NZ715029B2 - Crystalline polymorphs of the free base of 2-hydroxy-6-((2-(1-isopropyl-1h-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde - Google Patents

Abstract

Disclosed are crystalline free base ansolvate forms of 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde (or Compound 1), such as the free base Form I, Form II and Material N. Also disclosed are crystalline free base solvates of 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde (or Compound 1).

Description

WO 20133 PCT/U82015/014589 lline Polymorphs of the Free Base of 2-Hydroxy((2-(1-isopropyl- 1H-pyrazol-5—yl)pyridinyl)methoxy)benzaldehyde ound 2-Hydroxy((2-(1-isopropyl-1H-pyrazol-S-yl)pyridin yl)methoxy)benzaldehyde is a compound having the formula: \(N.\ N / N'\ l Sickle cell disease is a disorder of the red blood cells, found particularly among those ofAfrican and Mediterranean descent. The basis for sickle cell disease is found in sickle hemoglobin (HbS), which contains a point on relative to the prevalent peptide sequence ofhemoglobin (Hb).

Hemoglobin (Hb) transports oxygen les from the lungs to various tissues and organs throughout the body. Hemoglobin binds and releases oxygen through conformational changes. Sickle hemoglobin (HbS) contains a point mutation where glutamic acid is replaced with valinc, allowing HbS to become susceptible to polymerization to give the HbS containing red blood cells their characteristic sickle shape.

The sickled cells are also more rigid than normal red blood cells, and their lack of flexibility can lead to blockage ofblood vessels. A need exists for therapeutics that can treat disorders that are mediated by Hb or by abnormal Hb such as HbS, such as 2- hydroxy—6-((2—(1—isopropyl-1H-pyrazol—S—yl)pyridin—3-yl)methoxy)benzaldehyde.

When used for treating humans, it is important that a crystalline form of a therapeutic agent, like 2-hydroxy((2-( l -isopropy|-1 H-pyrazolyl)pyridin yl)methoxy)benzaldehyde, or a salt thereof, retains its polymorphic and chemical stability, solubility, and other physicochemical ties over time and among various manufactured batches of the agent. If the physicochemical properties vary with time and among batches, the administration of a therapeutically effective dose becomes problematic and may lead to toxic side s or to ineffective therapy, ularly if a given polymorph decomposes prior to use, to a less active, inactive, or toxic compound. Therefore, it is important to choose a form of the crystalline agent that is stable, is manufactured ucibly, and has physicochemical ties favorable for its use as a therapeutic agent.

However, the art s unable to predict which crystalline form of an agent will have a combination of the desired properties and will be suitable for human administration, and how to make the agent in such a crystalline form.

Summary [0005a] According to a first aspect, the present invention provides a lline form of Compound 1: Compound 1 characterized by an X-ray powder diffraction pattern (Cu Kα radiation) having X-ray powder diffraction peaks at , 14.37°, 19.95°, and 23.92°2θ, each ±0.2 °2θ. The crystalline form may further be characterized by an endothermic peak at 97 ±2 °C as measured by differential scanning metry. [0005b] Further provided are pharmaceutical compositions comprising a ceutically acceptable excipient and the crystalline form above. In certain embodiments the pharmaceutical compositions comprise less than 5 mole % of crystalline Form I, wherein: Form I is characterized by an X-ray powder diffraction pattern (Cu Kα radiation) having X- ray powder diffraction peaks at 12.82°, 15.74°, 16.03°, 16.63°, 17.60°, 25.14°, 25.82°, and 26.44°2θ, each ±0.2 °2θ. Still more preferably, the pharmaceutical composition ses less than 5 mole % of crystalline Form I as defined above, less than 5 mole % crystalline Material N and less than 5 mole % of amorphous forms of Compound 1, wherein: Material N is characterized by an X-ray powder diffraction pattern (Cu Kα radiation) having X-ray powder diffraction peaks at 11.65°, 11.85°, 12.08°, 16.70°, 19.65°, and 23.48°2θ, each ±0.2 °2θ. [0005c] In another , the present invention provides a lline form of Compound 1: (followed by page 2A) Compound 1 characterized by at least three X-ray powder diffraction peaks (Cu Kα radiation) selected from , , 12.08°, 16.70°, 19.65° and 23.48°2θ, each ±0.2 °2θ, for example a crystalline form characterized by an X-ray powder diffraction pattern (Cu Kα radiation) ntially similar to The crystalline form may be further terized by an endothermic peak at 95 ± 2 °C as measured by differential scanning calorimetry. Further provided are pharmaceutical compositions comprising a pharmaceutically acceptable excipient and the crystalline form above. [0005d] The present invention further provides for the use of any of the crystalline forms described above for the manufacture of a medicament, for example for increasing oxygen affinity of hemoglobin S, for treating a disorder mediated by hemoglobin e.g. sickle hemoglobin and/or for treating sickle cell disease. In certain embodiments the treatment of sickle cell comprises administration in combination with another active agent. [0005e] These and other s and embodiments are described in further detail below.

Certain aspects and embodiments may not form the subject of the present application but are heless included herein for completeness.

Ansolvates This invention arises in part out the ery that an HCl salt of Compound 1 disproportionates or loses HCl, and a disproportionation of the HCl salt of Compound 1 in water generates the free base and disproportionation was facile upon exposure to elevated humidity, with wet milling, and in direct contact with water (e.g. slurry). The sulfate salt of nd 1 also disproportionates from certain solvents such as dimethyl sulfoxide and methanol when itated with water. The volatilization of HCl was evident within hours of exposure to drying conditions. For example, partial conversion to the free base was observed within 12 hours at 30 °C. Accordingly, the free base of Compound 1 provides a stabler chemical entity ed to the corresponding HCl or sulfate and such other salt. (followed by page 2B It has now been discovered that 2-hydroxy((2-(1-isopropyl-1H-pyrazol yl)pyridinyl)methoxy)benzaldehyde (or Compound 1) i.e., the free base of Compound 1, can be obtained as one or more crystalline ansolvate forms, several of which are ed to here as crystalline Form I, Form II and Material N. In preferred embodiments, the free base of Compound 1 is a lline ansolvate, such as a crystalline anhydrous form. The free base of Compound 1, can be obtained from its corresponding salt form, such as the HCl salt of Compound 1.

Three anhydrous crystalline forms of the free base were fied, termed Free Base Forms I, II, and Material N. It has been discovered that nucleation of Free Base Form I generally occurs first from a slurry. Extending the slurry time can induce the transformation of Free Base Form I to Free Base Form II, a thermodynamically more stable phase relative to Form I. It has further been discovered that Free Base Material N can be stable relative to Forms I and II, at room ature.

Free Base Material N was found to be enantiotropically related to Form II, and will orm reversibly at a specific transition temperature (estimated herein near 40-42 °C). (followed by page 3) PCT/U82015/014589 Above the transition temperature, Free Base Form II appears to be the most stable form, relative to Form 1 and Material N. Thus, under operating temperatures below 40 °C, c.g., at °C, the free base of Compound 1 exists primarily as Material N, which may have some residual Form II. Thus, at operating temperatures above 40 °C, e.g., at 50 °C, the free base of Compound 1 exists primarily as Form 11, which may have some residual Material N. At 40 0C little appreciable conversion is seen n Material N and Form 11. This is contemplated to be true for slurries of the free base in certain solvents and in the solid state.

In one embodiment, the one or more crystalline free base forms of Compound I do not o polymorphic transformation under conditions le for manufacturing and storing the crystalline forms.

Form I In one embodiment, the crystalline free base of Compound 1 comprises crystalline Form I, which is characterized by an endothermic peak at (97d:2) C as measured by differential scanning calorimetry. In another embodiment, the lline Form I of the free base of crystalline Compound 1 is characterized by the substantial absence of thermal events at temperatures below the endothermic peak at (97i2) °C as measured by differential scanning calorimetry. In r embodiment, the crystalline Form I of the free base of crystalline Compound 1 is characterized by an X-ray powder diffraction peak (Cu Ka radiation at one or more of 12.82°, , l6.03°, , , , 25.82° and 26.44° $0.2 °20. In another ment, the crystalline Form I of the free base of crystalline Compound 1 is characterized by an X—ray powder diffraction pattern (Cu Ka radiation) substantially similar to that of In another embodiment, the crystalline Form I of the free base of crystalline Compound I is characterized by at least one X-ray powder diffraction peak (Cu K0. radiation) selected from , 15.74°, l6.03°, 16.63°, 17.60°, 25.14°, 25.82° and 26.44° (each £02 °20). In another embodiment, the lline Form I of the free base of crystalline Compound 1 is characterized by at least two X-ray powder diffraction peaks (Cu Ka radiation) selected from 12.82°, 15.74°, l6.03°, 16.63°, 17.60°, 25.14°, 25.82° and 26.44° (each 1:02 °20). In another embodiment, the crystalline Form I of the free base of crystalline nd I is characterized by at least three X-ray powder diffraction peaks (Cu Ka radiation) selected from 12.82°, 15.74°, l6.03°, 16.63°, 17.60°, 25.14°, 25.82° and 26.44° (each 10.2 °20).

In another embodiment, Form I is characterized by 1, 2, 3, 4, or more peaks as tabulated below.

Observed peaks for Form 1, XRPD file 609973. ‘20 0’ space (A) Intensity (%) .52 :t 0.20 16.021 :t 0.602 68 12.82 i 0.20 6.906 t 0.109 74 .03 i 0.20 5.897 i 0.079 38 .74 :t 0.20 5.629 i 0.072 46 16.03 i 0.20 5.530 d: 0.069 46 16.63 i: 0.20 5.331 i 0.064 61 17.60 :1: 0.20 5.040 t 0.057 100 18.74 i 0.20 4.736 i 0.051 24 19.07 3: 0.20 4.654 d: 0.049 17 19.35 i 0.20 4.587 :t 0.047 23 .32 i 0.20 4.370 t 0.043 18 21.64 i 0.20 4.106 t 0.038 23 22.80 i 0.20 3.901 t 0.034 26 23.28 :t 0.20 3.821 i 0.033 34 .14 i 0.20 3.543 :1: 0.028 52 .82 i 0.20 3.451 :1: 0.026 81 26.44 i: 0.20 3.371 1: 0.025 51 27.91 3: 0.20 3.197 i 0.023 17 28.19 :t 0.20 3.165 :t 0.022 26 Form 11 In another embodiment, the crystalline Compound 1 free base comprises crystalline Form 11, which is characterized by an endothermic peak at (97i2) C as measured by differential scanning metry. In another embodiment, the lline Form 11 of the free base of crystalline Compound 1 is characterized by the substantial absence of thermal events at temperatures below the endothermic peak at (97:1:2) °C as ed by differential ng calorimetry. In another embodiment, the crystalline Form 11 of the free base of crystalline Compound 1 is characterized by an X—ray powder diffraction peak (Cu Ka radiation at one or more of 13.37°, 14.37°, 19.95° or 23.92 °20. In another embodiment, the crystalline Form 11 of the free base of crystalline Compound 1 is characterized by an X-ray powder ction n (Cu Ka radiation) substantially similar to that of In r embodiment, the crystalline Form 11 of the free base of crystalline Compound 1 is terized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from l3.37°, l4.37°, l9.95° and23.92°20 (each $0.2 °20). In another embodiment, the crystalline Form 11 of the free base of crystalline Compound I is characterized by at least two X-ray powder diffraction peaks (Cu Ka radiation) selected from l3.37°, l4.37°, l9.95° and23.92°20 (each $0.2 °20). In another embodiment, the crystalline Form 11 of the free base of crystalline Compound 1 is characterized by at least three X-ray powder diffraction peaks (Cu Ka radiation) selected from 13.37°, I4.37°, l9.95° and23.92°20 (each $0.2 °20).

In another embodiment, Form 11 is characterized by l, 2, 3, 4, or more peaks as ted below.

Observed peaks for Form 11, XRPD file 613881.

C20 (1’ space (A) Intensity (%) .62 :l: 0.20 15.735 :1: 0.581 24 12.85 :1: 0.20 6.888 d: 0.108 22 12.97 i: 0.20 6.826 i 0.106 21 13.37 :1: 0.20 6.622 :1: 0.100 100 14.37 :1: 0.20 6.162 :1: 0.087 56 .31 :1: 0.20 5.788 :1: 0.076 21 16.09 i: 0.20 5.507 t 0.069 23 16.45 :1: 0.20 5.390 :1: 0.066 69 16.75 :1: 0.20 5.294 :1: 0.064 32 16.96 :t 0.20 5.227 i 0.062 53 19.95 :1: 0.20 4.450 :1: 0.045 39 .22 :1: 0.20 4.391 :1: 0.043 20 23.18 :1: 0.20 3.837 :1: 0.033 38 23.92 i: 0.20 3.721 :1: 0.031 41 24.40 :1: 0.20 3.648 :1: 0.030 44 24.73 :1: 0.20 3.600 :t 0.029 22 24.99 :1: 0.20 3.564 :t 0.028 50 .12 i 0.20 3.545 t 0.028 28 .39 i 0.20 3.509 t 0.027 5] .70 i 0.20 3.466 i 0.027 21 26.19 i 0.20 3.403 i 0.026 27 26.72 :t 0.20 3.336 t 0.025 30 27.02 i: 0.20 3.300 t 0.024 25 27.34 i: 0.20 3.262 t 0.024 23 28.44 i 0.20 3.138 i 0.022 20 In some embodiments, the free base of crystalline Compound 1 comprises the crystalline Form 11. In some preferred embodiments, the free base of crystalline Compound 1 comprises the lline Form 11 and less than 25 mole %, 10 mole % or 5 mole % of crystalline Form 1, crystalline Material N or amorphous forms of Compound 1.

In a preferred embodiment, the crystalline Form 11 is prepared from a slurry comprising the free base of Compound 1 in e, from which the crystalline Form 11 is formed and filtered. Thus, in some embodiments, the crystalline Form 11 comprises al (1-500 ppm) heptane. In another preferred embodiment, the crystalline Form 11 is prepared from a slurry comprising the free base of Compound 1 in water, from which the crystalline Form II is formed and filtered.

There are several advantages of crystalline Form 11 relative to crystalline Form I or Material N. For example, the crystalline Form 11 can be prepared from a slurry comprising the free base of Compound 1 in heptane, which is suitable for good manufacturing practices (GMP) protocols. Further, in a most preferred embodiment, the crystalline Form 11 can be prepared from a slurry comprising the free base of Compound 1 in water or the HCl salt of Compound 1 in water, thus reducing or ating the need for solvent during recrystalization. Thus, in some ments, crystalline Form 11 of Compound 1 comprises less than 500 ppm, 100 ppm, less than 50 ppm or less than 10 ppm organic solvent. Also, Form 11 has less of a sity than al N to agglomerate upon size reduction, e.g., upon milling. As such, Form II has r flowability than Material N.

Certain illustrative and non-limiting advantages of Form 11 over Material N (i.e., Form N) are shown in the table below.

PCT/U82015/014589 DATA/EXPERIMENT RESULTS/STATUS fy suitable solvent Form N: for scale-up 0 Limited number of suitable solvents compared to Form 11 o MTBE identified (suitable for GMP; Class III solvent) 0 Scale-up s look good Form 11: o More solvent options than Form N, including H20 0 Current solvent is heptane (suitable for GMP; Class III solvent) produced on 5 kg scale Formation time faster than N (could translate to 2-3 day saving in production time) Better recove than N Size/Morphology ofN Acicular morphology observed for form N; material and II composed of small and large particles erates are an issue for Form N relative to Form 11 (less agglomeration seen with energy—reduced ) PK ison ofN and Oral administrations of GBT440 Forms N and II to rats 11 resulted in comparable exposure at 100 & 500 mg/kg Material N In another embodiment, the crystalline Compound 1 free base comprises crystalline Material N, which is characterized by an endothermic peak at (95i2) C as measured by differential scanning calorimetry. The terms "Material N", "form N" and "polymorphic form N" are used interchangeably herein. In another embodiment, the crystalline Material N of the free base of crystalline Compound 1 is characterized by the substantial e of thermal events at temperatures below the endothermic peak at (95i2) °C as measured by ential scanning metry. In another embodiment, the crystalline Material N of the free base of crystalline Compound 1 is characterized by an X- ray powder diffraction peak (Cu Ker radiation at one or more of 1 1.65°, l 1.85°, 12.08°, 16.70°, l9.65° or 23.48 °20. In another embodiment, the crystalline Material N of the free base of crystalline Compound 1 is characterized by an X-ray powder diffraction pattern (Cu Ka ion) substantially similar to that of In another embodiment, the crystalline Material N of the free base of crystalline Compound 1 is characterized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 1 1.65°, 1 1.85°, 12.08°, , 19.65° and 23.48 °20 (each $0.2 °20). In another embodiment, the crystalline Material N of the free base of crystalline Compound 1 is characterized by at least two X-ray powder diffraction peaks (Cu Ka radiation) ed from 1 1.65°, 1 1.85°, 12.08°, 16.70°, 19.65° and 23.48 °20 (each $0.2 °20). In another embodiment, the lline Material N of the free base of crystalline Compound 1 is characterized by at least three X-ray powder diffraction peaks (Cu K01 radiation) selected from 1 1.65°, 11.85°, 12.08°, 16.70°, 19.65° and 23.48 °20 (each $0.2 °20).

In another embodiment, Material N is characterized by 1, 2, 3, 4, or more peaks as tabulated below.

Observed peaks for al N, XRPD file 615765. °29 (1' space (A) Intensity (%) .55 :1: 0.20 15.924 :1: 0.595 54 1 1.65 :t 0.20 7.597 t 0.132 31 11.85 :1: 0.20 7.468 :l: 0.128 50 12.08 i 0.20 7.324 d: 0.123 31 12.67 i: 0.20 6.987 d: 0.1 12 29 13.12 i 0.20 6.748 :t 0.104 83 14.94 :1: 0.20 5.929 d: 0.080 34 .19 i 0.20 5.832 :1: 0.077 56 .76 :t 0.20 5.623 :1: 0.072 20 16.70 i 0.20 5.310 i: 0.064 100 17.35 i 0.20 5.1 12 i 0.059 52 19.65 i 0.20 4.517 :1: 0.046 60 23.48 3: 0.20 3.789 :t 0.032 72 23.68 i: 0.20 3.757 1: 0.032 29 .25 i 0.20 3.527 d: 0.028 20 .47 i 0.20 3.497 d: 0.027 20 .70 i 0.20 3.466 i 0.027 85 26.04 :1: 0.20 3.422 :t 0.026 35 26.37 i 0.20 3.380 :1: 0.025 55 PCT/U82015/014589 In some embodiments, the free base of crystalline Compound 1 comprises the crystalline Material N and less than 25 mole %, 10 mole % or 5 mole % of crystalline Forms 1 or II or amorphous forms of Compound 1.

In r embodiment, the crystalline Material N is prepared from a slurry sing the free base of Compound 1 in methyl tertiary butyl ether (MTBE), from which the crystalline - Material N is formed and filtered. Thus, in some embodiments, the crystalline Material N comprises residual (1-500 ppm) MTBE.

There are several advantages of crystalline Material N relative to crystalline Forms I or II. For example, the crystalline Material N can be prepared from a slurry comprising the free base of Compound 1 in MTBE, which is suitable for good manufacturing practices (GMP) protocols.

In some embodiments, the crystalline ansolvate forms are stable to contact with water, heptane, iso propyl ether (IPE), MTBE, and toluene, and such other solvents.

In r of its composition embodiments, this invention provides for a pharmaceutical ition comprising a pharmaceutically acceptable excipient and a crystalline Compound 1 free base, comprising one or more of Form 1, Form 11 or Material In one of its method embodiments, this invention es a method of preparing the solid crystalline free base of Compound 1 comprising, e.g., Form 1, Form 11 and/or al N.

In yet another of its method embodiments, there are provided methods for increasing oxygen y of hemoglobin S in a subject, the method comprising administering to a subject in need thereof a therapeutically ive amount of a crystalline free base of Compound 1, comprising, e.g., Form 1, Form 11 and/or al N.

In yet another of its method embodiments, there are provided methods for treating oxygen deficiency associated with sickle cell anemia in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crystalline free base of Compound 1, comprising, e.g., Form 1, Form 11 and/or al N.

In all of such treatments, the effective amount of free base of Compound 1, comprising e.g., Form 1, Form II and/or Material N to the treated patient is already disclosed in the art.

PCT/U82015/014589 Solvates This invention arises in part out of the discovery that ansolvate polymorphs of the free base of Compound 1 form solvate polymorphs with a variety of solvents, preferably other than certain hydrocarbon solvents, water and .

Solvates of the crystalline free base of Compound 1 (e.g., from acetone, acetonitrile, dichloromethane, e, ethanol, ethyl acetate, isopropyl alcohol, methyl ethyl ketone (MEK) and tetrahydrofuran) are also contemplated to be used e.g., as intermediates to regenerate the free base crystalline ansolvate of Compound 1. Such methods can include, t tion, subjecting the solvate to vacuum conditions; and/or generating a salt and disproportionating it in water to form the ansolvate ', and/or slurrying or washing the e with a solvent less prone to e formation such as c, di-isopropyl ether (IPE), tort-methyl butyl ether (MTBE) and toluene.

In r of its composition embodiments, this invention provides for a pharmaceutical composition comprising a pharmaceutically acceptable cxcipicnt and one or more of the solvated crystal forms provided herein.

In one of its method embodiments, this invention provides a method of preparing the solvated crystal forms provided herein.

In yet another of its method embodiments, there are ed methods for increasing oxygen affinity of hemoglobin S in a subject, the method sing administering to a subject in need thereof a therapeutically effective amount of one or more of the solvated crystal forms provided herein.

In yet another of its method ments, there are provided methods for treating oxygen deficiency associated with sickle cell anemia in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more of the solvated crystal forms provided herein.

In all of such treatments, the effective amount of the free base of Compound 1, to the treated patient is already disclosed in the art.

Brief Description of the Drawings is a XRPD profile of the crystalline HCl salt before (top) and afier (bottom) minutes slurried in water.

PCT/U82015/014589 is a XRPD profile ofthe free base Form I (top). FOrm 11 (middle), and Material N (bottom). is a XRPD profile and contemplated indexing for free base Form 1. is a l characterization for free base Form 1. is a XRPD profile and contemplated indexing for free base Form 11. is a thermal characterization for free base Form 11. is a XRPD profile for free base Material N. is a thermal characterization for free base Material N. s an Energy—Temperature Diagram between the Free Base Forms 1, II, and Material N. The enthalpy (H) and free energy (G) isobars for each form are depicted as a function of temperature. AHfIS the heat of fusion; T is transition ature; m is melt temperature; superscripts I, II, and N refer to the polymorphs. *Under the test conditions, not enough information was available to graphically represent the free energy isobar of Form I below 6 °C and above the estimated transition ature TV'"; the isobar likely intersects G], at a temperature below In", allowing the possibility that Form 1 may be enantiotropie with Form [I (where T"" occurs below 6 °C) and/or Material N (where either 74w occurs below TL" or TV" occurs above TV‘", but not both). Free energy isobars can only intersect each other once. depicts 13C Solid State NMR spectra for Free Base Forms 1 (bottom), I] (middle), and Material N (top). Form I contains one le per asymmetric unit.

Material N contains four molecules per asymmetric unit. As observed by '3C Solid State NMR a, Forms 1] and N did not undergo a transition over 250 K to 340 K. Chemical shifts change slightly with temperature (not rated graphically). 1 s l5N Solid State NMR spectra for Free Base Forms 1 (bottom), 1] (middle), and Material N (top). depicts a differential scanning calorimetry (DSC) curve for Free Base Material N. depicts a DSC curve for Free Base Form 11. depicts a DSC curve for Free Base Form 1.

PCT/U82015/014589 depicts a XRPD profile of tion experiments for the free base of Compound 1 at multiple temperatures. depicts a contemplated XRPD profile for solvated Material E. depicts a contemplated XRPD profile for solvated Material F. depicts a contemplated XRPD profile for solvated Material G. s a contemplated XRPD profile for solvated Material H. depicts a contemplated XRPD profile for solvated Material J. s a contemplated XRPD profile for solvated Material K. depicts a plated XRPD profile for solvated Material L. depicts a contemplated XRPD profile for solvated Material M. depicts a contemplated XRPD profile for solvated Material 0. depicts an XRPD profile comparison of plated isostructural solvates of the free base of Compound I. From top to bottom: Material E from acetone; Material F from ACN; Material G from DCM; Material H from dioxane; Material J from EtOH; Material K from IPA/water (also obtained from IPA); and Material L from THF, Material M from MEK.

Detailed ption As noted above, this invention is directed, in part, to a stable free base of Compound 1 and, in particular, the free base Form 1, Form II or Material N. However, prior to discussing this invention in further detail, the following terms will be defined.

Definitions As used herein, the following terms have the ing meanings.

The singular forms "a," "an," and "the" and the like include plural referents unless the context clearly dictates ise. Thus, for example, reference to "a compound" includes both a single compound and a plurality of different compounds.

The term "about" when used before a numerical designation, e.g., temperature, time, amount, and concentration, including a range, indicates approximations which may vary by 3:10 %, :l:5 %oril %.

PCT/U82015/014589 "Administration" refers to introducing an agent into a patient. A therapeutic amount can be administered, which can be determined by the treating physician or the like.

An oral route of administration is preferred. The related terms and phrases administering" and "administration of", when used in connection with a compound or pharmaceutical composition (and grammatical equivalents) refer both to direct administration, which may be stration to a patient by a medical professional or by dministration by the patient, and/or to indirect administration, which may be the act of prescribing a drug. For example, a physician who cts a patient to self-administer a drug and/or provides a patient with a prescription for a drug is stering the drug to the patient. In any event, administration entails delivery to the patient of the drug.

The "crystalline ansolvate" of Compound 1 is a crystalline solid form of the free base of 2-hydroxy—6-((2-( 1 -isopropyl- 1 zolyl)pyridinyl)methoxy)benzaldehyde, such as, e.g., crystalline Form 1, Form 11 or Material N as disclosed herein. Each of the Form 1, Form 11 or Material N l lattices is substantially free of ts of crystallization. r, any solvent present is not included in the crystal lattice and is randomly distributed outside the crystal lattice. Therefore, Form 1, Form II or Material N crystals in bulk may contain, outside the crystal lattice, small s of one or more ts, such as the solvents used in its synthesis or crystallization. As used above, "substantially free of" and "small amounts," refers to the presence of solvents preferably less that 10,000 parts per million (ppm), or more preferably, less than 500 ppm.

The "crystalline solvate" of Compound 1 is a crystalline solid form of the free base of 2-hydroxy((2—(1-isopropyl-lH—pyrazol-S-yl)pyridin-3—yl)methoxy)benzaldehyde, where the crystal lattices comprises one or more solvents of crystallization.

"Characterization" refers to obtaining data which may be used to identify a solid form of a compound, for example, to identify whether the solid form is amorphous or crystalline and whether it is ated or solvated. The process by which solid forms are characterized involves analyzing data collected on the polymorphic forms so as to allow one of ordinary skill in the art to distinguish one solid form from other solid forms containing the same material. Chemical identity of solid forms can often be ined with solution- state techniques such as l3C NMR or 'H NMR. While these may help fy a material, and a solvent molecule for a solvate, such solution-state techniques themselves may not provide information about the solid state. There are, however, solid-state analytical PCT/U82015/014589 techniques that can be used to provide information about solid-state structure and differentiate among polymorphic solid forms, such as single crystal X-ray diffraction, X-ray powder diffraction (XRPD), solid state nuclear magnetic resonance (SS-NMR), and infrared and Raman spectroscopy, and thermal techniques such as differential scanning calorimetry (DSC), Solid state l3C-NMR, thermogravimetry (TG), melting point, and hot stage microscopy.

To "characterize" a solid form of a compound, one may, for example, collect XRPD data on solid forms of the compound and compare the XRPD peaks of the forms. For example, when only three solid forms, c.g., Forms 1 and I] and Material N, are compared and the Form I pattern shows a peak at an angle where no peaks appear in the Form 11 or Material N pattern, then that peak, for that compound, distinguishes Form I from Form 11 and Material N and r acts to characterize Form 1. The collection of peaks which distinguish e.g., Form I from the other known forms is a collection of peaks which may be used to characterize Form 1. Those of ordinary skill in the art will ize that there are often multiple ways, including multiple ways using the same analytical technique, to characterize solid forms. Additional peaks could also be used, but are not necessary, to terize the form up to and including an entire diffraction pattern. Although all the peaks within an entire XRPD pattern may be used to characterize such a form, a subset of that data may, and typically is, used to characterize the form.

An XRPD pattern is an x—y graph with diffraction angle (typically ° 20) on the x-axis and intensity on the y—axis. The peaks within this pattern may be used to characterize a crystalline solid form. As with any data measurement, there is ility in XRPD data.

The data are often represented solely by the diffraction angle of the peaks rather than including the intensity of the peaks because peak ity can be particularly sensitive to sample preparation (for example, particle size, moisture t, solvent content, and preferred orientation effects influence the sensitivity), so samples of the same material prepared under different conditions may yield slightly different patterns; this variability is usually greater than the variability in ction . Diffraction angle variability may also be sensitive to sample preparation. Other sources of variability come from instrument ters and sing of the raw X-ray data: different X-ray instruments operate using ent parameters and these may lead to slightly different XRPD patterns from the same solid form, and similarly different re packages process X-ray data differently and this also leads to variability. These and other sources of variability are known to those of 2015/014589 ordinary skill in the pharmaceutical arts. Due to such s of variability, it is usual to assign a variability of i0.2° 20 to diffraction angles in XRPD patterns.

"Comprising" or "comprises" is intended to mean that the compositions and s include the d elements, but not exclude others. "Consisting essentially of" when used to define compositions and methods, shall mean excluding other elements of any essential cance to the combination for the stated e. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps. ments defined by each of these transition terms are within the scope of this invention.

Form 11 and Material N are enantiotropic at a transition temperature (of approximately 42 °C). Below this transition temperature, Material N of the free base of Compound 1 is the thermodynamically more stable form ve to Forms 1 and 11. Above this transition temperature, Form 11 of the free base of Compound 1 is the thermodynamically more stable form relative to Form I and Material N.

"Room temperature" refers to (22:5) °C.

"Therapeutically effective amount" or "therapeutic amount" refers to an amount of a drug or an agent that when administered to a patient ing from a condition, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of the condition in the patient. The therapeutically effective amount will vary depending upon the t and the condition being treated, the weight and age of the subject, the severity of the ion, the particular composition or excipient chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can be ined readily by one of ordinary skill in the art. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. For example, and without limitation, a therapeutically effective amount of an agent, in the context of treating disorders related to hemoglobin S, refers to an amount of the agent that alleviates, ameliorates, palliates, or eliminates one or more manifestations of the disorders d to hemoglobin S in the patient.

WO 20133 PCT/U82015/014589 ment", "treating", and "treat" are defined as acting upon a disease, disorder, or condition with an agent to reduce or ameliorate the harmful or any other undesired effects of the disease, disorder, or condition and/or its symptoms. Treatment, as used herein, covers the treatment of a human t, and includes: (a) ng the risk of occurrence of the ion in a patient determined to be predisposed to the disease but not yet diagnosed as having the condition, (b) impeding the development of the condition, and/or (c) relieving the condition, i.e., causing regression of the condition and/or relieving one or more ms of the ion. For purposes of this invention, beneficial or desired clinical results e, but are not limited to, multilineage hematologic improvement, decrease in the number of required blood transfusions, decrease in infections, decreased bleeding, and the like.

Identifling Forms 1, II and Material N When the HCl salt of Compound 1 was subjected to various stress conditions, disproportionation of the HCl salt in water was observed to generate the free base. At least three ous crystalline forms of the free base were identified, termed Free Base Forms 1, II, and al N. It was discovered that nucleation of Free Base Form 1 generally occurs first and that extending the slurry time induces the transformation of Free Base Form I to Free Base Form II, a more thermodynamically stable phase relative to Form I. It was further discovered that Frcc Base al N appears to be most stable form, relative to Forms I and II, at room temperature. Frcc Base Material N was found to be enantiotropically active relative to Form II, and will transform reversibly at a specific transition temperature ated herein near 42 °C). Above the transition temperature, Free Base Form 11 appears to be the most stable form, relative to Form 1 and Material N.

Based in part on solid-state nuclear magnetic resonance data, all three forms are crystalline and are distinct polymorphic forms. Sec FIGS 10 and 1 1. Form I contains one molecule per asymmetric unit, Form 11 contains two molecules per asymmetric unit and Form N contains four molecules per asymmetric unit. See the '5 N spectra in 1.

Ansolvatcs of Forms I II and Material N In one ment, this invention provides the free base crystalline ansolvate of Compound I. The free base crystalline ansolvate of Compound 1 may include one or more of Form 1, Form [1 and/or Material N polymorphs. In some embodiments, the free base crystalline ansolvate of Compound 1 may include the Form II polymorph. Preferably, the PCT/U82015/014589 free base crystalline ansolvatc of Compound I may include Form [1 and/or Material N polymorphs. More preferably, the free base crystalline ansolvatc of Compound I may include the Material N polymorph. Yet more preferably, the free base crystalline ansolvatc of Compound 1 is substantially free of a solvatcd rph of Compound 1 free base. r yet more preferably, the free base crystalline ansolvate of Compound 1 is substantially free of other ansolavte polymorphs of Compound 1 free base. "Substantially free" of a component as used herein refers to contain up to about 5%, more preferably about 3%, and still more preferably about 1% of that coponent. As used herein, solvate includes a hydrate form as well.

Solvates of Compound 1 In one aspect, provided is a crystalline e of Compound 1: ,N / N\ l o o Compound 1 In some embodiments, the crystalline e is ntially free of an ansolvatcd rph of Compound 1.

Many of the solubility and screen ments with the free base of Compound 1 resulted in precipitation of solids characterized as solvate formation with some solvents.

Under the conditions, solvates were not observed from the free base of Compound 1 with four ts, including heptanc, di-isopropyl ether (IPE), tert-methyl butyl ether (MTBE) and toluene. Solvates were observed from the free base of Compound 1 in nine solvents including acetone (Material E), itrile (Material F), dichloromethane (Material G), dioxane (Material H), ethanol (Material J), isopropyl alcohol or a mixture of water and isopropyl alcohol (Material K), tetrahydrofuran ial L), methyl ethyl ketone "MEK" (Material M), ethyl acetate (Material 0) and dimethyl sulfoxide "DMSO" ial P). The majority of the solvates (i.e., Materials E-H, J-M, O and P are contemplated to be isostructural. In some embodiments, the crystalline e includes one or more of Material E, Material F, Material G, Material H, Material J, Material K, Material L, Material M, Material 0 or Material P. -1589 Material E can be characterized by at least one X-ray powder diffraction peak (Cu K01 radiation) selected from 8.69, 11.73, 12.10, 15.26, 16.11, 17.45, 22.39, 22.55 and 23.70 i: 0.20. Material F can be characterized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 8.47, 8.81, 12.75, 13.17, 14.92, 15.63, 17.01 23.73, and 24.07 i 0.20. Material G can be terized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 8.47, 11.45, 12.62, 14.66, 15.69, 17.01, 18.47, 20.32, 22.61, 23.08, 23.43 and 23.70 i: 0.20. Material H can be characterized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 8.61 1 1.67, 15.33, 16.28, 17.28, 22.58, 23.51 and 25.77 :1: 0.20. Material J can be terized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 8.52, 8.88, 12.79, 15.04, 15.61, 17.11, 22.81, 23.87, 24.17, 24.62 and 26.44 i 0.20. Material K can be characterized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 8.52; 8.83, l 1.35, 15.04, .74, 17.1 1, 23.46, 23.58, 24.08 and 25.99 d: 0.20. Material L can be terized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 8.61, 8.78, 1 1.67, 14.94, 15.28, 16.14, 17.30, 22.75, 23.71 and 26.05 d: 0.20; and Material M can be terized by at least one X-ray powder diffraction peak (Cu Ka radiation) selected from 7.74, 10.05, 12.82, 15.33, 16.80, 20.82, 21.14, 25.80 and 26.97 i 0.20.

The solvates (such as, of acetone, acetonitrile, dichloromethane, dioxane, ethanol, ethyl acetate, pyl alcohol, MEK, tetrahydrofuran or DMSO) could be used c.g., as ediates to regenerate the free base crystalline ansolvate of Compound 1 by several methods including ting the solvate to vacuum conditions; and/or regenerating the HCl salt and disproportionating HCl; and/or washing the solvate with a solvent less prone to solvate formation such as heptane, di—isopropyl ether (IPE), tert-methyl butyl ether (MTBE) and toluene.

PCT/U82015/014589 Table 1. Data Related to Solvatcs of the Free Base of nd 1 Estimated Volume Crystallization V0 umel Number of Formula per Formula Unit* Indexing Identifier Solvent (As/Cell) Units per Cell Result (A3) Material E acetone Material F ACN Material (i DCM Material H dioxanc i | Material J Et()H al K [PA \0 O'\ b) Material L THF \l k) Material M MEK J}.\O.1; Material () Et()Ac Material DMSO * The value is obtained by dividing the volume of the cell, derived from the ive indexing solution, by the number of formula units within the cell. ** Material P ed as a mixture with a "sulfate form 1".

Certain contemplated peaks of the various solvates provided herein are tabulated below. Certain peaks, which are preferably non-overlapping, low-angle peaks, with strong ity, were not identified. The peaks were determined to the extent that the state of preferred orientation in the samples were unknown.

Table 2. Observed peaks for Material E. °29 d space (A) Intensity (%) 8.41 :t 0.20 10.517 :l: 0.256 13 8.69 i 0.20 10.174 i 0.239 100 11.73 i 0.20 7.543 i 0.130 17 12.10i020 7.31410.122 20 13.00 i 0.20 6.809 :t 0.106 15 14.02 1: 0.20 6.316 2*: 0.091 5 14.77 i: 0.20 5.996 a: 0.082 16 .26 is 0.20 5.807 :l: 0.077 34 .81 :l: 0.20 5.605 d: 0.071 7 16.11 i: 0.20 5.501 2!: 0.069 20 16.48 :t 0.20 5.379 d: 0.066 11 16.65 i 0.20 5.326 t 0.064 11 16.88 i 0.20 5.253 t 0.063 17.26 i: 0.20 5.136 t 0.060 17.45 i 0.20 5.083 3: 0.058 32 .02 :t 0.20 4.435 t 0.044 .92 i: 0.20 4.246 t 0.041 13 21.91 i 0.20 4.057 i 0.037 20 22.39 i 0.20 3.970 :t 0.035 49 22.55 d: 0.20 3.944 3: 0.035 37 22.81 i: 0.20 3.898 i 0.034 16 23.36 :t 0.20 3.807 i 0.032 12 23.70 i 0.20 3.755 i 0.032 61 24.37 i 0.20 3.653 i 0.030 12 24.85 i 0.20 3.583 :t 0.029 .42 d: 0.20 3.504 t 0.027 .89 :t 0.20 3.442 t 0.026 26.19 i 0.20 3.403 i 0.026 40 26.97 :t 0.20 3.306 i 0.024 27.61 i: 0.20 3.231 :t 0.023 16 28.24 i 0.20 3.160 :1: 0.022 28.48 i 0.20 3.134 :1: 0.022 28.69 i 0.20 3.1 1 l i 0.021 29.83 i: 0.20 2.995 i 0.020 Auk/1N Table 3. Observed peaks for al F. °20 0' space (A) Intensity (%) 8.47 :1: 0.20 10.434 3: 0.252 100 8.81 :1: 0.20 10.039 :1: 0.233 49 11.42 i: 0.20 7.752 d: 0.138 1 5 12.75 i: 0.20 6.942 :t 0.1 10 27 13.17 i 0.20 6.723 d: 0.103 2 l 13.87 3: 0.20 6.384 t 0.093 7 14.61 d: 0.20 6.064 i 0.084 13 14.92 i: 0.20 5.936 t 0.080 43 .51 i 0.20 5.713 t 0.074 24 .63 i: 0.20 5.67] i 0.073 43 .96 3: 0.20 5.553 t 0.070 15 17.01 :1: 0.20 5.212 3: 0.062 17.26 i 0.20 5.136 t 0.060 17.70 i 0.20 5.01 1 i 0.057 18.17 i 0.20 4.883 i 0.054 18.79 i 0.20 4.724 i 0.050 19.35 i 0.20 4.587 3: 0.047 19.49 i: 0.20 4.555 i 0.047 .02 :1: 0.20 4.435 i 0.044 .29 :1: 0.20 4.377 i 0.043 21.06 :1: 0.20 4.219 i 0.040 11 21.33 i 0.20 4.167 :t 0.039 22.71 3: 0.20 3.915 t 0.034 27 23.11 :t 0.20 3.848 t 0.033 15 23.73 :1: 0.20 3.749 :1: 0.031 42 24.07 :t 0.20 3.698 i 0.031 59 24.65 :1: 0.20 3.612 :t 0.029 87 24.95 :1: 0.20 3.569 :1: 0.028 .20 i 0.20 3.534 :1: 0.028 .69 i: 0.20 3.468 t 0.027 15 26.52 i: 0.20 3.361 i 0.025 61 26.79 :t 0.20 3.328 :1: 0.025 10 27.02 i 0.20 3.300 :1: 0.024 Table 4. Observed peaks for al G. °20 (1' space (A) Intensity (%) 8.47 :1: 0.20 10.434 :1: 0.252 45 8.76 i: 0.20 10.096 i 0.235 12 1 1.45 i: 0.20 7.729 :t 0.137 76 12.62 i 0.20 7.015 d: 0.1 13 36 13.09 3: 0.20 6.765 i 0.105 10 13.87 i: 0.20 6.384 t 0.093 5 14.66 3: 0.20 6.044 d: 0.083 39 14.92 i 0.20 5.936 t 0.080 26 .33 i 0.20 5.782 t 0.076 .69 i: 0.20 5.647 t 0.072 88 16.01 i 0.20 5.536 d: 0.070 16.76 :t 0.20 5.289 t 0.063 15 17.01 i: 0.20 5.212 t 0.062 29 17.50 i 0.20 5.068 i 0.058 17.60 i 0.20 5.040 :t 0.057 18.13 d: 0.20 4.892 3: 0.054 18.47 i: 0.20 4.804 i 0.052 19.55 :t 0.20 4.540 t 0.046 .01 i 0.20 4.439 i 0.044 .32 i 0.20 4.370 i 0.043 21.1 1 i 0.20 4.209 :t 0.040 22.61 i: 0.20 3.932 t 0.035 22.88 i 0.20 3.887 t 0.034 23.08 i 0.20 3.854 t 0.033 23.43 :t 0.20 3.797 i 0.032 23.70 i: 0.20 3.755 :t 0.032 24.12 i 0.20 3.690 :1: 0.030 24.42 i 0.20 3.646 :1: 0.030 100 .05 i: 0.20 3.555 i 0.028 .40 i: 0.20 3.506 i 0.027 26 26.36 :t 0.20 3.382 :1: 0.025 50 26.57 :1: 0.20 3.355 :1: 0.025 26.82 i 0.20 3.324 i 0.025 27 27.07 i 0.20 3.294 1: 0.024 10 Table 5. ed peaks for Material H. °20 0' space (A) Intensity (%) 8.61 d: 0.20 10.273 i 0.244 48 8.81 :1: 0.20 10.039 d: 0.233 20 11.67 3: 0.20 7.586i0.132 32 12.10 i: 0.20 7.314i0.122 1 l 12.79 i: 0.20 6.924i0.110 9 14.56 i 0.20 6.085 t 0.084 14.87 i 0.20 5.956 t 0.081 22 .33 i: 0.20 5.782 t 0.076 42 .76 i 0.20 5.623 d: 0.072 18 16.28 i 0.20 5.445 at 0.067 51 16.73 i: 0.20 5.299 t 0.064 17.28 i 0.20 5.132 i 0.060 61 17.68 i 0.20 5.016 :t 0.057 .47 i 0.20 4.338 :1: 0.042 12 21.38 i 0.20 4.157 i 0.039 21.83 :t 0.20 4.072 i 0.037 22.23 i 0.20 3.999 i 0.036 22.58 i 0.20 3.938 i 0.035 100 22.95 i 0.20 3.876 :1: 0.034 23.11 i: 0.20 3.848 t 0.033 14 23.51 i 0.20 3.783 t 0.032 88 24.37 i 0.20 3.653 t 0.030 13 24.65 :t 0.20 3.612 i 0.029 10 .77 i: 0.20 3.457 :t 0.027 A 26.67 i 0.20 3.342 1: 0.025 26.97 :1: 0.20 3.306 i 0.024 27.66 i: 0.20 3.225 i 0.023 28.1 1 i 0.20 3.174 t 0.022 28.61 :t 0.20 3.120 :1: 0.022 28.96 :1: 0.20 3.083 :1: 0.021 29.23 i: 0.20 3.055 i 0.021 29.63 i 0.20 3.015 d: 0.020 WWAOAUJUIN Table 6. ed peaks for Material J. °20 0' space (A) Intensity (%) 8.52 i: 0.20 10.373 3: 0.249 100 8.88 :1: 0.20 9.964 d: 0.229 39 11.33 d: 0.20 7.809 i 0.140 22 12.79 i: 0.20 6.924 d: 0.1 10 25 13.12 i: 0.20 6.748 i 0.104 24 2015/014589 13.94 i: 0.20 6.354 t 0.092 14.47 i 0.20 6.120 t 0.085 14 .04 3: 0.20 5.890 i 0.079 42 .61 :1: 0.20 5.677 3: 0.073 56 .84 :1: 0.20 5.594 t 0.071 16 17.1 1 i 0.20 5.18] i 0.061 33 17.40 i 0.20 5.097 i 0.059 17.82 i 0.20 4.979 i 0.056 18.12 i 0.20 4.897 3: 0.054 18.90 :1: 0.20 4.695 i 0.050 19.39 i 0.20 4.579 i 0.047 19.62 :1: 0.20 4.525 i 0.046 .16 i 0.20 4.406 i 0.044 .96 :1: 0.20 4.239 :t 0.040 12 22.81 3: 0.20 3.898 t 0.034 27 23.15 :t 0.20 3.843 t 0.033 23.28 :1: 0.20 3.821 t 0.033 23.87 :t 0.20 3.729 i 0.031 34 24.17 i: 0.20 3.683 :t 0.030 52 24.62 i 0.20 3.616 :1: 0.029 95 .20 i 0.20 3.534 :1: 0.028 .77 i 0.20 3.457 t 0.027 13 26.44 i: 0.20 3.371 i 0.025 70 26.71 :t 0.20 3.338 :1: 0.025 10 27.21 i 0.20 3.278 :1: 0.024 Table 7. Observed peaks for GBT000440, Material K. °20 (1' space (A) Intensity (%) 8.52 :1: 0.20 10.373 :1: 0.249 75 8.83 i: 0.20 10.020 i 0.232 33 11.35 i: 0.20 7.797 :t 0.139 29 12.52 i: 0.20 7.071 d: 0.1 14 2 l 12.90 3: 0.20 6.86] i 0.108 24 13.92 i: 0.20 6.361 t 0.092 4 14.49 i: 0.20 6.1 13 i 0.085 1 8 .04 i: 0.20 5.890 t 0.079 41 .34 i: 0.20 5.775 t 0.076 17 .74 3: 0.20 5.629 i 0.072 57 .93 :1: 0.20 5.564 d: 0.070 13 16.61 :1: 0.20 5.336 t 0.065 17.1 1 i 0.20 5.18] i 0.061 33 17.70 i 0.20 5.01 1 i 0.057 18.00 :t 0.20 4.928 i 0.055 18.38 i 0.20 4.826 3: 0.053 13 19.04 :1: 0.20 4.662 i 0.049 19.74 :1: 0.20 4.498 i 0.046 .21 :1: 0.20 4.395 i 0.043 11 .99 :1: 0.20 4.232 i 0.040 12 22.70 :1: 0.20 3.918 :t 0.034 22 22.90 3: 0.20 3.884 t 0.034 17 23.46 :t 0.20 3.791 t 0.032 45 23.58 :1: 0.20 3.773 i 0.032 70 24.08 :t 0.20 3.695 i 0.030 100 24.75 i: 0.20 3.597 :t 0.029 .19 i 0.20 3.536 :1: 0.028 21 .99 i 0.20 3.429 :1: 0.026 71 26.71 d: 0.20 3.338 i 0.025 11 27.36 a: 0.20 3.260 i 0.024 28.11 :t 0.20 3.174 :1: 0.022 28.69 i 0.20 3.1 11 :1: 0.021 Table 8. Observed peaks for al L. °20 (1' space (A) Intensity (%) 8.61 :1: 0.20 10.273 :1: 0.244 79 8.78 i: 0.20 10.077 i 0.235 38 11.67 i: 0.20 7.586 :t 0.132 35 12.17 i: 0.20 7.274 d: 0.121 19 12.94 3: 0.20 6.844 i 0.107 14 14.07 i: 0.20 6.293 i 0.090 3 14.62 i: 0.20 6.057 t 0.084 5 2015/014589 14.94 i: 0.20 5.929 t 0.080 25 .28 i 0.20 5.800 t 0.076 50 .93 3: 0.20 5.564 i 0.070 18 16.14 i 0.20 5.490 d: 0.068 49 16.33 :1: 0.20 5.429 t 0.067 16.70 :1: 0.20 5.310 t 0.064 16.85 :t 0.20 5.263 i 0.063 17.30 :t 0.20 5.127 :t 0.060 17.63 :1: 0.20 5.030 3: 0.057 18.37 i 0.20 4.830 t 0.053 .14 :1: 0.20 4.409 i 0.044 .59 :1: 0.20 4.314 i 0.042 14 21.53 i 0.20 4.128 i 0.038 11 22.01 :1: 0.20 4.038 :t 0.037 22.44 i: 0.20 3.961 t 0.035 27 22.75 :t 0.20 3.910 t 0.034 72 23.10 :1: 0.20 3.851 i 0.033 20 23.31 :t 0.20 3.816 i 0.033 19 23.48 :1: 0.20 3.789 :t 0.032 12 23.71 i 0.20 3.752 :1: 0.031 100 24.48 i 0.20 3.636 :1: 0.029 20 24.70 i: 0.20 3.604 i 0.029 24.93 a: 0.20 3.571 i 0.028 .59 :t 0.20 3.482 :1: 0.027 .72 i 0.20 3.464 :1: 0.027 LII 26.05 i 0.20 3.420 i 0.026 26.59 :1: 0.20 3.352 3: 0.025 27.14 a: 0.20 3.286 :1: 0.024 27.83 i 0.20 3.206 i 0.023 28.38 i 0.20 3.145 :1: 0.022 28.78 i 0.20 3.102 :t 0.021 29.05 3: 0.20 3.074 d: 0.021 29.36 3: 0.20 3.042 1 0.020 UJ-P-OOUJOCOCON Table 9. ed peaks for Material M. °26 (1 space (A) Intensity (%) 7.74 i 0.20 11.424 i 0.303 100 8.34 i 0.20 10.601 i 0.260 4 .05 :1: 0.20 8.806 t 0.178 17 12.82 :1: 0.20 6.906 t 0.109 46 13.05 :t 0.20 6.783 i 0.105 4 14.17 i 0.20 6.249 i 0.089 14.54 i 0.20 6.092 :1: 0.085 14.99 i 0.20 5.910 :t 0.079 .33 i 0.20 5.782 :1: 0.076 .53 :1: 0.20 5.707 :t 0.074 16.80 i 0.20 5.278 :t 0.063 18.33 i 0.20 4.839 :1: 0.053 19J7i020 4.630 :1: 0.048 .19 :t 0.20 4.399 i 0.044 .82 :1: 0.20 4.266 :1: 0.041 21.14 :t 0.20 4.202 d: 0.040 21.29 :1: 0.20 4.173 :t 0.039 22.01 i 0.20 4.038 :1: 0.037 22.28 i 0.20 3.991 :1: 0.036 22.93 i: 0.20 3.879 i 0.034 23.35 a: 0.20 3.810 i 0.032 24.00 :t 0.20 3.708 :1: 0.031 24.25 i 0.20 3.670 :1: 0.030 24.88 i 0.20 3.578 :t 0.029 .54 :1: 0.20 3.488 3: 0.027 .80 3: 0.20 3.453 :1: 0.027 26.97 i 0.20 3.306 :1: 0.024 27.63 i 0.20 3.229 :1: 0.023 28.41 i 0.20 3.142 :t 0.022 28.54 i 0.20 3.127 :1: 0.022 29.03 i 0.20 3.076 :1: 0.021 29.30 3: 0.20 3.049 :1: 0.020 \IUJOCNN 29.63 i 0.20 3.015 3: 0.020 15 Pharmaceutical Compositions In r of its composition embodiments, this ion provides for a pharmaceutical composition comprising a pharmaceutically acceptable excipient and crystalline free base ansolvate of Compound 1, preferably including one or more of the Form 1, Form ll and/or Material N polymorphs.

Such compositions can be formulated for different routes of administration.

Although compositions suitable for oral delivery will probably be used most frequently, other routes that may be used e intravenous, intraarterial, pulmonary, , nasal, vaginal, l, intramuscular, intraperitoneal, utaneous, intracranial, subcutaneous and transdermal routes. Suitable dosage forms for administering any of the compounds bed herein include tablets, capsules, pills, powders, aerosols, suppositories, parenterals, and oral liquids, including suspensions, solutions and emulsions. ned release dosage forms may also be used, for example, in a transdermal patch form. All dosage forms may be prepared using methods that are standard in the art (see e.g., Remington’s Pharmaceutical Sciences, 16lh ed., A. Oslo editor, Easton Pa. 1980).

Pharmaceutically acceptable excipients are non—toxic, aid administration, and do not adversely affect the therapeutic benefit of the compound of this invention. Such excipients may be any solid, liquid, semi-solid or, in the case of an l composition, s excipient that is lly available to one of skill in the art. Pharmaceutical compositions in accordance with the invention are prepared by conventional means using methods known in the art.

The compositions disclosed herein may be used in conjunction with any of the vehicles and excipients commonly employed in pharmaceutical preparations, e.g., talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Coloring and flavoring agents may also be added to ations, particularly to those for oral administration. Solutions can be prepared using water or physiologically compatible organic ts such as ethanol, 1,2-propylene glycol, polyglycols, dimcthylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerin and the like.

Solid pharmaceutical excipients include starch, cellulose, hydroxypropyl cellulose, talc, glucose, lactose, sucrose, n, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearatc, sodium chloride, dried skim milk and the PCT/U82015/014589 like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, ble or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. In n embodiments, the compositions provided herein comprises one or more of a-toeopherol, gum arabic, and/or hydroxypropyl cellulose.

In one embodiment, this invention es sustained release formulations such as drug depots or patches comprising an effective amount of a compound provided herein. In another embodiment, the patch further comprises gum Arabic or hydroxypropyl cellulose separately or in ation, in the presence of alpha-tocopherol. Preferably, the hydroxypropyl ose has an average MW of from 10,000 to 100,000. In a more preferred embodiment, the hydroxypropyl cellulose has an average MW of from 5,000 to 50,000.

Compounds and pharmaceutical compositions of this invention maybe used alone or in combination with other compounds. When administered with another agent, the co- administration can be in any manner in which the pharmacological effects of both are manifest in the t at the same time. Thus, co-administration does not require that a single pharmaceutical composition, the same dosage form, or even the same route of stration be used for administration of both the compound of this invention and the other agent or that the two agents be stered at precisely the same time. However, co- stration will be accomplished most conveniently by the same dosage form and the same route of administration, at substantially the same time. Obviously, such administration most advantageously proceeds by delivering both active ingredients simultaneously in a novel pharmaceutical ition in accordance with the present invention.

Preparative and Treatment Methods Ansolvate In another aspect, the present invention provides a method of preparing the crystalline free base ansolvate of nd 1. In one embodiment, provided herein is a method of preparing the crystalline free base of Compound 1 comprising slurrying or WO 20133 PCT/U82015/014589 contacting the HCl salt of the Compound 1 with water and allowing dissociation of I-ICl to produce the free base of Compound 1. In one embodiment, the crystalline free base ansolavte of Compound I prepared comprises one or more of Form I, Form II and Material In yet another of its method ments, there are provided methods for increasing oxygen affinity of hemoglobin S in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crystalline free base of Compound I. In some embodiments, the crystalline free base of Compound 1 is an ansolvate. In one embodiment, the crystalline free base of Compound 1 ses one or more of Form 1, Form 11 and Material N.

In yet another of its method embodiments, there are provided methods for treating oxygen deficiency associated with sickle cell anemia in a subject, the method comprising administering to a t in need thereof a therapeutically effective amount of a crystalline free base of Compound 1. In some embodiments, the crystalline free base of Compound ] is an ansolvate. In one embodiment, the crystalline free base of nd 1 comprises one or more of Form 1, Form 11 and Material N.

In further aspects of the invention, a method is provided for ng sickle cell disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crystalline free base of Compound 1. In some embodiments, the crystalline free base of Compound 1 is an ansolvate. In one embodiment, the crystalline free base of nd 1 comprises one or more of Form 1, Form 11 and Material N. In still further aspects of the invention, a method is provided for treating cancer, a ary disorder, stroke, high altitude sickness, an ulcer, a pressure sore, mer's disease, acute respiratory disease syndrome, and a wound, the method comprising stering to a subject in need thereof a therapeutically effective amount of a crystalline free base of Compound 1.

In some ments, the crystalline free base of Compound 1 is an ansolvate. In one embodiment, the crystalline free base of Compound 1 comprises one or more of Form 1, Form II and Material N.

In such treatments, the dosing of the crystalline free base of nd 1 to the treated patient is already disclosed in the art. 2015/014589 Solvates In another aspect, the present invention provides a method of preparing the crystalline free base solvates of Compound 1. In some embodiments, a free base ansolvate, as bed herein (e.g, as obtained by slurrying an HCI salt of Compound 1 in water) of Compound 1 is contacted with a solvent as provided herein, including a mixture of solvents, to prepare the solvate. the solvent or the mixture of solvents. Thus, a solvent can be a single solvent or substantially a single solvent or a e of solvents. When a mixture of solvents is used, a solvate can be produced having one or more of the individual constituent solvents of the solvent mixture. In some embodiments, the solvent includes alcoholic solvents such as mono di or higher ls or alkanols. In some embodiments, the solvent includes chlorinated solvents such as dichloromethane chloroform, et cetera. In some embodiments, the solvent includes ketone solvents such as alkanones and cycloalkanones.

Certain solvents include without limitation, methanol, ethanol, 2-propanol, 2-methyl propanol, 1-butanol, acetonitrile, acetone, romethane, dioxane, or tetrahydrofuran, or combinations thereof, optionally including water.

In another aspect, a method is provided for sing oxygen affinity of hemoglobin S in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crystalline solvate of Compound 1.

In another , a method is provided for treating oxygen deficiency associated with sickle cell anemia, the method sing administering to a subject in need f a therapeutically effective amount of a crystalline solvate of Compound 1.

The following examples describe the ation, characterization, and properties of the free base of Compound 1 Form I ansolvate. Unless otherwise stated, all temperatures are in s Celcius (°C) and the following abbreviations have the following definitions: DSC Differential scanning metry DVS Dynamic vapor sorption HPLC High performance liquid tography NA Not applicable ND Not determined Q Percent dissolved per unit time PCT/U52015/014589 RH Relative humidity RSD Residual standard ion RRT Relative retention time SS-NMR Solid state nuclear ic resonance TGA Thermogravimetric analysis TG-lR Thennogravimetric infra red analysis XRPD X-ray powder diffraction VT-XRPD Variable temperature X-ray powder ction Synthetic Routes for Preparing Compound 1 The compound of formula (I) was synthesized as schematically described below and elaborated thereafter. wages»: as; an. serum" 533.. «(V :34"\ ..: .g < :q:\\ "9‘" ‘32". "arr u:- "‘_sssés. it\ . i \‘:‘ «is: \t{mr‘ am: .

-M fl vs '1\‘ {:\C.. \- "a . i‘x’.?¥t’.§i.$fi~il~?% '24: l-ECl N / O" O Y N \ K2003 EQNNCI N\ l I ' H + N / o —’T C O N: our. 65C -HF OMCM. \ 81/»"1 H E.\ (ii)"3 H 16 10 Cl 17 OMOM 0" Example 1: Synthesis of Compound 15 OH DWEA OMGM Br meme: Br OH 90% OMOM 4. 4 4, 5 To a solution of 2-bromobcnzenc-1,3-diol (5 g, 26.45 mmol) in DCM (50 ml) at 0 °C was added DIPEA (l 1.54 mL, 66.13 mmol) and MOMCl (4.42 mL, 58.19 mmol). The -32_ SUBSTITUTE SHEET (RULE 26) WO 20133 PCT/U82015/01-1589 mixture was stirred at 0 °C for 1.5 h, and then warmed to room temperature. The solution was diluted with DCM, washed with sat. NaHC03, brine, dried and concentrated to give crude product, which was d by column (hexanes/EtOAc=4: 1) to give desired product .58 g (90%).

Example 2: Synthesis of Compound 13 from 15 OMOM OMOM (53% BuLi. DMF CECHO OMOM -73 to 0 °C OMOM 13 To a solution of 2-bromo-1,3-bis(methoxymethoxy)benzene (15) (19.9g, 71.8 mmol) in THF (150 mL) at -78 °C was added BuLi (2.5 M, 31.6 mL, 79.0 mmol) dropwisc.

The solution was stirred at -78 °C for 25 min (resulting white cloudy mixture), then it was warmed to 0 °C and stirred for 25 min. The reaction mixture slowly turns homogenous. To the on was added DMF at 0 °C. After 25 min, HPLC showed reaction completed.

The mixture was quenched with sat. NH4C1 (150 mL), diluted with ether (300 mL). The c layer was separated, aq layer was further extracted with ether (2x200 mL), and organic layer was combined, washed with brine, dried and concentrated to give crude product, which was triturated to give 14.6 g desired product. The e was then concentrated and purified by column to give additional 0.7 g, total mass is 15.3 g.

Example 3: Synthesis of Compound 13 from resorcinol 11 1.1 RzTMEDA. R:BuLI. SzTHF. 2 h. —10°C 0" NaH OMOM 1.2 1h, 0°C; 83% OMOM MOMCI 0rgLett14(10). 2516-2519: 2012 OR: CHO 0" f SSQZ'IDMF OMOM 1.1 RzTMEDA.R:BuLi.S:Me(CH2)4Me.n?-10°C OMOM 11 -10°C;2h.-10°C;-10°C?0°C 12 1.3 0°C;1h.O°C 13 1.4 R:HC|_$:H20 Journal of Organic Chemistry. 74(11), 43114317: 2009 A three-necked round-bottom flask ed with mechanical stirrer was charged with 0.22 mol ofNaH (50 % suspension in mineral oil) under nitrogen atmosphere. NaH was washed with 2 portions (100 mL) of n—hexane and then with 300 mL of dry l ether; then 80 mL of anhydrous DMF was added. Then 0.09 mol of inol 11, dissolved in 100 mL of diethyl ether was added dropwisc and the mixture was left under stirring at rt for 30 min. Then 0.18 mol of MOMCl was slowly added. After 1 h under stirring at rt, 250 mL of water was added and the organic layer was ted with diethyl ether. The extracts -1589 were washed with brine, dried (Na2SO4), then concentrated to give the crude product that was purified by silica gel chromatography to give compound 12 (93 % yield).

A three-necked bottom flask was charged with 1 10 mL of n-hexane, 0.79 mol of BuLi and 9.4 mL of tetramethylethylendiamine (TMEDA) under nitrogen atmosphere. The mixture was cooled at —10 °C and 0.079 mol of bis-phenyl ether 12 was slowly added. The resulting mixture was left under magnetic stirring at —1 0 °C for 2 h. Then the temperature was raised to 0 °C and 0.067 mol of DMF was added dropwise. After 1 h, aqueous HCl was added until the pH was ; the mixture was then extracted with ethyl ether. The combined extracts were washed with brine, dried (Na2S04), and concentrated to give aldehyde 13 (84%). 2,6-bis(methoxymethoxy)benzaldehydc (13): mp 58—59 °C (n-hexane) ; IR (KBr) n: 1685 (C=O) cm‘l; lH-NMR (400 MHz, CDC13)5 3.51 (s, 6H, 2 OCHg), 5.28 (s, 4H, 2 OCHzO), 6.84 (d, 2H, J = 8.40 Hz, H-3, H-5), 7.41 (t, 1H, J = 8.40 Hz, H-4), 10.55 (s, 1H, CHO); MS, m/e (relative intensity) 226 (M+, 3), 180 (4), 164 (14), 122 (2), 92 (2), 45 (100); Anal. Calc’d. for C11H1405:C,58.40;H, 6.24. Found: C, 57.98; H, 6.20. e 4: The Synthesis of Compound 16 OMOM OH CHO 12N HCI CHO OMOM THF OMOM 13 15 To a solution of 2,6-bis(mcthoxymethoxy)benzaldehyde (13) (15.3 g, 67.6 mmol) in THF (105 mL) (solvent was purged with N2) was added conc. HCl (12N, 7 mL) under N2, then it was further stirred under N2 for 1.5 h. To the solution was added brine (100 mL) and ether (150 ml). The organic layer was separated and the aqueous layer was further ted with ether (2x200 mL). The organic layer was combined, washed with brine, dried and concentrated to give crude product, which was purified by column (300g, hexanes/EtOAc=85:15) to give desired product 16 (9.9 g) as yellow liquid. e 5: Synthesis of nd 17 CHO \(CIHNI \ K2003 N\ l + ,N / o o N\ l DMF OMOM H CI 65°C 16 10 31% OMOM PCT/U82015/014589 To a solution of 2-hydroxy(methoxymethoxy)benzaldehyde (16) (10.88 g, 59.72 mmol) in DMF (120 mL) (DMF solution was purged with N2 for 10 min) was added K2C03 (32.05 g, 231.92 mmol) and 3-(chloromethyl)(l-isopropyl-lH-pyrazol-S-yl)pyridine hydrochloride (10) (15.78 g, 57.98 mmol). The mixture was heated at 65 °C for 1.5 h, cooled to rt, poured into ice water (800 mL). The itated solids were isolated by filtration, dried and concentrated to give desired product ( 17, 18 g).

Example 6: Synthesis of Compound (1) Y N \ \( N \ I I ,N / ,N / N\ 1 12M HCI N\ I o o —> o o H H 17 96% (I) OMOM OH To a solution of 2-((2-(1-isopropyl-1H-pyrazol—S—yl)pyridinyl)methoxy) (methoxymethoxy)benzaldehyde (17) (18 g, 47.19 mmol) in THF (135 mL, solution was purged with N2) was added conc. HCl (12N, 20 mL). The solution was d at rt for 3 h when HPLC showed the on complete. The mixture was added to a solution of NaHCO; (15 g) in water (1.2 L), and the resulting precipitate was collected by filtration, dried to give crude solid, which was further purified by column (DCM/EtOAc=60:40) to give pure product (15.3 g).

Example 7: Synthesis of Compound 1 (free base) and its HCI salt form Compound (1) free base (40g) was obtained from the coupling of the alcohol intermediate 7 and 2,6—dihydroxybenzaldedhye 9 under Mitsunobu conditions. A procedure is also provided below: DIAD. PPH3 THF. RT e 8: Synthesis of Compound (I) by Mitsunobu coupling Into a 2000-mL three neck round-bottom flask, which was purged and maintained with an inert atmosphere of nitrogen, was placed a solution of [2-[1-(propanyl)-1H- lyl]pyridinyl]methanol (7) (70 g, 322.18 mmol, 1.00 equiv) in tetrahydrofuran (1000 mL). 2,6-Dihydr0xybenzaldchyde (9) (49.2 g, 356.21 mmol, 1.10 equiv) and PPh3 (101 g, 385.07 mmol, 1.20 equiv) were added to the reaction mixture. This was followed by the addition of a solution of DIAD (78.1 g, 386.23 mmol, 1.20 equiv) in tetrahydrofiiran (200 ml) dropwise with stirring. The resulting solution was d overnight at room temperature. The resulting on was diluted with 500 ml of H20. The resulting solution was extracted with 3x500 m1 of diehloromethane and the combined organic layers were dried over sodium sulfate and concentrated under . The e was applied onto a silica gel column with EAzPE (1 :50-1 :3) as elucnt to yield the crude product. The crude t was re-erystallized from i-propanol/Hzo in the ratio of 1/1.5. This resulted in 40 g (37%) of 2-hydroxy([2-[1-(propan—2-yl)-lH-pyrazolyl]pyridin yl]methoxy)benzaldehyde as a light yellow solid. The compound exhibited a melting point of80-82 °C. MS (ES, m/z): 338.1 [M+l]. lH NMR (300 MHz, DMSO-d6) 6 l 1.72(s, 1H), .21(s, 1H), 8.76(d, J=3.6Hz, 1H), 8.24(d, J=2.7Hz, 1H),7.55(m, 3H), 6.55(m,3H) ,5.21 (s, 2H), 4.65 (m, 1H), 1.37 (d, z, 6H). 1H NMR (400 MHz, CDC13)5 11.96 (s, 1H), .40 (s, 1H), 8.77 (dd, J: 4.8, 1.5 Hz, 1H), 8.00 (d, J: 7.8 Hz, 1H), 7.63 (d, J= 1.8 Hz, 1H), 7.49 — 7.34 (m, 2H), 6.59 (d, ./= 8.5 Hz, 1H), 6.37 (d, J= 1.8 Hz, 1H), 6.29 (d, ./= 8.2 Hz, 1H), 5.10 (s, 2H), 4.67 (sep, J= 6.7 Hz, 1H), 1.50 (d, J: 6.6 Hz, 6H).

In another approach, multiple batches of Compound (1) free base are prepared in multi gram quantities (20g). The advantage of this route is the use of mono-protected 2,6- dihydroxybenzaldehyde (16), which effectively eliminates the possibility of bis-alkylation side product. The mono-MOM ether of hydroxybenzaldehyde (16) can be obtained from two starting points, bromoresorcinol (14) or rcsorcinol (1 1) [procedures described in the Journal of Organic Chemistry, 74(1 1), 431 1-4317; 2009 ]. All steps and procedures are provided below. Due to the presence of phenolic aldehyde group, precautions (i.e., carry out all reactions under inert gas such as nitrogen) should be taken to avoid oxidation of the phenol and/or aldehyde group.

Preparation of compound 1 HCI salt: A solution of compound 1 (55.79 g, 165.55 mmol) in acctonitrile (275 mL) was flushed with nitrogen for 10 min, then to this on was added 3N aqueous HCl (62 mL) at room temperature. The mixture was stirred for additional 10 min after the on, most of the itrile (about 200 mL) was then removed by evaporation on a rotary evaporator at around 32 0C, the remaining solution was frozen by g in an acetone-dry ice bath and lyophilized to afford compound I HCl salt (59.4 g).

PCT/U82015/014589 Example 9: Characterization of the HCI salt of Compound 1 XRPD HCl salt of Compound 1 __pale yellow solids, thin blades/tablets, birefringent H NMR 6 consistent with structure, <0.01 moles MEK XRPD - HCl salt ofCompound 1 0.03% gain upon equilibration at 5% RH 0.10% gain from 5 to 95% RH 0.09% loss from 95 to 5%RH post XRPD HCl 1 + Free Base Form I Example 10: Physical Stability of the HCl salt of Compound 1 Exposed to Water Time "3" times are Condition ation XRPD Result appr0x1mated) t' t . . water ng yellow solids convert Free base (FB) water slu‘ "y about 5 min to white solids upon isolation l (indexed) water slurry_about 6days :31)th thln blades, blrefrlngent FB I + FB 11 Example 11: Physical Stability of the HCl salt of Compound 1 with Grinding grinding, dry offwhite/palc yellow HCl 1 offwhite/palc yellow paste HCl 1 + FB 1 PCT/U82015/014589 Example 12: Physical Stability of the HCI salt of Compound 1 d to ed Temperature and/or Vacuum _Condition Time ation XRPDResult _RT vacuum 6 days pale yellow, blades/plates, B HCll+FBI s pale yellow, blades/tablets, B HCll °C 12 hrs pale yellow, blades/tablets, B HCll+FBI 24 hrs pale yellow, /tablets, B HC11+FBI O\ hrs pale yellow, blades/tablets, B HCll+FBI .— N hrs pale yellow, blades/tablets, B HC11+FBI NA hrs pale yellow, blades/tablets, B HC11+FB1 0\ hrs pale yellow, blades/tablets, B HCll+FBI .— N hrs pale , blades/tablets, B HCll+FBl N4:. hrs pale yellow, blades/tablets, B HCll+FBI 60 °C 6 days pale yellow blades, B HClI+FBI 60 °C pale YCHOW, bladCS, 3; HCI 1 + FB 1 + other free 0.m"<0 vacuum irregular residue base form I100 to 125 20 pH paper above sample indicate HCI I + FB I + other free 0 min acidic volatiles base form Example 13: Generation of the Free Base of Compound 1 from the Disproportionation of the HCI salt of Compound 1 in Water (The ng material is the HCI salt of Compound 1). 1. contacted with water 1. pale yellow, wets 2. sonicated poorly 2' white 3. filtered and rinsed with water FB I 4. dried under N; for l0 minutes ' . vacuum RT, overnight ' . contacted with water 1. — . sonicated for 5 minutes 2. pale yellow, turned . slurried for 10 s . filtered, rinsed with water FB 1 + other free base form . dried under N2 for 10 minutes . vacuum RT, overnight . stored in freezer 1. slurry in water, RT, 8 days; seeded with FB [1 2. filtered, rinsed with water 3. vacuum RT, overnight 2. sub sample ofslurry 2. - FB ll (indexed). 3. rinsed w1th water. . 3. - e 14: terization Form l of the Free Base of Compound 1 XRPD Free Base Form I XRPD —Free Base Form 1 3,0850 0.2% weight loss up to 100 °C 20850 endothermic event with onset near 97 °C 22.7 °C initial, fines, birefringent 91.2 °C increase in particle size and birefringence 94.2 °C increase in particle size and birefringence 95.7 °C melt onset, larger particles from l heating Hot Stage 96.1 °C melt continuation Microscopy 96.3 °C melt complete, no crystallization upon melting 68.7 °C fresh preparation, larger magnification 91.1 °C increase in birefringence 94.8 °C melt onset, larger particles, birefringent 95.4 °C melt continuation PCT/U82015/014589 95.9 °C only few crystals remain, cooled to 92.6 °C held for 2 to 3 minutes crystal growth to larger blades,- 92.6 C0 began heating. 0.02% loss upon equilibration at 5% RH 0.22% gain from 5 to 95% RH 0.22% loss from 95 to 5%RH post XRPD Free Base Form I + other Free Base Material Example 15: Characterization of Form 11 of the Free Base of Compound 1 Free Base Form 11 XRPD after 7 days Free Base Form 11 to 350 °C 0.1% weight loss up to 100 °c to 350 °C endothermic event with onset near 97 °C H NMR DMSO-d6 consistent with structure Example 16: terization of Material N of the Free Base of Compound 1 XRPD -Free Base Material N TGA 25 to 350 °C 0.2% weight loss up to 100 °C DSC 25 to 350 °C endothermic event with onset near 94 °C H NMR 6 consistent with structure, no residual reaction solvent observed PCT/U82015/014589 Example 17: Competitive Interconversion Slurries Between Free Base Forms 1 and II 6 °C, 6 days faint pale yellow RT 6 d' .rror. ’00 mar not to me .

RT, 6 days pale yellow FB N 57 °C, 2 days fines, off white, B FB 11 + FB 1 57 °C, ght blades and tablets, B FB 11 57 ' ~ . » , blades, laminated FB 1] , pale yellow B, Example 18: Competitive Interconversion Slurries between Free Base Form 11 and Material N °C, 3 days heptane pale yellow fines, B FB N 57 °C, 3 days larger blades, and rosettes of blades, B FB ll Example 19: Selected Experimental Methods Indexing: XRPD patterns are indexed by using proprietary SSCI re.

Agreement between the allowed peak positions, marked with red bars within the figures, and the observed peaks indicates a consistent unit cell determination. Indexing and structure refinement are computational studies which are performed under the "Procedures for SSC I Non-CGMP Activities." To confirm the ive indexing solution, the lar packing motifs within the crystallographic unit cells must be ined. No attempts at molecular packing were performed.

PCT/U82015/01-1589 Differential Scanning Calorimetry (DSC): DSC was med using a TA Instruments Q2000 differential scanning calorimeter. Temperature calibration was performed using NlST-traceable indium metal. The sample was placed into an aluminum DSC pan, covered with a lid, and the weight was accurately recorded. A weighed aluminum pan red as the sample pan was placed on the reference side of the cell.

The data acquisition parameters and pan configuration for each thermogram are displayed in the image in the Data section of this report. The method code on the gram is an abbreviation for the start and end temperature as well as the heating rate; e.g., 250-10 means "from —30 °C to 250 °C, at 10 °C/min". The following summarizes the abbreviations used in each image for pan configurations: Tzcro crimped pan (TOC); and Lid not crimped (NC).

Dynamic Vapor Sorption (DVS): Dynamic vapor sorption (DVS) data were collected on a VTI SGA-lOO Vapor Sorption er. NaCl and PVP were used as calibration standards. Samples were not dried prior to analysis. Adsorption and desorption data were collected over a range from 5 to 95% RH at 10% RH ents under a nitrogen purge. The brium criterion used for analysis was less than 0.0100% weight change in minutes with a maximum equilibration time of 3 hours. Data were not corrected for the initial moisture content of the samples.

Microscopy Hot Stage Microscopy: Hot stage microscopy was performed using a Linkam hot stage (FTIR 600) mounted on a Leica DM LP microscope equipped with a SPOT InsightTM color digital camera. Temperature calibrations were performed using USP melting point rds. Samples were placed on a cover glass, and a second cover glass was placed on top of the . As the stage was heated, each sample was visually observed using a 20x 0.40 NA. long working distance objective with crossed polarizers and a first order red compensator. Images were captured using SPOT software (v. 4.5.9).

Polarized Light copy: During the course of mentation generated samples were viewed utilizing a microscope with cross polarized light to observe morphology and birefringence. Samples were visually ed at 40x magnification. 2015/01-1589 lH Solution Nuclear Magnetic Resonance ('H NMR) SSCI: Samples were prepared for NMR spectroscopy as ~5-50 mg solutions in the appropriate deuterated solvent. The specific acquisition parameters are listed on the plot of the first full um of each sample in the data section for samples run at 88C].

Spectral Data Solutions: For samples run using Spectral Data Solutions (subcontractor), the solution 1H NMR spectra were acquired at ambient temperature on a Varian melNOVA-400 spectrometer ('H Larmor Frequency = 399.8 MHz). The specific acquisition parameters are listed on the spectral data sheet and on each data plot of the spectrum of the sample.

Thermogravimetric Analysis (TGA) TG analyses were performed using a TA Instruments 2950 thermogravimetric analyzer. Temperature calibration was performed using nickel and Alumel'm. Each sample was placed in an aluminum pan and inserted into the TG furnace. The e was heated under a en purge. The data acquisition parameters are displayed above each thermogram in the Data section of this report. The method code on the thermogram is an abbreviation for the start and end temperature as well as the g rate; e.g., 25-350—10 means "from 25 °C to 350 °C, at 10 °C/min". The use of 0 as the initial temperature tes sample run initiated from ambient.

XRPD Analysis INEL: XRPD patterns were collected with an Incl 00 diffractomctcr. An incident beam of Cu Ka radiation was produced using a fine-focus tube and a lically graded multilayer mirror. Prior to the analysis, a silicon standard (NIST SRM 640d) was analyzed to verify the Si 1 11 peak position. A en of the sample was packed into a thin-walled glass capillary, and a beam-stop was used to ze the background from air.

Diffraction patterns were collected in transmission geometry using Windif v. 6.6 software and a curved position-sensitive x or with a 20 range of 120°. The data- acquisition parameters for each pattern are displayed above the image in the Data section of this report.

PANa/ytical Transmission: XRPD patterns were collected with a PANalytieal X'Pert PRO MPD diffractometer using an incident beam of Cu radiation produced using an Optix long, fine-focus source. An elliptically graded multilayer mirror was used to focus Cu PCT/U82015/014589 Ka X-rays through the specimen and onto the detector. Prior to the analysis, a silicon specimen (NIST SRM 640d) was analyzed to verify the Si 1 l 1 peak position. A specimen of the sample was sandwiched between 3 pm thick films and analyzed in transmission geometry. A beam-stop, short antiscatter extension, and an antiscatter knife edge were used to minimize the background generated by air. Soller slits for the incident and diffracted beams were used to minimize broadening from axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) located 240 mm from the specimen and Data Collector software v. 2.2b. The data-acquisition parameters for each pattern are displayed above the image in the Data section of this report ing the ence slit (DS) before the mirror and the incident—beam antiscatter slit (SS).

PANa/ytical Reflection: XRPD patterns were collected with a tical X'Pert PRO MPD diffractometer using an incident beam of Cu Ka radiation produced using a long, fine-focus source and a nickel filter. The ctometer was configured using the symmetric Bragg-Brentano geometry. Prior to the analysis, a silicon specimen (NIST SRM 640d) was ed to verify the observed position of the Si 1 1 1 peak is consistent with the NIST—ccrtificd pesition. A en of the sample was prepared as a thin, circular layer centered on a silicon ackground substrate. Antiscatter slits (SS) were used to minimize the background generated by air. Soller slits for the incident and diffracted beams were used to minimize broadening from axial divergence. Diffraction ns were ted using a scanning position-sensitive detector (X'Celerator) located 240 mm from the sample and Data Collector software v. 2.2b. The data acquisition ters for each n are displayed above the image in the Data section of this report including the divergence slit (DS) and the incident—beam SS.

Approximate Solubility: A weighed sample was treated with aliquots of the test solvent at room temperature. The mixture was sonicated between additions to facilitate dissolution. te dissolution of the test al was determined by visual inspection.

Solubility was estimated based on the total solvent used to provide complete dissolution.

Some samples were then heated and observed visually for te dissolution. The actual solubility may be greater than the value calculated because of the use of solvent aliquots that were too large or due to a slow rate of dissolution. The solubility is sed as "less than" if dissolution did not occur during the experiment. If complete dissolution was achieved as a result of only one aliquot addition, the solubility is expressed as "greater than". 2015/01-1589 olvent Additions: Compound l/organic t ons were ted with solvents that Compound 1 was determined to be poorly soluble or insoluble in. These anti solvent additions were added to help lower the solubility of the solvent system and induce crystallization. g and Slow Cools: Solutions were prepared in the selected solvent or solvent/anti-solvcnt system. These solutions were chilled below room temperature within a refrigerator for varying lengths of time in an attempt to induce nucleation. The presence or absence of solids was noted. Upon observation of solids, in quantities sufficient for analysis, isolation of material was conduction. If cient quantities were present further cooling was performed in a freezer. Samples were either isolated for analysis wet or as dry powders.

Compression: Selected samples were compressed utilizing a KBr die and a Carver hydraulic press. An applied load of 10000 lbs was applied to the die shaft for approximately 20 minutes.

Crystallizationfi'om Solution: ted solutions were generated at ambient and then capped. Nucleation was observed to occur from these systems during evaluation of the Free Base of Compound 1.

Fast Evaporation: ons were prepared in selected solvents and agitated between aliquot additions to assist in ution. Once a mixture reached complete dissolution, as judged by visual observation, the solution was allowed to evaporate at t temperature in an ed vial or at ambient under nitrogen. The solids that formed were isolated for evaluation.

Milling: Selected material was milled utilizing a Reitch Mill. The material was loaded into an agate lined milling vessel followed by the addition of an agate ball. The vessel was then placed on to the mill and milled for approximately 30 minutes at frequency of 1/30 seconds. The milling was stopped approximately every 10 minutes and material scraped from the wall before further milling.

Slurry: Solutions were prepared by adding enough solids to a given solvent so that excess solids were present. The mixture was then agitated in a sealed vial at either ambient or an elevated temperature. After a given amount of time, the solids were isolated for analysis.

PCT/U82015/014589 Temperature and Relative Humidity Stress: Selected materials were stressed at elevated related humidity and/or temperature. Relative humidity jars (saturated salt solutions used to generate desired ve humidity) were utilized to store ed samples.

The following relative humidity jars were utilized during evaluation: 75% RH (NaCl) and 60% (NaBr), to investigate the effects of humidity. Temperatures utilized were ambient, 30, 40, 60, and 100-125 °C.

Vacuum: Selected materials were stressed under reduced pressure for a set time period. Initial stressing was conducted with the in-house vacuum system with te pressure readings <500 mTorr, typically 30 to 50 mTorr (0.030 to 0.05 mm Hg). Additional vacuum ing was conducted at 48 mmHg utilizing a portable lab vacuum and bleed to simulate conditions similar to those expected during process.

Example 20: Disproportionation of the HCI salt The disproportionation of the HCI salt in water was utilized to generate free base.

The nucleation of Free Base Form I occurs first. Extending the slurry time induces the transformation to a more thermodynamically stable phase relative to Form 1, Free Base Form II.

Three anhydrous materials of the free base were fied; Free Base Forms I, II, and Material N. Free Base Material N appears to be most stable form, relative to Forms I and II, at room temperature. Free Base Material N is otropic relative to Form II, and will transform ibly at a specific transition temperature ated near 42 °C). Above the transition temperature, Free Base Form II appears to be the most stable form, relative to Form I and Material N.

The HCI salt (termed "HCI Form I") was subjected to various stress conditions and monitored by XRPD to evaluate physical stability. As discussed, disproportionation occurred during the DVS ment of the HCI salt, indicating ility upon exposure to elevated humidity. Disproportionation is further evident with wet milling or in direct contact with water (e.g. slurry) as shown by the presence of Free Base Forms I or 11, identified by XRPD. The volatilization and loss of HCI upon heating and/or vacuum is shown by the presence of Free Base Form 1, identified by XRPD, and also indicates instability at these ions. 0 t with water resulted in a visual color change of the material from pale yellow to white; physical changes were also observed microscopically. Immediate PCT/U82015/014589 disproportionation occurs. XRPD is identified the resulting material from a water slurry (~5 minutes) as Free Base Form 1. Free Base Form 11 also becomes evident if the amount of time in the slurry is extended.

The volatilization of HCl was evident within hours of exposure to drying conditions. sion to Free Base Form 1 was observed by XRPD at 30 °C (after 12 hrs), 40 °C (after 6 hrs), and at 40 °C/48 mmHg (after 6 hrs).

Free Base Material C becomes evident at more extreme conditions involving elevated temperatures. Heating HCl Form 1 up to 125 °C induces the loss of acidic volatiles (judged visually by use of pH paper held above sample). XRPD analysis identifies the resulting sample as a mixture of HCl Form 1, Free Base Form 1, and Free Base Material C. Exposing the HCl salt to 60 °C under vacuum for 6 days provides the same result. The nature of Material C is not established The HCl salt was shown to disproportionate immediately in water. This phenomenon was utilized to generate free base. The nucleation of Free Base Form 1 occurs first. Extending the slurry time induces the transformation to a more thermodynamically stable phase relative to Form 1, Free Base Form 11.

A 20 ml vial was Charged with 266.4 mg of HCl Form I and contacted with 10 ml of water. The sample was sonicated until the pale yellow material changed color to white. The resulting solids were collected by filtration (water aspirated) and rinsed with 10 ml of water. A nitrogen purge was blown over the sample for imately 10 minutes prior to exposure to vacuum at t temperature to dry overnight. The resulting material was analyzed by XRPD and determined to be Free Base Form 1.

A 250 ml Erlenmeyer flask was charged with 6.0250 grams of HCl Form I and contacted with 220 mL of water. The sample was sonicated for approximately 5 minutes to disperse the material. The yellow al changed color to white during tion. A stir bar was added and the sample was stirred at 700 RPM for approximately 10 minutes. The solids were ted by filtration and rinsed with 220 ml of water ed by a nitrogen purge over the sample for approximately 10 minutes prior to exposure to vacuum at ambient ature. The sample was dried at this condition for approximately 24 hours yielding 5.1834 grams of material. The resulting material was analyzed by XRPD and determined to be a mixture of Free WO 20133 PCT/U82015/014589 Base Form 1 and Free Base Material D. (The nature of Material D is not established.) The procedure used to generate Free Base Form 11 is described below. 0 A 20 ml vial was charged with 477.5 mg of HCI Form I lot 20 and contacted with 20 ml of water. The sample was sonicated until the pale yellow material changed color to white. A small amount of sample (mixture of Free Base Forms 1 and 11) was added as seeds. A stir bar was added and the sample was stirred at 200 RPM for 8 days. The resulting solids were collected by filtration (water aspirated) and rinsed with 15 ml of water. The sample was exposed to vacuum at ambient ature to dry overnight. The resulting material was analyzed by XRPD and ined to be Free Base Form 11.

Example 21: Additional Procedures for the Preparation of the free base of Form 1, Form 11, and From N Conversion ofthefi'ee base ofCompound 1 to the HCI salt General Procedure: Slowly treat a solution of the free base of Compound 1 in MEK (5 vol) with cone HCI (1.5 eq). Cool the ing slurry to 0—5 °C for 1 h and filter.

Wash solids with MEK (1 vol). Dry under vacuum at 30—35 °C.

Preparation A: Following the l procedure above, 35 g of crude Compound 1 was processed to provide the HCI salt as a pale yellow solid (32.4 g, 82% yield, 99.8% purity by HPLC).

Preparation oft/zefi'ee base Form I.from the HCI Salt ofCompound 1 General Procedure: Vigorously stir a slurry of the HCI salt of nd 1 in DIW (10 vol) for 5 min to 2 h. Filter the slurry, wash with D1W(2><1 vol), dry on funnel, then further dry under vacuum at 30—35 °C.

Preparation A: Following the general procedure above, after stirring for 1h, 32 g of the HCI salt of Compound 1 was processed to provide the free base as a pale yellow solid (27.3 g, 95% yield, 99.8% purity by HPLC; DSC indicates Form 1).

Preparation B: ing the general procedure above, after stirring for 1h, 39 g of the HCI salt of Compound 1 was processed to provide the free base as a pale yellow solid (31.8 g, 90% yield, >99.9% purity by HPLC)).

PCT/U82015/01-1589 Preparation C: Thus, the HCl salt of Compound 1 (134 g) was vigorously stirred in water (10 vol) until the material appeared as a finely dispersed white slurry. After ion and drying, a white crystalline solid (1 16 g, 96% recovery, >99.9% purity by HPLC) was isolated.

Preparation D: The purpose of this experiment was to e the free base from Compound 1, HCl. Thus, the HCl salt of Compound 1 (65.3 g) was usly stirred in water (10 vol) until the material appeared as a finely dispersed white slurry. Afier filtration and , a white crystalline solid (57.5 g, 97.6% ry, >99.9% purity by HPLC) was isolated.

Preparation ofGBT000440fi'ee base Form "from GBT000440free base Form 1 General Procedure: Stir a slurry of the free base of nd 1 Form I in an appreciate solvent (e.g. heptane or water) (10 vol) for 1-7 days. Filter the slurry, wash with DIW (2X1 vol), dry on funnel, then further dry under vacuum at 30—35 °C.

Preparation A: Thus, the free base of Compound 1, Form 1 (114 g) was stirred in n-heptane (10 vol) at 35 °C. Afier 4 days, XRPD indicated the material was Form 11. The slurry was filtered and dried to provide 1 10 g off white solid.

Preparation 8: the free base of Compound 1 (5 g) was slurricd in hcptancs (10 vol 50mlL) at room temperature. After 4 days, the slurry was filtered to provide an off-white solid. ation C: the free base of Compound 1 (5.8 kg) was slurricd in hcptancs (10 vol) at room temperature. After 2 days, the slurry was filtered and washed with 2x2 vol n-hcptanc to provide 4.745 kg of Form [1 as an off—white solid.

Preparation D: the free base of Compound 1 (5 g) was slurricd in water. After 4 days, the slurry was filtered to provide an off-white solid.Preparation QI'GBT000440fi‘ee base Form Nfrom GBT000440free base Form 1 or Form 11 General Procedure: Stir a slurry of the free base of nd 1, Form 1 in MTBE (4 vol) at room temperature for at least 4 days. After 4 days, filter the slurry to provide an off-white solid. Obtain XRPD to confirm polymorph as Material N.

Preparation A: Following the general procedure above, 27 g of the free base of nd 1, Form I (48TRSO79) was stirred in MTBE at 18—23 °C for 4 days. DSC PCT/U82015/01-1589 ted it should be Material N. Isolated 22.2 g cream colored solid (82% recovery, 99.9 purity by HPLC). XRPD analysis planned.

Preparation B: Following the general procedure above, 31 g of the free base of nd 1, Form l was d in 3 vol MTBE at 18—23 °C for 4 days.

Preparation C: the free base of Compound 1, Form I (13KWBO23, 1 g) was ed in MTBE (5 vol) at room temperature. Slurry was seeded with Material N (50 mg).

After 4 days, the slurry was filtered to provide a off-white solid. DSC indicated the melting point was the same as Material N.

Preparation D: The purpose of this experiment was to convert the the free base of Compound 1, Form 11 to Material N. Thus, the free base of Compound I (0.5 g) was stirred in 5 vol of di-n-propyl ether at 18—23 °C. After 2 days, DSC corresponded to the pattern observed for al N. XRPD analysis confirmed Material N had been formed.

Preparation E: To the free base of Compound 1, Form 11 (5 g) was added diisopropyl ether (5 vol, 25 mL) at room temperature. Afier 4 days, the slurry was filtered to provide a off-white solid. DSC indicates Material N.

Example 22: inary Solvent-based Screens Rapid, t-based screens were conducted in an attempt to determine the most stable form of the free base of Compound 1. The study also provides a preliminary assessment of the propensity of these als to exist in s l forms. Generated solids were observed by polarized light microscopy (PLM) and/or analyzed by X-ray powder diffraction (XRPD), comparing the resulting XRPD patterns to known patterns of Compound 1.

If possible, XRPD patterns were indexed. Indexing is the process of determining the size and shape of the crystallographic unit cell given the peak positions in a ction pattern. The term gets its name from the assignment of Miller index labels to individual peaks. XRPD indexing scrvcs several purposes. If all of the peaks in a pattern are indexed by a single unit cell, this is strong evidence that the sample contains a single crystalline phase. Given the indexing solution, the unit cell volume may be calculated directly and can be useful to determine their solvation states. Indexing is also a robust description of a crystalline form and provides a concise summary of all available peak positions for that phase at a particular thermodynamic state point.

PCT/U82015/01-1589 Materials ting unique lline XRPD patterns, based on visual inspection of peaks ated with these materials, were given letter ations. The letter designation is tentatively associated with the word 'Material' if insufficient characterization data is available. The nomenclature is used only to aid in the identification of unique XRPD patterns and does not imply that the stoichiometry, crystalline phase , or chemical purity of the material is known. Materials are further ated as forms with Roman numeral designations (i.e., Free Base Material A = Free Base Form 1), when phase purity ned through indexing of the XRPD pattern or single crystal structure elucidation) and chemical identity/purity (obtained through proton NMR spectroscopy) of the material is determined.

Three anhydrous materials were identified: Forms 1, II, and Material N. Material N appears to be most stable form, relative to Forms 1 and II, at room temperature. Material N is enantiotropic relative to Form 11, and will transform reversibly at a specific transition temperature (estimated near 42 °C). Above the transition temperature, Form 1] appears to be the most stable form, relative to Form 1 and Material N.

Materials C and D are used to fy a few additional, low intensity peaks observed in XRPD patterns which were predominantly composed of the Free Base Form 1 of Compound 1 or mixtures of the HCl Form 1 and Free Base Form I of nd 1.

Example 23: Anhydrous Ansolvate Forms Farm I Free Base Form I is a metastable, anhydrous phase of the free base that is formed immediately from the portionation of the HCl salt in water. A representative XRPD pattern of Form I was successfully indexed and the unit cell volume is tent with anhydrous free base. Visual comparison of the XRPD pattern to the historical pattern of the free base provided indicates the material may be similar; however, the historical pattern appears to exhibit additional peaks from a potential mixture.

The lH NMR spectrum is consistent with the chemical structure of Compound 1.

The chemical shift at approximately 2.5 ppm is ed to DMSO (due to residual protons in the NMR solvent). Peaks that could be associated with residual ts were not visible, consistent with the anhydrous unit cell volume determined from the indexing solution above and the negligible wt% loss observed by TGA discussed below.

PCT/U82015/01-1589 Thermograms (TG) data shows negligible weight loss, 0.2%, up to 100 °C, consistent with an anhydrous form. The DSC exhibits a single endotherm with an onset near 97 °C (similar to what is observed for Form II). The endotherm is consistent with a melt by hot stage microscopy. However, s in particle size and birefringence were evident prior to the melt; a possible phase change occurred. Consequently, if a phase change occurred and an endotherm similar to that of Free Base Form 11 was observed, it can be inferred that the observed melt is truly not of Form 1 but of the resulting phase, most likely Form 11.

The DVS isotherm indicates Form 1 is not hygroscopic. Negligible weight gain and loss, 0.2%, was ed through sorption/desorption. By XRPD, the material recovered from the DVS experiment was predominately Free Base Form 1 with a few onal peaks. The additional peaks were termed Free Base Material D. The nature of Material D is unknown; however, the ance of another s) indicates that Form 1 is not likely physically stable at ed humidity conditions (at ambient temperature).

Form [1 Free Base Form [1 is an anhydrous phase of the free base. Form 11 is enantiotropically related to al N, where it is the thermodynamically stable form above an estimated transition temperature of 42 °C. Form II can be generated in solvents that do not form known solvates; such as heptane, IPE, MTBE, or e; through short-term slurry conversions of Form 1 (where the crystallization kinetics delay the nucleation of the more stable form) or elevated temperature slurries (above 42 °C). A representative XRPD pattern of Form 11 was successfully indexed and the unit cell volume is consistent with ous free base of Compound 1.

The lH NMR um is consistent with the chemical structure of Compound 1.

The chemical shift at approximately 2.5 ppm is assigned to DMSO (due to residual protons in the NMR solvent). Peaks that could be associated with residual solvents were not visible, consistent with the anhydrous unit cell volume determined from the indexing solution above and the negligible wt% loss observed by TGA discussed below.

Thermograms (TG) data show negligible weight loss, 0.1%, up to 100 °C, consistent with an anhydrous form. The DSC exhibits a single endotherm (80.1 J/g) with an onset near 97 °C.

PCT/U82015/01-1589 Form 11 remained unchanged aflcr 7 days at ambient storage, through reanalysis by XRPD. The form is known to be thermodynamically metastable, relative to Material N, at this condition; however, the cs of polymorph conversion may be slow at ambient conditions in the solid state.

Material N Free Base Material N is an anhydrous phase of the free base. al N is enantiotropically related to Form 11, where it is the thermodynamically stable form below an ted transition temperature of 42 °C. Given the opportunity, Material N can be ted through slurries in solvents that do not form known solvates; such as heptane, IPE, MTBE, or toluene; at temperatures below 42 oC. The following is an example of a laboratory scale ure used to generate Free Base Material N. o 53.0 mg of Free Base Form 1 was contacted with 2 ml of an IPE/free base solution (concentration 13 mg/ml). A stir bar was added and the sample was slurried for 7 days at ambient. The solution was decanted from the sample and the remaining solids briefly dried under nitrogen. Characterization Data tes Material N is a unique crystalline phase.

The 'H NMR spectrum is consistent with the chemical structure of Compound 1.

The chemical shift at approximately 2.5 ppm is assigned to DMSO (due to residual protons in the NMR solvent). Peaks that could be associated with residual solvents were not visible, consistent with the negligible wt% loss observed by TGA sed below.

Thermograms (TG) data show negligible weight loss, 0.2%, up to 100 °C, consistent with an anhydrous form. The DSC exhibits a single errn (82.8 J/g) with an onset at 94 °C.

Tentative Determination of the Thermodynamic Relationship between Free Base Forms I, II, and Material N Characterization data indicates that Forms 1, 11, and Material N are unique crystalline phases; however, only the XRPD ns of Forms 1 and l] were successfully indexed to confirm phase . Therefore, any proposed thermodynamic relationship between these materials is a working hypothesis, where the phase purity of Material N is assumed.

PCT/U82015/01-1589 Phase transitions of solids can be thermodynamically reversible or irreversible.

Crystalline forms which transform ibly at a specific transition temperature (7",) are called enantiotropic rphs. If the crystalline forms are not interconvertable under these conditions, the system is monotropic (one thermodynamically stable form). Several rules have been developed to t the ve thermodynamic stability of polymorphs and whether the relationship n the polymorphs is enantiotropic or monotropic. The heat of fusion rule is applied within this study. The heat of fusion rule states that if the higher melting form has the lower heat of fusion then the two forms are enantiotropic, otherwise they are monotropic. al N appears to be most stable form, relative to Forms 1 and II, at room temperature. Based on the heats of fusion and melts determined by DSC, Material N is enantiotropic relative to Form 11, and will transform reversibly at a specific transition temperature (TW’). Due to a possible phase change of Form 1 to Form 11 that occurred prior to the observed endotherm in the DSC, the onship of Form 1 with either Material N or Form 11 cannot be conclusively determined through the heat of fusion rule. However, through various interconversion es, it was shown that Form 1 is the least thermodynamically stable form between 6 °C and TV'". In addition, assuming that Form 1 spontaneously converted to Form 11 in the DSC at elevated temperatures (prior to the observed melt), it must follow that Form 11 is also more stable than Form 1 above TV'".

Example 24: Estimated Transition Temperature The estimated transition temperature between two enantiotropically related forms can be calculated from their melt onsets and heats of fusion based on the equation shown below.

PCT/U82015/014589 AHm 'AHm +(Cpiiq 'Cp.l)'(Tf.l -Tr.2) p Mfr; AH" ...i ,__i-i__+ C( . _Cp‘l) nln ,_‘__ Tu Tu p‘l'q [Th2] Where.

(Cinq -Cp_l) = k' AH" andk=0.005 Between Material N and Form II, the equation estimates a transition temperature of 42 °C. To summarize, the relative stability of the forms from most to least stable is shown below.

Temperature Relative Comments Ran e9: Stabilit _Below6 °C Relationships to Form I are not established below this tem (11 > N) and (11 Relationship between Form 1 and Material N is not Above TN." > 1) ished above this tem 0 *TN'" is estimated to be near 42 °C Example 25: Energy — Temperature Diagram The Energy — Temperature Diagram of is a semi-quantitative graphical solution of the Gibbs — Helmholtz equation, where the enthalpy (H) and free energy (G) isobars for each form are depicted as a function of temperature.

Example 26: Competitive lnterconversion Slurry Experiments lnterconversion ments were performed to support the thermodynamic relationship between polymorphs illustrated by the Energy — Temperature Diagram above. lnterconversion or itive slurry experiments are a solution-mediated process that provides a y for the less soluble (more stable) l to grow at the expense of the more soluble crystal form. Outside the ion of a e or degradation, the resulting more stable polymorph from an interconversion ment is contemplated to be independent of the solvent used because the more thermodynamically stable polymorph has a lower energy and therefore lower solubility. The choice of solvent affects the kinetics of polymorph conversion and not the thermodynamic relationship n polymorphic forms.

The results of the interconversion studies are consistent with the tentative Energy — Temperature Diagram shown herein. Binary es were prepared at ambient, 6, and 57 °C using Forms I and 11. Form [1 resulted from the majority of these experiments, confirming that Form 11 is more stable relative to Form I within this temperature range.

A few of the experiments conducted at ambient and 6 °C resulted in Material N.

Although this does not provide specific clarification n Forms I and I], it does provide evidence that Material N is the most stable form relative to both Forms I and II at these atures (which were conducted below the estimated transition temperature of 42 °C).

Additional interconversion slurries between Form 1] and Material N were conducted at temperatures which bracket this estimated transition temperature and confirm that Form 11 and Material N are enantiotropically related.

Example 27: Solid-state r Magnetic Resonance 13C and lsN spectra acquired for the three rphic forms I, II and Material N.

See FIGS. 10 and l l. Spectra were ed at 253K to prevent any low temperature transitions occurring during measurement and acquisition parameters optimised for each polymorphic form.

Based on solid-state nuclear magnetic nce, all three forms are crystalline and are distinct polymorphic forms. Form I contains one molecule per asymmetric unit, Form 11 contains two molecules per asymmetric unit and Form N contains four molecules per asymmetric unit. See the [SN spectra in 1.

Example 28: Chemical and Physical Stability Evaluation of the Free Base Form I of Compound 1 A mixture predominately composed of Free Base Form I (with Free Base Material D) were d to stability conditions to assess al and chemical stability. Three conditions were used; open to 25 °C / 60% RH, open to 40 °C / 75% RH, and closed to 60 °C. Physical stability was evaluated by XRPD. Chemical ity was determined through UPLC and 'H NMR, when applicable. Materials were tested after I, 7, and 14 days of exposure.

Chemical Stability ofFree Base Form 1 For the free base stability , UPLC showed very low levels of impurities present. The level of impurities did not rise significantly afier 14 days of age. This would 2015/014589 seem to indicate good chemical stability against the conditions used for stability assessment.

The 1H NMR spectra of samples exposed to 60 °C (14 days) were also consistent with this conclusion.

Physical Stability ofFree Base Farm I The free base of nd I remained unchanged, by XRPD, at 25 °C / 60% RH.

However, differences were observed at the other two conditions. The few, minor peaks attributed to Free Base Material D were no longer observed, indicating that Material D is metastable and is not sustained at elevated temperatures. In addition, Free Base Form 11 was observed after 7 days of age. This is consistent with the conclusions discussed herein, where Free Base Form II is more stable relative to Free Base Form 1 at these temperatures.

Example 29: Physical Stability tion of the Free Base Form 1] and Material N (Form N) of Compound 1 DSC was modulated at low underlying heating rate, followed by X-ray powder diffraction. A low ying g rate was used of of 0.02 °C min". The temperature was 80 °C for form N and 90 °C for form 1]. Exposure was essentially isothermal, covering a temperature range with sensitivity to detect changes in physical form. The resultant materials were examined by X-ray powder diffraction. No changes in physical form were observed for either polymorphic form I] or polymorphic form N (i.e., material N).

Forms 11 and N were exposed to 40 °C/75% relative ty (RH), 80 °C, 80 °C /80%RH for 9 days followed by X-ray powder diffraction. No changes in al form were observed for either polymorphic form 11 or polymorphic form N.

The thermodynamic barrier for inter-conversion between polymorphic form I] and form N is high, and physical stability is good for both forms. Thermally induced inter- conversion between form 11 and form N is unlikely to occur.

Example 30: The Relative Thermodynamic Stability of rphic Forms ll and N.

Extended t mediated maturation studies were conducted with 1:] w/w mixtures of polymorphic form 11 and form N. Hexanc provided a good medium for solvent assessments. The temperatures used include -20 °C, -10 °C, 0 °C, 10 °C, 20 °C, 30 °C, 40 °C and 50 °C. Significantly increased lity was observed at 30 °C, 40 °C and 50 °C.

Solids derived from maturation at -20 °C, -10 °C, 0 °C, 10 °C, 20 °C were analyzed by X- ray powder ction. In each case, significant conversion to Form N was observed.

PCT/U82015/01-1589 Form N is thermodynamically more stable than form 11 at temperatures of 20 °C and lower. An enantiotropic relationship between the two forms is likely to exhibit equivalence in thermodynamic stability at ca. 30-40 °C .

Example 31: Morphology of Form N Initial ation of a batch of polymorphic form N indicates an acicular morphology.

While this invention has been bed in conjunction with specific embodiments and examples, it will be apparent to a person of ry skill in the art, having regard to that skill and this disclosure, that equivalents of the specifically disclosed materials and methods will also be applicable to this invention; and such lents are intended to be included within the following claims.

Claims (5)

What we claim is:

1. A crystalline form of Compound 1: Compound 1 characterized by an X-ray powder diffraction pattern (Cu Kα radiation) having X-ray powder diffraction peaks at 13.37°, 14.37°, 19.95°, and 23.92°2θ, each ±0.2 °2θ.

2. 2 The crystalline form of claim 1, characterized by an endothermic peak at 97 ±2 °C as measured by differential scanning calorimetry.

3. A pharmaceutical composition sing a pharmaceutically acceptable excipient and the crystalline form of any one of claims 1-2.

4. The pharmaceutical composition of claim 3, n the ceutical composition comprises less than 5 mole % of crystalline Form I, wherein: Form I is characterized by an X-ray powder ction pattern (Cu Kα radiation) having X-ray powder diffraction peaks at 12.82°, 15.74°, 16.03°, 16.63°, 17.60°, 25.14°, 25.82°, and 26.44°2θ, each ±0.2 °2θ.

5. The pharmaceutical composition of claim 3, wherein the ceutical composition ses less than 5 mole % of crystalline Form I, less than 5 mole % crystalline Material N and less than 5 mole % of amorphous forms of Compound 1, wherein: Form I is characterized by an X-ray powder diffraction pattern (Cu Kα radiation) having X-ray powder ction peaks at 12.82°, 15.74°, 16.03°, 16.63°, , 25.14°, 25.82°, and 26.44°2θ, each ±0.2 °2θ; and Material N is characterized by an X-ray powder diffraction pattern (Cu Kα radiation) having X-ray powder diffraction peaks at 11.65°, 11.85°, 12.08°, 16.70°, 19.65°, and 23.48°2θ, each ±0.2 °2θ.