有较好的溶解度，与游离碱起始样品相比，盐的晶型在水和FaSSIF的溶解度显著提高(>8毫克/毫升)。
实施例24引湿性实验
将晶型C-1和晶型E预先在0％RH条件下干燥去除吸附的溶剂或水后开始测试，晶型B从40％RH开始测试。
晶型B的DVS测试结果如图25所示，在25℃时当湿度降低到20％RH后，样品失重速率增加，DVS测试前后样品的XRPD对比见图26，表明晶型B在DVS测试前后晶型不变。
晶型C-1和晶型E的DVS测试结果分别如图27和图28所示。在25℃条件下当湿度增加到80％RH时，晶型C-1和晶型E分别吸收了0.3％和0.5％的水分，表明两种无水晶型均略有引湿性。两种晶型DVS测试前后样品的晶型不变。
实施例25马来酸盐溶解度实验
室温条件下，将适量实施例20条件2制备的马来酸盐加入容量瓶中，少量多次加入单一有机溶剂或混合溶剂，直至目测溶液澄清或无固体颗粒存在，以此测定马来酸盐在不同溶剂中的粗略溶解度，结果如下表6所示。
表6
溶剂 溶解度S(毫克/毫升) 溶剂 溶解度S(毫克/毫升)
甲醇 7.0<S<10.5 1,4-二氧六环 S<1.0
乙醇 S<1.1 乙腈 1.4<S<1.5
异丙醇 S<0.9 二氯甲烷 4.6<S<5.8
丙酮 S<1.1 醋酸 3.8<S<4.8
甲基异丁基酮 S<0.9 正己烷 S<1.0
乙酸乙酯 S<0.9 甲苯 S<1.1
乙酸异丙酯 S<1.0 二甲基亚砜 S>22.0
甲基叔丁基醚 S<1.0 N,N-二甲基乙酰胺 S>20.0
四氢呋喃 S<1.2 N-甲基吡咯烷酮 S>23.0
2-甲基四氢呋喃 S<0.9 水 10.0<S<20.0
实施例26晶型II的稳定性研究
在25℃恒温条件下对晶型II样品进行DVS测试，DVS结果(图29)显示，当湿度高于10％RH时样品基本无失重，表明晶型II具有较好的稳定性。
实施例27对野生型EGFR和突变型EGFR激酶的活性抑制测试
以下z’-lyte测试方法中所用试剂均可购自Invitrogen。
利用z’-lyte方法测定待测物对双突变型EGFR激酶(EGFR T790M/L858R激酶)(Invitrogen，PV4879)、野生型EGFR激酶(EGFR WT)(Invitrogen，PV3872)活性的抑制作用。
10μL T790M/L858R激酶反应体系中各组分的工作浓度为：25μM ATP,0.08(或0.1)ng/μL EGFR T790M/L858R激酶,2μM Tyr04底物(Invitrogen，PV3193，下同)。加入本发明上述实施例制备的化合物(即待测物)后DMSO的浓度为2vol％。
10μL EGFR WT激酶反应体系中各组分的工作浓度为：10μM ATP,0.8ng/μL EGFR WT激酶,2μM Tyr04底物。加入待测物后DMSO的浓度为2vol％。
测试方法：
室温溶解10mM的待测化合物储存液经4vol％DMSO的水梯度稀释至终浓度10-0.005μM。每孔中加入2.5μL的待测物溶液以及5μL经反应缓冲液稀释的EGFR T790M/L858R激酶(或EGFR WT激酶)与Tyr04底物的混合物，再加入2.5μL的ATP启动反应。C1孔用反应缓冲液代替ATP，C2孔不加入任何药物，C3孔按说明书描述加入磷酸化的底物。
在室温摇床反应60min后。加入5μL Development Reagent B(Invitrogen，用TR-FRET稀释缓冲液进行稀释)，于室温摇床反应60min。在VictorX5荧光酶标仪(PerkinElmer)上读板，测定激发波长为405nm，发射波长为450nm和520nm的光吸收。(例如，C3520nm表示C3孔在520nm的读值)。
抑制率计算方法(参照Invitrogen，PV3193的说明书)如下：
1、ER＝Coumarin Emission(450nm)/Fluorescein Emission(520nm)
2、磷酸化率＝(1-((ER×C3520nm-C3450nm)/((C1450nm-C3450nm)+ER×(C3520nm-C1520nm))))×100％
3、抑制率(IR)＝(1-(测试化合物的磷酸化率)/(C2的磷酸化率))×100％
用XLFIT 5.0软件(英国IDBS公司)拟合计算半数抑制浓度IC50。
酶抑制活性与选择抑制活性结果如下表7所示：
表7
Figure PCTCN2016091591-appb-000035
从上表中可以看出，本发明的式X化合物游离碱对EGFR突变型酶(T790M/L858R)表现出较强的抑制活性，而对EGFR野生型酶(T790M WT)抑制活性较弱，与阳性对照物BIBW2992(Afatinib)相比，本发明的化合物对EGFR突变型酶具有明显的选择抑制活性。选择性比对比化合物1(具体结构如下所示，并可参见WO2013014448A1)提高了8.5倍。
Figure PCTCN2016091591-appb-000036
实施例28MTT(3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐)方法检测细胞抑制活性
MTT测试方法步骤采用本领域技术人员熟知的方法进行，方法中所用试剂均可市购得到。
2.1测试方法：
首先，移除培养基并加入1mL 0.25％的胰酶/EDTA(Gibco,25200-056)。洗一次后，再加入1.5mL胰酶/EDTA消化贴壁细胞，至细胞分离，然后加入3.5mL培养基终止消化。将消化完的细胞悬浮液移至15mL离心管，1300rpm离心3min后弃上清，并用新鲜的培养基悬浮细胞。然后细胞计数，并稀释细胞至以下浓度：A431和H1975细胞每mL 2.78万，NIH3T3每mL 3.33万。将细胞种入96孔板(BD 3072)，每孔90μL，培养过夜。
A431细胞培养基为：含10％FBS(Gibco，10099-141)的DMEM(Hyclone SH30243.01B)；
NIH3T3细胞培养基：含10％FBS(Gibco，10099-141)的DMEM(Hyclone SH30243.01B)；
H1975细胞培养基：含10％FBS(Gibco，10099-141)的RPMI-1640(Hyclone SH30809.01B)；
取20μL 10mM待测化合物，按照如下浓度梯度(2000，666.67，222.22，74.07，24.69，8.23，2.74，0.91μM)稀释10X药品，再加入无血清培养基(终浓度为：10，3.333，1.111，0.370，0.123，0.041，0.014，0.005μM)，并加入每孔10μL药品到细胞培养板内，其中DMSO终浓度为0.5％。
加药后将细胞放入培养箱，培养72h后，每孔加入10μL的5mg/ml的MTT(Sigma,M5655)溶液，然后将96孔板放入37℃5％CO2培养箱孵育4h。
再在2000rpm，5min的条件下离心平板，移除上清后，每孔加入150μL DMSO，并在摇床中震荡平板至所有结晶紫溶解(约10-20min)。最后使用酶标仪测定492nm光吸收，使用XLFIT 5.0软件(英国IDBS公司)计算IC50。
化合物对细胞生长的抑制活性和选择性结果如下表8所示：
表8
Figure PCTCN2016091591-appb-000037
化合物对NIH3T3细胞的毒性测试结果如下表9所示：
表9
化合物编号 NIH3T3细胞MTT测试(IC50/nM)
式X化合物游离碱 >10000
BIBW2992 2750
对比化合物1 3552
从上表可以看出，游离碱对EGFR突变型细胞(H1975细胞)表现出较强的抑制活性，而对EGFR野生型细胞(A431细胞)表现出较弱抑制活性，与阳性对照物BIBW2992和对比化合物1相比，本发明的化合物对EGFR突变型细胞生长具有明显的选择抑制活性。而研究发现将吡唑环换成吡啶环或喹啉环后，对H1975细胞活性和对细胞生长的选择抑制活性明显降低。
从上表化合物对NIH3T3细胞的毒性测试结果中可以看出，式X化合物游离碱对NIH3T3细胞具有较高的IC50值，因此显示出较小的毒性。
实施例29EGFR T790M抑制剂细胞活性ELISA法测定
以下方法中的试剂、溶液的配置方法以及细胞处理和裂解液制备步骤、ELISA检测步骤均按照R&D DYC3570，R&D DYC1095E以及R&D DYC1095BE的说明书进行操作。
一、试剂和溶液
细胞裂解缓冲液：1％(W/V)NP-40，20mM Tris(pH 8.0)，137mM NaCl，10％(V/V)glycerol，1mM NaVO3，2mM EDTA。
细胞裂解液：细胞裂解缓冲液+10μg/mL抑肽酶(Aprotinin)(Sigma)+10μg/mL亮抑蛋白肽酶(Leupeptin)(Sigma)，现配现用。
1x PBS缓冲液：NaCl:0.137M，KCl:0.0027M，Na2PO4-12H2O:0.01M，KH2PO4:0.0015M，pH7.4。
洗涤缓冲液：含有0.05％(V/V)Tween-20的PBS缓冲液。
检测抗体稀释液：20mM Tris，137mM NaCl，0.05％(V/V)Tween-20，0.1％(W/V)BSA，pH 7.2-7.4。
封闭液：含有1％(W/V)BSA的PBS缓冲液。
ELISA试剂盒：R&D DYC3570，R&D DYC1095E和R&D DYC1095BE。
二、H1975细胞
2.1H1975细胞处理和裂解液制备
(1)将H1975细胞(购自中国科学院典型培养物保藏委员会细胞库)以1×104/孔的密度种到96孔板中，每孔90微升10％(V/V)FBS,RPMI1640培养基，37℃、5％(V/V)CO2培养过夜。
(2)将待测化合物按照MTT实验中药物稀释方法稀释，将10μL稀释后的化合物或稀释后的DMSO加入到细胞培板的对应孔中，DMSO终浓度为0.5％(V/V)，37℃、5％(V/V)CO2培 养1小时。以纯DMSO处理的细胞培养体系作为细胞对照。
(3)吸掉培养基后加入100μL细胞裂解液，封板模(Nunc)封置于-80℃冰箱中过夜。以细胞裂解缓冲液作为空白对照。
2.2ELISA检测步骤
按照R&D DYC1095E或R&D DYC1095BE给定说明书进行操作。
(1)R&D捕获抗体((DYC1095BE或DYC1095E))用PBS 1:180稀释，稀释好的抗体100μL/孔加入ELISA反应板(Corning costar 42592)，25℃摇床包被过夜；
(2)360μL洗涤缓冲液洗3次；
(3)加入300μL封闭液，25℃摇床孵育2小时；
(4)360μL洗涤缓冲液洗3次；
(5)加入40μL细胞裂解缓冲液和60μL细胞裂解液，25℃摇床孵育2小时；
(6)360μL洗涤缓冲液洗3次；
(7)检测抗体用检测抗体稀释液以试剂盒说明规定比例稀释，每孔加入100μL，25℃摇床避光孵育1小时；
(8)360μL洗涤缓冲液洗3次；
(9)将TMB底物(R&D DY999)中的A试剂和B试剂以1:1进行混合，每孔100μL，25℃摇床避光孵育20分钟；
(10)2N H2SO4每孔加入50μL；
(11)用酶标仪读板(Thermo Multiskan K3)分别测定细胞对照、空白对照以及药物处理情况下的OD 450值和OD570值，并用相同孔的OD 450值减去相应OD570值分别得到OD细胞、OD空白和OD药物处理。
2.3数据分析
抑制率(％)＝100％×(OD细胞-OD药物处理)/(OD细胞-OD空白)
2.4将计算得到的抑制率用XLFIT 5.0软件计算出IC50值，参见表10。
三、A431细胞
3.1A431细胞的处理和测试步骤
(1)将A431细胞(购自中国科学院典型培养物保藏委员会细胞库)以1×104/孔的密度种到96孔板中，每孔90微升含有10％FBS的DMEM培养基37℃、5％CO2培养过夜。
(2)将A431细胞培养基更换为90微升无血清DMEM培养基，继续培养过夜。
(3)将待测化合物按照MTT实验中药物稀释方法稀释，将10μL稀释后的化合物或稀释后的DMSO加入到细胞培板的对应孔中，DMSO终浓度为0.5％，37℃、5％CO2培养1小时。然后在除细胞对照孔外的每孔中加入10微升2μg/L的EGF(R&D，236-EG-01M)，在细胞孔加入10微升无血清DMEM培养45分钟；以不加入EGF与药物处理的细胞作为细胞对照，以不加入药物的只加入EGF处理的细胞作为EGF对照。
(4)吸掉培养基后加入100μL细胞裂解液，封板模封置于-80℃冰箱中过夜。
3.2ELISA检测步骤
参照R&D DYC3570E说明书进行操作。
(1)R&D捕获抗体(DYC3570E)用PBS 1:180稀释，稀释好的抗体100μL/孔加入ELISA反应板(Corning costar 42592)，25℃摇床包被过夜；
(2)360μL洗涤缓冲液洗3次；
(3)加入200μL封闭液，25℃摇床孵育2小时；
(4)360μL洗涤缓冲液洗3次；
(5)加入40μL细胞裂解缓冲液和60μL细胞裂解液，25℃摇床孵育2小时；
(6)360μL洗涤缓冲液洗3次；
(7)检测抗体用检测抗体稀释液以试剂盒说明规定比例稀释，每孔加入100μL，25℃摇床避光孵育1小时；
(8)360μL洗涤缓冲液洗3次；
(9)将TMB底物(R&D DY999)中的A试剂和B试剂以1:1进行混合，每孔100μL，25℃摇床避光孵育20分钟；
(10)2N H2SO4每孔加入50μL；
(11)用酶标仪读板(Thermo Multiskan K3)分别测定细胞对照、空白对照以及药物处理情况下的OD 450值和OD570值，并用相同孔的OD 450值减去相应OD570值分别得到ODEGF、OD药物、OD细胞。
3.3数据分析
抑制率(％)＝100％×(ODEGF-OD药物)/(ODEGF-OD细胞)
3.4将计算得到的抑制率用XLFIT 5.0软件计算出IC50值，参见表10。
细胞活性ELISA法测定结果如下表10所示：
表10
Figure PCTCN2016091591-appb-000038
从细胞活性ELISA法测定结果表可以看出，与阳性对照物BIBW2992和对比化合物1相比，本发明的式X化合物对细胞水平靶点具有明显的选择抑制活性。而研究发现将吡唑环换成吡啶环或喹啉环后，对H1975细胞活性和细胞水平靶点的选择抑制活性明显降低，甚至没有选 择抑制活性。
实施例30大鼠体内试验
应用LC/MS/MS法测定了大鼠灌胃给予实施例化合物后不同时刻血浆中的药物浓度，研究本发明化合物在大鼠体内的药代动力学行为，评价其药动学特征。
实验方案：
试验动物：健康成年雄性SD大鼠(体重200-225g，21只，禁食)，由斯莱克公司提供；
给药方式与剂量：灌胃给药(5mg/kg，5mL/kg，0.5％CMC-Na水溶液)
血样采集：首先对给药前挑选符合实验要求的动物，称重标记。采集血样前，绑定大鼠，每一只给药的大鼠在预定的采血时间点(灌胃给药：分别于给药前，给药后的0.083,0.25,0.5,1,2,4,8,24h采血,共9个时间点)，通过尾静脉采血，或经心脏采血(终末采血)约150μL。通过眼眶采血，或经心脏采血(终末采血)约150μL。血液转移至预先加入K2EDTA的1.5mL试管中。采完的血样放在湿冰上，离心5min(2000g,4℃)，取出血浆，整个过程在采血后15min内完成。所有的样品都需要存放于-70℃冰箱直到样品分析。
应用LC/MS/MS法测定药物浓度，本发明部分实施例化合物在相同剂量和给药方式下，大鼠体内的药代动力学性质参数如下表11所示：
表11
Figure PCTCN2016091591-appb-000039
从化合物在大鼠体内药代动力学参数表可以看出，本发明的盐的药代吸收好，具有明显的药代吸收效果，同时表现出良好的生物利用度。
实施例31药物组合物
由以下表12所示的组分制备式X化合物己二酸盐的片剂：
表12
式X化合物己二酸盐(实施例20条件2) 20g
淀粉 40g
乳糖 32g
PVP 3g
羧甲基淀粉钠 3g
十二烷基硫酸钠 1g
硬脂酸镁 1g
按常规方法，将式X化合物己二酸盐、淀粉混合过筛，再与上述其它组分混合均匀，直接压片。
实施例32药物组合物
由以下表13所示的组分制备晶型C-1的片剂：
表13
晶型C-1 15g
淀粉 40g
乳糖 37g
PVP 3g
羧甲基淀粉钠 3g
十二烷基硫酸钠 1g
硬脂酸镁 1g
按常规方法，将晶型C-1、淀粉混合过筛，再与上述其它组分混合均匀，直接压片。
实施例33药物组合物
由以下表14所示的组分制备晶型II的胶囊：
表14
晶型II 20g
淀粉 40g
乳糖 32g
PVP 3g
羧甲基淀粉钠 3g
十二烷基硫酸钠 1g
硬脂酸镁 1g
按常规方法，将晶型II、淀粉混合过筛，再与上述其它组分混合均匀，装入普通明胶胶囊。
实施例34马来酸盐晶型C-1与晶型C-2之间的稳定性关系研究
为了验证马来酸盐晶型C-1与晶型C-2在有溶剂条件下的相互转变关系，分别在室温及50℃下设置了混悬竞争试验。试验中，先在两种温度下分别配制马来酸盐晶型C-1的丙酮悬浊液，平衡约一小时后过滤，称取等质量的马来酸盐晶型C-1和晶型C-2(各约5毫克)加入到滤液中磁力搅拌。根据图30，在室温和50℃下搅拌5分钟后混合物均转变为晶型C-1，表明在室温到50℃的范围内，马来酸盐晶型C-1比晶型C-2有更高的热力学稳定性。
实施例35晶型I和晶型II的稳定性关系研究
1)DVS表征：
为了评估无水晶型I和水合物晶型II在不同湿度条件下的稳定性，在25℃恒温条件下对无水晶型I样品和水合物晶型II样品进行了DVS测试。晶型I样品的DVS结果(图31)显示，样品随着湿度的增大而持续吸收水分。
当湿度达到20％RH时，DVS等温图中出现一个转折点，结合图32中DVS测试前后样品的XRPD对比图，可以看出此转折点是由无水晶型I转变成水合物晶型II而引起。实际吸收的水分低于晶型II样品的水分含量，是由固相转变速率慢导致。图29中水合物晶型II的等温DVS曲线显示，当湿度高于10％RH时样品基本无失重，当湿度低于10％RH时样品迅速脱去水分，当湿度再次升高时样品也逐渐再次吸收水分，95％RH时共吸回6.9％的水。晶型II样品在DVS测试前后XRPD的表征结果对比如图33所示，其中晶型I的特征衍射峰同样可能由较低的固相转变速率引起。
2)混悬竞争试验
为了进一步确认室温下无水晶型I和水合物晶型II在不同水活度条件下的稳定性关系，晶型I和晶型II的混合物分别在不同水活度(aw～0.2/0.4/0.6/0.8)的丙酮/H2O溶剂体系中混悬搅拌。根据图34中试验结果可知，室温下混合物在所选的溶剂体系中搅拌三天后均转变成水合物晶型II，表明室温中水合物晶型II在水活度(aw)等于或大于0.2的条件下更为稳定。
实施例36晶型II和晶型IV的稳定性关系研究
为了研究不同水合物晶型II和IV在室温到50℃之间的稳定性关系，在含水体系中分别设置了室温和50℃下的混悬竞争试验。首先在室温和50℃条件下，分别配制了丙酮/H2O(aw～0.6)的游离碱悬浊液，在相应温度下平衡约1小时后，过滤获得相应温度下的近饱和溶液。将等质量的晶型II和IV样品(各约6毫克)分别加到0.8毫升的饱和溶液中形成悬浊液，分别在室温和50℃下磁力搅拌(～800转/分)，约5分钟后取样测试XRPD。
根据图35中对比结果显示，晶型IV在室温和50℃下迅速转变成水合物晶型II，表明含水条件下水合物晶型II在室温到50℃区间内比水合物晶型IV更稳定。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种结构如式X所示的化合物、或其药学上可接受的盐或其前药:
    Figure PCTCN2016091591-appb-100001
  2. 如权利要求1所述的化合物、或其药学上可接受的盐、或其前药，其特征在于，所述的药学上可接受的盐选自下组：盐酸盐、硫酸盐、磷酸盐、乙酸盐、L-乳酸盐、马来酸盐、富马酸盐、琥珀酸盐、L-苹果酸盐、己二酸盐、L-酒石酸盐、马尿酸盐、柠檬酸盐、粘酸盐、乙醇酸盐、D-葡萄糖醛酸盐、苯甲酸盐、龙胆酸盐、烟酸盐、乙二磺酸盐、草酸盐、甲磺酸盐、苯磺酸盐、2-羟基乙磺酸盐和氢溴酸盐。
  3. 如权利要求1所述的化合物、或其药学上可接受的盐、或其前药，其特征在于，所述的式X化合物或其药学上可接受盐为晶体。
  4. 如权利要求3所述的化合物、或其药学上可接受的盐、或其前药，其特征在于，所述的药学上可接受盐选自下组：盐酸盐、磷酸盐、马来酸盐、L-苹果酸盐、己二酸盐、硫酸盐、富马酸盐、琥珀酸盐、L-酒石酸盐、柠檬酸盐、甲磺酸盐、苯甲酸盐、和苯磺酸盐。
  5. 如权利要求3所述的化合物、或其药学上可接受的盐、或其前药，其特征在于，所述的晶体选自下组：
    式X化合物盐酸盐的A型结晶，即晶型A，其X射线粉末衍射图具有下组A1的衍射角2θ(°)值：8.47±0.10、20.32±0.10、23.31±0.10(最高峰)和25.98±0.10；
    式X化合物磷酸盐的B型结晶，即晶型B，其X射线粉末衍射图具有下组B1的衍射角2θ(°)值：11.94±0.10、19.92±0.10(最高峰)、22.27±0.10、23.93±0.10；
    式X化合物马来酸盐的C-1型结晶，即晶型C-1，其X射线粉末衍射图具有下组C-1-1的衍射角2θ(°)值：8.73±0.10、13.37±0.10、18.08±0.10和25.55±0.10(最高峰)；
    式X化合物马来酸盐的C-2型结晶，即晶型C-2，其X射线粉末衍射图具有下组C-2-1的衍射角2θ(°)值：7.48±0.10(最高峰)、8.60±0.10、20.63±0.10和23.27±0.10；
    式X化合物L-苹果酸盐的D型结晶，即晶型D，其X射线粉末衍射图具有下组D1的衍射角2θ(°)值：7.47±0.10、18.75±0.10、22.69±0.10(最高峰)和24.39±0.10；和
    式X化合物己二酸盐的E型结晶，即晶型E，其X射线粉末衍射图具有下组E1的衍射角2θ(°)值：7.43±0.10、18.45±0.10、21.64±0.10(最高峰)和24.22±0.10。
  6. 如权利要求3所述的化合物、或其药学上可接受的盐、或其前药，其特征在于，所述的晶体选自下组：
    式X化合物的晶型Ⅰ，其X射线粉末衍射图具有组I-1的衍射角2θ(°)值：8.74±0.10、9.80±0.10、15.63±0.10、和21.38±0.10(最高峰)；
    式X化合物的晶型II，其X射线粉末衍射图具有组II-1的衍射角2θ(°)值：9.47±0.10(最高峰)、17.34±0.10、18.87±0.10、和23.89±0.10；
    式X化合物的晶型Ⅲ，其X射线粉末衍射图具有组III-1的衍射角2θ(°)值：9.72±0.10、18.41±0.10、23.89±0.10(最高峰)和28.02±0.10；
    式X化合物的晶型Ⅳ，其X射线粉末衍射图具有组IV-1的衍射角2θ(°)值：7.69±0.10(最高峰)、18.90±0.10；和
    式X化合物的晶型V，其X射线粉末衍射图具有组V-1的衍射角2θ(°)值：8.28±0.10、8.89±0.10(最高峰)、9.44±0.10、17.76±0.10。
  7. 如权利要求5或6所述的化合物、或其药学上可接受的盐、或其前药，其特征在于，所述的晶体X射线粉末衍射图谱基本如图1、3、5、7、9、11、13、15、17、19、21中任一衍射图谱所表征。
  8. 一种制备权利要求1所述的式X化合物或其药学上可接受盐的方法，其特征在于，包括步骤：
    (1)在惰性溶剂中，将化合物I-2与化合物3a进行反应，从而形成式X化合物；和
    Figure PCTCN2016091591-appb-100002
    (2)任选地将式X化合物与酸进行成盐反应，从而形成药学上可接受的盐；
    (3)任选地将步骤(1)或(2)所形成的式X化合物或其药学上可接受的盐进行结晶处理，从而获得晶体。
  9. 一种药物组合物，其特征在于，所述药物组合物包括：
    (a)权利要求1-7中任一所述的式X化合物、其药学上可接受盐、或其前药；以及(b)药学可接受的载体。
  10. 权利要求1-7中任一所述的式X化合物、其药学上可接受盐、或其前药，或权利要求9所述药物组合物的用途，其特征在于，用于制备调控EGFR酪氨酸激酶或者治疗EGFR相关疾病的药物。
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WO2021089791A1 (en) 2019-11-08 2021-05-14 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment of cancers that have acquired resistance to kinase inhibitors
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US10513509B2 (en) 2016-05-26 2019-12-24 Recurium Ip Holdings, Llc EGFR inhibitor compounds
US11098030B2 (en) 2016-05-26 2021-08-24 Recurium Ip Holdings, Llc EGFR inhibitor compounds
WO2020125391A1 (zh) * 2018-12-21 2020-06-25 深圳市塔吉瑞生物医药有限公司 用于抑制蛋白激酶活性的氨基嘧啶类化合物
CN111349084A (zh) * 2018-12-21 2020-06-30 深圳市塔吉瑞生物医药有限公司 用于抑制蛋白激酶活性的氨基嘧啶类化合物
CN111349084B (zh) * 2018-12-21 2022-11-25 深圳市塔吉瑞生物医药有限公司 用于抑制蛋白激酶活性的氨基嘧啶类化合物
WO2020188015A1 (en) 2019-03-21 2020-09-24 Onxeo A dbait molecule in combination with kinase inhibitor for the treatment of cancer
WO2020245208A1 (en) 2019-06-04 2020-12-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Use of cd9 as a biomarker and as a biotarget in glomerulonephritis or glomerulosclerosis
WO2021089791A1 (en) 2019-11-08 2021-05-14 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment of cancers that have acquired resistance to kinase inhibitors
WO2021148581A1 (en) 2020-01-22 2021-07-29 Onxeo Novel dbait molecule and its use
WO2022100641A1 (zh) * 2020-11-11 2022-05-19 上海海雁医药科技有限公司 包含egfr抑制剂的药物组合物及其制备方法
WO2023040876A1 (zh) * 2021-09-15 2023-03-23 上海海雁医药科技有限公司 氮杂芳环类化合物及其药学上可接受的盐的多晶型物、药物组合物和应用

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