阅读说明：本技术 制备呋咱并苯并咪唑及其晶型的方法 (The method for preparing furazano benzimidazole and its crystal form ) 是由 G·韦尔蒂 M·厄贝斯 D·塔格里亚费里 于 2018-04-24 设计创作，主要内容包括：本发明提供了用于制备具有式(I)的化合物和其药学上可接受的盐的方法,该方法包括使具有式(II)的化合物脱保护,其中每个R<Sup>3</Sup>独立地表示叔烷基基团,优选地其中每个R<Sup>3</Sup>是叔丁基。本发明还提供了可用于制备具有式(I)的化合物的中间体和用于制备这些中间体的方法。另外,本发明提供了具有式(I)的化合物的二氯化物盐的多晶型及其在治疗增殖性病症中的用途。<Image he="451" wi="700" file="DDA0002238481190000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present invention provides the method for the compound being used to prepare with formula (I) and its pharmaceutically acceptable salt, this method includes being deprotected the compound with formula (II), wherein each R 3 Independently indicate tertiary alkyl groups, preferably wherein each R 3 It is tert-butyl.The present invention also provides the methods that can be used for preparing the intermediate of the compound with formula (I) and be used to prepare these intermediates.In addition, the present invention provides the polymorphic of the dichloride salt of the compound with formula (I) and its purposes in treatment proliferative disorders.)

1. a kind of method being used to prepare with the compound of Formulas I or its pharmaceutically acceptable salt

This method includes being deprotected the compound with Formula II

Wherein each R³Independently indicate tertiary alkyl groups.

2. according to the method described in claim 1, wherein each R³It is tert-butyl.

3. according to claim 1 or method as claimed in claim 2, wherein this method includes being prepared in the following manner with formula The step of compound of II: make the compound with formula III

Wherein R¹Indicate leaving group；And

Wherein each R³Independently indicate tertiary alkyl groups；

It is reacted with the compound with formula IV

4. according to the method described in claim 3, wherein R¹Indicate chlorine, bromine, iodine or sulphonic acid ester.

5. according to the method described in claim 3, wherein R¹Indicate chlorine.

6. the method according to any one of claims 1 to 5, wherein each R³It is tert-butyl.

7. method according to any one of claim 3 to 6, wherein this method also comprises prepares tool in the following manner There is the wherein R of formula III¹The step of indicating the compound of chlorine: make the compound with Formula V

Wherein R²Indicate OH；And

Wherein each R³Independently indicate tertiary alkyl groups

It is reacted with the compound with Formula IV

Wherein R^1aIndicate chlorine.

8. a kind of method for being used to prepare the compound with Formula II, this method includes making the compound with formula III and having The compound of formula IV reacts, as defined in any one of claim 3 to 7.

9. according to the method described in claim 8, wherein R¹It is chlorine.

10. a kind of be used to prepare with formula III, wherein R¹The method for indicating the compound of chlorine, this method include making with Formula V Compound reacted with the compound of Formula IV, as defined in claim 7.

11. method according to any one of claims 7 to 10, wherein making that there is the compound of Formula V and there is Formula IV Compound reacts in the presence of dicyclohexylcarbodiimide (DCC).

12. method according to any one of claims 7 to 10, wherein making that there is the compound of Formula V and there is Formula IV Compound is in 2,4,6- tripropyl -1,3,5,2,4,6- trioxatriphosphinane -2,4,6- trioxide's In the presence of react.

13. according to the method for claim 12, wherein the compound with Formula V exists with the compound with Formula IV In the presence of react, with pass through one pot reaction generate have formula III compound.

14. this method includes the chemical combination for making to have Formula II according to claim 1 to method described in any one of 7 and 11 to 13 Object is deprotected and obtains as the tool for crystallizing dichloride salt as defined in any one of claim 19 to 25 and 28 to 44 There is the compound of Formulas I.

15. the compound with Formula II

Wherein each R³Independently indicate tertiary alkyl groups.

16. the compound with formula III

Wherein R¹Indicate chlorine, bromine, iodine or sulphonic acid ester, and

Each R³Independently indicate tertiary alkyl groups.

17. the compound according to claim 16 with formula III, wherein R¹Indicate chlorine.

18. compound according to claim 15 with Formula II or according to claim 16 or claim 17 described in Compound with formula III, wherein each R³Indicate tert-butyl.

19. the crystallization dichloride salt of the compound with Formulas I

20. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has in 6.0 degree of 2 θ (± 0.2 degree of 2 θ),.

21. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has at 6.0,9.4 and 9.9 degree of 2 θ (± 0.2 degree of 2 θ),.

22. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the x-ray powder at peak that salt, which has in 6.0,9.4,9.9,10.7,17.4,21.4,25.8 and 28.4 degree of 2 θ (± 0.2 degree of 2 θ), Diffraction pattern.

23. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride Salt has at 6.0,9.4,9.9,10.7,11.6,11.9,17.4,21.4,22.4,23.0,24.2,24.6,25.8 and 28.4 degree 2 θ (± 0.2 degree of 2 θ) include the X-ray powder diffraction figure at peak.

24. crystallization dichloride salt described in any one of 9 to 23 according to claim 1, wherein the orthorhombic primitive unit cell parameter is

25. crystallization dichloride salt described in any one of 9 to 24 according to claim 1, which has 1701,1665,1335,1241,1170,942,924,864,699 and 628cm^-1(±2cm^-1) at comprising peak IR compose, and/or With reference TMS's¹³C CP MAS (14kHz) H NMR spectroscopy and/or in [D6]-DMSO¹³C H NMR spectroscopy, these H NMR spectroscopies include Peak in following table:

There is identical upper target signal can exchange by [a], [b], [c], [d].

26. a kind of be used to prepare the method for crystallizing dichloride salt as defined in any one of claim 19 to 25, the party Method the following steps are included: from or mixtures thereof acetonitrile, methanol, ethyl alcohol, ethyl acetate or isopropanol, or comprising acetonitrile, methanol, The solvent mixture of ethyl alcohol, ethyl acetate and/or isopropanol crystallizes the dichloride salt of the compound with Formulas I.

27. a kind of be used to prepare the method for crystallizing dichloride salt as defined in any one of claim 19 to 25, packet Include following steps: from or mixtures thereof acetonitrile, methanol or ethyl alcohol, or the solvent mixture knot comprising acetonitrile, methanol and/or ethyl alcohol Brilliant this has the dichloride salt of the compound of Formulas I.

28. crystallization dichloride salt according to claim 19 works as use when the crystal salt is substantially free of moisture When CuK α actinometry, it includes the X-ray powder diffraction at peak which, which has in 3.9 degree of 2 θ (± 0.2 degree of 2 θ), Figure.

29. crystallization dichloride salt according to claim 19 works as use when the crystal salt is substantially free of moisture When CuK α actinometry, it includes the X-ray at peak which, which has at 3.9,7.9 and 9.7 degree of 2 θ (± 0.2 degree of 2 θ), Powder diagram.

30. crystallization dichloride salt according to claim 19 works as use when the crystal salt is substantially free of moisture When CuK α actinometry, which has wraps in 3.9,7.9,9.7,11.2 and 23.9 degree of 2 θ (± 0.2 degree of 2 θ) X-ray powder diffraction figure containing peak.

31. crystallization dichloride salt according to claim 19 works as use when the crystal salt is substantially free of moisture When CuK α actinometry, the crystallization dichloride salt have 3.9,7.9,9.7,11.2,23.9,25.0 and 25.5 degree of 2 θ (± 0.2 degree of 2 θ) X-ray powder diffraction figure comprising peak.

32. crystallization dichloride salt according to claim 19, when the crystal salt be exposed to 100% humidity for a period of time with When it being made not reabsorb any other moisture, when using CuK α actinometry, which has at 2.7 degree 2 θ (± 0.2 degree of 2 θ) include the X-ray powder diffraction figure at peak.

33. crystallization dichloride salt according to claim 19, when the crystal salt be exposed to 100% humidity for a period of time with When it being made not reabsorb any other moisture, when using CuK α actinometry, which has 2.7, 8.3 and 9.4 degree of 2 θ (± 0.2 degree of 2 θ) includes the X-ray powder diffraction figure at peak.

34. crystallization dichloride salt according to claim 19, when the crystal salt be exposed to 100% humidity for a period of time with When it being made not reabsorb any other moisture, when using CuK α actinometry, which has 2.7, 8.3,9.4,14.8 and 19.7 degree of 2 θ (± 0.2 degree of 2 θ) include the X-ray powder diffraction figure at peak.

35. crystallization dichloride salt according to claim 19, when the crystal salt be exposed to 100% humidity for a period of time with When it being made not reabsorb any other moisture, when using CuK α actinometry, which has 2.7, 8.3,9.4,14.8,19.7 and 24.1 degree of 2 θ (± 0.2 degree of 2 θ) include the X-ray powder diffraction figure at peak.

36. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has in 3.6 degree of 2 θ (± 0.2 degree of 2 θ),.

37. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has at 3.6,4.0 and 8.1 degree of 2 θ (± 0.2 degree of 2 θ),.

38. crystallization dichloride salt according to claim 19, which has 3.6,4.0,8.1, 9.4,11.0,21.1 and 24.5 degree of 2 θ (± 0.2 degree of 2 θ) include the X-ray powder diffraction figure at peak.

39. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has in 3.4 degree of 2 θ (± 0.2 degree of 2 θ),.

40. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has at 3.4,4.0 and 8.1 degree of 2 θ (± 0.2 degree of 2 θ),.

41. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has in 3.4,4.0,8.1,11.1,16.5 and 24.0 degree of 2 θ (± 0.2 degree of 2 θ),.

42. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has in 3.0 degree of 2 θ (± 0.2 degree of 2 θ),.

43. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the X-ray powder diffraction figure at peak that salt, which has at 3.0,3.6 and 9.4 degree of 2 θ (± 0.2 degree of 2 θ),.

44. crystallization dichloride salt according to claim 19, when using CuK α actinometry, the crystallization dichloride It includes the x-ray powder at peak that salt, which has in 3.0,3.6,9.4,11.1,12.7,15.3,23.6 and 24.5 degree of 2 θ (± 0.2 degree of 2 θ), Diffraction pattern.

45. a kind of pharmaceutical composition, it includes be combined with pharmaceutically acceptable carrier, diluent or excipient, pharmacy The crystallization dichloride of upper a effective amount of compound as defined in any one of claim 19 to 25 and 28 to 44 with Formulas I Object salt.

46. the crystallization dichloride of the compound as defined in any one of claim 19 to 25 and 28 to 44 with Formulas I Salt is used to treat proliferative disorders or disease.

47. the crystallization dichloride of the compound as defined in any one of claim 19 to 25 and 28 to 44 with Formulas I Salt is preparing the purposes in the drug for treating proliferative disorders or disease.

48. a kind of method for treating proliferative disorders or disease, this method include by therapeutically effective amount such as claim 19 to The crystallization dichloride salt of compound defined in any one of 25 and 28 to 44 with Formulas I is applied to trouble in need thereof Person.

49. the crystallization dichloride salt of the compound with Formulas I for being used according to claim 46 or according to claim The purposes or treatment according to claim 48 proliferation of the crystallization dichloride salt of compound described in 47 with Formulas I Property conditions or diseases method, wherein the proliferative disorders or disease are the tumor diseases selected from following item: epithelial tumour, Squamous cytoma, basal cell tumor, transitional cell papilloma and cancer, adenoma and gland cancer, appendicle and appendages of skin tumour are glued Liquid epiderm-like tumour, cystoma, mucus and serous tumor, conduit, leaflet and medullary substance tumour, acinic cell tumor are multiple Miscellaneous epithelial tumour, specialization gonadal tumor, Chromaffionoma and glomangioma, mole and melanoma, soft tissue neoplasm and meat Tumor, fibromatous tumour, myxoma tumour, lipoma tumour, myomata tumour, complicated mixing and stromal tumors, it is fine Tie up epithelial tumour, synovial membrane sample tumour, mesothelial tumor, germinoma, trophoblastic tumor, mesonephroma, hemangioma, lymph Tuberculation, bone and chondroma tumour, giant-cell tumor, miscellany bone tumour, odontogenic tumor, glioma, diktoma Property tumour, meningioma, neurinoma, granular cell tumor and alveolar soft tissue sarcoma, Huo Qijin and non-Hodgkin lymphoma, other Lymphoreticular tissue tumour, plasmacytoma, mastocytoma, immunoproliferation disease, leukaemia, miscellany bone marrow proliferation venereal disease Disease, lympho-proliferative illness and myelodysplastic syndrome.

50. the crystallization dichloride salt of the compound with Formulas I for being used according to claim 46 or according to claim The purposes or treatment according to claim 48 proliferation of the crystallization dichloride salt of compound described in 47 with Formulas I The method of property conditions or diseases, wherein the proliferative disorders or disease are cancers.

51. the crystallization dichloride salt of the compound with Formulas I for being used according to claim 46 or according to claim The purposes or treatment according to claim 48 proliferation of the crystallization dichloride salt of compound described in 47 with Formulas I The method of property conditions or diseases, wherein the proliferative disorders or disease are cancers, wherein the organ of the body just involved and position For, which is selected from brain, breast, cervix, ovary, colon, rectum (including colon and rectum, i.e. colorectal cancer), lung (packet Include Small Cell Lung Cancer, non-small cell lung cancer, maxicell lung cancer and celiothelioma), endocrine system, bone, adrenal gland, thymus gland, liver, (such as lymthoma, leukaemia, myeloma or lymphoid malignant are swollen for stomach, intestines (including gastric cancer), pancreas, marrow, hematologic malignancies Tumor), bladder, urinary tract, kidney, skin, thyroid gland, brain, head, neck, prostate and testis.

52. the crystallization dichloride salt of the compound with Formulas I for being used according to claim 46 or according to claim The purposes or treatment according to claim 48 proliferation of the crystallization dichloride salt of compound described in 47 with Formulas I The method of property conditions or diseases, wherein the proliferative disorders or disease are cancers selected from the group below, which is made up of: brain Cancer, breast cancer, prostate cancer, cervix cancer, oophoroma, gastric cancer, colorectal cancer, cancer of pancreas, liver cancer, the cancer of the brain, neuroendocrine Cancer, lung cancer, kidney, hematologic malignancies, melanoma and sarcoma.

53. the crystallization dichloride salt of the compound with Formulas I for being used according to claim 46 or according to claim The purposes or treatment according to claim 48 proliferation of the crystallization dichloride salt of compound described in 47 with Formulas I Property conditions or diseases method, wherein the proliferative disorders or disease are tumor diseases, which is selected from following Brain tumor: neuroglial tumor and non-neuroglial tumor, astrocytoma (including glioblastoma multiforme and do not refer to Fixed glioma), oligodendroglioma, ependymoma, meningioma, hemangioblastoma, acoustic neurinoma, craniopharyhgeal canal Tumor, primary central nervous system lymphoma, germinoma, hypophysoma, tumors of pineal region, intramedullary primitive neuroectodermal tumor (PNET), medulloblastoma, hemanyiopericytoma, the spinal cord of the brain tumor including meningioma, chordoma and heredity driving The brain tumor of tumour, heredity driving includes neurofibromatosis, Peripheral Nerve Sheath Tumors and tuberous sclerosis.

54. the crystallization dichloride salt of the compound with Formulas I for being used according to claim 46 or according to claim The purposes or treatment according to claim 48 proliferation of the crystallization dichloride salt of compound described in 47 with Formulas I The method of property conditions or diseases, wherein the proliferative disorders or disease are tumor diseases, which is pleomorphism glue Matter blastoma.

55. the crystallization dichloride salt of the compound with Formulas I for being used according to any one of claim 50 to 54 or According to any one of claim 50 to 54 with Formulas I compound crystallization dichloride salt purposes or according to The method for the treatment of proliferative disorders or disease described in any one of claim 50 to 54, wherein the cancer to be treated is real Body tumor.

56. the crystallization dichloride salt of the compound with Formulas I for being used according to any one of claim 46 to 55 or According to any one of claim 46 to 55 with Formulas I compound crystallization dichloride salt purposes or according to The method for the treatment of proliferative disorders or disease described in any one of claim 46 to 55, wherein to proliferative disorders or disease The treatment be the proliferative disorders to people or disease treatment.

57. the crystallization dichloride salt of the compound with Formulas I for being used according to any one of claim 46 to 56 or According to any one of claim 46 to 56 with Formulas I compound crystallization dichloride salt purposes or according to The method for the treatment of proliferative disorders or disease described in any one of claim 46 to 56, the wherein compound with Formulas I Crystallization dichloride salt be as defined in any one of claim 20 to 25.

58. the crystallization dichloride salt of the compound with Formulas I for being used according to any one of claim 46 to 56 or According to any one of claim 46 to 56 with Formulas I compound crystallization dichloride salt purposes or according to The method for the treatment of proliferative disorders or disease described in any one of claim 46 to 56, the wherein compound with Formulas I Crystallization dichloride salt be as defined in any one of claim 28 to 44.

Fig. 1

Fig. 1 shows the atom number of NMR distribution.

Fig. 2

Fig. 2 shows the X-ray powder diffraction of the crystal form E of the dichloride salt of the compound with Formulas I at room temperature (XRPD) diffraction pattern.

Fig. 3

Fig. 3 shows the figure table that the Pawley (WPPD) of the crystal form E of the dichloride salt of the compound with Formulas I is calculated Show.The graphical representation of full coatings decomposition computation is presented, wherein line above shows the observed number from high-resolution XRPD According to.Black middle line indicates calculated coatings, and the rodlike peak value for indicating to have h, k, l index of black of the bottommost of figure Position.Grey baseline indicates the difference between (baseline correction) point of observation calculated.

Fig. 4

Fig. 4 shows the thermogravimetric analysis (TGA) of the crystal form E of the dichloride salt of the compound with Formulas I, has about Endothermic peak at 130 DEG C (± 2 DEG C) and 276 DEG C (± 2 DEG C).

Fig. 5

Fig. 5 shows the differential scanning calorimetry (DSC) of the crystal form E of the dichloride salt of the compound with Formulas I, tool There is endothermic peak at about 130 DEG C (± 2 DEG C) and 276 DEG C (± 2 DEG C) and higher than the decomposition of this temperature.

Fig. 6

Fig. 6 is shown using 25 → 200 → 25 DEG C of temperature profile；The rate of heat addition of 10 DEG C/min and having for rapid cooling The circulation DSC of the crystal form E of the dichloride salt of the compound of Formulas I.Heat absorption (130 DEG C ± 2 DEG C) shows that solid-solid changes, this It is reversible (in 97 DEG C of ± 2 DEG C of heat releases when cooling).

Fig. 7

Fig. 7 shows the XRPD diffraction that crystallizes high temperature crystal form E1 of the dichloride salt of the compound with Formulas I at 180 DEG C Figure.

Fig. 8

Fig. 8 shows the FTIR spectrum of the compound with Formulas I of the crystal form E of the dichloride salt of the compound with Formulas I.

Fig. 9

The FTIR that Fig. 9 shows the crystal form E of the dichloride salt of the compound with Formulas I is composed in 1830cm^-1With 400cm^-1 Between amplification.

Figure 10

Figure 10 shows the Magic angle spinning solid carbon 13 of the crystal form E of the dichloride salt of the compound with Formulas I, and { proton is gone Coupling } nuclear magnetic resonance (¹³C{¹H } MAS-NMR) spectrum.

Figure 11

Isothermal (24.1 DEG C) Dynamic Vapor that Figure 11 shows the crystal form E of the dichloride salt of the compound with Formulas I is inhaled Fufen analysis.

Figure 12

Figure 12 shows the XRPD diffraction pattern of crystal form A0.

Figure 13

Figure 13 shows the XRPD diffraction pattern of crystal form A1.

Figure 14

Figure 14 shows the XRPD diffraction pattern of mixture A1+M1.

Figure 15

Figure 15 shows the XRPD diffraction pattern of mixture A1+M4.

Figure 16

Figure 16 shows the XRPD diffraction pattern of mixture M 3+M5.

Figure 17

Figure 17 shows the XRPD diffraction patterns of mixture A2+M4.

Figure 18

Figure 18 shows the XRPD diffraction pattern of mixture A2+M11.

Figure 19

Figure 19 shows stacking chart's (from top to bottom) of XRPD diffraction pattern below: F: crystal form A1+M4, E: at 40 DEG C 75% RH (M3+M5) is after 1 week, D: after 40 DEG C/75%RH (M3+M5) 2.5 weeks, C: and after 40 DEG C/75%RH (M5) 4 weeks, B: 40 DEG C/after 75%RH 4 weeks and 25 DEG C/95%RH (A2+M4) after 2 days, after A:40 DEG C/75%RH 4 weeks and 25 DEG C/95%RH (A2+ M11) after 1 week.

Figure 20

Figure 20 shows the XRPD diffraction pattern of crystal form A2.

Figure 21

Figure 21 shows the XRPD diffraction pattern of mixture A2+A3.

Figure 22

Figure 22 shows the XRPD diffraction pattern of crystal form M1.

Figure 23

Figure 23 shows the XRPD diffraction pattern of crystal form M2.

Figure 24

Figure 24 shows the XRPD diffraction pattern of crystal form M3+M5.

Figure 25

Figure 25 shows the XRPD diffraction pattern of crystal form M4.

Figure 26

Figure 26 shows the XRPD diffraction pattern of crystal form M5.

Figure 27

Figure 27 shows the XRPD diffraction pattern of crystal form M8.

Figure 28

Figure 28 shows the XRPD diffraction pattern of crystal form M9.

Figure 29

Figure 29 shows the XRPD diffraction pattern of mixture M 10+M4.

Figure 30

Figure 30 shows the XRPD diffraction pattern of crystal form M11.

Figure 31

Figure 31 shows the XRPD diffraction pattern of crystal form M12.

Figure 32

Figure 32 shows the XRPD diffraction pattern of crystal form M13.

Figure 33

Figure 33 shows the XRPD diffraction pattern of crystal form F.

Figure 34

Figure 34 shows the XRPD diffraction pattern of crystal form G.

Figure 35

Figure 35 shows isothermal (24.9 DEG C) the dynamic vapor sorption measurement of the compound with Formulas I, and relative sample is presented Weight (%) is to relative humidity.Starting form is mixture A1+M4, and moisture profile is 0 → 95 → 0%RH, and every step is 10%RH, until every step reaches mass balance.Biggest quality variation is 34% when 95%RH.Hysteresis is not observed.

Figure 36

Figure 36 shows the thermodynamics pH dependent solubility of crystal form E.

Figure 37

Figure 37 A shows the thermodynamics pH dependent solubility of crystal form A1+M4.Figure 37 B shows the heat of crystal form A2+M11 Mechanics pH dependent solubility.

Figure 38

Figure 38 shows generating according to the method for WO 2011/012577 and being described in WO 20,11/,012,577 the The XRPD diffraction pattern of the dichloride salt of the compound with Formulas I in the final stages of page 36.The XRPD figure on top is from storage There are 5 DEG C of sample, lower part XRPD figure is from the sample for being stored in -60 DEG C.

Example

Example 1- has the synthesis of the compound of formula III

Example 1a: there is formula III (R¹=Cl, R³=tert-butyl) compound pass through the synthesis that is activated with DCC

At room temperature, N2, the bis- (t-butoxies of N6- will be added in phosphoric acid (85%, 57mL) solution in water (280mL) Carbonyl) the suspension of-L-lysine dicyclohexyl amine salt (438g, 0.831mol, 2.5 equivalent) in diisopropyl ether (DIPE, 1L) In liquid and stir until solid dissolves.The organic phase mixture of phosphoric acid (85%, 20mL) and water (160mL) is washed, so It is washed afterwards with water (4 × 160mL).After being dried over anhydrous sodium sulfate, it is (free that bis- (t-butoxy carbonyl)-L-lysines are concentrated Acid) solution.Concentrate is diluted with methylene chloride (DCM, 421mL).Dicyclohexylcarbodiimide is added at room temperature (88.5g, 0.429mol, 1.25 equivalent) solution in DCM (100mL) and reaction mixture is stirred into 15min.By gained Suspension filtering, cake is washed with DCM (3 × 50mL).By 4- Aminophenacyl chloride (56.2g, 0.331mol, 1.0 equivalents) it is added in the filtrate of merging and stirs the mixture for 4h.Insoluble matter is filtered out, and filtrate is dense under vacuum Contracting.Concentrate is diluted with 4-methyl-2 pentanone (MIBK, 279mL), is heated to about 45 DEG C.Heptane is added while cooling (836mL).Suspension is cooled to 10 DEG C, stirs and filters.By solid MIBK/ heptane and heptane wash and drying. Crude product is crystallized from MIBK/ heptane and drying is to provide 119.4g title compound (72%), purity is >=99.5%, with And >=99%ee.

Example 1b: the compound (R with formula III¹=Cl, R³=tert-butyl) pass through useThe synthesis of activation

By N2, bis- (the tert-butoxycarbonyl)-L-lysines of N6- (85%w/w, 216g, 531mmol, 1.5 equivalent) are dissolved in In toluene (1500g).Add the 4- Aminophenacyl chloride (60g, 354mmol, 1.0 equivalent) in toluene (600g) With the solution of 4- (dimethylamino) pyridine (DMAP, 4.32g, 35.4mmol, 0.1 equivalent).Mixture is cooled to -15 DEG C To -10 DEG C.It adds triethylamine (143g, 1.42mol, 4.0 equivalent), adds 2,4,6- 3 third at -15 DEG C to -10 DEG C through 2h after Base -1,3,5,2,4,6- trioxatriphosphinane -2,4,6- trioxide (The 50% of 495g in toluene Solution, 778mmol, 2.2 equivalents) solution in toluene (360g).It stirs the mixture for 17h and is warming up to about -5 DEG C. It adds water (1524g) and separates phase at room temperature.Organic phase is washed with hydrochloric acid (pH 1.0), then uses hydrochloric acid (pH= 0.5,5%w/w ethyl alcohol) and washed with saturated sodium bicarbonate aqueous solution.Solution is filtered and allows its standing.Suspension is existed 30 DEG C -35 DEG C, 50mbar concentration is cooled to about 20 DEG C and stirs.Solid is filtered, is washed with toluene and dry to provide The title compound (79%) of 138.5g, purity 99.3%, and >=99%ee.

Example 2- has the compound (R of Formula II³Tert-butyl) synthesis

By 3- { [- 1,2,5- oxadiazoles -3- base of 4- (1H- benzimidazolyl-2 radicals-yl)] amino } propionitrile (47g, 185mmol, 1.00 equivalents) it is dissolved in DMF (1.6L).Add N- [4- (2- chloracetyl) phenyl]-N2, bis--Boc-L- lysine acyl of N6- Amine (98g, 197mmol, 1.06 equivalent) and potassium carbonate (49.5g, 358mmol, 1.94 equivalent).40 DEG C are heated the mixture to hold Continuous 5h.Suspension is filtered at 0-5 DEG C and filtrate is added in aqueous ammonium chloride solution (2.5%w/w, 7L).By suspension It filters and solid is dry.Crude product is suspended in THF (188mL) and water (100mL).It is added under reflux (about 65 DEG C) Methanol (3.4L).The suspension is stirred 1 hour and is cooled to room temperature.Filtration product washs solid and drying with methanol.It will consolidate Body is heated to flowing back in THF (188mL) and methanol (3.4L), and about 10 DEG C are cooled in 2h.Suspension is filtered, is used Methanol washs and drying is to provide the title compound (91%) of 121g, purity 99.8%.

Example 3- has the synthesis of the compound (dihydrochloride) of Formulas I

There to be Formula II (R³It is tert-butyl) compound of (119g, 166.4mmol, 1.00 equivalent) is suspended in tetrahydrofuran In (785mL) and it is heated to 30 DEG C.Aqueous hydrochloric acid (30%w/w, 170g) is added in 3h.48h is stirred the mixture for, it is cooling To 10 DEG C, and add tetrahydrofuran (785mL).Resulting suspension is filtered, cake is washed with tetrahydrofuran and in height It is dry to provide 95.8g (97.8%) crude product to 55 DEG C.Crude product (75g) is dissolved in water (75mL) and tetrahydro at about 43 DEG C In furans (112mL).It is stirred 1 hour in about 40 DEG C of addition tetrahydrofurans (2.85L) and by suspension at about 50 DEG C.It is cooled to After 10 DEG C, product is filtered, is washed with tetrahydrofuran and dry to provide the product of the purifying of 68g at about 50 DEG C.It will purifying Product (67g) be dissolved in water (201mL) and filter resulting solution.By water evaporation.By product further up to 50 DEG C of dryings are to provide the title compound (83%) of 62.9g, purity 99.6%.

Comparison example 1 is (according to WO 2011/012577)

S- { 5- benzyloxycarbonyl amino -5- [4- (2- { 2- [4- (2- cyanoethyl amino) furazan -3- base]-benzimidazole -1- 1- yl }-acetyl group)-phenylcarbamoyl]-amyl }-benzyq carbamate uses hydrogen in the mixture of THF/MeOH/HCl Gas hydrogenates about 5h in the presence of Pd/C 10%.After processing, chromatography and salt are formed, the compound with Formulas I is generated Dihydrochloride, purity 90%-91%, 81%ee (yield: 50%).

Example 4- has the preparation of the crystallization dichloride salt (crystal form E) of the compound of Formulas I

Following some examples, which are described, prepares crystal form E using crystal seed.The main purpose of addition crystal seed is to accelerate formation more Crystal form.It is considered as in the case where no crystal seed, which still can generate crystal form E.It should be noted that example 4d, 4f, 4g, 4h, 4i and 4k do not use crystal seed, and example 4l, 4m, 4n, 4o and 4p are also such.

It is crystallized by slurries

Example 4a: from methanol/methyl t-butyl ether (MTBE)

At 65 DEG C, the compound with Formulas I of 0.20g is dissolved in 8mL methanol, which is filtered.Add 10mg The crystal seed of crystal form E and mixture is stirred through 30min.Through 2h-3h, 12mL MTBE is added dropwise, the mixture of acquisition is cold But to 5 DEG C -15 DEG C and in 5 DEG C of -15 DEG C of stir about 40h.Mixture is filtered and cake is dried under vacuum, to provide The crystalline solid form E of 0.18g.

Example 4b: from methanol/acetonitrile

At 30 DEG C -45 DEG C, there is the compound (mixture A1+M1) of Formulas I to be dissolved in 40mL methanol 4g.By solution mistake It filters and fills the crystal seed of 200mg crystal form E into solution.After stirring, suspension is formd, it is heated to back through about 15h It flows and is concentrated into 12mL.20mL acetonitrile is added, suspension is slowly cooled to 0-10 DEG C and is filtered.By cake at about 50 DEG C It is dried under vacuum, the crystal form E of 3.4g solid is provided.

Example 4c: from methanol/toluene

At 30 DEG C -45 DEG C, there is the compound (mixture A1+M1) of Formulas I to be dissolved in 20mL methanol 2g and neutralize from most In the mother liquor of later batch.Solution is filtered, be inoculated with 100mg crystal form E and is added dropwise to (80 DEG C -90 of the hot toluene of 50mL DEG C) in.(distilling out about 20mL) is concentrated in resulting suspension, boiling point is further heated to and then slowly cools to 0-10 ℃.Suspension is filtered and is dried in vacuo cake at 50 DEG C, to provide the crystal form E of 1.5g.

Example 4d: from methanol (room temperature slurries)

The compound (mixture A1+M1) with Formulas I of 65g is dissolved in 485mL methanol and is stirred at 15 DEG C -25 DEG C It mixes.Solution was stirred for about 14 days.In whipping process, suspension is formd.Suspension is filtered, cake is washed with methanol And it is dried in vacuo at about 50 DEG C, to provide the crystal form E of 46g.

Example 4e: from methanol (slurries of reflux)

At 30 DEG C -45 DEG C, the compound (mixture A1+M4) with Formulas I of 2g is dissolved in 20mL methanol.By solution Filtering is inoculated with crystal form E and is flowed back for about 15h.Suspension is concentrated into the volume of about 10mL, be cooled to 0-10 DEG C and Filtering.Cake is dried in vacuo at 50 DEG C, to provide the crystal form E of 1.37g.

Example 4f: from ethyl alcohol

The compound (mixture A1+M1) with Formulas I of 5g is flowed back in 100mL ethyl alcohol and persistently amounts to 11h.It will mixing Object is cooled to room temperature, and is filtered and is dried in vacuo cake at 45 DEG C, to provide the crystal form E of 4.45g.

Example 4g: from acetonitrile, reflux

The compound (mixture A1+M1) with Formulas I of 15g is flowed back in the acetonitrile of 300mL and persistently amounts to 11h.It will Suspension is cooled to room temperature and filters, and cake is dried in vacuo at 65 DEG C, to provide the crystal form E of 13g.

Example 4h: from ethyl acetate, slurries are in room temperature (RT) and 50 DEG C

The compound (mixture A1+M1) with Formulas I of 20.4mg is stirred at room temperature in the ethyl acetate of 1mL and is continued Two weeks.Sample is centrifuged later, and separates solid and mother liquor.Moist solid analyzed as being the mixing of crystal form E and crystal form F Object is in small polymorphic.Moist solid in room temperature in vacuo (5mbar) dry about 18h and is analyzed as crystal form E.

The compound (mixture A1+M1) with Formulas I of 28.4mg is held in the ethyl acetate of 1mL in about 50 DEG C of stirrings It is two weeks continuous.Sample is centrifuged later, and separates solid and mother liquor.Moist solid analyzed as being the mixture of crystal form E and F, In small polymorphic.Moist solid in room temperature in vacuo (5mbar) dry about 18h and is analyzed as crystal form E.

Example 4i: from 2- propyl alcohol

The compound (mixture A1+M1) with Formulas I of 27.5mg is continued in the 2- propyl alcohol of 0.9mL in 50 DEG C of stirrings About two weeks.Sample is centrifuged later, and separates solid and mother liquor.Moist solid analyzed as being crystal form E.By moist solid In room temperature in vacuo (5mbar) dry about 18h and analyze as crystal form E.

Example 4j: from ethyl acetate

By the compound (mixture A1+M1) with Formulas I of 19.8mg in 20 DEG C of stir abouts in the ethyl acetate of 0.6mL Two weeks.Sample is centrifuged later, and separates solid and mother liquor.Moist solid analyzed as being crystal form E.Moist solid is existed 40 DEG C/75%RH is further processed 2 days and is analyzed as crystal form E.

Example 4k: from acetonitrile, 20 DEG C

By the compound (crystal form A1+M1) with Formulas I of 18.0mg in the acetonitrile of 0.6mL 20 DEG C stir about two weeks. Sample is centrifuged later, and separates solid and mother liquor.Moist solid analyzed as being crystal form E.By moist solid 40 DEG C/ 75%RH is further processed 2 days and is analyzed as crystal form E.

In second test, the solid of the humidity is the compound with Formulas I of 18.0mg, by its 0.6mL second In nitrile 20 DEG C stir about two weeks.Sample is centrifuged later, and separates solid and mother liquor.Moist solid analyzed as being crystal form E.Moist solid in room temperature in vacuo (5mbar) dry about 18h and is analyzed as crystal form E.

Example 4l is from acetonitrile, and 50 DEG C

By the compound (crystal form A1+M1) with Formulas I of 18.0mg in the acetonitrile of 0.6mL 50 DEG C stir about two weeks. Sample is centrifuged later, and separates solid and mother liquor.Moist solid analyzed as being crystal form E.By moist solid 40 DEG C/ 75%RH is further processed 2 days and is analyzed as crystal form E.

Pass through crystallisation by cooling

Example 4m: from 2- butanol/methanol

The compound (mixture A1+M1) with Formulas I of 35.5mg is added to 2- butanol/methanol mixing of 1.2mL In object, slurries are generated, which is stirred one hour at about 60 DEG C.Later, sample is kept for 1 hour at 60 DEG C and allows it About 5 DEG C are cooled to, cooling rate is about 1 DEG C/h.Sample is maintained at about 5 DEG C for about for 24 hours.Moist solid is filtered and divided Analysis is crystal form E.

Example 4n: from 4- dioxanes/methanol

The compound (mixture A1+M1) with Formulas I of 32.5mg is added to methanol/1,4- dioxanes in 0.5mL In mixture, slurries are generated, which is stirred one hour at about 60 DEG C.Later, sample is kept for 1 hour and is permitted at 60 DEG C Perhaps it is cooled to about 5 DEG C, and cooling rate is about 1 DEG C/h.Sample is maintained at about 5 DEG C for about for 24 hours.Moist solid is filtered And analyzing is crystal form E.

Example 4o: from ethyl acetate/methanol

The compound (mixture A1+M1) with Formulas I of 32.5mg is added to the mixed of the ethyl acetate/methanol of 0.75mL It closes in object, generates slurries, which is stirred one hour at about 60 DEG C.Later, by sample about 60 DEG C keep one hour and It is allowed to be cooled to about 5 DEG C, cooling rate is about 1 DEG C/h.Sample is maintained at about 5 DEG C for about for 24 hours.By moist solid mistake It filters and analyzes as crystal form E.

The one pot of deprotection and crystallization of compound with Formula II

Example 4p

By the compound (R with Formula II of 0.5g³It is tert-butyl) it is suspended in 5mL methanol.In 20 DEG C of -25 DEG C of additions The HCl in MeOH of 2.4 molar equivalents, and at about 5 DEG C by suspension stir about 9 days.Suspension is filtered and will be obtained The cake obtained is dried under vacuum to provide the crystal form E of 0.3g.

From crystalline free base

Example 4q

The dichloride salt (mixture A1+M4) of the compound with Formulas I of 76g is dissolved in 280mL water and 280mL first In the mixture of alcohol.At 10 DEG C -15 DEG C, add the solution in the solution of 24.2g potassium carbonate, 140mL water and 140mL methanol. Reaction mixture is stirred at room temperature about 2 hours.Suspension is filtered, cake is washed with methanol, and in the water of 350mL and Pulp in the methanol of 350mL.Suspension is filtered, is dried in vacuo by the cake water washing of 70mL and at 45 DEG C, to provide 65g Compound (free alkali) with Formulas I.

The compound (free alkali) for making 1g have Formulas I is reacted with the hydrochloric acid in methanol solution at 65 DEG C.It is brilliant to add 10mg Mixture is slowly cooled to 8 DEG C -10 DEG C, stir about 16h by the crystal seed of type E, filters and the cake of acquisition is dried in vacuo to mention For the crystal form E of 0.44g.

Example 5- has the characterization of the crystallization dichloride salt (crystal form E) of the compound of Formulas I

Example 5a: it is characterized by XRPD

It is arranged using high-throughput XRPD and obtains XRPD figure.Plate is mounted on equipped with Hi-Star area detector On Bruker GADDS diffractometer.The calibration of XRPD platform is to use Silver behenate as long d- spacing and corundum as short D- spacing.2 θ regions of the data collection between 1.5 ° and 41.5 ° are carried out using monochromatic CuKa radiation in room temperature, this is XRPD figure In most unique part.The diffraction pattern in each hole collects (1.5 °≤2 θ≤21.5 ° of first frame and second within the scope of two 2 θ 19.5 °≤2 θ≤41.5 ° of frame), every exposed frame time is 90s.Background subtraction or curve smoothing are not schemed applied to XRPD.In Carrier material used in XRPD analytic process only slightly facilitates background to X-ray transparent.

Shown in Fig. 2 at room temperature with Formulas I compound dichloride salt crystal form (crystal form E) XRPD and its Diffraction pattern peak is shown in Table 2.It is indexed using P222 space group to the evaluation of high-resolution XRPD figure.To the anti-of pure form It penetrates intensity and indexs and generate rhombic system, and allow to extract cell parameter.

Crystallographic parameter is that the Pawley of the crystal form of the dichloride salt based on the compound with Formulas I calculates (full coatings point Solution, WPPD).All intensity at the peak of powder diagram and 2 θ values, which can specify, gives orthorhombic primitive unit cell (P), cell parameter are as follows: The coatings of the crystal form can also be to index compared with low-symmetry, example Such as monocline It is oblique with several three.But commonly used highest symmetry.In this case, highest symmetry is orthorhombic.Calculate and The comparison of the diffraction pattern of measurement shows fabulous consistency as shown in Figure 3.

Table 2. have Formulas I compound dichloride salt crystal form E 27 most abundant peaks diffraction pattern peak positions set, d- X-ray powder diffraction (XRPD) list of spacing and relative intensity

Similarly with crystal form E, the XRPD of high temperature polysilicon type E1 is measured, and diffraction pattern peak (Figure 10) is shown in table 3.

Table 3. have Formulas I compound dichloride salt crystallization high temperature crystal form E1 diffraction pattern peak positions set, d- spacing With X-ray powder diffraction (XRPD) list of relative intensity

Example 5b: pass through differential scanning calorimetry (DSC), the characterization of thermogravimetric analysis (TGA) and alternating temperature XRPD

Thermogravimetric analysis (TGA, Fig. 4) shows a large amount of heat absorptions, shows at about 276 DEG C (± 2 DEG C) along with the molten of decomposition Change event.Mean to occur before melting turn that solid-solid change to crystal form in about 130 DEG C (± 2 DEG C) of a small amount of heat absorptions Become, which formed in high-temperature and reversible.This behavior is studied by differential scanning calorimetry (DSC, Fig. 5) and alternating temperature XRPD It confirms.

Circulation DSC (Fig. 6) is carried out to study the property in the heat absorption of about 130 DEG C (± 2 DEG C).200 DEG C are heated to, it is then fast Speed is cooled to room temperature (RT) (25 DEG C -> 200 DEG C -> 25 DEG C).DSC thermogram after cooling is shown at about 97 DEG C (± 2 DEG C) A small amount of heat release, it means that solid form reverse becomes crystal form E1 (XRPD figure, Fig. 7).The XRPD data of solid show 25 DEG C Solid form does not change, and is solid reverted austenite which demonstrate heat release when cooling.Alternating temperature (VT) XRPD data are (about VT XRPD experimental detail confirms above-mentioned characteristic referring to example 8a).

Example 5c: experiment heat analysis (including DSC, TGA, TGA SDTA, TGA MS)

Melting characteristic is obtained by DSC thermogram, with heat flux DSC822e instrument (Mettler-Toledo GmbH [plum Teller support benefit company], Switzerland) record.With a fritter indium calibrated DSC822e temperature and (m.p.=156.6 DEG C of enthalpy；Δ Hf=28.45J.g-1).Sample is sealed in 40 μ L aluminium dishes of standard, thread a needle hole simultaneously with the rate of heat addition of 10 DEG C/min 300 DEG C are heated to from 25 DEG C in DSC.In measurement process, dry N is used₂Gas cleans DSC with the flow velocity of 50mL/min Equipment.

By thermogravimetric analysis/differential thermometric/heat analysis (TGA/SDTA) measurement simultaneously since solvent or moisture loss cause Slave crystal in mass loss.In TGA/SDTA851e instrument, ([Mei Tele-support benefit is limited by Mettler-Toledo GmbH Company], Switzerland) in heating during monitor example weight, obtain weight to temperature curve.TGA/SDTA851e indium and aluminium calibration Temperature.Sample weighing into 100 μ L aluminium crucibles and is sealed.By sealing element thread a needle hole and by crucible with the heating of 10 DEG C/min speed Rate is heated to 300 DEG C from 25 DEG C in TGA.It is swept using dry N2 air-blowing.

Pass through 301 T2 of mass spectrograph Omnistar GSD (Pfeiffer Vacuum GmbH [Pu Fa vacuum company], moral State) analyze the gas released from TGA sample.The latter is a kind of quadrupole mass spectrometer, can analyze the matter within the scope of 0-200amu Amount.

Example 5d: it is characterized by FTIR

Use 6700 spectrometer of Thermo Fischer Scientific FT-IR Nicolet equipped with ATR probe Record FT-IR spectrum.

FTIR analysis confirms the structure of the compound with Formulas I, is such as described in detail and describes in table 4 in fig. 8, and If Fig. 9 is in about 1800cm^-1With 400cm^-1Between amplify.The feature IR of the crystal form of the dichloride salt of compound with Formulas I shakes It is dynamic to be accredited as 1701cm^-1、1665cm^-1、1335cm^-1、1241cm^-1、1171cm^-1、942cm^-1、924cm^-1、864cm^-1、 699cm^-1、628cm^-1(±2cm^-1)。

Table 4. has the main IR vibration of the crystal form of the dichloride salt of the compound of Formulas I

* several possible distribution.

Example 5e: pass through solid-state¹³C{¹H } MAS-NMR characterization:

Magic angle spinning solid-state carbon-13 magnetic resonance is carried out on Bruker Avance III 400MHz solid state NMR instrument (¹³C{¹H } MAS-NMR) (see Figure 10), which has wide aperture (89mm room temperature hole) 9.4 tesla's magnet.Double resonance evil spirit angle The rotor size that sample rotates (MAS) probe is 4.0mm for outer diameter.Double nuclear frequency-the In for being tuned to observe of the probe In this research¹³C is 100.61MHz,¹H is 400.13MHz.Magnetic field's regularity is by the ZrO in 4mm₂On rotator The setting of adamantane sample shimming,¹³C line width (half maximum height of overall with) is less than 2Hz.Chemical shift is referred to by using tetramethyl Silane¹H signal (CDCl₃In < 1%v/v) substitution technique carry out, chemical shift is set as 0ppm.This is that IUPAC recommends Program.All measurements are all that laterally blowing is other on MAS rotator It is carried out under nitrogen stream (in 5 DEG C of 1200L/h), to carry out temperature control.Due to frictionally heating for MAS air bearing, really Sample temperature is about 15 DEG C higher than this.For magic angle sample rotates, speed is set as 14kHz.Scanning times are 1024 times, are followed Ring delay time is 5s, time of contact 2ms, acquisition time 33ms, and processing parameter is tdeff=0 and lb=5Hz.

Table 5 lists 13 chemical shift of carbon of the crystal form of the dichloride salt for the compound with Formulas I studied.Fig. 1 Depict the atomicity of the NMR distribution of 13 chemical shift of carbon.

5. crystal form E's of table¹³C{¹H } MAS-NMR displacement (the 13C chemical shift of ± 0.2ppm), with reference to using tetramethylsilane Alkane¹H signal (TMS < 1%v/v, in CDCl₃In) substitution technique (its chemical shift is set as 0ppm).It also shows¹³C { 1H } NMR displacement, with reference to liquid [D6]-DMSO of [D6]-DMSO (its chemical shift is set as 39.52ppm*).

^{[a]、[b]、[c]、[d]}It can be exchanged with identical upper target signal.

* H.E.Gottlieb, V.Kotlyar, A.Nudelman J.Org.Chem [Journal of Organic Chemistry], volume 62, 1997,7512-7515

Example 5f: it is characterized by DVS

The measurement of its relative stability when the moisture absorption sex differernce of different form solid material provides relative humidity increase.Make It is obtained with the DVS-1 system from Surface Measurement Systems [Surface Measurement Systems company] (London) Moisture sorption isotherm.In about 25 DEG C of steady temperature, relative humidity is changed during absorption-desorption (see specific experiment). At the end of DVS experiment, sample is measured by XRPD.

Figure 11 depicts dynamic vapor sorption (DVS) analysis of the crystal form E of the dichloride salt of the compound with Formulas I. It shows: the water absorption rate of the up to compound of 85%RH is 1% and the water absorption rate of the compound of up to 95%RH is about 4%.

Example 5g: solubility

In non-buffered water and use standard MerckBuffer carries out the dissolution of thermodynamics pH dependence Degree measurement (MerckPH of buffer 3 has citrate and HCl；MerckPH of buffer 4, With citrate and HCl；MerckPH of buffer 5 has citrate and NaOH；MerckPH of buffer 6 has citrate and NaOH；MerckPH of buffer 7 has phosphate； In order to be buffered in pH 4.5,50/50 mixture of the buffer of pH 4 and 5 is used；In order to be buffered in pH 5.5,5 He of pH is used 50/50 mixture of 6 buffer).

For each experiment, 8mL spiral is prepared with polycrystalline material, according to the buffer solvent and magnetic stirring bar of target pH Cap bottle.Each pH data point measures in triplicate, and target pH is 3,4,4.5,5,5.5 and 7.It measures pH and (flies generation that pH meter (Fisherbrand pH meter Hydrus 400), carries out three point calibrations before measuring) and adjusted with 1M NaOH solution. Balance mixture at room temperature for 24 hours.After for 24 hours, monitors pH and slurries are centrifuged 10min with 3000rpm to divide From solid and liquid and filter (0.45 micron of disc filter).If it is necessary, isolated filtrate is dilute in sample solvent It releases to fall into the calibration curve of HPLC test.The concentration of compound with Formulas I with Diode Array Detector by analyzing (HPLC-DAD) high effective liquid chromatography for measuring.Calibration curve is from the sample in water/THF/TFA (50/50/0.05v/v/v) It is obtained in the stock solution of two kinds of independently prepared compounds with Formulas I in product solution.

The HPLC test of 280nm wavelength is carried out on the Agilent 1100 with DAD detector.11 μ g/mL have been determined LOQ, the linearity be up to about 0.7mg/mL.Each sample be diluted to about 0.5mg/mL or if concentration less than or equal to about 0.5mg/mL is also measured as whole value.

Example 6- has the preparation of the crystallization dichloride salt (A+M) of the compound of Formulas I

Example 6a: the thick dichloride salt of the compound with Formulas I

The 111.6g (156mmol) of the program provided according to example 2 preparation is had to the compound (R of Formula II³It is tertiary fourth Base) it is suspended in the THF of 738mL and is heated to about 33 DEG C.It adds the 30% aqueous HCl of 160g and stirs the mixture for about 18h.Mixture is cooled to about 10 DEG C and adds the THF of 738mL.Suspension is filtered, filter cake is washed with the THF of 120mL It washs and in about 40 DEG C of vacuum drying to provide the compound with Formulas I of 90g.

Example 6b: purifying and crystallization

The crude compound (2.6kg) with Formulas I is dissolved in water (2.7L) and tetrahydrofuran (5.5L) at about 40 DEG C -50 DEG C In.At about 40 DEG C -50 DEG C, it is slowly added tetrahydrofuran (90L).Resulting suspension is stirred, is subsequently cooled to about 10 DEG C simultaneously And it futher stirs.Suspension is filtered, filter cake is washed and dried with THF.Resulting solid (2.4kg) is dissolved in In 7.3L water, solution is filtered and by the water washing of filter 2.3L.The solution of filtering and cleaning solution are subtracted at about 30 DEG C Pressure is evaporated to drying.Residue is further dried under reduced pressure at 50 DEG C, is in mixture A1+M1 with Formulas I with provide 2.2kg Compound.

In general, the starting point for generating other crystal forms in system A+M is mixture A1+M1 (Figure 14) and mixture A1+M4 (figure 15).Figure 19 gives the stacking chart for the XRPD figure observed when mixture A1+M4 is exposed to climatic chamber condition.40 DEG C/ 75%RH observes mixture M 3+M5 (Figure 24) after 1 week and also after 2.5 weeks.Mixture A1+ is handled in 40 DEG C/75%RH After M4 4 weeks, crystal form M5 (Figure 26) is observed.After 40 DEG C/75%RH4 weeks and after 25 DEG C/95%RH is 2 days, obtain Mixture A2+M4 (Figure 17).After 40 DEG C/75%RH is 4 weeks and after 25 DEG C/95%RH is 1 week, mixture A2+ is obtained M11 (Figure 18).

Example 7- has the preparation of the specific crystal of the crystallization dichloride salt in the system A+M of the compound of Formulas I

The preparation of crystal form A0

Example 7a

Continue 2.5h to 195 DEG C by heating mixture A1+M1 and obtains crystal form A0 (Figure 12, table 6).

Example 7b

Continue 4h to 195 DEG C by heating crystal form M1 and obtains crystal form A0.

The preparation of crystal form A1

Example 7c

Crystal form A1 (Figure 13, table 7) was obtained for about 11 days by allowing crystal form A0 to stand at ambient conditions.

Example 7d

By obtaining crystal form A1 to mixture A1+M1 crystallisation by cooling in following solvent system: water and methanol/water (50: 50).The 80 respective solvents of μ L are added in the mixture A1+M1 of about 4mg.It raises the temperature to 60 DEG C and is kept at 60 DEG C 60min.After being cooled to 20 DEG C with the cooling rate of 20 DEG C/min, so that mixture is maintained 20 DEG C under stiring and continue for 24 hours.Pass through Solvent evaporation under vacuum (5mbar) obtains crystal form F.Crystal form F is exposed to 67h under the conditions of the climatic chamber of 40 DEG C/75%RH, is produced Raw crystal form A1.

Example 7e

By obtaining crystal form A1 to mixture A1+M1 crystallisation by cooling in methyl alcohol.

The methanol of 80 μ L is added in the mixture A1+M1 of about 4mg.It raises the temperature to 60 DEG C and is kept at 60 DEG C 60min.After being cooled to 2 DEG C with the cooling rate of 20 DEG C/min, so that mixture is maintained 2 DEG C under stiring and continue for 24 hours.By true Solvent evaporation under empty (5mbar) obtains crystal form F.Crystal form F is exposed to 67h under the conditions of the climatic chamber of 40 DEG C/75%RH, is generated Crystal form A1.

The preparation of mixture A1+M1

XRPD diffraction pattern is depicted in Figure 14 and table 19.

Example 7f

By the compound with Formulas I of 23.2mg, i.e. mixture A1+M4 is added in the ether of 0.60mL, generates slurries, The slurries are stirred two weeks at 20 DEG C.Later, sample is centrifuged, liquid is isolated by filtration, and under vacuum by solid portion Dry (5mbar).To solid analysis and find it for mixture A1+M1.

Example 7g

By the compound with Formulas I of 22.7mg, i.e. mixture A1+M4 is added in the t-butyl methyl ether of 0.60mL, Slurries are generated, which is stirred two weeks at 20 DEG C.Later, sample is centrifuged, is isolated by filtration liquid, and by solid portion (5mbar) is dried under vacuum.To solid analysis and find it for mixture A1+M1.

The preparation of mixture A1+M4

XRPD diffraction pattern is depicted in Figure 15 and table 20.

Example 7h

Continue at least 3min by the way that 20mg mixture A1+M1 is exposed at least 40%RH and forms mixture A1+M4.

Example 7i

At 20 DEG C, the mixture A1+M1 pulp of 23.2mg is continued two weeks in the ether of 0.60mL.Pass through centrifugation and mistake Isolated moist solid is filtered, and analyzes and is found to be mixture A1+M4.

Example 7j

Continue two weeks in 20 DEG C of t-butyl methyl ethers by the mixture A1+M1 pulp of 22.7mg in 0.60mL.Pass through The moist solid for being centrifuged and being separated by filtration, and analyze and be found to be mixture A1+M4.

Example 7k

Continue two weeks in 20 DEG C of normal heptanes by the mixture A1+M1 pulp of 24.2mg in 0.60mL.By centrifugation and The moist solid being separated by filtration, and analyze and be found to be mixture A1+M4.

Example 7l

At 20 DEG C, the mixture A1+M1 pulp of 18.9mg is continued two weeks in the toluene of 0.60mL.Pass through centrifugation and mistake Isolated moist solid is filtered, and analyzes and is found to be mixture A1+M4.

Example 7m

Continue two weeks in 50 DEG C of diisopropyl ethers by the mixture A1+M1 pulp of 18.9mg in 0.40mL.Pass through centrifugation With the moist solid being separated by filtration, and analyzes and be found to be mixture A1+M4.

Example 7n

Continue two weeks in 50 DEG C of normal heptanes by the mixture A1+M1 pulp of 22.8mg in 0.40mL.By centrifugation and The moist solid being separated by filtration, and analyze and be found to be mixture A1+M4.

Example 7o

At 50 DEG C, the mixture A1+M1 pulp of 24.9mg is continued two weeks in the toluene of 0.40mL.Pass through centrifugation and mistake Isolated moist solid is filtered, and analyzes and is found to be mixture A1+M4.

The preparation of mixture A1+M4+M5

Example 7p

Mixture A1+M4+M5 is formed by the way that mixture A1+M4 is exposed to 60% to 80%RH about 3min.

The preparation of mixture A2+M4

XRPD diffraction pattern is depicted in Figure 17 and table 21.

Example 7q

40 DEG C/75%RH storing mixture A1+M4 it is for 4 weeks and 25 DEG C/95%RH store 2 days after, obtain mixed Close object A2+M4.

The preparation of mixture M 3+M5

XRPD diffraction pattern is depicted in Figure 16 and table 11.

Example 7r

40 DEG C/75%RH storing mixture A1+M4 it is for 1 week and 2.5 weeks between time after observe mixture M 3+ M5。

The preparation of mixture A2+M11

XRPD diffraction pattern is depicted in Figure 18 and table 22.

Example 7s

40 DEG C of 75%RH storing mixture A1+M4 it is for 4 weeks and 25 DEG C/95%RH store 1 week after mixed Object A2+M11 (Figure 19).

The preparation of crystal form A2

XRPD diffraction pattern is depicted in Figure 20 and table 20.

Example 7t

By obtaining crystal form A2:1,4- to mixture A1+M1 crystallisation by cooling in following all different solvent systems Dioxanes/water (50:50), isopropanol/water (50:50), acetonitrile/water (50:50), ethanol/water (50:50), isopropanol and third Ketone/water (50:50).The 80 respective solvents of μ L are added in the mixture A1+M1 of about 4mg.Raise the temperature to 60 DEG C and In 60 DEG C of holding 60min.After being cooled to 20 DEG C with the cooling rate of 20 DEG C/min, so that mixture is maintained 20 DEG C under stiring and hold Continue for 24 hours.Crystal form F is obtained by the solvent evaporation under vacuum (5mbar).Crystal form F is exposed to the climatic chamber item of 40 DEG C/75%RH 67h under part generates crystal form A2.

Example 7u

By obtaining crystal form A2: methanol and ethyl alcohol to mixture A1+M1 crystallisation by cooling in following solvent system.By 80 The respective solvent of μ L is added in the mixture A1+M1 of about 4mg.Raise the temperature to 60 DEG C and in 60 DEG C of holding 60min.With After the cooling rate of 20 DEG C/min is cooled to 20 DEG C, so that mixture is maintained 20 DEG C under stiring and continue for 24 hours.Pass through vacuum Solvent evaporation under (5mbar) obtains crystal form G.Crystal form G is exposed to 67h under the conditions of the climatic chamber of 40 DEG C/75%RH, is generated brilliant Type A2.

The preparation of crystal form M1

XRPD diffraction pattern is depicted in Figure 22 and table 9.

Example 7v

By obtaining crystal form M1: water, 1 to mixture A1+M1 crystallisation by cooling in following all different solvent systems, 4- dioxanes/water (50:50), ethyl acetate/dimethyl sulfoxide (50:50), isopropanol/water (50:50), acetonitrile/water (50:50), Ethanol/water (50:50) and tetrahydrofuran/water (50:50).The 80 respective solvents of μ L are added to the mixture A1+M1 of about 4mg In.Raise the temperature to 60 DEG C and in 60 DEG C of holding 60min.After being cooled to 2 DEG C with the cooling rate of 2 DEG C/min, stirring Under make mixture maintain 2 DEG C to continue for 24 hours.Crystal form F is obtained by the solvent evaporation under vacuum (5mbar).Crystal form F is exposed to 67h under the conditions of the climatic chamber of 40 DEG C/75%RH generates crystal form M1.

Example 7w

By obtaining crystal form M1 to mixture A1+M1 crystallisation by cooling in following different solvent system: paraxylene/ Methanol (50:50) and 2- butanone/methanol (50:50).The 80 respective solvents of μ L are added in the mixture A1+M1 of about 4mg.It will Temperature is increased to 60 DEG C and in 60 DEG C of holding 60min.After being cooled to 2 DEG C with the cooling rate of 2 DEG C/min, make to mix under stiring Conjunction object, which maintains 2 DEG C, to be continued for 24 hours.Crystal form G is obtained by the solvent evaporation under vacuum (5mbar).By crystal form G be exposed to 40 DEG C/ 67h under the conditions of the climatic chamber of 75%RH generates crystal form M1.

Example 7x

By obtaining crystal form M1 to mixture A1+M1 crystallisation by cooling in following different solvent system: tetrahydrofuran/ Methanol (50:50) and 2 tetrahydrofurans/ethyl acetate (50:50).The 80 respective solvents of μ L are added to the mixture A1+ of about 4mg In M1.Raise the temperature to 60 DEG C and in 60 DEG C of holding 60min.After being cooled to 20 DEG C with the cooling rate of 20 DEG C/min, In Make mixture maintain 20 DEG C under stirring to continue for 24 hours.Crystal form G is obtained by the solvent evaporation under vacuum (5mbar).By crystal form G It is exposed to 67h under the conditions of the climatic chamber of 40 DEG C/75%RH, generates crystal form M1.

Example 7y

By obtaining crystal form M1 to mixture A1+M1 crystallisation by cooling in following all different solvent systems: acetonitrile/ Water (50:50), tetrahydrofuran/water (50:50), methanol/water (50:50), acetone/water (50:50), 2 butanone/water (50:50), second Acetoacetic ester/methanol (50:50) and tetrahydrofuran/methanol (50:50).The 80 respective solvents of μ L are added to the mixture of about 4mg In A1+M1.Raise the temperature to 60 DEG C and in 60 DEG C of holding 60min.After being cooled to 2 DEG C with the cooling rate of 20 DEG C/min, Make mixture maintain 2 DEG C under stiring to continue for 24 hours.Crystal form F is obtained by the solvent evaporation under vacuum (5mbar).By crystal form F It is exposed to 67h under the conditions of the climatic chamber of 40 DEG C/75%RH, generates crystal form M1.

The preparation of crystal form M2

From mixture A1+M4, obtained by carrying out collision crystallization (crash-crystallisation) with anti-solvent addition Crystal form M2 (Figure 23, table 10).

Example 7z

In all following different solvent systems, by carrying out collision crystallization with the addition of the anti-solvent of mixture A1+M1 Obtain crystal form M2: solvent: n-butyl alcohol/water (9.6:90.4v/v) and each anti-solvent: acetonitrile, 2- butanone, tetrahydrofuran or acetic acid Ethyl ester.Stock solution is prepared in 200 μ L solvents, with Formulas I compound concentration balance before filtration 24 hours after Reach saturation under environment temperature, or the retention concentration with 170mg/mL.

For each experiment, anti-solvent is added in each solvent bottle, solvent and anti-solvent ratio are 1:0.25.In In the case where not precipitating, which increases to 1:1, and if the ratio increases to 1:4 again without precipitating (for all crystal form M2 preparations) wait the time (until third time is added) of 60min between addition.Due to not enough Solid precipitation and separation, sample are maintained at 5 DEG C three days.Do not precipitate.Solvent is evaporated to dryness in 200mbar.

Using different solvent systems, obtain different intermediate polymorphics, i.e., it is armorphous (from anti-solvent acetonitrile, 2- butanone), crystal form M1 (tetrahydrofuran) and mixture F+M1 (ethyl acetate).By measurement plate accelerated ageing conditions (40 DEG C/ After storing 65h under 75%RH), all these samples are converted to polymorphic M2.

The preparation of crystal form M4

From mixture A1+M4, crystal form M4 (Figure 25, table 12) is mainly obtained by the slurry test at pH 4.

Example 7aa

The compound (mixture A1+M4) with Formulas I of 151.4mg is suspended in the 4 buffer (Merck of pH of 600 μ LPH of buffer 4 has citrate and HCl) in.Initial pH is about 3.2.After 15min, by pH with 25 μ L 0.1M NaOH is adjusted to about 4.1.After 2h-4h, pH is adjusted to 3.8.The pH 4 for adding 10 μ L 0.1M NaOH and 200 μ L is slow Fliud flushing.(including addition number) for 24 hours is stirred at room temperature in slurries.The slurries of acquisition show about pH 4.0.Use 1 micron of disc type mistake Filter is filtered.Obtain the crystal form M4 in filter cake.

Example 7bb

The mixture A1+M4 of 198.3mg is suspended in the 4 buffer (Merck of pH of 1000 μ LBuffer PH 4 has citrate and HCl) in.Initial pH is about 2.9.After 15min, pH is adjusted to about with 50 μ L 0.1M NaOH 3.8.(including addition number) for 24 hours is stirred at room temperature in slurries.Obtain the muddy solution of about pH 3.8.Use 1 micron of disk Formula filter is filtered.Obtain the crystal form M4 in filter cake.

Example 7cc

The mixture A1+M4 of 245.4mg is suspended in the pH4 buffer (Merck of 1000 μ LPH of buffer 4, there is citrate and HCl) in.Initial pH is about 3.1.After 15min, pH is adjusted to about 3.9 with 50 μ L 0.1M NaOH. It slurry is stirred for 30-45 minutes and pH is adjusted to about 3.9.The 0.1M NaOH of 10 μ L is added to generate about pH 4.1.By slurries (including addition number) for 24 hours is stirred at room temperature.The slurries of acquisition show about pH 4.0.It was carried out using 0.2 μm of centrifugal filter Filter.Obtain the crystal form M4 in filter cake.

The preparation of crystal form M5

XRPD diffraction pattern is depicted in Figure 26 and table 13.

Example 7dd

By having the compound of Formulas I, that is, mixture A1+M1 or A1+M4 is for 4 weeks obtains in 40 DEG C/75%RH storage Crystal form M5.

The preparation of crystal form M8

From mixture A1+M4, crystal form M8 (Figure 27, table 14) is mainly obtained by the slurry test at pH 7.5.It wants It is noted that these experiments use the buffer for containing alternative counter ion.Exist in polymorphic although cannot ignore completely Trace counter ion, but there is no in XRPD diffraction pattern the visible diffraction maximum for being attributable to these inorganic substances (inorganic matter be logical 2 angle θ Chang Gao is just high-visible and is usually very sharp peak).

Example 7ee

By MerckPH of buffer 7 (there is phosphate) and Merck(the tool of pH of buffer 8 Have borate and HCl) it is mixed with the ratio of 1:1 (v/v) to provide the buffer with pH 7.5.By by the mixing of 26.9mg Object A1+M4 is added in 7.5 buffer of above-mentioned pH of 5.0mL and prepares suspension.Resulting pH is about 7.3.After 15min, by pH About pH7.4 is adjusted to 10 μ L 0.1M NaOH.(including addition number) for 24 hours is stirred at room temperature in mixture.Obtain pH About 7.5 slurries.It is filtered using 1 micron of disc filter.Obtain the crystal form M8 in filter cake.

Example 7ff

It is prepared for suspension of the mixture A1+M4 of 16.4mg in 7.5 buffer of above-mentioned pH of 5.0mL.Initially pH is About 7.5.Resulting mixture is stirred at room temperature for 24 hours.Obtain the slurries of about pH 7.4.Use 1 micron of disc filter It is filtered.Obtain the crystal form M8 in filter cake.

The preparation of crystal form M9

From mixture A1+M4, crystal form M9 (figure is mainly obtained by slurry test in the range of pH 4.5 to 5.5 28, table 15).It should be noted that these experiments use the buffer for containing alternative counter ion.Although cannot ignore completely more Trace counter ion present in crystal form, but there is no the visible diffraction maximum for being attributable to these inorganic substances in XRPD diffraction pattern (inorganic matter is usually just high-visible at the angle high 2 θ and is usually very sharp peak).

Example 7gg

The mixture A1+M4 of 150.5mg is suspended in the Merck of 5.0mL(pH 5, contains lemon to buffer Acid and NaOH) in.Initial pH is about 4.2.After 15min, pH is adjusted to about pH 4.9 with 70 μ L 0.1M NaOH.By mixture (including addition number) for 24 hours is stirred at room temperature.Obtain the slurries of about pH 5.1.It was carried out using 1 micron of disc filter Filter.Obtain the crystal form M9 in filter cake.

Example 7hh

The mixture A1+M4 of 32mg is suspended in the Merck of 5.0mL(pH 5, contains citric acid to buffer And NaOH) in.Initial pH is about 5.0.(including addition number) for 24 hours is stirred at room temperature in mixture.Obtain about pH 5.0 Slurries.It is filtered using 1 micron of disc filter.Obtain the crystal form M9 in filter cake.

Example 7ii

By MerckPH of buffer 5 (containing citrate and NaOH) and MerckBuffer PH 6 (containing citrate and NaOH) generates the buffer of pH 5.5 with the ratio mixing of 1:1 (v/v).By having for 34mg The compound (mixture A1+M4) of Formulas I is suspended in 5.5 buffer of above-mentioned pH of 5.0mL.Initial pH is about 5.6.It will mixing (including addition number) for 24 hours is stirred at room temperature in object.Obtain the slurries of about pH 5.5.It is carried out using 1 micron of disc filter Filtering.Obtain the crystal form M9 in filter cake.

The preparation of crystal form M11

By the way that pH is changed into 7 from 3 in supersaturation experiment, crystal form M11 (figure is obtained from mixture A1+M4 and crystal form E 30, table 16).It should be noted that these experiments use the buffer for containing alternative counter ion.Although cannot ignore completely more Trace counter ion present in crystal form, but there is no the visible diffraction maximum for being attributable to these inorganic substances in XRPD diffraction pattern (inorganic matter is usually just high-visible at the angle high 2 θ and is usually very sharp peak).

Example 7kk

The crystal form E of about 210mg is suspended in 1.00mL MerckPH of buffer 3 (containing citrate and HCl in) and 20 μ L 0.1M NaOH are added.Filter the saturated solution (0.2 μm of centrifugal filter).Passing through 270 μ L of addition 0.1M NaOH be adjusted to before pH 7 for solution to be kept at room temperature and continue for 24 hours.There is solid precipitating.It was centrifuged with 0.2 μm Filter filters suspension and obtains the crystal form M11 in filter cake.When pH is adjusted to pH 7 by the 0.1M NaOH using 350 μ L, make Identical result is obtained with unfiltered solution.

Example 7ll

The mixture A1+M4 of about 420mg is suspended in the 0.1M in 3 buffer of 1.00mL pH and adding 40 μ L NaOH.Filtering saturated solution (0.2 μm of centrifugal filter) is simultaneously kept for 24 hours at room temperature, passes through the 0.1M of 300 μ L of addition later NaOH is adjusted to pH 7.There is solid precipitating.With 0.2 μm of centrifugal filter filtering suspension and acquisition is in the crystal form M11 of filter cake. When pH is adjusted to pH 7 by the 0.1M NaOH using 350 μ L, identical result is obtained using unfiltered solution.

The preparation of crystal form M12

Crystal form M12 (Figure 31, table 17) is observed from mixture A1+M4 in the different slurry runs of about pH 7.It infuses Meaning, these experiments use the buffer for containing alternative counter ion.Although cannot ignore completely present in the polymorphic Trace counter ion, but in XRPD diffraction pattern there is no the visible diffraction maximum for being attributable to these inorganic substances (inorganic matter is usual At the just high-visible and usually very sharp peak in the angle high 2 θ).

Example 7mm

The mixture A1+M4 of about 30mg or crystal form E are suspended in the Merck of 5.0mLPH of buffer 7 (contains Have phosphate) in.Initial pH is about 6.9.After stirring 15min, pH is adjusted to about 7.0 with 10 μ L 0.1M NaOH.It will mixing (including addition number) for 24 hours is stirred at room temperature in object.Obtain the slurries of about pH 7.0.Using 0.45 micron of disc filter into Row filtering.Obtain the crystal form M12 in filter cake.

The preparation of crystal form M13

Crystal form M13 is obtained by the way that pH is changed into 5 from mixture A1+M4 and crystal form E from 3 in supersaturation experiment (Figure 32, table 18).It should be noted that these experiments use the buffer for containing alternative counter ion.Although cannot ignore completely The trace counter ion present in polymorphic, but do not have in XRPD diffraction pattern visible to be attributable to spreading out for these inorganic substances Penetrate peak (inorganic matter is usually just high-visible at the angle high 2 θ and is usually very sharp peak).

Example 7nn

The crystal form E of about 210mg is suspended in 1.0mL MerckPH of buffer 3 (containing citrate and HCl in) and 20 μ L 0.1M NaOH are added.Filtering saturated solution (0.2 μm of centrifugal filter) is simultaneously kept for 24 hours at room temperature, PH 5 is adjusted to by adding the 0.1M NaOH of about 50 μ L later.There is solid precipitating.It is outstanding with 0.2 μm of centrifugal filter filtering Supernatant liquid and acquisition are in the crystal form M13 of filter cake.When pH is adjusted to pH 5 by the 0.1M NaOH using 70 μ L, use is unfiltered Solution obtains identical result.

Example 7oo

The mixture A1+M4 of about 410mg is suspended in 1.00mL MerckPH of buffer 3 (contains lemon Hydrochlorate and HCl) in and add 40 μ L 0.1M NaOH.Filtering saturated solution (0.2 μm of centrifugal filter) is simultaneously protected at room temperature It holds for 24 hours, is adjusted to pH 5 by adding the 0.1M NaOH of 60 μ L later.There is solid precipitating.With 0.2 μm of centrifugal filter mistake It filters suspension and obtains the crystal form M13 in filter cake.When pH is adjusted to pH 5 by the 0.1M NaOH using 80 μ L, non-mistake is used The solution of filter obtains identical result.

Note that while crystal form F and G in the above-described example in A+M system when preparing some polymorphics as intermediate Form description, but solvent is apparent in its physical stability and plays an important role.According to used solvent, crystal form F and G be can be Solvation or anhydrous form.

Example 8- has the characterization of the crystallization dichloride salt (A+M) of the compound of Formulas I

Example 8a: it is characterized by XRPD

The carry out XRPD analysis such as described in example 5a.These be included in spontaneous mixture in A+M system, with And the separated polymorphous peak XRPD specific A or M as mentioned.Data include polymorphic A0, A1, A2, M1, M2, M3+ M5, M4, M5, M8, M9, M10+M4, M11, M12, M13 and mixture A1+M4, A2+M4 and the A2+M11 often observed.Also it observes To crystal form M6 and M7, but only as other polymorphous mixtures with a part for not being A+M system.

The list of the peak position XRPD of 6. crystal form A0 of table.

The list of the peak position XRPD of 7. crystal form A1 of table.

The list of the peak position XRPD of 8. crystal form A2 of table.

The list of the peak position XRPD of 9. crystal form M1 of table.

The list of the peak position XRPD of 10. crystal form M2 of table.

The list of the peak position XRPD of 11. mixture M 3+M5 of table.

The list of the peak position XRPD of 12. crystal form M4 of table.

The list of the peak position XRPD of 13. crystal form M5 of table.

The list of the peak position XRPD of 14. crystal form M8 of table.

The list of the peak position XRPD of 15. crystal form M9 of table.

The list of the peak position XRPD of 16. crystal form M11 of table.

The list of the peak position XRPD of 17. crystal form M12 of table.

The list of the peak position XRPD of 18. crystal form M13 of table.

The list of the peak position XRPD of 19. mixture A1+M1 of table.

The list of the peak position XRPD of 20. mixture A1+M4 of table.

The list of the peak position XRPD of 21. mixture A2+M4 of table.

The list of the peak position XRPD of 22. mixture A2+M11 of table.

The list of the peak position XRPD of 23. crystal form F of table.

The list of the peak position XRPD of 24. crystal form G of table.

Example 8b: experiment high-resolution X-ray powder diffraction (including variable humidity and alternating temperature XRPD experiment)

Variable humidity (VH) and alternating temperature (VT) are tested, using the room ANSYCO HT, are mounted on cloth loudspeaker lattice-Boulogne tower Promise geometry (Bragg-Brentano geometry), which designs and is equipped with the D8 Advance system of LynxEye solid-state detector, to spread out It penetrates in instrument (Bruker [Brooker company]).Radiation for collecting data is the CuK α 1 by germanium crystal monochromatizationMaterial is placed on and is installed on indoor fixed specimen holder.

VH-XRPD: humidity locally applies, and changes to 70% (dew point) from 10%.Figure is collected in 4 ° -30 ° (2 θ) ranges, The step of VH-XRPD is 0.0145 ° (2 θ), and every step time of measuring is 1.2 seconds.It opens within 60 seconds after the moisture stable of each step Dynamic data collection (the data collection time about 40min of each RH value).All figures acquire at room temperature, about 295K.

VT-XRPD: rate of temperature change is 10 DEG C/min, and the equilibration time before each temperature starts data collection is 8min.In following scale collection figure: 4 ° -34.5 ° (2 θ), step is 0.0107 ° (2 θ) and the time of measuring of every step is 1 second (being directed to T=25 DEG C, 50 DEG C, 80 DEG C, 100 DEG C and 110 DEG C) or 1.5sec (being directed to T=40 DEG C, 60 DEG C, 115 DEG C -180 DEG C).Often The data collection time of a temperature is 48min or 70min, this depends on the time of measuring of every step.

Crystal form A1+M4 is placed in climatic chamber in 40 DEG C/75%RH to test 4 weeks, then stores two in 25 DEG C/95%RH Week.During studying herein, initial crystal form A1+M4 is changed into M3+M5 after 1 week, is changed into crystal form M5 after 4 weeks, and at 4 weeks It is changed into crystal form A2+M4 after with 2 days, is finally just changed into crystal form A2+M11 (Figure 19).

Example 8c: it is characterized by DVS

Related experimental detail refers to example 5f.Figure 35 depicts the crystallization of the dichloride salt of the compound with Formulas I The DVS of system A+M is analyzed.It shows: the water absorption rate of the up to compound of 85%RH is the change of about 22% and up to 95%RH The water absorption rate for closing object is below about 34%.

Example 8d: solubility

If example 5g is for as described in crystal form E, the thermodynamics pH dependent solubility of crystal form A1+M4 is determined, in addition to target PH is 1,2,3 (two different buffers), 4,4.5,5,5.5,6,6.5,7.5,8,9.5,10.5,11.5 and 12.5.In addition The buffer used is MerckPH of buffer 1 (has glycine and HCl)；MerckBuffer PH 2 (has citrate and HCl)；MerckPH of buffer 8 (has borate and HCl)；MerckPH of buffer 9 (has boric acid, KCl and NaOH)；MerckPH of buffer 10 (have boric acid, KCl and NaOH)；MerckPH of buffer 11 (has boric acid, KCl and NaOH)；MerckBuffering Liquid pH 12 (has phosphate and NaOH)；MerckPH of buffer 13 (has KCl and NaOH)；For not having The second buffer of the pH 3 of HCl, by the citric acid (21.01g monohydrate potassium is in 1L deionized water) of 80.3mL with The 0.2M disodium hydrogen phosphate (35.6g is in 1L deionized water) of 19.7mL mixes.In order to buffer in pH 6.5, pH 6 and 7 is used Buffer 50/50 mixture；In order to be buffered in pH 7.5,50/50 mixture of the buffer of pH 7 and 8 is used；In order to It is buffered in pH 9.5, uses 50/50 mixture of the buffer of pH 9 and 10；In order to be buffered in pH 10.5,10 He of pH is used 50/50 mixture of 11 buffer；In order to be buffered in pH 11.5,50/50 mixture of the buffer of pH 11 and 12 is used； In order to be buffered in pH 12.5,50/50 mixture of the buffer of pH 12 and 13 is used.The LOQ of about 8ug/mL has been determined.

(in addition to determining 18 μ g/ as described in the thermodynamics pH dependent solubility such as example 5g of crystal form A2+M11 is directed to crystal form E The LOQ of mL) it is measured.