PROCESSES FOR THE PREPARATION OF FURAZANOBENZIMIDAZOLES AND CRYSTALLINE FORMS THEREOF

Abstract

The present invention provides processes for preparing a compound of formula (I) and pharmaceutically acceptable salts thereof, comprising deprotecting a compound of formula (II), wherein each R.sup.3 independently represents a tertiary alkyl group, preferably wherein each R.sup.3 is tertiary butyl. The invention also provides intermediates useful for preparing compounds of formula (I) and processes for preparing these intermediates. Additionally the invention provides polymorphic forms of the dichloride salt of the compound of formula (I) and their use in the treatment of proliferative disorders.

##STR00001##

Claims

1. A process for preparing a compound of formula I: ##STR00013## or a pharmaceutically acceptable salt thereof, comprising step of deprotecting a compound of formula II: ##STR00014## wherein each R.sup.3 independently is a tertiary alkyl group.

2. The process according to claim 1, wherein each R.sup.3 is tertiary butyl.

3. The process according to claim 1, wherein the process further comprises the step of preparing a compound of formula II by reacting a compound of formula III: ##STR00015## wherein R.sup.1 is a leaving group; and wherein each R.sup.3 independently is a tertiary alkyl group; with a compound of formula IV: ##STR00016##

4. The process according to claim 3, wherein R.sup.1 represents chloro, bromo, iodo or a sulfonate ester.

5. The process according to claim 3, wherein R.sup.1 is chloro.

6. The process according to claim 1, wherein each R.sup.3 is tertiary butyl.

7. The process according to claim 3, wherein the process further comprises the step of preparing a compound of formula III wherein R.sup.1 is chloro by reacting a compound of formula V ##STR00017## wherein R.sup.2 is OH; and wherein each R.sup.3 independently is a tertiary alkyl group with a compound of formula VI ##STR00018## wherein R.sup.1a is chloro.

8. The process for preparing a compound of formula II, comprising reacting a compound of formula III with a compound of formula IV as defined in any one of claims 3 to 7.

9. The process according to claim 8, wherein R.sup.1 is chloro.

10. A process for preparing a compound of formula III, wherein R.sup.1 is chloro, comprising the step of reacting a compound of formula V with a compound of formula VI as defined in claim 7.

11. The process according to claim 7, wherein the compound of formula V is reacted with a compound of formula VI in the presence of dicyclohexyl carbodiimide (DCC).

12. The process according to claim 7, wherein the compound of formula V is reacted with a compound of formula VI in the presence of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide.

13. The process according to claim 12, wherein the compound of formula V is reacted with a compound of formula VI in the presence of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide to produce the compound of formula III via a one-pot reaction.

14. The process according to claim 1, further comprising the step of deprotecting the compound of formula II and obtaining the compound of formula I as a crystalline dichloride salt.

15. A compound of formula II: ##STR00019## wherein each R.sup.3 independently is a tertiary alkyl group.

16. A compound of formula III: ##STR00020## wherein R.sup.1 is chloro, bromo, iodo or a sulfonate ester, and each R.sup.3 independently is a tertiary alkyl group.

17. The compound of formula III according to claim 16, wherein R.sup.1 is chloro.

18. The compound of formula II according to claim 15 or a compound of formula III according to claim 16 wherein each R.sup.3 is tertiary butyl.

19. A crystalline dichloride salt of the compound of formula I: ##STR00021##

20. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising a peak at 6.0 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

21. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 6.0, 9.4 and 9.9 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

22. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 6.0, 9.4, 9.9, 10.7, 17.4, 21.4, 25.8 and 28.4, degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

23. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks 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 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

24. The crystalline dichloride salt according to claim 19, wherein the orthorhombic primitive cell parameters are a=4.813±0.001 Å, b=20.02±0.01 Å, c=59.40±0.02 Å, V=57245 Å.sup.3.

25. The crystalline dichloride salt according to claim 19, having an IR spectrum comprising peaks at 1701, 1665, 1335, 1241, 1170, 942, 924, 864, 699 and 628 cm.sup.−1 (±2 cm.sup.−1) and/or having a .sup.13C CP MAS (14 kHz) NMR spectrum referenced to TMS and/or a .sup.13C NMR spectrum in [D6]-DMSO comprising the peaks in the following table: TABLE-US-00025 [D6]-DMSO CP MAS 14 kHz — — 140.9 137.4 [a] — — 141.5 141.4 [a] 119.9 118.8 [b] 123.3 121.8 [b] 124.8 124.2 [b] 111.2 109.5 136.1 134.8 [a] 137.7 137.4 [a] — — — — 155.8 156.2 — — 40.1 40.3 16.7 19.0 119.1 119.6 — — 51.8 49.1 191.3 196.2 129.6 128.1 129.6 131.2 [c] 119.0 121.2 143.6 144.0 [a] 119.0 121.2 129.6 128.9 [c] — — 168.3 167.1 52.7 55.2 30.3 34.6 [d] 21.1 25.0 [d] 26.2 26.6 [d] 38.1 39.5 — — — — .sup.[a], .sup.[b], .sup.[c], .sup.[d] Signals with the same superscript might be exchanged.

26. A process for preparing the crystalline dichloride salt as defined in claim 19, comprising the step of crystallizing the dichloride salt of the compound of formula I from acetonitrile, methanol, ethanol, ethylacetate, or isopropanol or mixture thereof, or a solvent mixture comprising acetonitrile, methanol, ethanol, ethylacetate and/or isopropanol.

27. A process for preparing the crystalline dichloride salt as defined in claim 19, comprising the step of crystallizing the dichloride salt of the compound of formula I from acetonitrile, methanol or ethanol or mixture thereof, or a solvent mixture comprising acetonitrile, methanol and/or ethanol.

28. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising a peak at 3.9 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt contains essentially no moisture.

29. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.9, 7.9 and 9.7 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt contains essentially no moisture.

30. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.9, 7.9, 9.7, 11.2 and 23.9 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt contains essentially no moisture.

31. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.9, 7.9, 9.7, 11.2, 23.9, 25.0 and 25.5 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt contains essentially no moisture.

32. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising a peak at 2.7 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt is exposed to 100 percent humidity for a period of time such that it does not take up any additional moisture.

33. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 2.7, 8.3 and 9.4 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt is exposed to 100 percent humidity for a period of time such that it does not take up any additional moisture.

34. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 2.7, 8.3, 9.4, 14.8 and 19.7 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt is exposed to 100 percent humidity for a period of time such that it does not take up any additional moisture.

35. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 2.7, 8.3, 9.4, 14.8, 19.7 and 24.1 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation, when the crystalline salt is exposed to 100 percent humidity for a period of time such that it does not take up any additional moisture.

36. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising a peak at 3.6 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

37. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.6, 4.0 and 8.1 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

38. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.6, 4.0, 8.1, 9.4, 11.0, 21.1 and 24.5 degrees 2θ (±0.2 degrees 2θ).

39. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising a peak at 3.4 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

40. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.4, 4.0 and 8.1 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

41. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.4, 4.0, 8.1, 11.1, 16.5 and 24.0 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

42. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising a peak at 3.0 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

43. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.0, 3.6, and 9.4 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

44. The crystalline dichloride salt according to claim 19, having an X-ray powder diffraction pattern comprising peaks at 3.0, 3.6, 9.4, 11.1, 12.7, 15.3, 23.6, and 24.5 degrees 2θ (±0.2 degrees 2θ) when measured using CuKα radiation.

45. A pharmaceutical composition, comprising a pharmaceutically effective amount of the crystalline dichloride salt of the compound of formula I as defined in claim 19 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

46-47. (canceled)

48. A method of treating a proliferative disorder or disease, comprising the step of administering a therapeutically effective amount of a crystalline dichloride salt of the compound of formula I as defined in claim 19 to a patient in need thereof.

49. The method according to claim 48, wherein the proliferative disorder or disease is a neoplastic disease selected from epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ducal-, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, specialized gonadal neoplasms, paragangliomas and glomus tumors, naevi and melanomas, soft tissue tumors and sarcomas, fibromatous neoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed and stromal neoplasms, fibroepithelial neoplasms, synovial like neoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblastic neoplasms, mesonephromas, blood vessel tumors, lymphatic vessel tumors, osseous and chondromatous neoplasms, giant cell tumors, miscellaneous bone tumors, odontogenic tumors, gliomas, neuroepitheliomatous neoplasms, meningiomas, nerve sheath tumors, granular cell tumors and alveolar soft part sarcomas, Hodgkin's and non-Hodgkin's lymphomas, other lymphoreticular neoplasms, plasma cell tumors, mast cell tumors, immunoproliferative diseases, leukemias, miscellaneous myeloproliferative disorders, lymphoproliferative disorders and myelodysplastic syndromes.

50. The method according to claim 48, wherein the proliferative disorder or disease is cancer.

51. The method according to claim 30, wherein the cancer in terms of the organs and parts of the body affected is selected from the brain, breast, cervix, ovaries, colon, rectum, (including colon and rectum i.e. colorectal cancer), lung (including small cell lung cancer, non-small cell lung cancer, large cell lung cancer and mesothelioma), endocrine system, bone, adrenal gland, thymus, liver, stomach, intestine (including gastric cancer), pancreas, bone marrow, hematological malignancies (such as lymphoma, leukemia, myeloma or lymphoid malignancies), bladder, urinary tract, kidneys, skin, thyroid, brain, head, neck, prostate and testis.

52. The method according to claim 50, wherein said cancer is selected from the group consisting of brain cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, brain cancer, neuroendocrine cancer, lung cancer, kidney cancer, hematological malignancies, melanoma and sarcomas.

53. The method according to claim 48, wherein the proliferative disorder or disease is a neoplastic disease, which neoplastic disease is a brain neoplasm selected from glial- and non-glial-tumors, astrocytomas (including glioblastoma multiforme and unspecified gliomas), oligodendrogliomas, ependydomas, menigiomas, haemangioblastomas, acoustic neuromas, craniopharyngiomas, primary central nervous system lymphoma, germ cell tumors, pituitary tumors, pineal region tumors, primitive neuroectodermal tumors (PNET's), medullablastomas, haemangiopericytomas, spinal cord tumors including meningiomas, chordomas and genetically-driven brain neoplasms including neurofibromatosis, peripheral nerve sheath tumors and tuberous sclerosis.

54. The method according to claim 53, wherein the neoplastic disease is glioblastoma multiforme.

55. The method according to claim 50, wherein the cancer is a solid tumor.

56. The method according to claim 48, wherein the patient is a human.

57. The method according to claim 48, wherein the crystalline dichloride salt of the compound of formula I is as defined in claim 20.

58. The method according to claim 48, wherein the crystalline dichloride salt of the compound of formula I is as defined in claim 28.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0089] FIG. 1 shows the atom numbering for NMR assignments.

[0090] FIG. 2 shows the X-ray powder diffraction (XRPD) diffractogram of the crystalline form E of the dichloride salt of the compound of formula I at room temperature.

[0091] FIG. 3 shows the graphical representation of the Pawley (WPPD) calculation for the crystalline form E of the dichloride salt of the compound of formula I. The graphical representation of the whole powder pattern decomposition calculation is presented, where the upper line shows observed data from a high resolution XRPD. The black middle line presents the calculated powder pattern and the black sticks at the very bottom of the figure are indicating the position of peaks with their h, k, l indices. The grey bottom line represents the difference between calculated and (baseline corrected) observed points.

[0092] FIG. 4 shows the thermogravimetric analysis (TGA) of the crystalline Form E of the dichloride salt of the compound of formula I with endothermic peaks at about 130° C. (±2° C.) and 276° C. (±2° C.).

[0093] FIG. 5 shows the differential scanning calorimetry (DSC) of the crystalline Form E of the dichloride salt of the compound of formula I with endothermic peaks at about 130° C. (±2° C.) and 276° C. (±2° C.) as well as decomposition above this temperature.

[0094] FIG. 6 shows the cyclic DSC for the crystalline Form E of the dichloride salt of the compound of formula I using the temperature profile 25.fwdarw.200.fwdarw.25° C.; a heating rate of 10° C./min and fast cooling. The endotherm (130° C. 2° C.) indicates a solid-solid transition, which is reversible (exotherm at 97° C. 2° C. upon cooling).

[0095] FIG. 7 shows the XRPD diffractogram of the crystalline high temperature Form E1 of the dichloride salt of the compound of formula I at 180° C.

[0096] FIG. 8 shows the FTIR spectrum of the compound of formula I for the crystalline Form E of the dichloride salt of the compound of formula I.

[0097] FIG. 9 shows the zoom between 1830 and 400 cm.sup.−1 of the FTIR spectrum for the crystalline Form E of the dichloride salt of the compound of formula I.

[0098] FIG. 10 shows the magic angle spinning solid state carbon 13 {proton decoupled} nuclear magnetic resonance (.sup.13C{.sup.1H} MAS-NMR) spectrum for the crystalline Form E of the dichloride salt of the compound of formula I.

[0099] FIG. 11 shows the isothermic (24.1° C.) dynamic vapor sorption analysis for the crystalline Form E of the dichloride salt of the compound of formula I.

[0100] FIG. 12 shows the XRPD diffractogram of Form A0.

[0101] FIG. 13 shows the XRPD diffractogram of Form A1.

[0102] FIG. 14 shows the XRPD diffractogram of Mixture A1+M1.

[0103] FIG. 15 shows the XRPD diffractogram of Mixture A1+M4.

[0104] FIG. 16 shows the XRPD diffractogram of Mixture M3+M5.

[0105] FIG. 17 shows the XRPD diffractogram of Mixture A2+M4.

[0106] FIG. 18 shows the XRPD diffractogram of Mixture A2+M11.

[0107] FIG. 19 shows an overlay of XRPD diffractograms of (from bottom to top) F: Forms A1+M4, E: after 1 week at 40° C. 75% RH (M3+M5), D: after 2.5 weeks at 40° C./75% RH (M3+M5), C: after 4 weeks at 40° C./75% RH (M5), B: after 4 weeks at 40° C./75% RH and 2 days 25° C./95% RH (A2+M4), A: after 4 weeks at 40° C./75% RH and 1 week at 25° C./95% RH (A2+M11).

[0108] FIG. 20 shows the XRPD diffractogram of Form A2.

[0109] FIG. 21 shows the XRPD diffractogram of Mixture A2+A3.

[0110] FIG. 22 shows the XRPD diffractogram of Form M1.

[0111] FIG. 23 shows the XRPD diffractogram of Form M2.

[0112] FIG. 24 shows the XRPD diffractogram of Form M3+M5.

[0113] FIG. 25 shows the XRPD diffractogram of Form M4.

[0114] FIG. 26 shows the XRPD diffractogram of Form M5.

[0115] FIG. 27 shows the XRPD diffractogram of Form M8.

[0116] FIG. 28 shows the XRPD diffractogram of Form M9.

[0117] FIG. 29 shows the XRPD diffractogram of Mixture M10+M4.

[0118] FIG. 30 shows the XRPD diffractogram of Form M11.

[0119] FIG. 31 shows the XRPD diffractogram of Form M12.

[0120] FIG. 32 shows the XRPD diffractogram of Form M13.

[0121] FIG. 33 shows the XRPD diffractogram of Form F.

[0122] FIG. 34 shows the XRPD diffractogram of Form G.

[0123] FIG. 35 shows the isothermic (24.9° C.) dynamic vapor sorption measurement of the compound of formula I presenting the relative sample weight (%) versus the relative humidity. The starting form was Mixture A1+M4 and the humidity profile was 0.fwdarw.95.fwdarw.0% RH with steps of 10% RH until mass equilibration was achieved per step. The maximum mass change was 34% at 95% RH. No hysteresis was observed.

[0124] FIG. 36 shows the thermodynamic pH-dependent solubility of Form E.

[0125] FIG. 37A shows the thermodynamic pH-dependent solubility of Form A1+M4. FIG. 37B shows the thermodynamic pH-dependent solubility of Form A2+M11.

[0126] FIG. 38 shows the XRPD diffractogram of the dichloride salt of the compound of formula I produced according to the methodology of WO 2011/012577 and which is described on page 36, final paragraph, of WO 2011/012577. The upper XRPD plot is from sample stored at 5° C., the lower XRPD plot is from sample stored at −60° C.

EXAMPLES

Example 1—Synthesis of the Compound of Formula III

Example 1a: Synthesis of the Compound of Formula III (R.SUP.1.=Cl, R.SUP.3.=Tert-Butyl) by Activation with DCC

[0127] A solution of phosphoric acid (85%, 57 mL) in water (280 mL) was added to a suspension of N2,N6-bis(tert-butoxycarbonyl)-L-lysine dicyclohexylamine salt (438 g, 0.831 mol, 2.5 eq.) in diisopropyl ether (DIPE, IL) at room temperature and stirred until dissolution of the solids. The organic phase was washed with a mixture of phosphoric acid (85%, 20 mL) and water (160 mL), then with water (4×160 mL). After drying over anhydrous sodium sulfate the solution of bis(tert-butoxycarbonyl)-L-lysine (free acid) was concentrated. The concentrate was diluted with dichloromethane (DCM, 421 mL). A solution of dicyclohexylcarbodiimide (88.5 g, 0.429 mol, 1.25 eq.) in DCM (100 mL) was added at room temperature and the reaction mixture was stirred for 15 min. The resulting suspension was filtered, the cake washed with DCM (3×50 mL). 4-aminophenacyl chloride (56.2 g, 0.331 mol, 1.0 eq.) was added to the combined filtrates and the mixture was stirred for 4 h. Insoluble matter was filtered off and the filtrate was concentrated in vacuo. The concentrate was diluted with 4-methyl-2-pentanone (MIBK, 279 mL), heated to ca. 45° C. Heptane (836 mL) was added with cooling. The suspension was cooled to 10° C., stirred and filtered. The solid was washed with MIBK/heptane and heptane and dried. The crude product was crystallized from MIBK/heptane and dried to provide 119.4 g of the title compound (72%) in a purity of ≥99.5% and ≥99% ee.

Example 1b: Synthesis of the Compound of Formula III (R.SUP.1.=Cl, R.SUP.3.=Tert-Butyl) by Activation with T3P®

[0128] N2,N6-bis(tert-butoxycarbonyl)-L-lysine (85% w/w, 216 g, 531 mmol, 1.5 eq.) was dissolved in toluene (1500 g). A solution of 4-aminophenacyl chloride (60 g, 354 mmol, 1.0 eq.) and 4-(dimethylamino)-pyridine (DMAP, 4.32 g, 35.4 mmol, 0.1 eq.) in toluene (600 g) was added. The mixture was cooled to −15 to −10° C. Triethylamine (143 g, 1.42 mol, 4.0 eq.) was added followed by dosing of a solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P, 495 g of a 50% solution in toluene, 778 mmol, 2.2 eq.) in toluene (360 g) over 2 h at −15 to −10° C. The mixture was stirred for 17 h and warmed to ca. −5° C. Water (1524 g) was added and phases were separated at room temperature. The organic phase was washed with hydrochloric acid (pH1.0), then with hydrochloric acid (pH=0.5, 5% w/w ethanol) and with saturated aqueous sodium bicarbonate solution. The solution was filtered and allowed to stand. The suspension was concentrated at 30-35° C., 50 mbar, cooled to ca. 20° C. and stirred. The solid was filtered, washed with toluene and dried to provide 138.5 g of the title compound (79%) in a purity of 99.3% and ≥99% ee.

Example 2—Synthesis of the Compound of Formula II (R.SUP.3 .is Tert-Butyl)

[0129] 3-{[4-(1H-Benzoimidazol-2-yl)-1,2,5-oxadiazol-3-yl]amino}propanenitrile (47 g, 185 mmol, 1.00 eq) was dissolved in DMF (1.6 L). N-[4-(2-chloroacetyl)phenyl]-N2,N6-di-Boc-L-lysinamide (98 g, 197 mmol, 1.06 eq.) and potassium carbonate (49.5 g, 358 mmol, 1.94 eq.) was added. The mixture was heated to 40° C. for 5 h. The suspension was filtered and the filtrate was dosed to aqueous ammonium chloride solution (2.5% w/w, 7 L) at 0-5° C. The suspension was filtered and the solid was dried. The crude product was suspended in THF (188 mL) and water (100 mL). Methanol (3.4 L) was added at reflux (ca. 65° C.). The suspension was stirred for 1 hour and cooled to room temperature. The product was filtered, the solids washed with methanol and dried. The solids were heated to reflux in THF (188 mL) and methanol (3.4 L), and cooled to ca. 10° C. within 2 h. The suspension was filtered, washed with methanol and dried to provide 121 g of the title compound (91%) in a purity of 99.8%.

Example 3—Synthesis of the Compound of Formula I (Dihydrochloride)

[0130] The compound of formula II (R.sup.3 is tert-butyl) (119 g, 166.4 mmol, 1.00 eq.) was suspended in tetrahydrofuran (785 mL) and heated to 30° C. Aqueous hydrochloric acid (30% w/w, 170 g) was added within 3 h. The mixture was stirred for 48 h, cooled to 10° C., and tetrahydrofuran (785 mL) was added. The resulting suspension was filtered, the cake is washed with tetrahydrofuran and dried at up to 55° C. to provide 95.8 g (97.8%) crude product. The crude product (75 g) was dissolved in water (75 mL) and tetrahydrofuran (112 mL) at ca. 43° C. Tetrahydrofuran (2.85 L) was added at ca. 40° C. and the suspension was stirred at ca. 50° C. for 1 hour. After cooling to 10° C. the product was filtered, washed with tetrahydrofuran and dried at ca. 50° C. to provide 68 g of purified product. The purified product (67 g) was dissolved in water (201 mL) and the resulting solution was filtered. Water was evaporated. The product was further dried at up to 50° C. to provide 62.9 g of the title compound (83%) in a purity of 99.6%.

Comparative Example 1 (According to WO 2011/012577)

[0131] S-{5-benzyloxycarbonylamino-5-[4-(2-{2-[4-(2-cyanoethylamino)furazan-3-yl]-benzoimidazo-1-1-yl}-acetyl)-phenylcarbamoyl]-pentyl}-carbamic acid benzylester was hydrogenated in a mixture of THF/MeOH/HCl with hydrogen in the presence of Pd/C 10% for ca. 5 h. After work-up, chromatography and salt formation this resulted in the dihydrochloride of the compound of formula I with a purity of 90-91%, 81% ee (yield: 50%).

Example 4—Preparation of the Crystalline Dichloride Salt (Form E) of the Compound of Formula I

[0132] Some of the Examples below describe preparation of Form E using seed crystals. The main purpose of adding seed crystals was to speed up formation of the polymorph. It is believed that without seed crystals the Examples would have still resulted in Form E. Note that Examples 4d, 4f, 4 g, 4 h, 4i and 4k did not use seed crystals, as well as 4l, 4m, 4n, 4o and 4p.

Crystallization by Slurry

Example 4a: From Methanol/Methyl Tert-Butylether (MTBE)

[0133] 0.20 g of the compound of formula I was dissolved in 8 mL methanol at 65° C., the solution was filtered. 10 mg seeds of Form E were added and the mixture was stirred over 30 min. 12 mL MTBE was added dropwise over 2-3 h, the mixture obtained was cooled to 5-15° C. and stirred for ca. 40 h at 5-15° C. The mixture was filtered and the cake was dried under vacuum, providing 0.18 g solid of Form E.

Example 4b: From Methanol/Acetonitrile

[0134] 4 g of the compound of formula I (Mixture A1+M1) is dissolved in 40 mL methanol and 30-45° C. The solution was filtered and 200 mg seeds of Form E were charged into the solution. After stirring a suspension formed which was heated to reflux over ca. 15 h and concentrated to 12 mL. 20 mL acetonitrile was added, the suspension cooled slowly to 0-10° C. and filtered. The cake was dried at ca. 50° C. under vacuum, providing 3.4 g solid of Form E.

Example 4c: From Methanol/Toluene

[0135] 2 g of the compound of formula I (Mixture A1+M1) was dissolved in 20 mL methanol and the mother liquid from last batch at 30-45° C. The solution was filtered, seeded with 100 mg Form E and added dropwise to 50 mL hot toluene (80-90° C.). The resulting suspension was concentrated (ca. 20 mL solvent distilled off), further heated to the boiling point and then slowly cooled to 0-10° C. The suspension was filtered and the cake was dried at 50° C. under vacuum, providing 1.5 g of Form E.

Example 4d: From Methanol (Room Temperature Slurry)

[0136] 65 g of the compound of formula I (Mixture A1+M1) was dissolved in 485 mL methanol and stirred at 15-25° C. The solution was stirred for ca. 14 days. During stirring a suspension was formed. The suspension was filtered, the cake was washed with methanol and dried at ca. 50° C. under vacuum, providing 46 g of Form E.

Example 4e: From Methanol (Slurry at Reflux)

[0137] 2 g of the compound of formula I (Mixture A1+M4) was dissolved in 20 mL methanol at 30-45° C. The solution was filtered, seeded with form E and refluxed for ca. 15 h. The suspension was concentrated to a volume of ca. 10 mL, cooled to 0-10° C. and filtered. The cake was dried at 50° C. under vacuum, providing 1.37 g of Form E.

Example 4f from Ethanol

[0138] 5 g of the compound of formula I (Mixture A1+M1) were refluxed in 100 mL ethanol for a total of 1 h. The mixture was cooled to room temperature, filtered and the cake was dried at 45° C. under vacuum, providing 4.45 g of Form E.

Example 4g: From Acetonitrile, Reflux

[0139] 15 g of the compound of formula I (Mixture A1+M1) were refluxed in 300 mL acetonitrile for a total of 11 h. The suspension was cooled to room temp and filtered, filtered and the cake was dried at 65° C. under vacuum, providing 13 g of Form E.

Example 4h: From Ethyl Acetate, Slurry at Room Temperature (RT) and 50° C.

[0140] 20.4 mg of the compound of formula I (Mixture A1+M1) were stirred for two weeks in 1 mL of ethyl acetate at room temperature. Afterwards the samples were centrifuged and solids and mother liquor were separated. The wet solid was analyzed to be a mixture of Form E and Form F as a minor polymorph. The wet solid was dried at room temperature under vacuum (5 mbar) for ca. 18 h and analyzed to be Form E.

[0141] 28.4 mg of the compound of formula I (Mixture A1+M1) were stirred for two weeks in 1 mL of ethyl acetate at ca. 50° C. Afterwards the samples were centrifuged and solids and mother liquor were separated. The wet solid was analyzed to be a mixture of Form E and F as a minor polymorph. The wet solid was dried at room temperature under vacuum (5 mbar) for ca. 18 h and analyzed to be Form E.

Example 4i: From 2-Propanol

[0142] 27.5 mg of the compound of formula I (Mixture A1+M1) were stirred for ca. two weeks in 0.9 mL of 2-propanol at 50° C. Afterwards the samples were centrifuged and solids and mother liquor were separated. The wet solid was analyzed to be Form E. The wet solid was dried at room temperature under vacuum (5 mbar) for ca. 18 h and analyzed to be Form E.

Example 4j: From Ethyl Acetate

[0143] 19.8 mg of the compound of formula I (Mixture A1+M1) were stirred for ca. two weeks in 0.6 mL of ethyl acetate at 20° C. Afterwards the samples were centrifuged and solids and mother liquor were separated. The wet solid was analyzed to be Form E. The wet solid was further treated for 2 days at 40° C./75% RH and analyzed to be Form E.

Example 4k: From Acetonitrile, 20° C.

[0144] 18.0 mg of the compound of formula I (Form A1+M1) were stirred for ca. two weeks in 0.6 mL of acetonitrile at 20° C. Afterwards the samples were centrifuged and solids and mother liquor were separated. The wet solid was analyzed to be Form E. The wet solid was further treated for 2 days at 40° C./75% RH and analyzed to be Form E.

[0145] In a second trial the wet solid was 18.0 mg of the compound of formula I were stirred for ca. two weeks in 0.6 mL of acetonitrile at 20° C. Afterwards the samples were centrifuged and solids and mother liquor were separated. The wet solid was analyzed to be Form E. The wet solid was dried at room temperature under vacuum (5 mbar) for ca. 18 h and analyzed to be Form E.

Example 4l from Acetonitrile, 50° C.

[0146] 18.0 mg of the compound of formula I (Form A1+M1) were stirred for ca. two weeks in 0.6 mL of acetonitrile at 50° C. Afterwards the samples were centrifuged and solids and mother liquor were separated. The wet solid was analyzed to be Form E. The wet solid was further treated for 2 days at 40° C./75% RH and analyzed to be Form E.

Crystallisation by Cooling

Example 4m: From 2-Butanol/Methanol

[0147] 35.5 mg of the compound of formula I (Mixture A1+M1) were added in 1.2 mL of a mixture of 2-butanol/methanol resulting in a slurry which was stirred at ca. 60° C. for one hour. Afterwards the sample was held for one hour at 60° C. and allowed to cool down to ca. 5° C. with a cooling rate of ca. 1° C./h. The sample was kept at ca. 5° C. for ca. 24 h. The wet solid was filtrated and analyzed to be Form E.

Example 4n: From 4-Dioxane/Methanol

[0148] 32.5 mg of the compound of formula I (Mixture A1+M1) were added in 0.5 mL of a mixture of methanol/1,4-dioxane resulting in a slurry which was stirred at ca. 60° C. for one hour. Afterwards the sample was held for one hour at 60° C. and allowed to cool down to ca. 5° C. with a cooling rate of ca. 1° C./h. The sample was kept at ca. 5° C. for ca. 24 h. The wet solid was filtrated and analyzed to be Form E.

Example 4o: from ethyl acetate/methanol

[0149] 32.5 mg of the compound of formula I (Mixture A1+M1) were added to 0.75 mL of a mixture of ethyl-acetate/methanol resulting in a slurry which was stirred at ca. 60° C. for one hour. Afterwards the sample was held for one hour at ca. 60° C. and allowed to cool down to ca. 5° C. with a cooling rate of ca. 1° C./h. The sample was kept at ca. 5° C. for ca. 24 h. The wet solid was filtrated and analyzed to be FormE.

One-Pot Deprotection of the Compound of Formula II and Crystallisation

Example 4p

[0150] 0.5 g of the compound of formula II (R.sup.3 is tert-butyl) was suspended in 5 mL methanol. 2.4 molar equivalents of HCl in MeOH was added at 20-25° C. and the suspension was stirred for ca. 9 days at ca. 5° C. The suspension was filtered and the cake obtained was dried under vacuum, providing 0.3 g of Form E.

Crystallisation from Free Base

Example 4q

[0151] 76 g of the dichloride salt of the compound of formula I (Mixture A1+M4) was dissolved in a mixture of 280 mL water and 280 mL methanol. The solution was added to a solution of 24.2 g potassium carbonate, 140 mL water and 140 mL methanol at 10-15° C. The reaction mixture was stirred for ca. 2 hours at room temperature. The suspension was filtered, the cake was washed with methanol, and slurried in 350 mL of water and 350 mL of methanol. The suspension was filtered, the cake was washed with 70 mL of water and dried under vacuum at 45° C., providing 65 g of the compound of formula I (free base).

[0152] 1 g of the compound of formula I (free base) was reacted with hydrochloric acid in methanol solution at 65° C. 10 mg seeds of Form E were added, the mixture was slowly cooled to 8-10° C., stirred for ca. 16 h filtered and the cake obtained was dried under vacuum to provide 0.44 g of Form E.

Example 5—Characterization of the Crystalline Dichloride Salt (Form E) of the Compound of Formula I

Example 5a: Characterization by XRPD

[0153] XRPD patterns were obtained using a high-throughput XRPD set-up. The plates were mounted on a Bruker GADDS diffractometer equipped with a Hi-Star area detector. The XRPD platform was calibrated using Silver Behenate for the long d-spacings and Corundum for the short d-spacings. Data collection was carried out at room temperature using monochromatic CuKα radiation in the 2θ region between 1.5° and 41.5°, which is the most distinctive part of the XRPD pattern. The diffraction pattern of each well was collected in two 20 ranges (1.5°≤2θ≤21.5° for the first frame, and 19.5°≤2θ≤41.5° for the second) with an exposure time of 90 s for each frame. No background subtraction or curve smoothing was applied to the XRPD patterns. The carrier material used during XRPD analysis was transparent to X-rays and contributed only slightly to the background.

[0154] The XRPD of the crystalline form of the dichloride salt of the compound of formula I (Form E) at room temperature is shown in FIG. 2 and its diffractogram peaks are shown in Table 2. The evaluation of the high-resolution XRPD pattern was indexed using a P222 space group. Indexing the intensities of reflections of the pure form resulted in an orthorhombic crystal system and allowed extraction of the cell parameters.

[0155] The crystallographic parameters are based on a Pawley calculation (whole powder pattern decomposition, WPPD) for the crystalline form of the dichloride salt of the compound of formula I. All intensities and 20 values for the peaks from the powder diffraction pattern could be assigned for the orthorhombic primitive cell (P), with the cell parameters: a=4.8 Å, b=20.02 Å, c=59.40 Å; V=5724 Å.sup.3 (a=4.813±0.001 Å, b=20.02±0.01 Å, c=59.40±0.02 Å, V=5724±5 Å.sup.3). The powder pattern of this form could also be indexed in the lower symmetries such as monoclinic (a=10.08 Å; b=59.42 Å; c=5.16 Å; beta=97.28 Å; V=3065 Å.sup.3) and several triclinic. However, as a general rule the highest symmetry is applied. In this case the highest symmetry is orthorhombic. A comparison of the calculated and measured diffractograms shows excellent agreement as depicted in FIG. 3.

TABLE-US-00002 TABLE 2 X-ray powder diffraction (XRPD) list of diffractogram peak positions, d-spacing, and relative intensities of the 27 most abundant peaks for the crystalline Form E of the dichloride salt of the compound of formula I Angle d-Spacing Intensity [2θ] [Å] [rel. %] 6.0 14.76 49 9.4 9.42 69 9.9 8.89 81 10.7 8.26 100 11.6 7.61 55 11.9 7.43 56 12.6 7.03 25 17.4 5.10 64 18.5 4.79 46 19.9 4.45 31 21.4 4.15 68 22.4 3.96 53 23.0 3.86 54 23.8 3.73 45 24.2 3.68 51 24.6 3.61 56 25.8 3.45 79 26.4 3.37 35 28.4 3.14 75 32.8 2.73 42 34.2 2.62 25

[0156] The XRPD of the high-temperature polymorph form E1 was determined similarly to form E and the diffractogram peaks (FIG. 10) are shown in Table 3.

TABLE-US-00003 TABLE 3 X-ray powder diffraction (XRPD) list of diffractogram peak positions, d-spacing, and relative intensities for the crystalline high-temperature Form E1 of the dichloride salt of the compound of formula I Angle d-Spacing Intensity [2θ] [Å] [rel. %] 6.0 14.79 55 9.0 9.85 9 9.4 9.46 57 9.9 8.91 77 10.7 8.29 100 11.6 7.64 53 11.9 7.41 72 12.6 7.02 24 17.4 5.10 89 18.5 4.79 50 19.9 4.45 42 20.5 4.32 26 21.0 4.23 30 21.2 4.18 42 21.4 4.15 70 22.4 3.97 78 23.0 3.86 65 23.8 3.74 72 24.2 3.68 84 24.6 3.62 77 24.8 3.59 39 25.4 3.50 46 25.8 3.46 67 25.9 3.44 65 26.4 3.38 51 26.8 3.32 27 27.8 3.21 25 28.4 3.14 86 29.1 3.07 20 29.5 3.03 33

Example 5b: Characterization by Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), and Variable Temperature XRPD

[0157] The thermogravimetric analysis (TGA, FIG. 4) showed a large endotherm indicated a melting event at about 276° C. (±2° C.) accompanied by decomposition. A small endotherm at about 130° C. (±2° C.) implied that a solid-solid transition to a crystalline form variation, built reversibly at high temperatures, occurred prior to melting. This behavior was confirmed by differential scanning calorimetry (DSC, FIG. 5) as well as by variable temperature XRPD studies.

[0158] A cyclic DSC (FIG. 6) was performed to investigate the nature of the endotherm at ca. 130° C. (±2° C.). Heating up to 200° C. was followed by fast cooling to room temperature (RT) (25° C.->200° C.->25° C.). The DSC thermogram upon cooling showed a small exotherm at ca. 97° C. (±2° C.), implying the reverse solid form transition to Form E1 (XRPD pattern FIG. 7). XRPD data of the solids showed no change of the solid form at 25° C., confirming that the exotherm upon cooling was the reverse solid transition. Variable temperature (VT) XRPD data (see Example 8a for VT XRPD experimental details) confirmed the above properties.

Example 5c: Experimental Thermal Analysis (Including DSC, TGA, TGA SDTA, TGA MS)

[0159] Melting properties were obtained from DSC thermograms, recorded with a heat flux DSC822e instrument (Mettler-Toledo GmbH, Switzerland). The DSC822e was calibrated for temperature and enthalpy with a small piece of indium (m.p. =156.6° C.; ΔHf=28.45 J.g−1). Samples were sealed in standard 40 μL aluminium pans, pin-holed and heated in the DSC from 25° C. to 300° C., at a heating rate of 10° C./min. Dry N.sub.2 gas, at a flow rate of 50 mL/min was used to purge the DSC equipment during measurement.

[0160] Mass loss due to solvent or water loss from the crystals was determined by Thermo Gravimetric Analysis/Simultaneous Differential Temperature/Thermal Analysis (TGA/SDTA). Monitoring the sample weight, during heating in a TGA/SDTA851e instrument (Mettler-Toledo GmbH, Switzerland), resulted in a weight vs. temperature curve. The TGA/SDTA851e was calibrated for temperature with indium and aluminum. Samples were weighed into 100 μL aluminum crucibles and sealed. The seals were pin-holed and the crucibles heated in the TGA from 25 to 300° C. at a heating rate of 10° C./min. Dry N2 gas was used for purging.

[0161] The gases evolved from the TGA samples were analyzed by a mass spectrometer Omnistar GSD 301 T2 (Pfeiffer Vacuum GmbH, Germany). The latter is a quadrupole mass spectrometer which analyses masses in the range of 0-200 amu.

Example 5d: Characterization by FTIR

[0162] FT-IR spectra were recorded using a Thermo Fischer Scientific FT-IR Nicolet 6700 spectrometer equipped with ATR probe.

[0163] The FTIR analysis confirmed the structure of the compound of formula I as detailed in Table 4 and depicted in FIG. 8 and in the zoom between ca. 1800 cm.sup.−1 and 400 cm.sup.−1 as FIG. 9. Characteristic IR vibrations of the crystalline form of the dichloride salt of the compound of formula I have been identified to be 1701, 1665, 1335, 1241, 1171, 942, 924, 864, 699, 628 cm.sup.−1 (±2 cm.sup.31 1).

TABLE-US-00004 TABLE 4 Main IR vibrations of the crystalline form of the dichloride salt of the compound of formula I IR vibration (in cm-1) and its assignment according to literature .sup.[1] Observed vibration [cm.sup.−1] 3500-3100 N-H (amide) stretching 3282,3183,3093* 3080-2840 C-H (aromatic and aliphatic) stretching 3093*, 3056, 3024, 2936 3000-2000 NH.sub.3.sup.+ stretching 2630, 2574, 2505 2260-2240 CN stretching 2250 1740-1630 C═O stretching 1701,1665 1630-1510 N-H deformation and 1626*, 1596*, 1543*, 1507* N-C═O stretching asymmetric 1690-1520 C═N stretching 1626*, 1596*, 1543*, 1507* 1625-1575 C-C (aromatic) skeletal vibrations 1626*, 1596* 1525-1450 1507*, 1457 *Several possible assignments.

Example 5e: Characterization by Solid State .SUP.13.C{.SUP.1.H} MAS-NMR

[0164] Magic angle spinning solid state carbon 13 nuclear magnetic resonance (.sup.13C{.sup.1H} MAS-NMR) (see FIG. 10) was performed on a Bruker Avance III 400 MHz solid-state NMR instrument equipped with a wide bore (89 mm room temperature bore) 9.4 Tesla magnet. A double resonance magic angle sample spinning (MAS) probe was used for a rotor size of 4.0 mm outer diameter. The probe was doubly tuned to the observe nucleus frequency—.sup.13C at 100.61 MHz in this study—and .sup.1H at 400.13 MHz. The homogeneity of the magnetic field was set by shimming on an adamantane sample in a 4 mm ZrO.sub.2 spinner, the .sup.13C line width (full width at half maximum height) was less than 2 Hz.

[0165] Chemical shift referencing was done by the substitution method using the .sup.1H signal of tetramethylsilane (<1% v/v in CDCl.sub.3) whose chemical shift was set to 0 ppm. This is the procedure recommended by the IUPAC. All measurements were performed with an additional flow of nitrogen gas (1200 L/h at 5° C.) blown laterally on the MAS spinner for temperature control. The true sample temperature was about 15° C. above this due to frictional heating in the MAS air bearings. For magic angle sample spinning the spinning frequency was set to 14 kHz. The number of scans was 1024, the recycle delay was 5 s, the contact time was 2 ms, the acquisition time was 33 ms, the processing parameters were tdeff=0 and lb=5 Hz.

[0166] The carbon 13 chemical shifts for the investigated crystalline form of the dichloride salt of the compound of formula I are listed in Table 5. The atom numbers for the NMR assignment of the carbon 13 chemical shifts is depicted in FIG. 1.

TABLE-US-00005 TABLE 5 .sup.13C{.sup.1H} MAS-NMR shifts (±0.2 ppm for 13C chemical shifts) of Form E referenced by the substitution method using the .sup.1H signal of tetramethylsilane (TMS <1% v/v in CDCl.sub.3) whose chemical shift was set to 0 ppm. Also shown are the .sup.13C{.sup.1H} NMR shifts in liquid [D6]-DMSO referenced to [D6]-DMSO whose chemical shift was set to 39.52 ppm*. .sup.13C chemical shifts High resolution (liquid) .sup.13C chemical shifts # Group in [D.sub.6]-DMSO CP MAS 14 kHz 1 N — — 2 C 140.9 137.4 [a] 3 N — — 4 C 141.5 141.4 [a] 5 CH ar 119.9 118.8 [b] 6 CH ar 123.3 121.8 [b] 7 CH ar 124.8 124.2 [b] 8 CH ar 111.2 109.5 9 C ar 136.1 134.8 [a] 10 C 137.7 137.4 [a] 11 N — — 13 N — — 14 C 155.8 156.2 15 NH — — 16 CH.sub.2 40.1 40.3 17 CH.sub.2 16.7 19.0 18 CN 119.1 119.6 19 CN — — 20 CH2 51.8 49.1 21 C═O 191.3 196.2 22 C ar 129.6 128.1 23 CH ar 129.6 131.2 [c] 24 CH ar 119.0 121.2 25 C ar 143.6 144.0 [a] 26 CH ar 119.0 121.2 27 CH ar 129.6 128.9 [c] 28 NH — — 29 C═O 168.3 167.1 30 CH 52.7 55.2 31 CH.sub.2 30.3 34.6 [d] 32 CH.sub.2 21.1 25.0 [d] 33 CH.sub.2 26.2 26.6 [d] 34 CH.sub.2 38.1 39.5 35 NH.sub.3.sup.+ — — 36 NH.sub.3.sup.+ — — .sup.[a], [b], [c], [d] Signals with the same superscript might be exchanged. *H.E. Gottlieb, V. Kotlyar, A. Nudelman J. Org. Chem, Vol 62, 1997, 7512-7515

Example 5: Characterization by DVS

[0167] Differences in hygroscopicity of the various forms of a solid material provided a measure of their relative stability at increasing relative humidity. Moisture sorption isotherms were obtained using a DVS-1 system from Surface Measurement Systems (London, UK). The relative humidity was varied during sorption-desorption (see specific experiment) at a constant temperature of ca. 25° C. At the end of the DVS experiment the sample was measured by XRPD.

[0168] The dynamic vapor sorption (DVS) analysis for the crystalline Form E of the dichloride salt of the compound of formula I is depicted in FIG. 11. It shows a 1% water absorption for the compound up to 85% RH and ca. 4% water absorption up to 95% RH.

Example 5g: Solubility

[0169] The thermodynamic pH-dependent solubility was performed in unbuffered water as well as using standard Merck Titriplex® buffers (Merck Titrisol® buffer pH 3 with citrate and HCl; Merck Titrisol® buffer pH 4 with citrate and HCl; Merck Titrisol® buffer pH 5 with citrate and NaOH; Merck Titrisol® buffer pH 6 with citrate and NaOH; Merck Titrisol® buffer pH 7 with phosphate; for buffering at pH 4.5 a 50/50 mixture of buffers for pH 4 and 5 was used; for buffering at pH 5.5 a 50/50 mixture of buffers for pH 5 and 6 was used).

[0170] For each experiment, an 8 mL screw cap vial was prepared with the polymorphic material, the buffer solvent according to the target pH and a magnetic stirring bar. Each pH data point was determined in triplicate with a target pH of 3, 4, 4.5, 5, 5.5 and 7. The pH was measured (Fisherbrand pH meter Hydrus 400, a three point calibration was performed prior to measurement) and adjusted with 1M NaOH solution. The mixtures were left to equilibrate for 24 h at room temperature while stirring. After 24 h the pH was monitored and the slurries were centrifuged for 10 min at 3000 rpm to separate the solids and liquids and filtered (0.45 micron disk filter). If necessary, the isolated filtrates were diluted in the sample solvent to fall within the calibration curve of the HPLC testing. Concentrations of the compound of formula I were determined by High Performance Liquid Chromatography with Diode Array Detection analysis (HPLC-DAD). The calibration curves were obtained from two independently prepared stock solutions of the compound of formula I in a sample solution of water/THF/TFA (50/50/0.05 v/v/v).

[0171] HPLC testing was performed on Agilent 1100 with DAD detector at 280 nm wavelength. A LOQ of 11 μg/mL was determined, linearity is given up to ca. 0.7 mg/mL. Each sample was diluted to ca. 0.5 mg/mL or measured as neat if the concentration was below or equal ca. 0.5 mg/mL.

Example 6—Preparation of the Crystalline Dichloride Salt (A+M) of the Compound of Formula I

Example 6a: Crude Dichloride Salt of the Compound of Formula I

[0172] 111.6 g (156 mmol) of the compound of formula II (R.sup.3 is tert-butyl) prepared according to the procedure provided in Example 2 was suspended in 738 mL of THF and heated to ca. 33° C. 160 g of 30% aqueous HCl was added and the mixture was stirred for ca. 18 h. The mixture was cooled to ca. 10° C. and 738 mL of THF was added. The suspension was filtered, the cake washed with 120 mL of THF and dried at ca. 40° C. under vacuum, providing 90 g of compound of formula I.

Example 6b: Purification and Crystallization

[0173] Crude compound of formula I (2.6 kg) was dissolved in water (2.7 L) and tetrahydrofuran (5.5 L) at ca. 40-50° C. Tetrahydrofuran (90 L) was slowly added at ca. 40-50° C. The resulting suspension was stirred, then cooled to ca. 10° C. and further stirred. The suspension was filtered, the cake was washed with THF and dried. The resulting solid (2.4 kg) was dissolved in 7.3 L water, the solution was filtered and the filter was washed with 2.3 L of water. The filtered solution and wash were evaporated to dryness at ca. 30° C. under reduced pressure. The residue was further dried at 50° C. under reduced pressure, providing 2.2 kg of compound of formula I as Mixture A1+M1.

[0174] Typically the starting point for generation of other crystal forms within System A+M was Mixture A1+M1 (FIG. 14) and Mixture A1+M4 (FIG. 15). FIG. 19 gives an overlay of XRPD patterns that were observed when Mixture A+M4 was exposed to climate chamber conditions. Mixture M3+M5 (FIG. 24) was observed after 1 week and also after 2.5 weeks at 40° C./75% RH. Form M5 was observed after 4 weeks of treating Mixture A1+M4 at 40° C./75% RH (FIG. 26). After 4 weeks at 40° C./75% RH and 2 days 25° C./95% RH Mixture A2+M4 was obtained (FIG. 17). After 4 weeks at 40° C./75% RH and 1 week at 25° C./95% RH Mixture A2+M11 was obtained (FIG. 18).

Example 7—Preparation of Specific Forms of the Crystalline Dichloride Salt within System A+M of the Compound of Formula I

Preparation of Form A0

Example 7a

[0175] Form A0 (FIG. 12, Table 6) was obtained by heating Mixture A1+M1 for 2.5 h to 195° C.

Example 7b

[0176] Form A0 was obtained by heating Form M1 for 4 h to 195° C.

Preparation of Form A1

Example 7c

[0177] Form A1 (FIG. 13, Table 7) was obtained by allowing form A0 to stand at ambient conditions for ca. 11 days.

Example 7d

[0178] Form A1 was obtained by cooling crystallization of Mixture A1+M1 in the following solvent systems: water and methanol/water (50:50). 80 μL of the respective solvent were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 20° C. with a cooling rate of 20° C./min, the mixture was allowed to remain at 20° C. under stirring for 24 h. Form F was obtained by solvent evaporation under vacuum (5 mbar). Form F was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form A1.

Example 7e

[0179] Form A1 was obtained by cooling crystallization of Mixture A1+M1 in methanol. 80 μL of the methanol were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 2° C. with a cooling rate of 20° C./min, the mixture was allowed to remain at 2° C. under stirring for 24 h. Form F was obtained by solvent evaporation under vacuum (5 mbar). Form F was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form A1.

Preparation of Mixture A1+M1

[0180] The XRPD diffractogram is depicted in FIG. 14 and Table 19.

Example 7f

[0181] 23.2 mg of the compound of formula I mixture A1+M4 were added in 0.60 mL of diethyl ether resulting in a slurry which was stirred at 20° C. for two weeks. Afterwards the sample was centrifuged, the liquid separated by filtration and the solid part was dried under vacuum (5 mbar). The solid was analyzed and found to be Mixture A1+M1.

Example 7g

[0182] 22.7 mg of the compound of formula I mixture A1+M4 were added in 0.60 mL of tert-butyl methyl ether resulting in a slurry which was stirred at 20° C. for two weeks. Afterwards the sample was centrifuged, the liquid separated by filtration and the solid part was dried under vacuum (5 mbar). The solid was analyzed and found to be Mixture A1+M1.

Preparation of Mixture A1+M4

[0183] The XRPD diffractorgam is depicted in FIG. 15 and Table 20.

Example 7h

[0184] Mixture A1+M4 was formed by exposing 20 mg Mixture A1+M1 for at least 3 min to 40% RH.

Example 7i

[0185] 23.2 mg of the Mixture A1+M1 were slurried in 0.60 mL of diethyl ether at 20° C. for two weeks. The resulting wet solid was separated by centrifugation and filtration and was analyzed and found to be Mixture A1+M4.

Example 7j

[0186] 22.7 mg of the Mixture A1+M1 were slurried in 0.60 mL of tert-butyl methyl ether at 20° C. for two weeks. The resulting wet solid was separated by centrifugation and filtration and was analyzed and found to be Mixture A1+M4.

Example 7k

[0187] 24.2 mg of the Mixture A1+M1 were slurried in 0.60 mL of n-heptane at 20° C. for two weeks. The resulting wet solid was separated by centrifugation and filtration and was analyzed and found to be Mixture A1+M4.

Example 7l

[0188] 18.9 mg of the Mixture A1+M1 were slurried in 0.60 mL of toluene at 20° C. for two weeks. The resulting wet solid was separated by centrifugation and filtration and was analyzed and found to be Mixture A1+M4.

Example 7m

[0189] 18.9 mg of the Mixture A1+M1 were slurried in 0.40 mL of diisopropylether at 50° C. for two weeks. The resulting wet solid was separated by centrifugation and filtration and was analyzed and found to be Mixture A1+M4.

Example 7n

[0190] 22.8 mg of the Mixture A1+M1 were slurried in 0.40 mL of n heptane at 50° C. for two weeks. The resulting wet solid was separated by centrifugation and filtration and was analyzed and found to be Mixture A1+M4.

Example 7o

[0191] 24.9 mg of the Mixture A1+M1 were slurried in 0.40 mL of toluene at 50° C. for two weeks. The resulting wet solid was separated by centrifugation and filtration and was analyzed and found to be Mixture A1+M4.

Preparation of Mixture A1+M4+M5

Example 7p

[0192] Mixture A1+M4+M5 was formed by exposing Mixture A1+M4 for ca. 3 min to 60% to 80% RH.

Preparation of Mixture A2+M4

[0193] The XRPD diffractogram is depicted in FIG. 17 and Table 21.

Example 7q

[0194] After storing Mixture A1+M4 for 4 weeks at 40° C./75% RH and 2 days at 25° C./95% RH Mixture A2+M4 was obtained.

Preparation of Mixture M3+M5

[0195] The XRPD diffractogram is depicted in FIG. 16 and Table 11.

Example 7r

[0196] Mixture M3+M5 is observed after storing Mixture A1+M4 for between 1 week and 2.5 weeks at 40° C./75% RH.

Preparation of Mixture A2+M11

[0197] The XRPD diffractogram is depicted in FIG. 18 and Table 22.

Example 7s

[0198] Mixture A2+M11 was obtained after storage of Mixture A1+M4 for 4 weeks at 40° C. 75% RH and 1 week at 25° C./95% RH (FIG. 19).

Preparation of Form A2

[0199] The XRPD diffractogram is depicted in FIG. 20 and Table 20.

Example 7t

[0200] Form A2 was obtained by cooling crystallization of Mixture A1+M1 in all of the following different solvent systems: 1,4-dioxane/water (50:50), isopropanol/water (50:50), acetonitrile/water (50:50), ethanol/water (50:50), isopropanol, and acetone/water (50:50). 80 μL of the respective solvent were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 20° C. with a cooling rate of 20° C./min, the mixture was allowed to remain at 20° C. under stirring for 24 h. Form F was obtained by solvent evaporation under vacuum (5 mbar). Form F was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form A2.

Example 7u

[0201] Form A2 was obtained by cooling crystallization of Mixture A1+M1 in the following solvent systems: Methanol and ethanol. 80 μL of the respective solvent were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 20° C. with a cooling rate of 20° C./min, the mixture was allowed to remain at 20° C. under stirring for 24 h. Form G was obtained by solvent evaporation under vacuum (5 mbar). Form G was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form A2.

Preparation of Form M1

[0202] The XRPD diffractogram is depicted in FIG. 22 and Table 9.

Example 7v

[0203] Form M1 was obtained by cooling crystallization of Mixture A1+M1 in all of the following different solvent systems: water, 1,4-dioxane/water (50:50), ethyl acetate/dimethylsulfoxide (50:50), isopropanol/water (50:50), acetonitrile/water (50:50), ethanol/water (50:50), and tetrahydrofuran/water (50:50). 80 μL of the respective solvent were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 2° C. with a cooling rate of 2° C./min, the mixture was allowed to remain at 2° C. under stirring for 24 h. Form F was obtained by solvent evaporation under vacuum (5 mbar). Form F was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form M1.

Example 7w

[0204] Form M1 was obtained by cooling crystallization of Mixture A1+M1 in the following different solvent systems: p-xylene/methanol (50:50) and 2-butanone/methanol (50:50). 80 μL of the respective solvent were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 2° C. with a cooling rate of 2° C./min, the mixture was allowed to remain at 2° C. under stirring for 24 h. Form G was obtained by solvent evaporation under vacuum (5 mbar). Form G was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form M1.

Example 7x

[0205] Form M1 was obtained by cooling crystallization of Mixture A+M1 in the following different solvent systems: tetrahydrofuran/methanol (50:50) and 2 tetrahydrofuran/ethyl acetate (50:50). 80 μL of the respective solvent were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 20° C. with a cooling rate of 20° C./min, the mixture was allowed to remain at 20° C. under stirring for 24 h. Form G was obtained by solvent evaporation under vacuum (5 mbar). Form G was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form M1.

Example 7y

[0206] Form M1 was obtained by cooling crystallization of Mixture A1+M1 in all of the following different solvent systems: acetonitrile/water (50:50), tetrahydrofuran/water (50:50), methanol/water (50:50), acetone/water (50:50), 2 butanone/water (50:50), ethyl acetate/methanol (50:50), and tetrahydrofuran/methanol (50:50). 80 μL of the respective solvent were added to ca. 4 mg of Mixture A1+M1. The temperature was increased to 60° C. and was kept for 60 min at 60° C. After cooling to 2° C. with a cooling rate of 20° C./min, the mixture was allowed to remain at 2° C. under stirring for 24 h. Form F was obtained by solvent evaporation under vacuum (5 mbar). Form F was exposed to climate chamber conditions of 40° C./75% RH for 67 h resulting in Form M1.

Preparation of Form M2

[0207] Form M2 (FIG. 23, Table 10) was obtained by crash-crystallisation with anti-solvent addition from Mixture A1+M4.

Example 7z

[0208] Form M2 was obtained by crash-crystallisation with anti-solvent addition of Mixture A1+M1 in all of the following different solvent systems: solvent: 1-butanol/water (9.6:90.4 v/v) with each anti-solvent: acetonitrile, 2-butanone, tetrahydrofuran or ethyl acetate. A stock solution was prepared in 200 μL solvent, the concentration of of the compound of formula I being that attained at saturation at ambient temperature after equilibration for 24 h before filtering or with a cut off concentration of 170 mg/mL. For each experiment, the anti-solvent was added to each solvent vial, with a solvent to anti-solvent ratio of 1:0.25. In the cases where no precipitation occurred, this ratio was increased to 1:1, and if again no precipitation occurred the ratio was increased to 1:4 (for all Form M2 preparations), with a waiting time of 60 min between the additions (up to the third addition). Since not enough solids precipitated for separation, samples were kept at 5° C. for three days. No precipitation occurred. The solvents were evaporated at 200 mbar until dry.

[0209] Using different solvent systems, different intermediate polymorphic forms, i.e. amorphous (from anti-solvent acetonitrile, 2-butanone), Form M1 (tetrahydrofuran) and Mixture F+M1 (ethyl acetate) were obtained. After storage of the measuring plate at accelerated ageing conditions (40° C./75% RH) for 65 h all these samples transformed to polymorphic form M2.

Preparation of Form M4

[0210] Form M4 (FIG. 25, Table 12) was mainly obtained by slurry experiments at pH of 4 from Mixture A1+M4.

Example 7aa

[0211] 151.4 mg of the compound of formula I (Mixture A1+M4) were suspended in 600 μL of pH4 buffer (Merck Titrisol® buffer pH4, with Citrate and HCl). The initial pH was ca. 3.2. After 15 min. the pH was adjusted with 25 μL 0.1M NaOH to ca. 4.1. After 2-4 h the pH was adjusted to 3.8. 10 μL 0.1M NaOH and 200 μL of the pH4 buffer were added. The slurry was stirred at RT for 24 h (including addition times). The slurry obtained showed ca. pH4.0. Filtration was performed using a 1 micron disk filter. Form M4 was obtained as the filter cake.

Example 7bb

[0212] 198.3 mg of Mixture A1+M4 were suspended in 1000 μL of pH4 buffer (Merck Titrisol® buffer pH4, with Citrate and HCl). The initial pH was ca. 2.9. After 15 min the pH was adjusted with 50 L 0.1M NaOH to ca. 3.8. The slurry was stirred at RT for 24 h (including addition times). A hazy solution is obtained with ca. pH3.8. Filtration was performed using a 1 micron disk filter. Form M4 was obtained as the filter cake.

Example 7cc

[0213] 245.4 mg of Mixture A1+M4 were suspended in 1000 μL of pH4 buffer (Merck Titrisol® buffer pH4, with Citrate and HCl). The initial pH was ca. 3.1. After 15 min the pH was adjusted with 50 μL 0.1M NaOH to ca. 3.9. The slurry was stirred for 30-45 min and the pH was adjusted to ca. 3.9. 10 μL of 0.1M NaOH were added to result in ca. pH4.1. The slurry was stirred at RT for 24 h (including addition times). The slurry obtained showed ca. pH4.0. Filtration was performed using a 0.2 μm centrifugal filter. Form M4 was obtained as the filter cake.

Preparation of Form M5

[0214] The XRPD diffractogram is depicted in FIG. 26 and Table 13.

Example 7dd

[0215] Form M5 was obtained by storage of the compound of formula I Mixture A1+M1 or A1+M4 for 4 weeks at 40° C./75% RH.

Preparation of Form M8

[0216] Form M8 (FIG. 27, Table 14) was mainly obtained by slurry experiments at the pH of 7.5 from Mixture A1+M4. Note that these experiments used buffers containing alternative counter ions. Although it cannot be entirely discounted that traces of the counter ions were present in the polymorph, no diffraction peaks that could be attributable to these inorganic substances were visible in the XRPD diffractograms (inorganic substances are usually clearly visible at high 20 angles and are usually very sharp peaks).

Example 7ee

[0217] Merck Titrisol® buffer pH7, with phosphate and Merck Titrisol® buffer pH8, with Borate and HCl were mixed in a ratio 1:1 (v/v) to give a buffer having a pH of 7.5. A suspension was prepared by adding 26.9 mg of Mixture A1+M4 to 5.0 mL of the above mentioned pH7.5 buffer. The resulting pH was ca. 7.3. After 15 min the pH was adjusted with 10 μL 0.1M NaOH to ca. pH7.4. The mixture was stirred at RT for 24 h (including addition times). A slurry was obtained with pH of ca. 7.5. Filtration was performed using a 1 micron disk filter. Form M8 was obtained as the filter cake.

Example 7ff

[0218] A suspension of 16.4 mg of Mixture A1+M4 in 5.0 mL of the above mentioned pH7.5 buffer was prepared. The initial pH was ca. 7.5. The resulting mixture was stirred at RT for 24 h. A slurry was obtained with ca. pH7.4. Filtration was performed using a 1 micron disk filter. Form M8 was obtained as the filter cake.

Preparation of Form M9

[0219] Form M9 (FIG. 28, Table 15) was mainly obtained by slurry experiments in the pH range 4.5 to 5.5 from Mixture A1+M4. Note that these experiments used buffers containing alternative counter ions. Although it cannot be entirely discounted that traces of the counter ions were present in the polymorph, no diffraction peaks that could be attributable to these inorganic substances were visible in the XRPD diffractograms (inorganic substances are usually clearly visible at high 20 angles and are usually very sharp peaks).

Example 7gg

[0220] 150.5 mg of Mixture A1+M4 was suspended in 5.0 mL of Merck Titrisol® buffer (pH5, containing Citrate and NaOH). The initial pH was ca. 4.2. After 15 min. the pH was adjust with 70 μL 0.1M NaOH to ca. pH4.9. The mixture was stirred at RT for 24 h (including addition times). A slurry was obtained with ca. pH5.1. Filtration was performed using a 1 micron disk filter. Form M9 was obtained as the filter cake.

Example 7hh

[0221] 32 mg of Mixture A1+M4 was suspended in 5.0 mL of Merck Titrisol® buffer (pH5, containing Citrate and NaOH). The initial pH was ca. 5.0. The mixture was stirred at RT for 24 h (including addition times). A slurry was obtained with ca. pH5.0. Filtration was performed using a 1 micron disk filter. Form M9 was obtained as the filter cake.

Example 7ii

[0222] Merck Titrisol® buffer pH5 (containing Citrate and NaOH) was mixed with Merck Titrisol® buffer pH6 (containing Citrate and NaOH) in a ratio 1:1 (v/v) to result in a buffer of pH5.5. 34 mg of the compound of formula I (Mixture A1+M4) were suspended in 5.0 mL of the above mentioned pH5.5 buffer. The initial pH was ca. 5.6. The mixture was stirred at RT for 24 h (including addition times). A slurry was obtained with ca. pH5.5. Filtration was performed using a 1 micron disk filter. Form M9 was obtained as the filter cake.

Preparation of Form M11

[0223] Form M11 (FIG. 30, Table 16) was obtained in supersaturation experiments by changing the pH from 3 to 7 from Mixture A1+M4 and Form E. Note that these experiments used buffers containing alternative counter ions. Although it cannot be entirely discounted that traces of the counter ions were present in the polymorph, no diffraction peaks that could be attributable to these inorganic substances were visible in the XRPD diffractograms (inorganic substances are usually clearly visible at high 20 angles and are usually very sharp peaks).

Example 7kk

[0224] Ca. 210 mg of Form E were suspended in 1.00 mL Merck Titrisol® buffer pH3 (containing Citrate and HCl) and 20 μL 0.1 M NaOH were added. The saturated solution was filtered (0.2 μm centrifugal filter). The solution was kept at RT for 24 h prior to adjustment to pH7 by addition of 270 μL of 0.1M NaOH. Precipitation of solids occurred. The suspensions were filtered with 0.2 μm centrifugal filter and Form M11 was obtained as the filter cake. The same result was obtained using the unfiltered solution when using 350 μL of 0.1M NaOH for the pH adjustment to pH7.

Example 7ll

[0225] Ca. 420 mg of Mixture A1+M4 were suspended in 1.00 mL pH3 buffer and 40 μL of 0.1M NaOH were added. The saturated solution was filtered (0.2 m centrifugal filter) and kept at RT for 24 h prior to adjustment to pH7 by addition of 300 μL of 0.1M NaOH. Precipitation of solids occurred. The suspension was filtered with 0.2 m centrifugal filter and Form M11 was obtained as the filter cake. The same result was obtained using the unfiltered solution when using 350 μL of 0.1M NaOH for the 10 pH adjustment to pH7.

Preparation of Form M12

[0226] Form M12 (FIG. 31, Table 17) was observed in different slurry experiments at ca. pH7 from Mixture A1+M4 and Form E. Note that these experiments used buffers containing alternative counter ions. Although it cannot be entirely discounted that traces of the counter ions were present in the polymorph, no diffraction peaks that could be attributable to these inorganic substances were visible in the XRPD diffractograms (inorganic substances are usually clearly visible at high 20 angles and are usually very sharp peaks).

Example 7 mm

[0227] Ca. 30 mg of Mixture A1+M4 or Form E were suspended in 5.0 mL of Merck Titrisol® buffer pH7 (containing phosphate). The initial pH was ca. 6.9. After stirring for 15 min the pH was adjust with 10 μL 0.1M NaOH to ca. 7.0. The mixture was stirred at RT for 24 h (including addition times). A slurry was obtained with ca. pH7.0. Filtration was performed using a 0.45 micron disk filter. Form M12 was obtained as the filter cake.

Preparation of Form M13

[0228] Form M13 (FIG. 32, Table 18) was obtained in supersaturation experiments by changing the pH from 3 to 5 from Mixture A1+M4 and Form E. Note that these experiments used buffers containing alternative counter ions. Although it cannot be entirely discounted that traces of the counter ions were present in the polymorph, no diffraction peaks that could be attributable to these inorganic substances were visible in the XRPD diffractograms (inorganic substances are usually clearly visible at high 20 angles and are usually very sharp peaks).

Example 7nn

[0229] Ca. 210 mg of Form E were suspended in 1.0 mL Merck Titrisol® buffer pH3 (containing Citrate and HCl) and 20 μL 0.1M NaOH were added. The saturated solution was filtered (0.2 m centrifugal filter) and was kept at RT for 24 h prior to an adjustment to pH5 by addition of ca. 50 μL 0.1 M NaOH. Precipitation of solids occurred. The suspensions were filtered with 0.2 m centrifugal filter and form M13 was obtained as the filter cake. The same result was obtained using the unfiltered solution when using 70 μL of 0.1M NaOH for the pH adjustment to pH5.

Example 7oo

[0230] Ca. 410 mg of Mixture A1+M4 were suspended in 1.00 mL Merck Titrisol® buffer pH3 (containing Citrate and HCl) and 40 μL 0.1 M NaOH were added. The saturated solution was filtered (0.2 m centrifugal filter) and was kept at RT for 24 h prior to an adjustment to pH5 by addition of 60 μL of 0.1M NaOH. Precipitation of solids occurred. The suspensions were filtered with 0.2 m centrifugal filter and form M13 was obtained as the filter cake. The same result was obtained using the unfiltered solution when using 80 μL of a 0.1M NaOH for the pH adjustment to pH5.

[0231] Note: Although Forms F and G are described above as intermediate forms in the preparation of some polymorphic forms within the A+M System in the Examples above, the solvent appears to play an important role in their physical stability. Forms F and G may be solvated or anhydrous forms that occur depending on the solvent used.

Example 8—Characterization of the Crystalline Dichloride Salt (A+M) of the Compound of Formula I

Example 8a: Characterization by XRPD

[0232] XRPD analysis was performed as described under Example 5a. These include XRPD peaks for mixtures that arise naturally in the A+M system, as well as specific A or M polymorphs, isolated as described. Data is included for polymorphs A0, A1, A2, M1, M2, M3+M5, M4, M5, M8, M9, M10+M4, M11, M12, M13 as well as the commonly observed mixtures of A1+M4, A2+M4 and A2+M11. Forms M6 and M7 were also observed but only as mixtures with other polymorphic forms not part of the A+M System.

TABLE-US-00006 TABLE 6 List of XRPD peak positions of Form A0. Angle d-Spacing Intensity [2θ] [Å] [rel. %] 3.9 22.40 100 7.9 11.18 91 9.7 9.11 79 11.2 7.90 82 23.9 3.72 75 25.0 3.55 83 25.5 3.48 82

TABLE-US-00007 TABLE 7 List of XRPD peak positions of Forms A1. Angle d-Spacing Intensity [2θ] [Å] [rel. %] 4.0 21.95 58 8.1 10.96 52 9.4 9.38 65 11.1 7.99 24 12.7 6.98 23 15.3 5.80 53 18.3 4.84 11 20.8 4.26 31 24.3 3.65 100 25.5 3.48 30

TABLE-US-00008 TABLE 8 List of XRPD peak positions of Form A2. Angle d-Spacing Intensity [2θ] [Å] [rel. %] 3.9 22.4 35 8.2 10.74 54 9.4 9.38 100 11.6 7.63 15 12.7 6.98 31 14.7 6.00 43 15.5 5.71 37 19.8 4.48 34 24.1 3.68 92 25.1 3.55 50 25.6 3.47 41

TABLE-US-00009 TABLE 9 List of XRPD peak positions of Forms M1. Angle d-Spacing Intensity [2θ] [Å] [rel. %] 3.6 24.38 100 7.9 11.23 25 9.5 9.34 19 15.5 5.72 17 24.5 3.62 34

TABLE-US-00010 TABLE 10 List of XRPD peak positions of Form M2. Angle d-Spacing Intensity [2θ] [Å] [rel. %] 3.5 24.93 100 9.4 9.42 15

TABLE-US-00011 TABLE 11 List of XRPD peak positions of Mixture M3 + M5. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.0 29.61 92 3.6 24.38 99 9.4 9.38 66 11.1 7.99 48 12.7 6.96 46 15.3 5.77 56 23.6 3.76 70 24.5 3.63 100

TABLE-US-00012 TABLE 12 List of XRPD peak positions of Form M4. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.2 27.41 55 6.5 13.5 34 8.6 10.25 38 9.8 9.00 34 11.2 7.90 40 11.9 7.43 29 13.3 6.63 34 16.5 5.38 58 18.7 4.75 57 20.5 4.32 39 23.7 3.76 100 25.2 3.53 45 27.8 3.20 41 31.7 2.82 31

TABLE-US-00013 TABLE 13 List of XRPD peak positions of Form M5. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.7 24.11 100 7.5 11.77 25 9.4 9.38 46 15.3 5.77 27 19.8 4.47 14 24.3 3.65 65

TABLE-US-00014 TABLE 14 List of XRPD peak positions of Form M8. Angle d-Spacing Intensity [2θ] [Å] [rel %] 7.3 12.03 100 9.6 9.22 60 10.8 8.17 69 13.1 6.77 70 15.1 5.88 51 16.0 5.53 47 16.5 5.35 34 19.3 4.59 27 20.8 4.26 28 24.2 3.67 66 25.5 3.49 60 26.2 3.40 43 27.7 3.22 43 31.7 2.82 30

TABLE-US-00015 TABLE 15 List of XRPD peak positions of Form M9. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.2 27.75 27 6.5 13.67 88 9.7 9.07 59 10.3 8.55 62 15.8 5.61 87 18.1 4.88 45 19.2 4.62 54 21.1 4.21 51 23.1 3.85 57 25.0 3.56 100 26.8 3.33 56

TABLE-US-00016 TABLE 16 List of XRPD peak positions of Form M11. Angle d-Spacing Intensity [2θ] [Å] [rel %] 2.7 32.21 100 15.5 5.71 21 20.4 4.34 25 23.6 3.76 35

TABLE-US-00017 TABLE 17 List of XRPD peak positions of Form M12. Angle d-Spacing Intensity [2θ] [Å] [rel %] 7.3 12.03 100 9.5 9.26 56 11.3 7.79 25 12.4 7.14 66 13.5 6.55 28 14.8 5.99 50 15.6 5.68 24 17.6 5.04 51 19.8 4.48 37 21.1 4.21 41 23.4 3.79 29 24.3 3.66 63 25.9 3.44 27 26.7 3.34 31 27.5 3.24 73 27.9 3.19 87 29.6 3.02 32 32.1 2.79 42

TABLE-US-00018 TABLE 18 List of XRPD peak positions of Form M13. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.1 28.1 73 8.6 10.29 36 11.0 8.05 32 13.3 6.63 28 16.3 5.43 53 17.5 5.07 20 18.4 4.82 44 23.5 3.77 100 25.5 3.49 34 28.0 3.18 63 28.6 3.12 57

TABLE-US-00019 TABLE 19 List of XRPD peak positions of Mixture A1 + M1. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.6 24.65 76 4.0 22.17 91 8.1 10.9 73 9.4 9.42 56 11.0 8.05 57 21.1 4.21 56 24.5 3.63 100

TABLE-US-00020 TABLE 20 List of XRPD peak positions of Mixture A1 + M4. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.4 25.8 92 4.0 22.17 67 8.1 10.85 50 11.1 7.93 50 16.5 5.38 54 24.0 3.7 100

TABLE-US-00021 TABLE 21 List of XRPD peak positions of Mixture A2 + M4. Angle d-Spacing Intensity [2θ] [Å] [rel %] 3.01 28.84 100 6.9 12.87 27 8.5 10.44 52 9.4 9.38 62 12.6 7.01 40 14.8 5.99 42 15.4 5.74 48 19.8 4.48 45 22.7 3.91 35 24.3 3.66 80 24.9 3.57 60

TABLE-US-00022 TABLE 22 List of XRPD peak positions of Mixture A2 + M11. Rel. Angle d-Spacing Intensity [2θ] [Å] [%] 2.7 32.21 100 8.3 10.69 31 9.4 9.38 39 14.8 5.99 31 19.7 4.49 30 24.1 3.69 37

TABLE-US-00023 TABLE 23 List of XRPD peak positions of Form F. Rel. Angle d-Spacing Intensity [2θ] [Å] [%] 2.3 39.0 45 8.0 11.0 58 8.8 10.1 65 11.0 8.1 15 13.4 6.6 20 14.1 6.3 32 15.6 5.7 47 16.9 5.3 21 17.7 5.0 19 19.5 4.6 27 20.5 4.3 12 21.5 4.1 26 23.5 3.8 100 24.3 3.7 41 25.1 3.5 41 26.1 3.4 35 27.1 3.3 21

TABLE-US-00024 TABLE 24 List of XRPD peak positions of Form G. Rel. Angle d-Spacing Intensity [2θ] [Å] [%] 2.3 39.04 42 2.5 34.74 44 5.3 16.53 37 7.9 11.12 100 8.7 10.11 49 9.4 9.38 18 10.1 8.71 25 10.7 8.29 54 12.3 7.21 42 13.4 6.59 82 14.3 6.17 35 16.1 5.51 50 17.7 4.99 40 18.9 4.70 47 19.4 4.57 34 20.0 4.43 43 20.6 4.31 63 21.6 4.11 65 22.3 3.97 33 23.0 3.86 74 23.7 3.75 51 24.4 3.64 45 25.4 3.51 45 26.3 3.39 55 26.9 3.31 26 31.3 2.85 22 32.3 2.76 44

Example 8b: Experimental High-Resolution X-Ray Powder Diffraction (Including Variable Humidity and Variable Temperature XRPD Experiments)

[0233] For variable humidity (VH) and variable temperature (VT) experiments a ANSYCO HT chamber was used, installed within a D8 Advance system diffractometer (Bruker) designed with Bragg-Brentano geometry and equipped with LynxEye solid state detector. The radiation used for collecting the data was CuKα1 (λ=1.54056 Å) monochromatized by germanium crystal. The material was placed on a fixed sample holder that was mounted inside the chamber.

[0234] VH-XRPD: The humidity was applied locally and varied from 10 to 70% (dew point). The patterns were collected in the range 4-30° (2θ), with a step of 0.01450 (2θ) for the VH-XRPD and measuring time per step of 1.2 sec. Data collection was initiated 60 sec following stabilization of humidity at each step (data collection time per RH value about 40 min). All patterns were taken at Room Temperature, ca. 295 K.

[0235] VT-XRPD: The temperature variation rate was 10° C./min and the equilibration time, prior to starting the data collection at each temperature, was 8 min. The patterns were collected in the range 4-34.5° (2θ), with a step of 0.0107° (2θ) and measuring time per step of 1 sec (for T=25, 50, 80, 100 and 110° C.) or 1.5 sec (for T=40, 60, 115-180° C.). The data collection time, per temperature, was 48 or 70 min, depending on the measuring time per step.

[0236] Form A1+M4 was put to a climate chamber experiment at 40° C./75% RH for 4 weeks followed by storage at 25° C./95% RH for two weeks. During this study the initial Form A1+M4 changed after one week into M3+M5, after 4 weeks into form M5 and after 4 weeks and two days into Form A2+M4 before eventually transforming into Form A2+M11 (FIG. 19).

Example 8c: Characterization by DVS

[0237] See Example 5f for experimental details. The DVS analysis for the crystalline System A+M of the dichloride salt of the compound of formula I is depicted in FIG. 35. It shows ca. 22% water absorption for the compound up to 85% RH and below ca. 34% water absorption up to 95% RH.

Example 8d: Solubility

[0238] The thermodynamic pH-dependent solubility of Form A1+M4 was determined as described in Example 5g for Form E, except that the target pHs were 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. The additionally used buffers were Merck Titrisol® buffer pH 1 with glycin and HCl; Merck Titrisol® buffer pH 2 with citrate and HCl; Merck Titrisol® buffer pH 8 with borate and HCl; Merck Titrisol® buffer pH 9 with boric acid, KCl and NaOH; Merck Titrisol® buffer pH 10 with boric acid, KCl and NaOH; Merck Titrisol® buffer pH 11 with boric acid, KCl and NaOH; Merck Titrisol® buffer pH 12 with phosphate and NaOH; Merck Titrisol® buffer pH 13 with KCl and NaOH; for a second buffer at pH 3 without HCl 80.3 mL of citric acid (21.01 g citric acid monohydrate in 1 L deionized water) were mixed with 19.7 mL of 0.2M disodiumhydrogenphosphate (35.6 g in 1 L deionized water). For buffering at pH 6.5 a 50/50 mixture of buffers for pH 6 and 7 was used; for buffering at pH 7.5 a 50/50 mixture of buffers for pH 7 and 8 was used; for buffering at pH 9.5 a 50/50 mixture of buffers for pH 9 and 10 was used; for buffering at pH 10.5 a 50/50 mixture of buffers for pH 10 and 11 was used; for buffering at pH 11.5 a 50/50 mixture of buffers for pH 11 and 12 was used; for buffering at pH 12.5 a 50/50 mixture of buffers for pH 12 and 13 was used). A LOQ of ca. 8 ug/mL was determined.

[0239] The thermodynamic pH-dependent solubility of Form A2+M11 was determined as described in Example 5g for Form E except that an LOQ of 18 g/mL was determined.

PROCESSES FOR THE PREPARATION OF FURAZANOBENZIMIDAZOLES AND CRYSTALLINE FORMS THEREOF

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Abstract

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