EA040317B1 - CRYSTALLINE POLYMORPHIC FORMS OF THE FREE BASE OF 2-HYDROXY-6-((2-(1-ISOPROPYL-1H-PYRAZOL-5-YL)PYRIDIN-3-YL)METHOXY)BENZALDEHYDE - Google Patents

Description

State of the art

2-Hydroxy-6-((2-( 1 -isopropyl-1 H-pyrazol-5-yl)pyridin-3 -yl)methoxy)benzaldehyde is a compound having the formula

Sickle cell anemia is a red blood cell disorder that occurs particularly in people of African and Mediterranean descent. Sickle cell anemia is based on sickle cell hemoglobin (HbS), which contains a point mutation relative to the predominant hemoglobin (Hb) peptide sequence.

Hemoglobin (Hb) carries oxygen molecules 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 by valine, allowing HbS to become susceptible to polymerization to give HbS-containing red blood cells their characteristic crescent shape. Sickle cells are also more rigid than normal red blood cells, and their lack of plasticity can lead to clogged blood vessels. There is a need for therapeutic agents such as 2-hydroxy-6-((2-(1isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde which can treat Hb mediated disorders or abnormal Hb, so so HbS.

When used to treat humans, it is important that the crystalline form of the therapeutic agent, such as 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde, or its salt, retained polymorphic and chemical stability, solubility and other physicochemical properties over time and among various industrial batches of the agent. If physicochemical properties change over time and between batches, administration of a therapeutically effective dose becomes problematic and may result in toxic side effects or therapy failure, especially if the polymorph degrades to a less active, inactive, or toxic compound prior to use. Thus, it is important to choose a form of crystalline agent that is stable, industrially reproducible, and has physicochemical properties favorable for use as a therapeutic agent.

However, the current state of the art still does not predict which crystalline form of an agent will have a combination of desired properties and be suitable for human administration, and how to obtain an agent in such a crystalline form.

Brief description of the invention

Desolvates.

This invention is partly the result of the discovery that the HCl salt of compound 1 disproportionates or loses HCl and disproportionation of the HCl salt of compound 1 in water leads to the formation of a free base and the disproportionation reaction was facilitated by exposure to high humidity during wet grinding and in direct contact with water (for example, in suspension). The sulfate salt of Compound 1 also disproportionates from some solvents such as dimethyl sulfoxide and methanol upon precipitation with water. Evaporation of HCl was observed within a few hours when kept under 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 has a more stable chemical entity compared to the corresponding chloride or sulfate and the like other salt.

It was found that 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde (or compound 1), i. the free base of compound 1 may be obtained as one or more desolvated crystalline forms, some of which are referred to herein as crystalline form I, form II, and substance N. In preferred embodiments of the invention, the free base of compound 1 is a crystalline desolvate, such as crystalline anhydrous form. The free base of Compound 1 can be prepared from the corresponding salt, such as the HCl salt of Compound 1.

Three anhydrous crystalline forms of the free base have been identified, named Forms I, II, and Substance N of the free base. It was found that the formation of form I of the free base in the beginning, mainly comes from suspension. Increasing the residence time in suspension can cause the free base Form I to transform into Free Base Form II, a thermodynamically more stable phase relative to Form I. In addition, it has been found that the free base of Substance N may be more stable to Forms I and II. at room temperature.

The free base of substance N has been found to be enantiotropically more closely related to form II and will reversibly convert at a certain transition temperature (estimated here at about 40-42°C). At a temperature above the temperature transition, the free base form II exhibits the properties of the most stable form in relation to form I and substance N. Thus, at operating temperatures below 40°C, for example at 30°C, the free base of compound 1 exists mainly in the form of substance N, which may have some residual form II. Thus, at operating temperatures above 40°C, for example at 50°C, the free base of compound 1 exists predominantly as Form II, which may have some residual amount of substance N. At 40°C, there is little conversion between substance N and form II. It is envisaged that this process can occur for suspensions of the free base in some solvents and in the solid state. In one embodiment of the invention, one or more crystalline forms of the free base of compound 1 do not undergo polymorph transformation under conditions suitable for the manufacture and storage of crystalline forms.

Form I

In one embodiment of the invention, the crystalline free base of compound 1 comprises crystalline form I, which is characterized by an endothermic peak at 97±2°C, as measured by differential scanning calorimetry. In another embodiment of the invention, crystalline form I of the free base of crystalline compound 1 is characterized by a significant absence of thermal events at temperatures below the endothermic peak at 97±2°C, as measured by differential scanning calorimetry. In another embodiment, crystalline form I of the free base of crystalline compound 1 is characterized by a powder x-ray diffraction (Cu Ka radiation) peak at one or more of 12.82, 15.74, 16.03, 16.63, 17.60, 25, 14, 25.82 and 26.44±0.2° 2θ. In another embodiment of the invention, crystalline form I of the free base of crystalline compound 1 exhibits an X-ray powder diffraction (Cu Ka radiation) substantially similar to that shown in FIG. 3.

In another embodiment, crystalline form I of the free base of crystalline compound 1 is characterized by at least one powder x-ray diffraction (Cu Ka radiation) peak selected from 12.82, 15.74, 16.03, 16.63, 17.60, 25.14, 25.82 and 26.44° (each ±0.2° 2θ). In another embodiment, crystalline form I of the free base of crystalline compound 1 is characterized by at least two powder x-ray diffraction (Cu Ka radiation) peaks selected from 12.82, 15.74, 16.03, 16.63, 17.60, 25.14, 25.82 and 26.44° (each ±0.2° 2θ). In another embodiment of the invention, crystalline form I of the free base of crystalline compound 1 is characterized by at least three powder x-ray diffraction (Cu Ka radiation) peaks selected from 12.82, 15.74, 16.03, 16.63, 17.60, 25.14, 25.82 and 26.44° (each ±0.2° 2θ).

In another embodiment of the invention, form I is characterized by the presence of 1, 2, 3, 4 or more peaks shown in the table below.

Observed peaks for Form I, X-ray powder diffraction (XRPD) file 609973.

°2Θ d distance (A) Intensity (%) 5.52±0.20 16.021±0.602 68 12.82±0.20 6.906±0.109 74 15.03±0.20 5.897±0.079 38 15.74±0.20 5.629±0.072 46 16.03±0.20 5.530 ±0.069 46 16.63±0.20 5.331±0.064 61 17.60±0.20 5.040±0.057 100 18.74±0.20 4.736±0.051 24 19.07±0.20 4.654±0.049 17 19.35±0.20 4.587±0.047 23 20.32±0.20 4.370±0.043 18 21.64±0.20 4.106±0.038 23 22.80±0.20 3.901±0.034 26 23.28±0.20 3.821±0.033 34 25.14±0.20 3.543 ± 0.028 52 25.82±0.20 3.451±0.026 81 26.44±0.20 3.371±0.025 51 27.91±0.20 3.197±0.023 17 28.19±0.20 3.165±0.022 26

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Form II.

In another embodiment of the invention, the crystalline free base of compound 1 comprises crystalline form II, which is characterized by an endothermic peak at 97±2°C, as measured by differential scanning calorimetry. In another embodiment of the invention, crystalline form II of the free base of crystalline compound 1 is characterized by a significant absence of thermal events at temperatures below the endothermic peak at 97±2°C, as measured by differential scanning calorimetry. In another embodiment, crystalline form II of the free base of crystalline compound 1 is characterized by a powder x-ray diffraction (Cu Ka radiation) peak at one or more of 13.37, 14.37, 19.95, or 23.92° 2θ. In another embodiment of the invention, crystalline form II of the free base of crystalline compound 1 exhibits powder X-ray diffraction (Cu Ka radiation) substantially similar to that shown in FIG. 5.

In another embodiment of the invention, crystalline form II of the free base of crystalline compound 1 is characterized by at least one peak of powder X-ray diffraction (Cu Ka radiation) selected from 13.37, 14.37, 19.95 and 23.92° 2Θ (each ± 0.2° 2θ). In another embodiment of the invention, crystalline form II of the free base of crystalline compound 1 is characterized by at least two powder x-ray diffraction (Cu Ka radiation) peaks selected from 13.37, 14.37, 19.95 and 23.92° 2Θ (each ± 0.2° 2Θ). In another embodiment of the invention, crystalline form II of the free base of crystalline compound 1 is characterized by at least three peaks of powder X-ray diffraction (Cu Ka radiation) selected from 13.37, 14.37, 19.95 and 23.92° 2Θ (each ± 0.2° 2Θ).

In another embodiment of the invention, form II is characterized by the presence of 1, 2, 3, 4 or more peaks shown in the table below.

Observed peaks for Form II, X-ray powder diffraction (XRPD) file 613881.

°2Θ d distance (A) Intensity (%) 5.62±0.20 15.735±0.581 24 12.85±0.20 6.888±0.108 22 12.97±0.20 6.826±0.106 21 13.37±0.20 6.622±0.100 100 14.37±0.20 6.162±0.087 56 15.31±0.20 5.788±0.076 21 16.09±0.20 5.507±0.069 23 16.45±0.20 5.390±0.066 69 16.75±0.20 5.294±0.064 32 16.96±0.20 5.227±0.062 53 19.95±0.20 4.450±0.045 39 20.22±0.20 4.391±0.043 20 23.18±0.20 3.837±0.033 38 23.92±0.20 3.721±0.031 41 24.40±0.20 3.648±0.030 44 24.73±0.20 3.600±0.029 22 24.99±0.20 3.564±0.028 50 25.12±0.20 3.545 ± 0.028 28 25.39±0.20 3.509±0.027 51 25.70±0.20 3.466 ± 0.027 21 26.19±0.20 3.403 ± 0.026 27 26.72±0.20 3.336±0.025 thirty 27.02±0.20 3.300±0.024 25 27.34±0.20 3.262 ± 0.024 23 28.44±0.20 3.138±0.022 20

In some embodiments, the free base of crystalline Compound 1 includes crystalline Form II. In some preferred embodiments, the free base of crystalline Compound 1 includes crystalline Form II and less than 25, 10, or 5 mole % of crystalline Form I, crystalline N, or amorphous forms of Compound 1.

In a preferred embodiment of the invention, crystalline form II is obtained from a suspension containing the free base of compound 1 in heptane, from which crystalline form II is formed and filtered. Thus, in some embodiments of the invention, crystalline Form II contains residual (1-500 ppm) heptane. In another preferred embodiment of the invention, crystalline form II is obtained from a suspension containing the free base of compound 1 in water, from which crystalline form II is formed and filtered.

Crystal Form II has several advantages over Crystal Form I or Substance N. For example, Crystal Form II can be prepared from a suspension containing Compound 1 free base in heptane that is suitable for Good Manufacturing Practice (GMP) protocols. In addition, in the most preferred embodiment of the invention, the crystalline form II can be obtained from a suspension containing the free base of compound 1 in water or the HCl salt of compound 1 in water, thereby reducing or eliminating the need for a solvent during recrystallization. Thus, in some embodiments of the invention, the crystalline form II of compound 1 contains less than 500, 100, less than 50, or less than 10 ppm organic solvent. In addition, form II has a lower tendency to agglomerate than substance N when the size is reduced, for example, when grinding. Thus Form II is more fluid than substance N. Some illustrative and non-limiting advantages of Form II over Substance N (ie, Form N) are shown in the table below.

DATA/TEST RESULTS/STATUS Determining the right solvent for scaling Form N: • Limited number of suitable solvents compared to Form II • Methyl tert-butyl ether (MTBE) defined (suitable for GMP; III Solvent Grade) • Scale-up results are promising Form II: • More options for solvents than Form N, including H ₂ O • Current solvent is heptane (suitable for GMP; Solvent Grade III) • produced at 5kg level • Formulation time is faster than Form N (which can result in 2-3 days shorter production time) • Better recovery than Form N Size/Morphology Forms N and II • Needle morphology observed for Form N; a substance made up of small and large particles • Agglomerates are a problem for Form N in relation to Form II (less agglomeration observed using the energy reduction method) Comparison of FC Form N and II • Oral administration of GBT440 Forms N and II to rats resulted in comparable effects at 100 & 500 mg/kg

Substance N.

In another embodiment of the invention, the crystalline free base of compound 1 comprises crystalline substance N, which is characterized by an endothermic peak at 95±2°C, as measured by differential scanning calorimetry. The terms substance N, form N and polymorph form N are used interchangeably in the present invention. In another embodiment of the invention, the crystalline substance N of the free base of the crystalline compound 1 is characterized by a significant absence of thermal events at temperatures below the endothermic peak at 95±2°C, as measured by differential scanning calorimetry. In another embodiment of the invention, the crystalline substance N of the free base of crystalline compound 1 is characterized by a powder x-ray diffraction (Cu Ka radiation) peak at one or more of 11.65, 11.85, 12.08, 16.70, 19.65, or 23, 48° 2θ. In another embodiment of the invention, the crystalline substance N of the free base of the crystalline compound 1 has an X-ray powder diffraction pattern (Cu Ka radiation) substantially similar to that shown in FIG. 7.

In another embodiment of the invention, the crystalline substance N of the free base of crystalline compound 1 is characterized by at least one peak of powder X-ray diffraction (Cu Ka radiation) selected from 11.65, 11.85, 12.08, 16.70, 19.65 and 23.48° 2Θ (each ±0.2° 2θ). In another embodiment of the invention, the crystalline substance N of the free base of crystalline compound 1 is characterized by at least two peaks of powder X-ray diffraction (Cu Ka radiation) selected from 11.65, 11.85, 12.08, 16.70, 19.65 and 23.48° 2Θ (each ±0.2° 2Θ). In another embodiment of the invention, the crystalline substance N of the free base of crystalline compound 1 is characterized by at least three peaks of powder X-ray diffraction (Cu Ka radiation) selected from 11.65, 11.85, 12.08, 16.70, 19.65 and 23.48° 2Θ

- 4 040317 (each ±0.2° 2θ).

In another embodiment of the invention, the substance N is characterized by the presence of 1, 2, 3, 4 or more of the peaks shown in the table below.

Observed peaks for substance N, X-ray powder diffraction (XRPD) file 615765.

°2Θ d distance (A) Intensity (%) 5.55±0.20 15.924±0.595 54 11.65±0.20 7.597±0.132 31 11.85±0.20 7.468±0.128 50 12.08±0.20 7.324±0.123 31 12.67±0.20 6.987±0.112 29 13.12±0.20 6.748±0.104 83 14.94±0.20 5.929±0.080 34 15.19±0.20 5.832±0.077 56 15.76±0.20 5.623±0.072 20 16.70±0.20 5.310 ±0.064 100 17.35±0.20 5.112±0.059 52 19.65±0.20 4.517±0.046 60 23.48±0.20 3.789±0.032 72 23.68±0.20 3.757±0.032 29 25.25±0.20 3.527±0.028 20 25.47±0.20 3.497±0.027 20 25.70±0.20 3.466 ± 0.027 85 26.04±0.20 3.422 ± 0.026 35 26.37±0.20 3.380±0.025 55

In some preferred embodiments, the free base of crystalline compound 1 comprises crystalline substance N and less than 25, 10, or 5 mole % of crystalline forms I or II or amorphous forms of compound 1.

In another embodiment, crystal N is obtained from a suspension containing compound 1 free base in methyl tert-butyl ether (MTBE), from which crystal N is formed and filtered. Thus, in some embodiments, crystal N contains residual (1-500 ppm) MTBE.

The crystalline substance N has several advantages over crystalline forms I or II. For example, crystalline substance N can be obtained from a suspension containing the free base of compound 1 in MTBE, which is suitable for good manufacturing practice (GMP) protocols.

In some embodiments, the crystalline forms of the desolvates are stable when contacted with water, heptane, isopropyl ether (IPE), MTBE, and toluene, and similar other solvents.

In one embodiment of this composition, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a crystalline free base of Compound 1 containing one or more of Form I, Form II, or Substance N.

In one embodiment of this process, the present invention relates to a process for the preparation of a solid crystalline free base of Compound 1, including, for example, Form I, Form II, and/or Substance N.

In yet another embodiment of this method, methods are provided for increasing the oxygen affinity of hemoglobin S in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1 crystalline free base, including, for example, Form I, Form II and/or substance n.

In yet another embodiment of this method, methods are provided 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 Compound 1 crystalline free base comprising, for example, Form I, Form II and/or substance N.

In all cases of such treatment, the effective amount of the free base of compound 1, including, for example, form I, form II and/or substance N, for the patient undergoing treatment, has already been disclosed in the art.

Solvates.

This invention is partly the result of the discovery that desolvated free base polymorphs of Compound 1 form solvated polymorphs with different

- 5 040317 solvents, preferably other than certain hydrocarbon solvents, water and ethers.

Solvates of the crystalline free base of Compound 1 (e.g. from acetone, acetonitrile, dichloromethane, dioxane, ethanol, ethyl acetate, isopropyl alcohol, methyl ethyl ketone (MEK) and tetrahydrofuran) are also considered for use, for example, as intermediates for the regeneration of the desolvated crystalline free base. compounds 1. Such methods may include, without limitation, subjecting the solvate to vacuum conditions; and/or obtaining salt and its disproportionation in water with the formation of desolvate; and/or suspending or washing the solvate with a less solvate-forming solvent such as heptane, diisopropyl ether (TPE), methyl tert-butyl ether (MTBE), and toluene.

In one embodiment of such a composition, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more of the solvated crystalline forms provided herein.

In one embodiment of this method, this invention relates to a method for obtaining solvated crystalline forms presented in the present invention.

In yet another embodiment of this method, methods are provided for increasing the oxygen affinity of hemoglobin S in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more solvated crystalline forms provided herein.

In yet another embodiment of this method, methods are provided 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 solvated crystalline forms provided herein.

In all cases of such treatment, the effective amount of Compound 1 free base for the patient being treated has already been disclosed in the art.

Brief description of the drawings

Fig. 1 is an XRPD profile of a crystalline HCl salt before (top) and after (bottom) 5 minutes of slurry in water.

Fig. 2 is an XRPD profile of Form I free base (top), Form II (center), and substance N (bottom).

Fig. 3 is the XRPD profile and suggested indexing for Form I free base.

Fig. 4 is the thermal curve for Form I free base.

Fig. 5 is the XRPD profile and suggested indexing for Form II free base.

Fig. 6 is the thermal response for Form II free base.

Fig. 7 is the XRPD profile for the free base of substance N.

Fig. 8 is the thermal characteristic for the free base of substance N.

Fig. 9 depicts an energy-temperature diagram between Forms I, II and substance N of the free base. The enthalpy (H) and free energy (G) isobars for each form are plotted as functions of temperature. AHf is the heat of fusion; T is the transition temperature; m is the melting point; the indices I, II, and N refer to polymorphs. * Sufficient information was not available under the test conditions to visualize the Form I free energy isobar below 6°C and above the assumed transition temperature T ^N 'ⁿ; the isobar most likely intersects G _L at a temperature below m ⁿ , allowing for the possibility that form I may be enantiotropic with form II (where TI'II occurs below 6°C) and/or substance N (where or T ^IN occurs below TI'II or T ^N 'I occurs above T ^N 'II, but not both). Free energy isobars intersect each other only once.

Fig. 10 shows the ¹³ C solid state NMR spectrum for Forms I (bottom), II (center), and substance N (top) of the free base. Form I contains one molecule per asymmetric unit. Substance N contains four molecules per asymmetric unit. As can be seen from the NMR spectrum of the ¹³ C solid state, Forms II and N do not undergo a transition of more than 250 to 340 K. Chemical shifts change slightly with temperature (not graphically illustrated).

Fig. 11 shows the ¹⁵ N solid state NMR spectrum for Forms I (bottom), II (center) and substance N (top) of the free base.

Fig. 12 depicts a differential scanning calorimetry (DSC) curve for the free base of substance N.

Fig. 13 depicts the DSC curve for Form II free base.

Fig. 14 depicts the DSC curve for Form I free base.

Fig. 15 depicts the XRPD profile of maturation experiments for Compound 1 free base at several temperatures.

Fig. 16 shows the proposed XRPD profile for solvated substance E.

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Fig. 17 depicts the proposed XRPD profile for solvated substance F.

Fig. 18 depicts the putative XRPD profile for solvated substance G.

Fig. 19 depicts the putative XRPD profile for solvated substance H.

Fig. 20 depicts the putative XRPD profile for solvated substance J.

Fig. 21 depicts the proposed XRPD profile for solvated substance K.

Fig. 22 depicts the proposed XRPD profile for solvated substance L.

Fig. 23 depicts the putative XRPD profile for solvated substance M.

Fig. 24 depicts the proposed XRPD profile for solvated substance O.

Fig. 25 depicts a comparison of the XRPD profile of putative isostructural solvates of the free base of Compound 1. From top to bottom: Substance E from acetone; substance F from ACN; Substance G from DCM; substance H from dioxane; substance J from ethanol; substance K from IPA/water (also derived from IPA) and substance L from THF, substance M from MEK.

Detailed description of the invention

As previously noted, this invention relates in particular to the stable free base of Compound 1, and in particular to Form I, Form II, or Substance N of the free base. However, before a more detailed discussion of the present invention, the following terms will be defined.

Definitions.

Used in the present invention, the following terms have the following meanings.

Singular forms include references to plural forms unless the context clearly dictates otherwise. For example, a connection reference includes both one connection and many different connections.

The term approximately when used in front of a numerical designation, for example, temperature, time, amount and concentration, including range, indicates approximations that may vary within ±10, ±5 or ±1%.

Administration refers to the introduction of a substance into the body of a patient. A therapeutic amount may be administered, which may be determined by the attending physician or the like. The oral route of administration is preferred. The relevant terms and phrases administration and use when used in relation to a compound or pharmaceutical composition (and their grammatical equivalents) refer to direct administration, which may be administration to a patient by a healthcare professional, or self-administration by a patient, and/or indirect administration, which may be an action as a result of drug administration. For example, the use of a drug by a patient corresponds to a situation where the doctor instructs the patient to self-administer the drug and/or provides the patient with a prescription for the drug. In either case, administration entails delivering the drug to the patient's body.

The crystalline desolvate of Compound 1 is the crystalline solid form of 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde free base, such as, for example, crystalline form I, form II or substance N, as described in the present invention. Each of the Form I, Form II, or Substance N crystal lattices is substantially free of crystallization solvents. However, any solvent present is not in the crystal lattice and is randomly distributed outside the crystal lattice. Thus, crystals of form I, form II or substance N, to a large extent, may contain, outside the crystal lattice, small amounts of one or more solvents, such as solvents used in their synthesis or crystallization. Previously used is largely free and small amounts refer to the presence of solvents, preferably less than 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-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde, where the crystal lattices contain one or more solvents for crystallization.

Characterization refers to obtaining data that can be used to identify the solid form of a compound, for example, to determine whether the solid form is amorphous or crystalline and whether it is desolvated or solvated. The method by which solid forms are characterized involves analyzing data collected on polymorphic forms so as to enable any person of ordinary skill in the art to distinguish one solid form from other solid forms containing the same substance. The chemical identity of solid forms can often be determined using solution analysis methods such as ¹³ C NMR or 1 H NMR. While they can help identify the substance and solvent molecules for a solvate, such methods of analysis into solution states cannot, by themselves, provide solid state information. However, there are solid-state analytical methods that can be used to obtain information about the solid-state structure and distinguish polymorphic solid forms from each other, 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 methods such as differential scanning calorimetry (DSC), solid state ¹³ C-NMR, thermogravimetry (TG), melting point and hot state microscopy.

In order to characterize the solid form of a compound, one can, for example, collect data on the XRPD of the solid forms of the compound and compare the XRPD peaks for the forms. For example, when only three solid forms are compared, e.g., Forms I and II and Substance N, and a Form I sample shows a peak at an angle where no peaks appear for a Form II sample or Substance N, then that peak for that compound distinguishes Form I from Form II and Substance N and henceforth serves to characterize Form I. The set of peaks that distinguish, for example, Form I from other known forms is the set of peaks that can be used to characterize Form I. Those skilled in the art it will be understood that there are often multiple methods, including multiple methods using the same analytical method, to characterize solid forms. Additional peaks can also be used, but they are not necessary in order to characterize the shape down to the entire diffraction pattern. Although all peaks within the entire XRPD sample can be used to characterize such a shape, a subset of these data can, and is usually used, to characterize the shape.

The XRPD sample is an x-y plot with diffraction angle (typically °2θ) on the abscissa and intensity on the y-axis. Peaks within this sample can be used to characterize the crystalline solid form. As with any data measurement, there is variability in the XRPD data. Often, data is presented solely as peak diffraction angle, rather than including peak intensities, because peak intensities can be particularly sensitive to sample preparation (e.g., particle size, moisture content, solvent content, and preferred orientation effects that affect sensitivity), thus, samples of the same material prepared under different conditions may result in slightly different diffractograms; this variability is generally greater than the variability in diffraction angles. Diffraction angle variability can also be sensitive to sample preparation. Other sources of variability come from the parameters of the instrument and the processing of the original X-ray data: different X-ray instruments operate using different parameters and this can lead to slightly different X-ray patterns of the same solid shape, in the same way that different software packages process X-ray data differently, which also leads to variability. These and other sources of variability are known to those skilled in the pharmaceutical arts. According to these sources of variability, the level of variability is usually assigned at ±0.2° 2θ with respect to the diffraction angles in XRPD samples.

Containing or including means that the compositions and methods include the listed elements, but do not exclude others. Consisting essentially of, when used to define compositions and methods, means the exclusion of other elements having any essential value for the combination to achieve the stated goal. Thus, a composition essentially consisting of elements as defined in the present invention does not exclude other substances or steps that do not significantly affect the main(s) and new(s) characteristic(s) of the claimed invention. Consisting of means the exclusion, more than at the micronutrient level, of other ingredients and essential process steps. The embodiments defined by each of these connecting terms are within the scope of the present invention.

Form II and substance N are enantiotropic at the transition temperature (approximately 42°C). Below this transition temperature, compound 1 free base substance N is thermodynamically more stable than Forms I and II. Above this transition temperature, form II of the free base of compound 1 is the thermodynamically more stable form with respect to form I and substance N.

Room temperature refers to a temperature of 22±5°C.

A therapeutically effective amount or therapeutic amount refers to the amount of a drug or substance that, when administered to a patient suffering from a condition, will produce the desired therapeutic effect, such as alleviating, ameliorating, alleviating, or eliminating one or more of the patient's symptoms of the condition. A therapeutically effective amount will vary depending on the subject and the condition being treated, the weight and age of the subject, the severity of the condition, the particular formulation or excipient chosen, the dosage regimen to be followed, the regularity of administration, the route of administration, and the like, all of the above. factors can be easily determined by a person skilled in the art. The emergence of a full therapeutic effect does not necessarily occur with the introduction of a single dose and may occur only after a series of doses. Thus, a therapeutically effective amount may be administered in one or more doses. For example, but not limited to disorders, a therapeutically effective amount of an agent, in the context of treating a hemoglobin S related disorder, refers to an amount of an agent that alleviates, ameliorates, alleviates, or eliminates one or more manifestations of a hemoglobin S related disorder, at the patient.

Treating, treating and treating are defined as acts on a disease, disorder or condition using a substance to reduce or mitigate the harmful or any other undesirable effects of the disease, disorder or condition and/or its symptoms. Treatment, in the context of use in the present invention, encompasses the treatment of a human patient and includes (a) reducing the risk of a condition in a patient with an identified predisposition to the disease, but not yet diagnosed as a condition, (b) preventing the development of the condition and/or (c) relief of the condition, i.e. regression of the condition and/or relief of one or more symptoms of the condition. To achieve the objectives of the present invention, beneficial or desirable clinical results include, but are not limited to, improvement in general hematological parameters, reduction in the number of blood transfusions required, reduction in infections, reduction in the incidence of bleeding, and the like.

Identification of forms I, II and substance N.

When the HCl salt of Compound 1 was exposed to various stress conditions, disproportionation of the hydrochloride in water was observed to form the free base. At least three anhydrous crystalline forms of the free base have been identified, named Forms I, II, and Substance N of the free base. Form I of the free base is generally found to form first and increasing residence time in suspension induces the conversion of Form I of the free base to Form II of the free base, a more thermodynamically stable phase relative to Form I. In addition, it has been found that that substance N of the free base appears to be the most stable form with respect to Forms I and II at room temperature. The free base substance N has been found to be enantiotropically active with respect to Form II and will reversibly convert at a certain transition temperature (estimated according to the present application at a temperature of about 40-42°C). Above the transition temperature, form II of the free base appears to be the most stable form with respect to form I and substance N.

Based in part on solid-state nuclear magnetic resonance data, it was found that all three forms are crystalline and have various polymorphic forms. See fig. 10 and 11. Form I contains one molecule per asymmetric unit, Form II contains two molecules per asymmetric unit, and Form N contains four molecules per asymmetric unit. See ¹⁵ N spectra in FIG. eleven.

Desolvates of forms I, II and substance N.

In one embodiment, the present invention provides a crystalline desolvate of Compound 1 free base. The crystalline desolvate free base of Compound 1 may include one or more Form I, Form II, and/or Substance N polymorphs. In some embodiments, the crystalline desolvate free base Compound 1 may include a Form II polymorph. Preferably, Compound 1 crystalline desolvate free base may include Form II and/or compound N polymorphs. More preferably, Compound 1 crystalline desolvate free base may include Compound 1 crystalline desolvate free base. from the solvated free base polymorph of Compound 1. Further, even more preferably, the free base of the crystalline desolvate of Compound 1 is substantially free of other desolvated free base polymorphs of Compound 1. Essentially free of a component, as used in the present invention, refers to the content up to about 5%, more preferably up to about 3%, and even more preferably up to about 1% of the component. The solvate used in the present invention also includes the hydrated form.

Solvates of compound 1.

In one aspect of the invention, a crystalline solvate of Compound 1 is provided.

Compound 1

In some embodiments, the crystalline solvate is substantially free of the desolvated compound 1 polymorph.

Many of the dissolution and sieving experiments using the free base of Compound 1 resulted in the precipitation of solid particles and were characterized as the formation

- 9 040317 solvate with some solvents. Under certain conditions, the formation of solvates from the free base of compound 1 was not observed using four solvents, including heptane, diisopropyl ether (IPE), methyl tert-butyl ether (MTBE) and toluene. The formation of solvates from the free base of compound 1 was observed in nine solvents, including acetone (substance E), acetonitrile (substance F), dichloromethane (substance G), dioxane (substance H), ethanol (substance J), isopropyl alcohol, or a mixture of water and isopropyl alcohol. alcohol (substance K), tetrahydrofuran (substance L), methyl ethyl ketone MEK (substance M), ethyl acetate (substance O) and dimethyl sulfoxide DMSO (substance P). Most of the solvates (ie substances E-H, J-M, O and P) are considered to be isostructural. In some embodiments, the crystalline solvate includes one or more of substance E, substance F, substance G, substance H, substance J, substance K, substance L, substance M, substance O, or substance P.

Substance E can be characterized by at least one X-ray powder diffraction (Cu Ka radiation) peak selected from 8.69, 11.73, 12.10, 15.26, 16.11, 17.45, 22.39, 22 .55 and 23.70±0.20. Substance 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, 17.0123, 73 and 24.07±0.20. Substance G can be characterized by at least one powder x-ray diffraction (Cu Ka radiation) peak 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±0.20. Substance H can be characterized by at least one powder x-ray diffraction (Cu Ka radiation) peak selected from 8.61, 11.67, 15.33, 16.28, 17.28, 22.58, 23.51 and 25 .77±0.20. Substance J can be characterized by at least one powder X-ray diffraction (Cu Ka radiation) peak 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±0.20. Substance K can be characterized by at least one powder x-ray diffraction (Cu Ka radiation) peak selected from 8.52; 8.83, 11.35, 15.04, 15.74, 17.11, 23.46, 23.58, 24.08 and 25.99±0.20. Substance L can be characterized by at least one powder x-ray diffraction peak (Cu Ka radiation) selected from 8.61, 8.78, 11.67, 14.94, 15.28, 16.14, 17.30, 22 .75, 23.71 and 26.05±0.20; and substance M can be characterized by at least one powder x-ray diffraction (Cu Ka radiation) peak selected from 7.74, 10.05, 12.82, 15.33, 16.80, 20.82, 21.14, 25.80 and 26.97±0.20.

Solvates (such as acetone, acetonitrile, dichloromethane, dioxane, ethanol, ethyl acetate, isopropyl alcohol, MEK, tetrahydrofuran, or DMSO) can be used, for example, as intermediates to regenerate the crystalline desolvate of Compound 1 free base by several methods, including including by exposing the solvate to vacuum conditions; and/or regeneration of the HCl salt and disproportionation of the HCl; and/or washing the solvate with a less solvate-forming solvent such as heptane, diisopropyl ether (IPE), methyl tert-butyl ether (MTBE), and toluene.

Table 1

Data relating to free base solvates of Compound 1

Name Solvent FOR crystallization Volume (A ³ /Cell) Number of formula units in a cell The volume of the formula unit * (A ³ ) indexed result Substance E acetone 968 2 484 FIG. 1 Substance F ACN 947 2 473 FIG. 2 Substance G DCM 959 2 480 FIG. 3 Substance H dioxane 977 2 488 FIG. 4 Substance J EtOH 943 2 472 FIG. 5 Substance K IPA 963 2 481 FIG. 6 Substance L THF 972 2 486 FIG. 7 Substance M MEK 3956 8 494 FIG. 8 Substance O EtOAc - - - FIG. 9 Substance P** DMSO - - - -

* Value obtained by dividing the cell volume resulting from the pre-indexing of the solution by the number of formula units in the cell.

** Substance P was observed as a mixture with sulfate form I.

Some expected peaks of the various solvates contemplated by the present invention are shown in the table below. Some peaks that are preferably non-crossing, small angle peaks, high intensity peaks were not identified. The peaks were determined on the assumption that the state of the preferred orientation in the samples was unknown.

- 10 040317

Observed peaks for substance E

table 2

°20 d distance (A) Intensity (%) 8.41±0.20 10.517±0.256 13 8.69±0.20 10.174±0.239 100 11.73±0.20 7.543±0.130 17 12.10±0.20 7.314±0.122 20 13.00±0.20 6.809±0.106 15 14.02±0.20 6.316±0.091 5 14.77±0.20 5.996±0.082 16 15.26±0.20 5.807±0.077 34 15.81±0.20 5.605±0.071 7 16.11±0.20 5.501±0.069 20 16.48±0.20 5.379±0.066 AND 16.65±0.20 5.326±0.064 AND 16.88±0.20 5.253±0.063 3 17.26±0.20 5.136±0.060 9 17.45±0.20 5.083±0.058 32 20.02±0.20 4.435 ± 0.044 2 20.92±0.20 4.246±0.041 13 21.91±0.20 4.057±0.037 20 22.39±0.20 3.970±0.035 49 22.55±0.20 3.944±0.035 37 22.81±0.20 3.898±0.034 16 23.36±0.20 3.807±0.032 12 23.70±0.20 3.755±0.032 61 24.37±0.20 3.653±0.030 12 24.85±0.20 3.583 ± 0.029 5 25.42±0.20 3.504±0.027 2 25.89±0.20 3.442 ± 0.026 8 26.19±0.20 3.403 ± 0.026 40 26.97±0.20 3.306±0.024 3 27.61±0.20 3.231±0.023 16 28.24±0.20 3.160±0.022 2 28.48±0.20 3.134±0.022 5 28.69±0.20 3.111±0.021 7 29.83±0.20 2.995±0.020 4 Table 3 Observed peaks for substance F °20 d distance (A) Intensity (%) 8.47±0.20 10.434±0.252 100 8.81±0.20 10.039±0.233 49 11.42±0.20 7.752±0.138 15 12.75±0.20 6.942±0.110 27 13.17±0.20 6.723±0.103 21 13.87±0.20 6.384±0.093 7 14.61±0.20 6.064±0.084 13

- 11 040317

14.92±0.20 5.936±0.080 43 15.51±0.20 5.713±0.074 24 15.63±0.20 5.671±0.073 43 15.96±0.20 5.553±0.070 15 17.01±0.20 5.212±0.062 31 17.26±0.20 5.136±0.060 4 17.70±0.20 5.011±0.057 9 18.17±0.20 4.883 ± 0.054 4 18.79±0.20 4.724±0.050 10 19.35±0.20 4.587±0.047 4 19.49±0.20 4.555±0.047 3 20.02±0.20 4.435±0.044 4 20.29±0.20 4.377±0.043 9 21.06±0.20 4.219±0.040 AND 21.33±0.20 4.167±0.039 4 22.71±0.20 3.915±0.034 27 23.11±0.20 3.848±0.033 15 23.73±0.20 3.749±0.031 42 24.07±0.20 3.698±0.031 59 24.65±0.20 3.612±0.029 87 24.95±0.20 3.569±0.028 6 25.20±0.20 3.534±0.028 5 25.69±0.20 3.468 ± 0.027 15 26.52±0.20 3.361±0.025 61 26.79±0.20 3.328±0.025 10 27.02±0.20 3.300±0.024 9

Table 4

Observed peaks for substance G

°2Θ d distance (A) 8.47±0.20 10.434±0.252 45 8.76±0.20 10.096±0.235 12 11.45±0.20 7.729±0.137 76 12.62±0.20 7.015±0.113 36 13.09±0.20 6.765±0.105 10 13.87±0.20 6.384±0.093 5 14.66±0.20 6.044 ± 0.083 39 14.92±0.20 5.936±0.080 26 15.33±0.20 5.782±0.076 7 15.69±0.20 5.647±0.072 88 16.01±0.20 5.536±0.070 8 16.76±0.20 5.289±0.063 15 17.01±0.20 5.212±0.062 29 17.50±0.20 5.068±0.058 5 17.60±0.20 5.040±0.057 4 18.13±0.20 4.892±0.054 5 18.47±0.20 4.804±0.052 21 19.55±0.20 4.540±0.046 4 20.01±0.20 4.439±0.044 5 20.32±0.20 4.370±0.043 20 21.11±0.20 4.209±0.040 15 22.61±0.20 3.932±0.035 42

Intensity (%)

- 12 040317

22.88±0.20 3.887±0.034 9 23.08 ± 0.20 3.854±0.033 28 23.43±0.20 3.797±0.032 56 23.70±0.20 3.755±0.032 48 24.12±0.20 3.690 ±0.030 13 24.42±0.20 3.646±0.030 100 25.05±0.20 3.555±0.028 7 25.40±0.20 3.506±0.027 26 26.36±0.20 3.382±0.025 50 26.57±0.20 3.355±0.025 7 26.82±0.20 3.324±0.025 27 27.07 ± 0.20 3.294 ± 0.024 10

Table 5

Observed peaks for substance H

Intensity (%)

°20 d distance (A) 8.61±0.20 10.273 ± 0.244 48 8.81±0.20 10.039±0.233 20 11.67±0.20 7.586±0.132 32 12.10±0.20 7.314±0.122 AND 12.79±0.20 6.924±0.110 9 14.56±0.20 6.085±0.084 4 14.87±0.20 5.956±0.081 22 15.33±0.20 5.782 ± 0.076 42 15.76±0.20 5.623±0.072 18 16.28±0.20 5.445 ± 0.067 51 16.73±0.20 5.299±0.064 9 17.28±0.20 5.132±0.060 61 17.68±0.20 5.016±0.057 3 20.47±0.20 4.338 ± 0.042 12 21.38±0.20 4.157±0.039 7 21.83±0.20 4.072 ± 0.037 4 22.23±0.20 3.999±0.036 9 22.58±0.20 3.938±0.035 100 22.95±0.20 3.876±0.034 6 23.11±0.20 3.848±0.033 14 23.51±0.20 3.783 ± 0.032 88 24.37±0.20 3.653±0.030 13 24.65±0.20 3.612±0.029 10 25.77±0.20 3.457±0.027 41 26.67±0.20 3.342±0.025 7 26.97±0.20 3.306±0.024 5 27.66±0.20 3.225±0.023 3 28.11±0.20 3.174±0.022 4 28.61±0.20 3.120 ±0.022 6 28.96±0.20 3.083 ± 0.021 4 29.23±0.20 3.055±0.021 3 29.63±0.20 3.015±0.020 3

Table 6

Observed peaks for substance J

Intensity (%)

°20 d distance (A) 8.52±0.20 10.373±0.249 100 8.88 ± 0.20 9.964±0.229 39 11.33±0.20 7.809±0.140 22 12.79±0.20 6.924±0.110 25

- 13 040317

13.12±0.20 6.748±0.104 24 13.94±0.20 6.354±0.092 4 14.47±0.20 6.120±0.085 14 15.04±0.20 5.890±0.079 42 15.61±0.20 5.677 ± 0.073 56 15.84±0.20 5.594±0.071 16 17.11±0.20 5.181±0.061 33 17.40±0.20 5.097±0.059 4 17.82±0.20 4.979±0.056 8 18.12±0.20 4.897±0.054 3 18.90±0.20 4.695±0.050 AND 19.39±0.20 4.579±0.047 5 19.62±0.20 4.525±0.046 4 20.16±0.20 4.406 ± 0.044 8 20.96±0.20 4.239±0.040 12 22.81±0.20 3.898±0.034 27 23.15±0.20 3.843 ± 0.033 9 23.28±0.20 3.821±0.033 7 23.87±0.20 3.729±0.031 34 24.17±0.20 3.683±0.030 52 24.62±0.20 3.616±0.029 95 25.20±0.20 3.534±0.028 5 25.77±0.20 3.457±0.027 13 26.44±0.20 3.371±0.025 70 26.71±0.20 3.338±0.025 10 27.21±0.20 3.278±0.024 7

Table 7

Observed peaks for GBT 000440, substance K

Intensity (%)

°20 d distance (A) 8.52±0.20 10.373±0.249 75 8.83±0.20 10.020 ±0.232 33 11.35±0.20 7.797±0.139 29 12.52±0.20 7.071±0.114 21 12.90±0.20 6.861±0.108 24 13.92±0.20 6.361±0.092 4

- 14 040317

14.49±0.20 6.113±0.085 18 15.04±0.20 5.890±0.079 41 15.34±0.20 5.775±0.076 17 15.74±0.20 5.629±0.072 57 15.93±0.20 5.564±0.070 13 16.61±0.20 5.336±0.065 7 17.11±0.20 5.181±0.061 33 17.70±0.20 5.011±0.057 7 18.00±0.20 4.928±0.055 4 18.38±0.20 4.826±0.053 13 19.04±0.20 4.662 ± 0.049 4 19.74±0.20 4.498 ± 0.046 5 20.21±0.20 4.395±0.043 AND 20.99±0.20 4.232 ± 0.040 12 22.70±0.20 3.918±0.034 22 22.90±0.20 3.884±0.034 17 23.46±0.20 3.791±0.032 45 23.58±0.20 3.773 ± 0.032 70 24.08 ± 0.20 3.695±0.030 100 24.75±0.20 3.597±0.029 6 25.19±0.20 3.536±0.028 21 25.99±0.20 3.429±0.026 71 26.71±0.20 3.338±0.025 AND 27.36±0.20 3.260 ±0.024 9 28.11±0.20 3.174±0.022 4 28.69±0.20 3.111±0.021 9

Table 8

Observed peaks for substance L

°20 d distance (A) 8.61±0.20 10.273 ± 0.244 79 8.78±0.20 10.077±0.235 38 11.67±0.20 7.586±0.132 35 12.17±0.20 7.274±0.121 19 12.94±0.20 6.844±0.107 14 14.07±0.20 6.293±0.090 3 14.62±0.20 6.057±0.084 5 14.94±0.20 5.929±0.080 25 15.28±0.20 5.800±0.076 50 15.93±0.20 5.564±0.070 18 16.14±0.20 5.490±0.068 49 16.33±0.20 5.429±0.067 9 16.70±0.20 5.310 ±0.064 9 16.85±0.20 5.263±0.063 6 17.30±0.20 5.127±0.060 52 17.63±0.20 5.030±0.057 6 18.37±0.20 4.830 ±0.053 3 20.14±0.20 4.409 ± 0.044 5

Intensity (%)

- 15 040317

20.59±0.20 4.314±0.042 14 21.53±0.20 4.128±0.038 AND 22.01±0.20 4.038±0.037 3 22.44±0.20 3.961±0.035 27 22.75±0.20 3.910±0.034 72 23.10±0.20 3.851±0.033 20 23.31±0.20 3.816±0.033 19 23.48±0.20 3.789±0.032 12 23.71±0.20 3.752±0.031 100 24.48 ± 0.20 3.636±0.029 20 24.70±0.20 3.604±0.029 4 24.93±0.20 3.571±0.028 3 25.59±0.20 3.482±0.027 5 25.72±0.20 3.464 ± 0.027 5 26.05±0.20 3.420±0.026 62 26.59±0.20 3.352±0.025 6 27.14±0.20 3.286 ± 0.024 8 27.83±0.20 3.206 ± 0.023 8 28.38±0.20 3.145±0.022 3 28.78±0.20 3.102±0.021 8 29.05±0.20 3.074±0.021 4 29.36±0.20 3.042 ± 0.020 3

Table 9

Observed peaks for matter M °2Θ d distance (A) Intensity (%)

7.74 ± 0.20 11.424±0.303 100 8.34±0.20 10.601±0.260 4 10.05±0.20 8.806±0.178 17 12.82±0.20 6.906±0.109 46 13.05±0.20 6.783±0.105 4 14.17±0.20 6.249±0.089 2 14.54±0.20 6.092±0.085 6 14.99±0.20 5.910±0.079 16 15.33±0.20 5.782±0.076 47 15.53±0.20 5.707±0.074 21 16.80±0.20 5.278 ± 0.063 27 18.33±0.20 4.839±0.053 3 19.17±0.20 4.630±0.048 22

- 16 040317

20.19±0.20 4.399±0.044 23 20.82±0.20 4.266±0.041 32 21.14±0.20 4.202±0.040 27 21.29±0.20 4.173±0.039 14 22.01±0.20 4.038±0.037 13 22.28±0.20 3.991±0.036 23 22.93±0.20 3.879±0.034 6 23.35±0.20 3.810 ±0.032 AND 24.00±0.20 3.708±0.031 14 24.25±0.20 3.670 ±0.030 3 24.88±0.20 3.578±0.029 AND 25.54±0.20 3.488 ± 0.027 9 25.80±0.20 3.453 ± 0.027 94 26.97±0.20 3.306±0.024 27 27.63±0.20 3.229±0.023 2 28.41±0.20 3.142±0.022 7 28.54±0.20 3.127±0.022 8 29.03±0.20 3.076±0.021 3 29.30±0.20 3.049±0.020 7 29.63±0.20 3.015±0.020 15

pharmaceutical compositions.

In other embodiments of this composition, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a crystalline free base of Compound 1, preferably containing one or more Form I, Form II, and/or Substance N polymorphs.

Such compositions may be formulated for various routes of administration. Although compositions suitable for oral administration are likely to be used more frequently, other routes of administration may be used, including intravenous, intra-arterial, pulmonary, rectal, nasal, vaginal, lingual, intramuscular, intraperitoneal, intradermal, intracranial, subcutaneous, and transdermal routes. Suitable dosage forms for administering any of the compounds described herein include tablets, capsules, pills, powders, aerosols, suppositories, parenteral and oral liquids including suspensions, solutions and emulsions. Sustained release dosage forms may also be used, for example in the form of a transdermal patch. All dosage forms can be prepared using methods that are standard in the art (see, for example, Remington's Pharmaceutical Sciences, ^16th ed., A. Oslo editor, Easton Pa. 1980).

Pharmaceutically acceptable excipients are non-toxic, facilitate administration, and do not interfere with the therapeutic benefit of the compound of the present invention. Such excipients can be any solid, liquid, semi-solid, or, in the case of an aerosol composition, a gaseous excipient that is generally available to those skilled in the art. Pharmaceutical compositions according to the present invention are prepared by conventional means using methods known in the art.

The compositions disclosed in the present invention can be used in combination with any of the carriers and excipients commonly used in pharmaceutical preparations, for example, talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, derivatives paraffin, glycols, etc. Dyes and flavors can also be added to preparations, in particular to preparations for oral administration. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2-propylene glycol, polyglycols, dimethyl sulfoxide, fatty alcohols, triglycerides, glycerol partial esters, and the like.

Solid pharmaceutical excipients include starch, cellulose, hydroxypropyl cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, skimmed milk powder, etc. Liquid and semi-liquid excipients may be selected from glycerin, propylene glycol, water, ethanol, and various oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. certain embodiments of the invention, the compositions provided in the present invention contain one or more of α-tocopherol, gum arabic and/or hydroxypropyl cellulose.

- 17 040317

In one embodiment, the present invention provides sustained release formulations, such as drug depots or patches, containing an effective amount of a compound of the present invention. In another embodiment, the patch further comprises gum arabic or hydroxypropylcellulose alone or in combination in the presence of alpha-tocopherol. Preferably, the hydroxypropyl cellulose has an average molecular weight of 10,000 to 100,000. In a more preferred embodiment, the hydroxypropyl cellulose has an average molecular weight of 5,000 to 50,000.

The compounds and pharmaceutical compositions of the present invention may be used alone or in combination with other compounds. When administered with another agent, co-administration may be in any manner in which the pharmacological effects of both agents appear in the patient at the same time. Thus, co-administration does not require that the same pharmaceutical composition, the same dosage form, or even the same route of administration be used to administer both the compound of the present invention and the other agent, or that the two agents be administered at exactly the same time.

However, in essence, co-administration will be most convenient when using the same dosage form and the same route of administration. Obviously, such administration is advantageously accomplished by delivering both active ingredients simultaneously in the new pharmaceutical composition according to the present invention.

Ways of preparation and treatment.

Desolvates.

In another aspect, the present invention relates to a process for preparing a crystalline desolvate of the free base of Compound 1. In one embodiment of the invention, presented in the present invention, a method is provided for obtaining a crystalline free base of Compound 1, comprising suspending or contacting an HCl salt of Compound 1 with water and dissociating the HCl with the purpose of obtaining the free base of compound 1. In one embodiment, the prepared crystalline desolvate of the free base of compound 1 comprises one or more of Form I, Form II, and Substance N.

In yet another embodiment of this method, methods are provided for increasing the oxygen affinity of hemoglobin S in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1 crystalline free base. In some embodiments, Compound 1 crystalline free base is a desolvate . In one embodiment, the crystalline free base of Compound 1 comprises one or more of Form I, Form II, and Substance N.

In yet another embodiment of this method, methods are provided 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 Compound 1 crystalline free base. In some embodiments, the Compound 1 crystalline free base 1 is a desolvate. In one embodiment, the crystalline free base of Compound 1 comprises one or more of Form I, Form II, and Substance N.

In additional aspects, the present invention provides a method for treating sickle cell disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1 crystalline free base. In some embodiments, Compound 1 crystalline free base is a desolvate. In one embodiment, the crystalline free base of Compound 1 comprises one or more of Form I, Form II, and Substance N. In further aspects, the present invention provides a method of treating cancer, pulmonary disorder, stroke, vertigo, ulcers, bedsores, Alzheimer's disease, acute respiratory disease and wounds, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of Compound 1 crystalline free base. In some embodiments, Compound 1 crystalline free base is a desolvate. In one embodiment, the crystalline free base of Compound 1 comprises one or more of Form I, Form II, and Substance N.

In all cases of such treatment, the dosage of Compound 1 crystalline free base for the patient undergoing treatment has already been disclosed in the art.

Solvates.

In another aspect, the present invention relates to a process for preparing a crystalline solvate of the free base of compound 1. In some embodiments, the desolvate of the free base of compound 1 described in the present invention (for example, obtained by suspending the HCl salt of compound 1 in water) is contacted with a solvent , as provided in the present invention, including mixtures of solvents for the preparation of solvate, solvent or mixture of solvents. Thus, the solvent can be one solvent, or

- 18 040317 essentially one solvent, or a mixture of solvents. When using a mixture of solvents, the solvate can be obtained in the presence of one or more separate constituents of the solvent mixture. In some embodiments, the solvent includes alcoholic solvents such as mono-, di-, or more alcohols or alkanols. In some embodiments, the solvent includes chlorinated solvents such as dichloromethane, chloroform, and so on. In some embodiments, the solvent includes ketone solvents such as alkanones and cycloalkanones. Some solvents include, without limitation, methanol, ethanol, 2-propanol, 2-methyl-1-propanol, 1-butanol, acetonitrile, acetone, dichloromethane, dioxane, tetrahydrofuran, or combinations thereof, optionally including water.

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

In yet another embodiment of this method, a method is provided for the treatment of oxygen deficiency associated with sickle cell anemia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crystalline solvate of Compound 1.

Examples

The following examples describe the preparation, characterization, and properties of Compound 1 Free Base Form I desolvate. Unless otherwise noted, all temperatures are in degrees Celsius (°C) and the following abbreviations have the following meanings:

DSC - differential scanning calorimetry;

DVS - dynamic vapor sorption;

HPLC - high performance liquid chromatography;

NA - not applicable;

ND - not determined;

Q is the percentage of dissolved per unit time;

RH - relative humidity;

RSD - residual standard deviation;

RRT - relative retention time;

SS-NMR - solid-state nuclear magnetic resonance;

TGA - thermogravimetric analysis;

TG-IR - thermogravimetric infrared analysis;

XRPD - X-ray powder diffraction;

VT-XRPD - x-ray powder diffraction at variable temperature.

Ways to obtain a synthetic compound 1.

The compound of formula (I) was synthesized according to the schematic description below and then the procedure was carefully worked out.

R WtDA RBuUSW,24-10X

1 u 0‘С 83%

Orgl nN 14{10) 2516-2519 201?

NaH

HE

OMOM

2

OR

84%

R TMEDA RBuli S Me(CH2IMek.t'-10 C

-10 C 24 -10'0 -Yu'S C

0‘S 1 H OH

R HC) S H2O

Bull. DMF

OMOM

Example 1: Synthesis of Compound 15

DIPEA (11.54 ml, 66.13 mmol) and MOMCl (4.42 ml, 58.19 mmol). The 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. NaHCO3, brine was dried and concentrated to give a crude product which was purified by column (hexane/EtOAc=4:1) to give 15.58 g (90%) of the desired product.

Example 2: Synthesis of compound 13 from 15 ohm ohm /AyBr BuLi, DMF 0y ^CHO ^^OMOM -78 to 0 °C ^TEMOM alo/, 13

BuLi (2.5 M, 31.6 ml, 79.0 mmol). The solution was stirred at -78°C for 25 minutes (getting a white cloudy mixture), then it was heated to 0°C and stirred for 25 minutes. The reaction mixture slowly becomes homogeneous. DMF was added to the solution at 0°C. After 25 minutes, HPLC indicated completion of the reaction. The mixture was quenched with sat. NH ₄ Cl (150 ml), diluted with ether (300 ml). The organic layer was separated, the aqueous layer was further extracted with ether (2x200 ml) and combined with the organic layer, washed with brine, dried and concentrated to give a crude product which was triturated to give 14.6 g of the desired product. The filtrate was then concentrated and purified on a column to obtain an additional 0.7 g, so the total weight was 15.3 g.

Example 3: Synthesis of compound 13 from resorcinol 11

He A . NaH MOMCI ohm 1.1 R.TMEDA, R:BuLi, S:THF, 2h, -10’C 1.2 1h, 0”C; 83% OrgLett 14(10), 2516-2519; 2012 OR OMOM A CHO ether/DMF 93% Υί^ΟΜΟΜ 1.1 R:TMEDA, R:BuLi, S:Me{CH2)4Me.K.T? -10X 1 Λ OMOM 4 4 1.2-10X; 2 4 _f -lO°C;-10°C?aX eleven 12 1.30C; 1h,0'S 13 1.4 R:HCI, S:H2O

84%

Journal of Organic Chemistry. 74(11), 4311-4317: 2009

In a three-necked round-bottom flask equipped with a mechanical stirrer, 0.22 mol NaH (50% suspension in mineral oil) was placed under a nitrogen atmosphere. NaH was washed with 2 portions (100 ml) of n-hexane and then with 300 ml of dry diethyl ether; then 80 ml of anhydrous DMF was added. Then 0.09 mol of resorcinol 11 dissolved in 100 ml of diethyl ether was added dropwise and the mixture was left under stirring at room temperature for 30 min. Then 0.18 mol MOMCl was added slowly. After 1 hour with stirring at room temperature, 250 ml of water was added and the organic layer was extracted with diethyl ether. The resulting extracts were washed with brine, dried (Na ₂ SO ₄ ), then concentrated to give the crude product, which was purified by silica gel chromatography to give compound 12 (93% yield).

110 ml of n-hexane, 0.79 mol of BuLi and 9.4 ml of tetramethylethylenediamine (TMEDA) were placed in a three-necked round bottom flask under a nitrogen atmosphere. The mixture was cooled at a temperature of -10°C and 0.079 mol of bis-phenyl ether 12 was slowly added. The resulting mixture was left under stirring on a magnetic stirrer at a temperature of -10°C for 2 hours. drop by drop. After 1 h, aqueous HCl was added until the pH became acidic; the mixture was then extracted with ethyl ether. The combined extracts were washed with brine, dried (Na2SO4) and concentrated to give aldehyde 13 (84%).

2,6-bis-(Methoxymethoxy)benzaldehyde (13): m.p. 58-59°C (n-hexane); IR (KBr) n: 1685 (C=O) cm ^-1 ;

¹ H-NMR (400 MHz, CDCl3) δ 3.51 (s, 6H, 2 OCH3), 5.28 (s, 4H, 2 OCH2 O), 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); analytical elemental analysis for С11Н14О5: C, 58.40; H, 6.24. Found: C, 57.98; H, 6.20. Example 4: Synthesis of a 16 ohm compound

81% ^1b

Conc. HCl (12N, 7 ml) under N2, then stirred further under N2 for 1.5 h. Brine (100 ml) and ether (150 ml) were added to the solution. The organic layer was separated and the aqueous layer was further extracted with ether (2x200 ml). The organic layer was combined, washed with brine, dried and concentrated to give the crude product, which was purified by column (300 g, hexane/EtOAc=85:15) to give the desired product 16 (9.9 g) as a yellow liquid.

- 20 040317

Example 5 Synthesis of Compound 17

K2CO3 (32.05 g, 231.92 mmol) and 3-(chloromethyl)2-(1-isopropyl-1H-pyrazol-5-yl)pyridine hydrochloride (10) (15.78 g, 57.98 mmol). The mixture was heated at 65°C for 1.5 h, cooled to room temperature, poured into ice water (800 ml). The precipitated solid was separated by filtration, my product 17 (18 g).

Example 6 Synthesis of Compound (I) was dried and concentrated to obtain the desired

To a solution of 2-((2-( 1 -isopropyl-1 H-pyrazol-5 -yl)pyridin-3 -yl)methoxy)-6-(methoxymethoxy)benzaldehyde (17) (18 g, 47.19 mmol) in THF (135 ml, solution purged with N ₂ ) was added with conc. HCl (12 N, 20 ml). The solution was stirred at room temperature for 3 hours until HPLC indicated that the reaction was complete. The mixture was added to a solution of NaHCO ₃ (15 g) in water (1.2 L) and the resulting precipitate was collected by filtration, dried to give a crude solid which was further purified by column (DCM/EtOAc=60:40) to give pure product (15.3 g).

Example 7: Synthesis of compound I (free base) and its HCl salt.

Compound (I) free base (40 g) was obtained from a mixture of alcohol intermediate 7 and 2,6-dihydroxybenzaldehyde 9 under Mitsunobu reaction conditions. The procedure is also below

Example 8: Synthesis of compound (I) under Mitsunobu reaction conditions.

Into a 2000 ml three-necked round bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed a solution of [2-[1-(propan-2-yl)-1H-pyrazol-5-yl]pyridin-3yl]methanol (7 ) (70 g, 322.18 mmol, 1.00 eq.) in tetrahydrofuran (1000 ml). 2,6 Dihydroxybenzaldehyde (9) (49.2 g, 356.21 mmol, 1.10 eq.) and PPh ₃ (101 g, 385.07 mmol, 1.20 eq.) 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 eq.) in tetrahydrofuran (200 ml) dropwise with stirring. The resulting solution was stirred overnight at room temperature. The resulting solution was diluted by adding 500 ml of H2O. The resulting solution was extracted with 3x500 ml dichloromethane and the combined organic layers were dried over sodium sulfate and concentrated in vacuo. The residue was applied to a silica gel column with EA:PE (1:50-1:3) as eluent to obtain a crude product. The crude product was recrystallized from isopropanol/H ₂ O in a ratio of 1/1.5. This resulted in 40 g (37%) of 2-hydroxy-6-([2-[1-(propan-2-yl)-1H-pyrazol-5-yl]pyridin-3-yl]methoxy)benzaldehyde as light yellow solid. The compound exhibited a melting point of 80-82°C. MS (ES, mass/charge): 338.1 [M+1].

¹ H NMR (300 MHz, DMSO-d6) δ 11.72 (s, 1H), 10.21 (s, 1H), 8.76 (d, J=3.6 Hz, 1H), 8.24 ( d, J=2.7 Hz, 1H), 7.55 (m, 3H), 6.55 (m, 3H), 5.21 (s, 2H), 4.65 (m, 1H), 1, 37 (d, J=5.1 Hz, 6H).

¹ H NMR (400 MHz, CDCl) δ 11.96 (s, 1H), 10.40 (s, 1H), 8.77 (dd, J=4.8, 1.5 Hz, 1H), 8, 00 (d, J=7.8Hz,

1H), 7.63 (d, J=1.8 Hz, 1H), 7.49-7.34 (m, 2H), 6.59 (d, J=8.5 Hz, 1H), 6, 37 (d, J=1.8 Hz, 1H), 6.29 (d, J=8.2 Hz,

1H), 5.10 (s, 2H), 4.67 (sep, J=6.7 Hz, 1H), 1.50 (d, J=6.6 Hz, 6H).

Using a different approach, several batches of compound (I) free base are obtained in multigram amounts (20 g). The advantage of this preparation route is the use of monoprotected 2,6-dihydroxybenzaldehyde (16), which effectively eliminates the possibility of bis-alkylation of the side product. Mono-MOM 2,6-dihydroxybenzaldehyde ether (16) can be prepared from two starting points, bromoresorcinol (14) or resorcinol (11) [using the procedures described in Journal of Organic Chemistry 74 (11), 4311-4317; 2009]. All steps and procedures are listed below. Due to the presence of a phenolic aldehyde group, precautions (i.e.

performing all reactions under an inert gas atmosphere such as nitrogen) must be observed to avoid oxidation of the phenol and/or aldehyde group.

Preparation of the HCl salt of compound I: a solution of compound I (55.79 g, 165.55 mmol) in acetonitrile (275 ml) was purged with nitrogen for 10 min, and then 3N aqueous HCl solution (62 ml) was added to this solution at room temperature . The mixture was stirred for an additional 10 min, after addition, most of the acetonitrile (200 ml) was removed by rotary evaporation at a temperature of about 32°C, the remaining solution was frozen under cooling in a dry ice-acetone bath and lyophilized to obtain the HCl salt of compound I (59.4 g).

Example 9: characterization of the HCl salt of compound 1.

Technology detailed information Result

XRPD indexed salt HC1 Compounds 1 Microscopic analysis - pale yellow hard, thin discs/tablets, birefringent Ή nmr DMSO-d6 corresponds to the desired structure, <0.01 mol MEK

XRPD - salt HC1 Compounds 1 DVS - 0.03% gain when balanced at 5% RH 0.10% gain from 5 to 95% RH 0.09% loss at 95 to 5% RH after XRPD HCl I + Form I Free Base

Example 10: Physical stability of the HCl salt of Compound 1 in contact with water.

State Time (all times are approximate) Observation XRPD result contact with water - layer formation after 5 min - Aqueous suspension about 5 min Floating solids turned into white solids when isolated Free base (FB)I (indexed) vacuum dried about 1 day staying in the same state FB I Aqueous suspension about 6 days white, thin discs, birefringent FB I + FB II (IN)

Example 11: physical stability of the HCl salt of compound 1 during grinding.

State Time Observation XRPD result grinding, drying 30 min whitish to pale yellow HCl I grinding, moisturizing thirty min whitish to pale yellow paste HC1I + FB I

- 22 040317

Example 12 Physical Stability of Compound 1 HCl Salt at High Temperatures and/or Vacuum.

State Time Observation XRPD result room temperature, vacuum 6 days pale yellow, discs/plates, V HC1I + FB I 30°С 6 hours pale yellow, discs/plates, V HC11 12 hours pale yellow, discs/plates, V HC1I + FB I 24 hours pale yellow, discs/plates, V HC1I + FB I 40°С 6 hours pale yellow, discs/plates, V HC1I + FB I 12 hours pale yellow, discs/plates, V HC1I + FB I 24 hours pale yellow, discs/plates, V HC1I + FB I 40°C, vacuum 6 hours pale yellow, discs/plates, V HCl I + FB I 12 hours pale yellow, discs/plates, V HCl I + FB I 24 hours pale yellow, discs/plates, V HC1I + FB I 60°С 6 days pale yellow, discs, B HC1I + FB I 60°C, vacuum 6 days pale yellow, discs, B; HCl I + FB I + another form irregular sediment free base from 100 to 125°C 20 minutes The pH indicator paper above the sample indicates the presence of acidic volatiles. HCl I + FB I + another form of free base

Example 13 Preparation of the free base of compound 1 by disproportionation of the HCl salt of compound 1 in water (starting material is the HCl salt of compound 1).

Way Observation XRPD result 1. contact with water 2. sonication 3. filtration and washing with water 4. drying under N ₂ atmosphere for 10 minutes 5. vacuum at room temperature overnight 1. pale yellow, poorly wetted 2. white 3.- 4. - 5. - FBI 1. Contact with water 2. Sonication for 5 minutes 3. Suspension for 10 minutes 4. Filtration and washing with water 5. Drying under N ₂ atmosphere for 10 minutes 6. Vacuum at room temperature overnight 7. Storage in freezer 1. - 2. pale yellow, turned white 3.- 4. - 5. white 6.- 7.- FB I + other form of free base 1. suspension in water, room temperature, 8 days; FB II was used as a seed 2. filtering and washing with water 3. Vacuum at room 1. thick white suspension 2.- 3.- FBII

- 23 040317

temperature during the night 2. suspension weighing 3. flushing with water 2.- 3.- FB II (indexed)

Example 14 Characterization of Form I Free Base of Compound 1.

Technology detailed information Result XRPD indexed Form I Free Base

XRPD - Form I free base TGA from 25 to 350°C 0.2% weight loss at temperatures up to 100°C DSC from 25 to 350°С an endothermic event that occurs at about 97°C Hot microscopy 22.7°С type of original product, finely divided, birefringent 91.2°С increase in particle size and birefringence 94.2°С increase in particle size and birefringence 95.7°C initial melting stage, larger particles compared to initial heating 96.1°C continued melting 96.3°С completion of melting, no crystallization during melting 68.7°С fresh preparation, greater magnification factor 91.1°C increase in birefringence 94.8°С initial stage of melting, larger particles, birefringence 95.4°С continued melting 95.9°C only a few crystals remain, cooled to 92.6°C 92.6°С incubated for a period of 2 to 3 minutes of crystal growth to larger discs, - start heating 96.3°С completion of melting Ή NMR DMSO-d6 corresponds to the structure DVS - 0.02% loss on balancing at 5% RH 0.22% gain from 5 to 95% RH 0.22% loss at 95 to 5% RH after XRPD Form I free base + other free base substance

Example 15 Characterization of Form II Free Base of Compound 1.

Technology detailed information Result XRPD indexed Form II free base

XRPD originally Form II free base after 7 days Form II free base TGA from 25 to 350°C 0.1% weight loss at temperatures up to 100°C DSC from 25 to 350°C an endothermic event that occurs at about 97°C Ή NMR DMSO-d6 corresponds to the structure

- 24 040317

Example 16: characterization of the substance N of the free base of compound 1.

Technology detailed information Result XRPD - Substance N free base TGA from 25 to 350°C 0.2% weight loss at temperatures up to 100°C DSC from 25 to 350°C an endothermic event that occurs at about 94°C 'H NMR DMSO-d6 conforms to the structure, no residual solvent reaction was observed

Example 17 Competitive Interconversion of Suspensions between Free Base Forms I and II.

State Solvent Observation XRPD result

6°С, 6 days water white FBI 6°С, 6 days heptane white FBII 6°С, 6 days GRE faint pale yellow FBN

room temperature, 6 days water white FB YY room temperature, 6 days heptane whitish FB YY room temperature, 6 days GRE pale yellow FBN room temperature, 6 days toluene pale yellow FBN

57°С, 2 days water finely chopped, whitish, V FB YY + FB Y 57°C, during the night heptane disks and tablets, FB YY 57°C, during the night GRE discs, laminated, pale yellow, B FB YY

Example 18 Competitive Interconversion of Suspensions between Form II and Substance N.

State 35°C, 3 days heptane pale yellow, finely ground, B FBN 57°С, 3 days heptane large disks and sockets from disks, V FB P

Example 19: Selected Experimental Methods.

Indexing: X-ray diffraction patterns are indexed using proprietary SSCI software. The correspondence between the resolved peak positions, marked with red stripes within the figures, and the observed peaks indicates an agreed definition of the unit cell. The indexing and processing of structure information are computational studies that were performed in accordance with the Procedures for SSCI without following existing Good Manufacturing Practice (cGMP) procedures. To confirm the preliminary indexing, it is necessary to determine the molecular stacking motifs in the crystallographic unit cells. There were no attempts to determine the molecular fold.

Differential Scanning Calorimetry (DSC): DSC was performed using a Q2000 Differential Scanning Calorimeter from TA Instruments. Temperature calibration was performed using indium, which was tracked by NIST (National Institute of Standards and Technology). The sample was placed in an aluminum DSC crucible, covered with a lid, and the exact weight was recorded. A weighed crucible, configured in the same way as the experimental sample, was placed in the cell, which was used as a reference. Data collection parameters and crucible configuration for each thermogram were displayed as part of the picture in the data section of the report. The thermogram coding method is an abbreviation for the initial and final temperature, as well as the heating rate; for example -30-250-10 means a temperature range of -30 to 250°C at a temperature rise of 10°C/min. The following are the abbreviations used in each image for crucible configurations: zero crucible crucible temperature (T0C); the cover is not crimped (NC).

Dynamic Vapor Sorption (DVS): Dynamic Vapor Sorption (DVS) data has been obtained on

- 25 040317 VTI SGA-100 Vapor Sorption Analyzer. NaCl and PVP were used as calibration standards. Samples were not dried prior to analysis. Adsorption and desorption data were obtained from 5 to 95% RH in 10% RH increments with a nitrogen purge. The equilibrium criterion used for analysis was less than 0.0100% weight change over 5 minutes with a maximum equilibration time of 3 hours. The data was not corrected for the initial moisture content of the samples.

Microscopy.

Hot microscopy: Hot microscopy was performed using a Linkam hot microscopy camera (FTIR 600) mounted on a Leica DM LP microscope equipped with a SPOT Insight™ color digital camera. Temperature calibrations were performed using USP melting point standards. Samples were placed on a coverslip and a second coverslip was placed on top of the sample. Upon reaching the heating stage, each sample was visually assessed using a 20x objective with a 0.40 long-range digital aperture with crossed polarizers and a first order red compensator. Images were obtained using the SPOT software (version 4.5.9).

Microscopy in polarized light: samples obtained during the experiments were observed using a polarizing microscope in cross-polarization mode to reveal morphology and birefringence. The samples were observed visually at 40x magnification.

1H nuclear magnetic resonance solution (1H NMR).

SSCI: Samples were prepared for NMR spectroscopy as ~5-50 mg solutions in an appropriate deuterated solvent. The specific parameters obtained are listed on the graph of the first full spectrum of each sample in the data section for samples processed using SSCI.

Spectral Data Solutions: For sampling using Spectral Data Solution Service (subcontractor), 1H NMR spectra of solutions were acquired at ambient temperature on a Varian ^unity INOVA-400 spectrometer (1H Larmor precession frequency=399.8 MHz). Specific obtained parameters are indicated in the spectral data sheet and on each graph of the obtained spectral data of the sample.

Thermogravimetric analysis (TGA).

TG analysis was performed using a TA Instruments 2950 thermogravimetric analyzer. Temperature calibration was performed using nickel and Alumel™. Each sample was placed in an aluminum tray and placed in a TG oven. The furnace was heated in a stream of nitrogen. The data collection options were displayed above each image in the Data section of this report. The thermogram coding method is an abbreviation for the initial and final temperature, as well as the heating rate; for example, 25-350-10 means a temperature range of 25 to 350° C. at a temperature increase of 10° C./min. Using 0 as the start temperature indicates the start of sample analysis from ambient temperature.

XRPD analysis.

INEL: diffraction patterns were obtained using an Inel XRG-3000 diffractometer. An incident beam of Cu Κα radiation was created using a fine focusing tube and a parabolic multilayer mirror. Prior to analysis, a silicon standard (NIST SRM 640d) was analyzed to confirm position 111 of the Si peak. The sample fragment was placed in a thin-walled glass capillary, and the primary beam shutter was used to minimize the air background. The transmission geometry of the diffractograms was obtained using version 6.6 of the WinDiff software and a curved Equinox position-sensitive detector with a range of 2θ 120°. The acquisition parameters of each diffractogram are shown above the picture in the data section of this report.

Transmission diffraction measurement using a PANalytical diffractometer: Diffraction patterns were obtained using a PANalytical X'Pert PRO MPD diffractometer using an incident beam from Cu radiation produced with an Optix long fine focusing source. An elliptical multilayer mirror was used to focus the Cu Κα X-rays through the sample and onto the detector. Prior to analysis, a silicon standard (NIST SRM 640d) was analyzed to confirm position 111 of the Si peak. A fragment of the sample was placed between films 3 μm thick and analyzed according to the throughput geometry. A primary beam shutter, a short anti-scatter extension, and an anti-scatter reference prism were used to minimize the background created by the air. Soller slots for incident and reflected beams were used to minimize expansion from axial divergence. The diffraction patterns were acquired with a scanning position sensitive detector (X'Celerator) located 240 mm from the sample and data acquisition software version 2.2b. The acquired data parameters for each diffractogram are presented above the image in the data section of this report, including the deflection slit (DS) in front of the mirror and the antiscatter slit for the incident beam (SS).

Reflection diffraction measurement using a PANalytical diffractometer: Diffraction patterns were obtained using a PANalytical X'Pert PRO MPD diffractometer using an incident Cu beam from Κα radiation produced with a fine focusing source and a nickel filter. The diffractometer was set up using a symmetric Bragg-Brentano geometry. Prior to analysis, a silicon standard (NIST SRM 640d) was analyzed to confirm the Si peak position 111 according to the position certified by NIST. A fragment of the sample was prepared as a thin round layer centered on a silicon substrate with zero background. Anti-scatter slots (SS) were used to minimize the background created by the air. Soller slots for incident and reflected beams were used to minimize expansion from axial divergence. The diffraction patterns were acquired with a scanning position sensitive detector (X'Celerator) located 240 mm from the sample and data acquisition software version 2.2b. The acquired data parameters for each diffractogram are presented above the image in the data section of this report, including the deflection slit (DS) and the incident beam antiscatter slit (SS).

Approximate solubility: The weighed sample was treated with aliquots of the test solvent at room temperature. The mixture was sonicated between additions to improve dissolution. Complete dissolution of the test substance was determined by visual inspection. Solubility was calculated based on the total amount of solvent used to achieve complete dissolution. Then some samples were heated and visually assessed for complete dissolution. The actual solubility may be greater than the specified value due to the use of too large solvent aliquots or due to the slow dissolution rate. Solubility is expressed as a value less than if dissolution does not occur during the experiment.

If complete dissolution was achieved by adding only one aliquot, then the solubility is expressed as greater than.

Antisolvent Additions: Compound 1/organic solvent solutions were contacted with solvents that have poor or no solubility of Compound 1 in them. These antisolvent additives were added to reduce the solubility of the solvent systems and promote crystallization.

Cooling, slow cooling: Solutions were prepared in the chosen solvent or solvent/antisolvent system. These solutions were cooled below room temperature in a refrigerator for various periods of time in an attempt to induce the formation of the respective substances. The presence or absence of particulate matter was noted. Based on the observation of solid particles in quantities sufficient for analysis, the isolation of substances was carried out. If insufficient quantities were present to isolate, further cooling was carried out in a freezer. Samples were isolated for analysis as wet or dry powders.

Pressing: Individual samples were pressed using a KBr mold and a Carver hydraulic press. A load of 4.53 tons was applied to the die shaft for approximately 20 minutes.

Crystallization from solution: Saturated solutions were prepared at ambient temperature and then stoppered. When evaluating the free base of compound 1, the formation of crystals from these systems was found.

Rapid Evaporation: Solutions were prepared in selected solvents and mixed between additional aliquots to promote dissolution. After the mixture had reached complete dissolution, as judged by visual observation, the solution was evaporated at ambient temperature in an open vial or at ambient temperature under nitrogen. The resulting solids were isolated for evaluation.

Grinding: Selected materials were ground using a Reitch grinder. The material was loaded into an agate grinding bowl followed by an agate pestle. The vessel was then placed on a grinder and crushed for about 30 min at a frequency of 1/30 s. Grinding was stopped approximately every 10 minutes and the material was removed from the walls before further grinding.

Suspension: Solutions were prepared by adding sufficient solids to the available solvent such that an excess of solids was present. The mixture was then stirred in a sealed tube at ambient or elevated temperature. After a certain time, the solids were isolated for analysis.

Temperature and Relative Humidity Stress: Selected substances were stressed at elevated relative humidity and/or temperature. Relative humidity vessels (saturated saline solutions were used to obtain the desired relative humidity) were used to store the selected samples. The following relative humidity vessels were used in the evaluation: 75% RH (NaCl) and 60% (NaBr) to investigate the effect of humidity. The ambient temperatures used were 30, 40, 60 and 100-125°C.

Vacuum: Selected substances were stressed at reduced pressure for a specified period of time. The initial stress exposure was conducted in an internal vacuum system with absolute pressure readings of <500 millitorr, typically between 30 and

- 27 040317 mitorr (0.030 to 0.05 mmHg). Additional stress exposure was carried out at 48 mm Hg. using a portable laboratory vacuum pump and starter to simulate conditions similar to those expected in the process.

Example 20: disproportionation of the HCl salt.

Disproportionation of the HCl salt in water has been used to obtain the free base. Form I of the free base is formed first. An increase in the residence time in suspension triggers the transformation into a more thermodynamically stable phase with respect to form I - form II of the free base.

Three anhydrous free base substances have been identified; forms I, II and substance N of the free base. Substance N, the free base, appears to be the most stable form, relative to Forms I and II, at room temperature. The free base substance N is enantiotropic with respect to form II and will reversibly convert at a certain transition temperature (estimated according to the present application at a temperature of about 40-42°C). Above the transition temperature, form II of the free base appears to be the most stable form with respect to form I and substance N.

Salt HCl (named form I HCl) was exposed to various stress conditions and monitored using XRPD to assess physical stability. As already discussed, disproportionation occurred during the DVS experiment of the HCl salt, indicating the presence of instability when exposed to high humidity. Disproportionation was further observed using wet milling or in direct contact with water (eg slurry) as indicated by the presence of Forms I or II of the free base as determined by XRPD. Evaporation and loss of HCl under heating and/or vacuum conditions are represented by the presence of form I of the free base, determined using XRPD and indicating instability under these conditions.

Contact with water resulted in a visual change in the color of the substance from pale yellow to white; physical changes were also observed under the microscope. There is an immediate disproportionation. The substance obtained from the aqueous suspension was determined by XRPD analysis (~ 5 min) as form I of the free base. Also determination of form II of the free base becomes possible if the residence time in suspension is increased.

Evaporation of HCl was observed within a few hours when kept under drying conditions. Conversion to form I of the free base was observed using XRPD at 30°C (after 12 h), at 40°C (after 6 h) and at 40°C/48 mm Hg. (after 6 h).

The determination of substance C free base becomes possible under more extreme conditions associated with elevated temperatures. Heating Form I HCl to 125° C. causes loss of acidic volatiles (visually assessed using a pH indicator paper placed over the sample). Using XRPD analysis, the resulting sample is defined as a mixture of Form I HCl, Form I free base and Substance C free base. Keeping the HCl salt at up to 60°C under vacuum for 6 days provides the same result. The nature of substance C has not been determined.

The HCl salt has been shown to disproportionate immediately upon contact with water. This phenomenon was used to obtain a free base. Form I of the free base is formed first. An increase in the residence time in suspension triggers the transformation into a more thermodynamically stable phase with respect to form I - form II of the free base.

A 20 ml ampoule was loaded with 266.4 mg Form I HCl and brought into contact 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 (removal of water) and washed with 10 ml of water. The sample was purged with nitrogen for approximately 10 minutes before being placed under vacuum overnight to dry at ambient temperature. The material obtained was analyzed by XRPD and found to be form I of the free base.

A 250 ml Erlenmeyeran conical flask was charged with 6.0250 g of Form I HCl and contacted with 220 ml of water. The sample was sonicated for about 5 minutes to disperse the material. The yellow substance changed color to white upon sonication. Using a stirring magnet, the sample was stirred at 700 rpm for approximately 10 minutes. The solids were collected by filtration and washed with 220 ml of water, followed by a nitrogen purge over the sample for approximately 10 minutes before being subjected to a vacuum at ambient temperature. The sample was dried under these conditions for approximately 24 hours, yielding 5.1834 g of material. The resulting material was analyzed by XRPD and identified as a mixture of form I free base and substance D free base (the nature of substance D is unknown).

The procedure used to obtain Form II of the free base is described below.

A 20 ml ampoule was loaded with 477.5 mg Form I HCl number 20 and contacted with 20 ml of water.

- 28 040317

The sample was sonicated until the pale yellow material changed color to white. A small amount of sample (a mixture of form I and II free base) was added as a seed. Using a stirring magnet, the sample was stirred at 200 rpm for 8 days. The resulting solids were collected by filtration (removal of water) and washed with 15 ml of water. The sample was exposed under vacuum at ambient temperature overnight to dry. The resulting substance was analyzed using XRPD, and it was found that the substance was form II of the free base.

Example 21: additional procedures for obtaining Form I, Form II and Form N of the free base.

Conversion of the free base of compound 1 to the HCl salt.

Basic procedure: Slowly add concentrated HCl (1.5 eq.) to a solution of compound 1 free base in MEK (5 volumes). Cool the resulting suspension to 0-5°C for 1 hour and filter. Wash the solid with MEK (1 volume). Dry under vacuum at 30-35°C.

Preparation A: Following the general procedure described above, 35 g of crude compound 1 was processed to give the HCl salt as a pale yellow solid (32.4 g, 82 yield, 99.8% pure by HPLC).

Obtaining form I of the free base from the HCl salt of compound 1.

Basic procedure: Stir vigorously a suspension of the HCl salt of compound 1 in DIW (10 volumes) for a period of 5 minutes to 2 hours. Filter the suspension, wash with DIW (2x1 volume), dry on a funnel, then additionally dry under vacuum at 35°C.

Preparation A: Following the general procedure described above, after stirring for 1 hour, 32 g of the HCl salt of compound 1 was treated to give the free base as a pale yellow solid (27.3 g, yield 95, 99.8 HPLC purity, DSC identified form I).

Preparation B: Following the general procedure described above, after stirring for 1 hour, 39 g of the HCl salt of Compound 1 was worked up to give the free base as a pale yellow solid (31.8 g, 90% yield, >99 .9% purity by HPLC).

Preparation Procedure C: Thus, the HCl salt of Compound 1 (134 g) was vigorously stirred in water (10 volumes) until the material was in the form of a fine white suspension. After filtration and drying, a white crystalline solid was obtained (116 g, 96% yield, >99.9% purity by HPLC).

Preparation Procedure D: The purpose of this experiment was to prepare the free base from the hydrochloride of compound 1. Thus, the HCl salt of compound 1 (65.3 g) was vigorously stirred in water (10 volumes) until the substance appeared as a fine white suspension. After filtration and drying, a white crystalline solid was obtained (57.5 g, 97.6% yield, >99.9% purity by HPLC).

Preparation of GBT000440 form II free base from GBT000440 form I free base.

Basic procedure: A free base suspension of Form I Compound 1 is stirred in an appropriate solvent (eg heptane or water) (10 volumes) for 1-7 days. The suspension is filtered, washed with DIW (2x1 volume), dried on a funnel, then dried further under vacuum at a temperature of 30-35°C.

Procedure for obtaining A: thus, form I of the free base of compound 1 (114 g) is stirred in n-heptane (10 volumes) at a temperature of 35°C. After 4 days, XRPD revealed form II. The suspension was filtered and dried to give 110 g of an off-white solid.

Preparation Procedure B: Compound 1 free base (5 g) was suspended in heptane (10 volumes, 50 ml) at room temperature. After 4 days, the suspension was filtered to give an off-white solid.

Preparation Procedure C: Compound 1 free base (5.8 kg) was suspended in heptane (10 volumes) at room temperature. After 2 days, the suspension was filtered and washed with 2x2 volumes of n-heptane to give 4.745 kg of Form II as an off-white solid.

Preparation D: Compound 1 free base (5 g) was suspended in water. After 4 days, the suspension was filtered to give an off-white solid.

Preparation of GBT000440 form N free base from GBT000440 form I or II free base.

Basic procedure: a suspension of Form I Compound 1 free base in MTBE (4 volumes) is stirred at room temperature for at least 4 days. After 4 days, the suspension is filtered to give an off-white solid. An XRPD is obtained to confirm that the polymorph is substance N.

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Production Procedure A: Following the basic procedure described above, 27 g of form I (48TRS079) of Compound 1 free base was stirred in MTBE at 18-23°C for 4 days.

DSC results indicate that substance N should be obtained. 22.2 g of a cream-colored solid isolated (82% recovery, 99.9% pure by HPLC). Analysis planned

XRPD.

Preparation Procedure B: Following the basic procedure described above, 31 g of compound 1 free base Form I was stirred in 3 volumes of MTBE at 18-23° C. for 4 days.

Preparation Procedure C: Form I (13KWB023, 1 g), Compound 1 free base was suspended in MTBE (5 volumes) at room temperature. Substance N was added as a seed (50 mg) to the suspension. After 4 days, the suspension was filtered to give an off-white solid. The melting point determined by DSC was the same as for substance N.

Procedure for Preparation D: The purpose of this experiment was to convert form II of the free base of compound 1 to substance N. Thus, the free base of compound 1 (0.5 g) was stirred in 5 volumes of di-p-propyl ether at a temperature of 18-23°C. After 2 days, the DSC results were consistent with the pattern observed for substance N. XRPD analysis confirmed the formation of substance N.

Preparation Procedure E: Diisopropyl ether (5 vols, 25 ml) was added to form II of the free base of Compound 1 (5 g) at room temperature. After 4 days, the suspension was filtered to give an off-white solid. DSC results confirmed the presence of substance N.

Example 22: Solvent based pre-selection procedures.

Rapid solvent-based procedures were carried out to determine the most stable free base form of Compound 1. The study also provides a preliminary assessment of the propensity of these substances to exist in various crystalline forms. Solids formed were observed by polarized light microscopy (PLM) and/or analyzed by X-ray solid matter analysis (XRPD), comparing the obtained diffraction patterns with known samples for compound 1.

If possible, the diffractograms are indexed. Indexing is the process of determining the size and shape of a crystallographic cell, given the positions of peaks in a diffraction pattern. The term derives its name from the assignment of a crystallographic Miller index to individual peaks. XRPD indexing serves several purposes. If all the peaks in a sample are indexed by the same unit cell, this is strong evidence that the sample contains a single crystalline phase. Given an indexing solution, the unit cell volume can be calculated directly and used to determine their solvation state. Indexing is also a reliable way to describe the crystal form and provide a concise summary of all available peak positions for that phase at a particular point in the thermodynamic state.

Substances with unique crystal diffraction patterns based on visual evaluation of the peaks associated with these substances were assigned letter designations. The letter designator is tentatively associated with the word Substance if insufficient characterization data is available. The nomenclature is used only for the purpose of facilitating the identification of unique diffractograms and does not imply that the stoichiometry, the purity of the crystalline phase, or the chemical purity of the substance is known. In the following, substances are reported as designations of the form with a Roman numeral (i.e. substance A of the free base = form I of the free base) when the purity of the phase (obtained by X-ray indexing or crystal structure elucidation) and chemical identity/purity (obtained by spectroscopy) are determined. proton magnetic resonance) material.

Three anhydrous species have been identified: Forms I, II and Substance N. Substance N appears to be the most stable form, relative to Forms I and II, at room temperature. Substance N is enantiotropic with respect to form II and will reversibly transform at a certain transition temperature (estimated at a temperature of about 42°C). Above the transition temperature, form II appears to be the most stable form with respect to form I and substance N.

Substances C and D are used to identify several additional low intensity peaks predominantly observed in the X-ray diffraction patterns of Compound 1 Form I free base or mixtures of HCl Form I and Compound 1 Free Base Form I.

Example 23 Anhydrous Desolvate Forms.

Form I

Form I of the free base is a metastable, anhydrous phase of the free base, which is formed immediately upon disproportionation of the HCl salt in water. The exemplary diffraction pattern of Form I was successfully indexed and the unit cell volume is consistent with the anhydrous free base. Visual comparison of the diffraction pattern with the free base factual data provided indicates that the substance may be similar; however, the actual data pattern provides additional peaks for the potential mixture.

The ¹ H-NMR spectrum is consistent with the chemical structure of compound 1. The chemical shift of approximately 2.5 ppm is due to the presence of DMSO (due to residual protons in the solvent

- 30 040317 for NMR). Peaks that may be associated with the presence of residual solvents were not visible, which corresponds to the anhydrous unit cell volume determined by indexing the solution without and taking into account the slight loss of wt% observed during TGA, discussed below.

Thermogram data (TG) shows a slight weight loss, 0.2%, at temperatures up to 100°C, which corresponds to the anhydrous form. DSC shows one endotherm with the onset of about 97°C (similar to what is observed for form II). Endotherm is consistent with melting by hot microscopy. However, changes in particle size and birefringence were evident up to the point of melting; a phase transition is possible. Therefore, if a phase change occurred and an endotherm similar to form II of the free base was detected, it can be concluded that the observed melting is characteristic of form I, and the resulting phase is most likely form II.

The DVS isotherm indicates that Form I is not hygroscopic. A slight increase and weight loss of 0.2% was observed during sorption/desorption. Based on the XRPD results, the material recovered from the DVS experiment was predominantly free base form I with a few additional peaks. Additional peaks were designated substance D free base. The nature of substance D is unknown; however, the appearance of other phase(s) means that Form I is unlikely to be physically stable under conditions of high humidity (ambient temperature).

Form II.

Form II of the free base is the anhydrous phase of the free base. Form II is enantiotropically bound to substance N, which is a thermodynamically stable form above the expected transition temperature of 42°C. Form II can be prepared in solvents that do not form known solvates, such as heptane, IPE, MTBE, or toluene, by short-term transformations of form I in suspension (where the crystallization kinetics retard formation of a more stable form) or by increasing the temperature of the suspensions (above 42° WITH). The illustrative diffraction pattern of Form II was indexed successfully and the unit cell volume is consistent with Compound 1 anhydrous free base.

The ¹ H-NMR spectrum is consistent with the chemical structure of compound 1. The chemical shift of approximately 2.5 ppm is due to the presence of DMSO (due to residual protons in the NMR solvent). Peaks that could be associated with the presence of residual solvents were not visible, which was consistent with the anhydrous unit cell volume determined by indexing the solution without and taking into account the slight loss of wt% observed during the TGA discussed below.

Thermogram data (TG) shows a slight weight loss, 0.1%, at temperatures up to 100°C, which corresponds to the anhydrous form. DSC results show one endotherm (80.1 J/g) starting at about 97°C.

Form II remained unchanged after 7 days of storage at room temperature, as shown by repeated XRPD analysis. The form is known to be thermodynamically metastable with respect to substance N in this state; however, the polymorph conversion kinetics may be slow at ambient solid state conditions.

Substance N.

The free base substance N is the anhydrous phase of the free base. Substance N is enantiotropically bound to form II, which is a thermodynamically stable form below the expected transition temperature of 42°C. Where possible, substance N can be prepared using suspensions in solvents that do not form known solvates such as heptane, IPE, MTBE or toluene at temperatures below 42°C. The following is an example of a procedure that is used to obtain substance N free base on a laboratory scale.

53.0 mg Form I free base was added to 2 ml IPE/free base solution (concentration 13 mg/ml). Using a stirring magnet, the sample was stirred for 7 days at ambient temperature. The solution was decanted from the sample and the remaining solid was quickly dried under nitrogen. These characteristics indicate that substance N is a unique crystalline phase.

The ¹ H-NMR spectrum is consistent with the chemical structure of compound 1. The chemical shift of approximately 2.5 ppm is due to the presence of DMSO (due to residual protons in the NMR solvent). Peaks that could be associated with the presence of residual solvents were not visible, which was consistent with the slight loss in wt % observed during the TGA discussed below.

Thermogram data (TG) shows a slight weight loss, 0.2%, at temperatures up to 100°C, which is consistent with the anhydrous form. DSC results show one endotherm (82.8 J/g) starting at about 94°C.

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Preliminary determination of the thermodynamic relationship between Forms I, II and substance N of the free base.

These characteristics show that Forms I, II and substance N are unique crystalline phases; however, only the diffraction patterns of Forms I and II were successfully indexed to confirm phase purity. Thus, any proposed thermodynamic relationship between these materials is a working hypothesis, where the purity of the substance N phase is conjectural.

Phase transitions in solids can be thermodynamically reversible or irreversible. Crystalline forms that reversibly transform at a certain transition temperature (Tp) are called enantiotropic polymorphs. If the crystalline forms are not interconvertible under these conditions, the system is monotropic (one thermodynamically stable form). Several rules have been developed with the aim of being able to predict the relative thermodynamic stability of polymorphs and the relationship between polymorphs (enantiotropic or monotropic). In this study, the fusion temperature rule is applied. The fusion temperature rule states that if the higher melting form has a lower fusion temperature, then the two forms are enantiotropic, otherwise they are monotropic.

Substance N appears to be the most stable form, relative to Forms I and II, at room temperature. Based on the fusion and melt temperatures determined using DSC, substance N is enantiotropic with respect to form II and will reversibly transform at a certain transition temperature (TN-II). Due to the possible phase change from form I to form II that occurs before the endotherm is observed in DSC, the relationship of form I to substance N or form II cannot be definitively determined by the fusion temperature rule. However, it has been shown through various suspension interconversions that Form I is the least thermodynamically stable form in the temperature range between 6°C and TN-II. In addition, from the assumption that Form I spontaneously converts to Form II when DSC is performed at elevated temperatures (before melting is observed), it should follow that Form II is also more stable than Form I above the TN-II temperature.

Example 24: Estimated junction temperature.

The calculated transition temperature between two enantiotropically related forms can be calculated from the melt onset temperature and the fusion temperature based on the equation below.

AHf.2

Tf.2 'TfJ ⁽

Where (Cp.liq ^- Cp.l) ⁼ ΔΗ _Π and k=ο,οοδ

The equation estimates the transition temperature at 42°C between substance N and form II. To summarize, the relative shape stability from most to least stable is shown below.

Temperature range* Relative stability Comments Below 6°С N>II Relationship with Form I not established below this temperature Between 6°C and T^' II N > II - Above T^' ¹¹ (II > N) and (II > I) The relationship between Form I and Substance N is not established above this temperature

* TN-II is approximately equal to 42°C.

Example 25: temperature-energy diagram.

The temperature-energy diagram of FIG. 17 is a semi-quantitative graphical solution of the Gibbs-Helmholtz equation, where the enthalpy (H) and free energy (G) isobars for each form are presented as a function of temperature.

Example 26 Competitive Suspension Interconversion Experiments.

Interconversion experiments are illustrated with temperature-energy diagrams and were performed to confirm the thermodynamic relationship between polymorphs. Suspension interconversion or competition experiments are a solution-mediated process that allows a less soluble (more stable) crystal to grow at the expense of a more soluble crystal form. Outside of solvate formation or degradation, it is assumed that the resulting more stable polymorph from the interconversion experiment is independent of the solvent used, since the more thermodynamically stable polymorph has lower energy and hence lower solubility. The choice of solvent affects the kinetics of polymorph transformation and does not affect the thermodynamic relationships between polymorphs.

The results of the interconversion studies are consistent with the preliminary energy-temperature diagram given in the present invention. Double solutions were prepared at ambient temperature, 6 and 57°C using Forms I and II. Form II, obtained from most of these experiments, confirms that form II is more stable with respect to form I within this temperature range.

Some of the experiments performed at ambient temperature and 6°C resulted in the formation of substance N. Although these experiments do not provide specific clarification regarding the differences between forms I and II, they provide evidence that substance N is the most stable form in relation to to both forms I and II at these temperatures (the experiments were carried out at temperatures below the calculated transition temperature, which is 42°C). Additional slurry interconversions between Form II and Substance N were performed at temperatures that did not correspond to the calculated transition temperature and confirmed that Form II and Substance N were enantiotropically related.

Example 27 Solid state NMR.

¹³ C and ¹⁵ N spectra were obtained for the three polymorphs I, II and substance N. See FIG. 10 and 11. Spectra were taken at 253K to prevent low temperature transitions occurring during parameter measurements and to obtain parameters optimized for each polymorph.

Based on solid state nuclear magnetic resonance data, all three forms are crystalline and are various polymorphic forms. Form I contains one molecule per asymmetric unit, form II contains two molecules per asymmetric unit, and form N contains four molecules per asymmetric unit. See ¹⁵ N spectra in FIG. eleven.

Example 28 Chemical and Physical Stability Form I of the Free Base of Compound 1.

A mixture predominantly consisting of Free Base Form I (with Free Base Substance D) was subjected to stable conditions to assess physical and chemical stability. Three conditions were used: open state, temperature up to 25°C/60% RH; open state, temperature up to 40°C/75% RH; closed state, temperature up to 60°С. Physical stability was assessed using XRPD. Chemical stability was determined using UPLC and 1H NMR where possible. Substances were tested after 1, 7 and 14 days of exposure.

Chemical stability of Form I free base.

For the free compound stability sample, UPLC showed very low levels of impurities present. Impurity levels did not increase significantly after 14 days of storage. This most likely indicates good chemical stability under the conditions used to evaluate the stability. The 1H NMR spectra of samples that were exposed to a temperature of 60°C (14 days) also agree with this conclusion.

Physical stability of Form I free base.

According to the XRPD results, the free base of compound 1 remained unchanged at 25°C/60% RH. However, differences were observed under two other conditions. Several minor peaks attributed to substance D of the free base were not observed, indicating that substance D is metastable and not stable at elevated temperatures. In addition, form II of the free base was found after 7 days of storage. This is in line with the findings of the present invention that Form II of the free base is more stable than Form I of the free base at these temperatures.

Example 29 Evaluation of the Physical Stability of Form II and Substance N (Form N) of Compound 1 Free Base.

During DSC, the temperature was modulated at a low heating rate followed by powder X-ray diffraction analysis. The heating rate used was 0.02° C. min ^-1 . The temperature was 80°C for form N and 90°C for form P°C. The exposure was essentially isothermal covering a sensitive range of temperatures to detect changes in physical form. The resulting materials were analyzed using X-ray powder diffraction analysis. No change in physical form was observed for either Form II polymorph or Form N polymorph (ie material N).

Forms II and N were exposed to 40°C/75% relative humidity (RH), 80°C, 80°C/80% RH for 9 days followed by X-ray diffraction analysis of the powder. No change in physical form was observed for either polymorph form II or polymorph form N.

The thermodynamic barrier to interconversion between polymorph form II and form N is high and the physical stability is good for both forms. Thermally induced

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Claims (11)

interconversion between form II and form N is unlikely. Example 30: relative thermodynamic stability of polymorphs II and N. Extensive studies on the effect of solvent on the formation of molecules were carried out using mixtures of 1:1 wt./wt. polymorph form II and form N. Hexane provides a favorable environment for evaluating solvents. The temperatures that have been used include -20, -10, 0, 10, 20, 30, 40 and 50°C. Significantly higher solubility was observed at 30, 40 and 50°C. Solids obtained by forming at -20, -10, 0, 10, 20° C. were analyzed by powder X-ray diffraction analysis. In each case, significant conversion to the N form was observed. Form N is thermodynamically more stable than form II at 20° C. and below. The enantiotropic relationship between the two forms is likely to be equivalent in terms of thermodynamic stability over the temperature range of 30-40°C. Example 31: Form N morphology. An initial evaluation of a batch of the polymorphic form N indicates its acicular morphology. Although the present invention has been described in conjunction with specific embodiments of the invention and examples, one of ordinary skill in the art will understand, given the skill and description provided, that equivalents of the specifically described materials and methods can also be applied to the present invention; and such equivalents will also be included in the following claims. CLAIM 1. Crystalline desolvate of compound 1 Compound 1 characterized by at least three powder X-ray diffraction (Cu Ka radiation) peaks selected from 11.65, 11.85, 12.08, 16.70, 19.65 and 23.48° 2Θ (each ±0.2 ° 2θ). 2. Crystalline desolvate according to claim 1, characterized by an endothermic peak at 95±2°C, measured by differential scanning calorimetry. 3. Pharmaceutical composition containing a pharmaceutically acceptable excipient and a crystalline desolvate of compound 1 Compound 1 characterized by at least three powder X-ray diffraction (Cu Ka radiation) peaks selected from 11.65, 11.85, 12.08, 16.70, 19.65 and 23.48° 2Θ (each ±0.2 ° 2θ). 4. Pharmaceutical composition according to claim 3, in which the crystalline desolvate of compound 1 is characterized by an endothermic peak at 95±2°C, measured by differential scanning calorimetry. 5. Use of a crystalline desolvate according to any one of claims 1, 2 to increase the oxygen affinity of hemoglobin S in a subject. 6. The use of a crystalline desolvate according to any one of claims 1, 2 for the treatment of sickle cell anemia. 7. The use of a crystalline desolvate according to any one of claims 1, 2 for the treatment of a hemoglobin-mediated disorder. 8. The use of a pharmaceutical composition according to any one of claims 3, 4 to increase the oxygen affinity of hemoglobin S in a subject. 9. The use of a pharmaceutical composition according to any one of claims 3, 4 for the treatment of sickle cell anemia. 10. The use of a pharmaceutical composition according to any one of claims 3, 4 for the treatment of a hemoglobin-mediated disorder. 11. Use according to claim 7 or 10, wherein the hemoglobin is sickle cell hemoglobin. -