EA041474B1 - METHODS FOR OBTAINING AG-10, ITS INTERMEDIATES AND THEIR SALTS - Google Patents

Description

Prior Art

Aberrant protein interaction and aggregation, either through protein misfolding or signaling pathway activation, is the underlying cause of a large number of human degenerative diseases. Thus, targeting protein-protein interactions (PPIs) is of therapeutic interest.

To date, approved PPI inhibitors are proteins, not small molecule inhibitors. For example, therapeutic monoclonal antibodies (mAbs) are used to treat cancer, autoimmune, infectious, and neurodegenerative diseases. Therapeutic mAbs are expensive to manufacture, require injection, and can elicit an immune response in the patient. For these reasons, the development of small molecule PPI inhibitors is of interest.

One such example of aberrant protein aggregation is the soluble protein transthyretin (TTR or prealbumin). TTR is a 55 kDa homotetrameric protein found in blood and cerebrospinal fluid. When TTR dimers dissociate from their homotetrameric form, they can become amyloidogenic monomers. This has been observed with wild-type TTR, and with over 100 different mutated variants. Studies have shown that stabilization of the tetrameric form of TTR inhibits the misfolding of amyloidogenic monomers and the subsequent formation of TTR amyloid.

Recent studies have identified 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4fluorobenzoic acid (AG-10) as a promising candidate for the treatment of TTR-amyloid related diseases such as TTR - amyloid cardiomyopathy. This compound has been disclosed in WO 2014/100227. Notably, the disclosure does not provide any additional forms of AG-10 and the synthesis method described is not suitable for industrial production.

Thus, there is a need to obtain improved methods for the synthesis of AG-10 and provide additional forms of AG-10 that provide beneficial pharmacokinetic properties. The present disclosure addresses these needs, and offers related benefits.

Short description

In one aspect, the present invention provides an improved process for the preparation of a compound of formula IX.

including:

(a) contacting a compound of formula II

with a compound of formula III

a first base and a first organic solvent to give a compound of formula IV

O _(IV) where each R ¹ is independently a halogen or a sulfonic acid ester;

(b) contacting a compound of formula IV with hydrazine and a second organic solvent to obtain a compound of formula V

HNA' (V) (c) contacting a compound of formula V with a sulphonating agent or a halogenating agent to obtain a compound of formula VI

where R ² represents a halogen or ester of sulfonic acid;

- 1 041474 (d) contacting a compound of formula VI with a compound of formula VII

a second base and a third organic solvent to give a compound of formula

VIII

COOR ³

NH(VIII), where R ³ is selected from the group consisting of C _1-2 αlkyl, C _2-12 αlkenyl, C _2-12 alkynyl, C _3-8 cycloαlkylα, heterocycloalkyl, aryl and heteroaryl, each of which is optionally substituted ;

(e) contacting a compound of formula VIII with a third base to obtain a compound of formula IX.

In a second aspect, the present disclosure relates to a pharmaceutically acceptable salt represented by formula I or Ib

where X is a pharmaceutically acceptable protic acid anion and Y is a multiprotic acid.

In a third aspect, the present invention provides crystalline forms A-K of formula IX.

Other features, elements and aspects of the present invention will be apparent from the accompanying drawings and from the detailed description that follows.

Brief description of the drawings

In FIG. 1 is a schematic of the preparation of AG-10 and its intermediates described herein.

In FIG. 2 shows pharmacokinetic results demonstrating high bioavailability of AG-10 in a variety of species.

In FIG. 3 shows pharmacokinetic results demonstrating high bioavailability of AG-10 in male and female dogs at different dosage levels.

In FIG. 4 shows the X-ray powder diffraction (XRPD) pattern of the mesylate salt of Formula IX.

In FIG. 5 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of the formula IX mesylate salt.

In FIG. 6 shows the X-ray powder diffraction (XRPD) pattern of the edisylate salt of formula IX.

In FIG. 7 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of the edisylate salt of formula IX.

In FIG. 8 shows the X-ray powder diffraction (XRPD) pattern of the formula IX besylate salt.

In FIG. 9 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of the formula IX besylate salt.

In FIG. 10 shows the X-ray powder diffraction (XRPD) pattern of the tosylate salt of formula IX.

In FIG. 11 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of the formula IX tosylate salt.

In FIG. 12 shows the X-ray powder diffraction (XRPD) pattern of the esylate salt of formula IX.

In FIG. 13 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of the esylate salt of formula IX.

In FIG. 14 shows the X-ray powder diffraction (XRPD) pattern of the bromide salt of formula IX.

In FIG. 15 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of the bromide salt of Formula IX.

- 2 041474

In FIG. 16 shows the X-ray powder diffraction (XRPD) pattern of Form a of the nitrate salt of formula IX.

In FIG. 17 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of form a of the nitrate salt of formula IX.

In FIG. 18 shows the X-ray powder diffraction (XRPD) pattern of Form b of the nitrate salt of formula IX.

In FIG. 19 shows the X-ray powder diffraction (XRPD) pattern of the sulfate salt of Formula IX.

In FIG. 20 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of the sulfate salt of formula IX.

In FIG. 21 shows an X-ray powder diffraction (XRPD) pattern of an oxalate salt of formula IX.

In FIG. 22 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of an oxalate salt of formula IX.

In FIG. 23 shows an X-ray powder diffraction (XRPD) pattern of Form a of the maleate salt of formula IX.

In FIG. 24 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of form a of the maleate salt of formula IX.

In FIG. 25 shows the X-ray powder diffraction (XRPD) pattern b of the maleate salt of formula IX.

In FIG. 26 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of form b of the maleate salt of Formula IX.

In FIG. 27 shows an X-ray powder diffraction (XRPD) pattern of an acetic acid salt of formula IX.

In FIG. 28 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of an acetic acid salt of formula IX.

In FIG. 29 shows an X-ray powder diffraction (XRPD) pattern of an L-malic acid salt of formula IX.

In FIG. 30 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of an L-malic acid salt of formula IX.

In FIG. 31 shows an X-ray powder diffraction (XRPD) pattern of crystalline form A of formula IX (3 different samples).

In FIG. 32, 33, 34 show thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline Form A of formula IX.

In FIG. 35 shows a polarized light microscopy (PLM) image of crystalline form A of formula IX.

In FIG. 36 shows an asymmetric unit of crystalline form A of formula IX.

In FIG. 37 shows dynamic vapor sorption (DVS) data for crystalline form A of formula IX.

In FIG. 38 shows X-ray powder diffraction (XRPD) patterns of crystalline form A of formula IX before (bottom) and after (top) DVS.

In FIG. 39 shows a diagram summarizing the interconversions between identified crystal forms A, B, C, E, G, H, I, J.

In FIG. 40 shows an X-ray powder diffraction (XRPD) pattern of crystalline Form B of formula IX.

In FIG. 41 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline form B of formula IX.

In FIG. 42 shows X-ray powder diffraction (XRPD) patterns of crystalline form B of formula IX before heating (top), heating to 100° C. (second from top), heating to 170° C. (second from bottom), and the reference crystalline Form I (bottom). When heated, Form B converts to Form I.

In FIG. 43 shows an X-ray powder diffraction (XRPD) pattern of crystalline Form C of Formula IX.

In FIG. 44 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline Form C of formula IX.

In FIG. 45 shows an X-ray powder diffraction (XRPD) pattern of crystalline form D of formula IX (upper panel) and crystalline form F of formula IX (lower panel).

In FIG. 46 shows an X-ray powder diffraction (XRPD) pattern of crystalline Form E of formula IX.

In FIG. 47 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline form E of formula IX.

In FIG. 48 shows X-ray powder diffraction (XRPD) radiographs of a crystalline

- 3 041474 form E of formula IX before heating (top), when heated to 195°C (center) and reference crystalline form I (bottom). When heated, Form E converts to Form I.

In FIG. 49 shows an X-ray powder diffraction (XRPD) pattern of crystalline form G of formula IX.

In FIG. 50 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline form G of formula IX.

In FIG. 51 shows an X-ray powder diffraction (XRPD) pattern of crystalline form H of formula IX.

In FIG. 52 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline form H of formula IX.

In FIG. 53 shows the 1H NMR spectrum of the crystalline form H of formula IX.

In FIG. 54 shows X-ray powder diffraction (XRPD) patterns of crystalline form H of formula IX before heating (top), when heated to 120° C. (center), and reference crystalline form I (bottom). When heated, form H transforms into form I.

In FIG. 55 shows an X-ray powder diffraction (XRPD) pattern of crystalline Form I of formula IX.

In FIG. 56 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of Formula IX crystalline Form I.

In FIG. 57 shows X-ray powder diffraction (XRPD) patterns of crystalline form I of formula IX (reference sample, top), before N2 purge (second from top), after 1.5 hour N2 purge (second from bottom), and reference crystalline Form B (bottom) . After purging with N2, form I turns into form B.

In FIG. 58 shows a thermogravimetric analysis (TGA) plot of crystalline Form I of formula IX.

In FIG. 59 shows an X-ray powder diffraction (XRPD) pattern of crystalline Form J of formula IX.

In FIG. 60 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline form J of formula IX.

In FIG. 61 shows the 1 H NMR spectrum of crystalline form J of formula IX.

In FIG. 62 shows X-ray powder diffraction (XRPD) patterns of Formula IX Crystal Form J before heating (top), heating to 130° C. (second from top), reference Crystal Form A (second from bottom), and reference Crystal Form I (bottom). When heated, Form J transforms into a mixture of Form A and Form I.

In FIG. 63 shows an X-ray powder diffraction (XRPD) pattern of crystalline form K of formula IX.

In FIG. 64 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) plots of crystalline form K of formula IX.

Detailed description

I. General.

The present disclosure specifically provides an improved process for the preparation of a compound of formula IX (AG-10) and its intermediates. The newly described process provides high yields and improved efficiency.

While a complete summary diagram is provided in the Summary of the Invention and in Scheme 1 (FIG. 1), one skilled in the art will appreciate that the selected steps of the present process are novel and can be performed regardless of the origin of the starting material or intermediates.

Also provided herein is a pharmaceutically acceptable salt of Formula I and Formula Ib. The pharmaceutically acceptable salts of formula I and formula Ib have surprising pharmacokinetic properties that improve the bioavailability of the compound of formula IX. Without wishing to be bound by any particular theory, it is believed that a pharmaceutically acceptable salt of formula I and formula Ib provides a protonated pyrazole in a compound of formula IX that combines with a protic acid or multiprotonic acid anion. Unlike the pharmaceutically acceptable salts of formula I and formula Ib, salts derived from alkali hydroxides such as NaOH or the zwitterion of a compound of formula IX do not provide the advantageous features described herein. In specific embodiments, a compound of formula I is a compound of formula Ia, an HCl salt of formula I.

II. Definitions.

The term compound of formula IX refers to 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4fluorobenzoic acid, also known as AG-10, a compound with the following structure

- 4 041474

The terms used in this document (in English) include not only aspects with one element, but also include aspects with more than one element. For example, singular forms include plural denominators unless the context clearly indicates otherwise. Thus, for example, a reference to a cell includes a plurality of such cells, and a reference to an agent includes a reference to one or more agents known to those skilled in the art, and so on.

The term alkyl refers to a linear or branched, saturated, aliphatic radical having the specified number of carbon atoms. Alkyl may include any number of carbon atoms ^{such as} C1-2, ^C1-3 , C1-4 ^, C1-5, C1-6, ^{C1-7 ,} C1-8, C1-9, C1-10, C2-3 , C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C _5-6 . For example, C ₁ _ ₆ αkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, hexyl, and the like. Alkyl may also refer to alkyl groups containing up to 20 carbon atoms such as, but not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl groups may be substituted or unsubstituted. Specific substituents include hydroxy, halo, alkoxy, and amino groups. One skilled in the art will appreciate that a number of substituents can be added to alkyl groups without deviating from the teachings herein.

The term alkenyl refers to a straight or branched chain hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl may include any number of carbon atoms such as C ₂ , C _2-3 , C _2-4 , C _2-5 , C _2-6 , C _2-7 , C _2-8 , C _2-9 , C2- 10, _C3 , _C3-4 , _C3-5 , C3-6, C4, _C4-5 , C4-6, C5, C5-6 and C6. Alkenyl groups may have any suitable number of double bonds, including, without limitation, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1, 4- pentadienyl, 1-hexenyl, 2hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl or 1,3,5-hexatrienyl. Alkenyl groups, like the alkyl groups described above, may be substituted or unsubstituted.

The term alkynyl refers to either a straight chain or branched chain hydrocarbon having at least 2 carbon atoms and at least one triple bond. The alkynyl may include any number of carbon atoms such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3 ₄ , C _3-5 , C _3-6 , C ₄ , C _4-5 , C _4-6 , C ₅ , C _5-6 and C ₆ . Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadienyl, 1,4-pentadiynyl , 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl or 1,3,5-hexatriynyl. Alkynyl groups, like the alkyl groups described above, may be substituted or unsubstituted.

The term cycloalkyl refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring system containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl may include any number of carbon atoms such as C _3-6 , C _4-6 , C _5-6 , C _3-8 , C ₄ _ ₈ , C ₅ _ ₈ , C _6-8 , C _3-9 , C ₃ _ ₁₀ , C _3-11 and C _3-12 . Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. When cycloalkyl is a saturated monocyclic C ₃ _ ₈ cycloalkyl, exemplary groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be substituted or unsubstituted. As will be appreciated by one of skill in the art, many different substituents on cycloalkyl groups may be included without departing from the teachings herein.

The term heterocycloalkyl refers to a saturated ring system having 3 to 12 ring members and 1 to 4 N, O, and S heteroatoms. Additional heteroatoms may also be useful, including, but not limited to, B, Al, Si, and P. Heteroatoms can also be oxidized, for example, without limitation, -S(O)- and -S(O) ₂ -. Heterocycloalkyl groups may include any number of ring atoms, for example 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11 or 3 to 12 ring elements. Any suitable number of heteroatoms may be included in the heterocycloalkyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Heterocycloalkyl groups such as the cycloalkyl groups described above may be substituted or unsubstituted.

The term aryl refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups may include any sub

- 5 041474 a suitable number of ring atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, and 6 to 10, 6 to 12, or 6 to 14 ring elements. Aryl groups may be monocyclic, fused to form bicyclic or tricyclic groups, or bonded to form a biaryl group. Typical aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl having a methylene linking group. Some aryl groups have 6 to 12 ring members such as phenyl, naphthyl or biphenyl. Other aryl groups have 6 to 10 units per ring, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups such as the cycloalkyl groups described above may be substituted or unsubstituted.

The term heteroaryl refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where 1 to 5 ring atoms are a heteroatom such as N, O, or S. Additional heteroatoms may also be useful, including, without limitation, B, Al, Si and P. Heteroatoms can also be oxidized, for example, -S(O)- and -S(O) ₂ -, without limitation. Heteroaryl groups may include any number of ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11 or 3 to 12 ring elements. Any suitable number of heteroatoms may be included in heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups may have 5 to 8 ring units and 1 to 4 heteroatoms, or 5 to 8 ring units and 1 to 3 heteroatoms, or 5 to 6 ring elements and 1 to 4 heteroatoms, or 5 to 6 ring elements and 1 to 3 heteroatoms. The heteroaryl group may include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazinethiophene, furan, thiazole, isothiazole, oxazole and isoxazole. Heteroaryl groups such as the cycloalkyl groups described above may be substituted or unsubstituted.

The term halogen means fluorine, chlorine, bromine and iodine.

The term hydrated refers to a chemical that includes water. Hydrated in the context of chemical conversion in step (a) refers to a chemical with sufficient water to complete said chemical conversion. In specific embodiments, the hydrated reagent comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20% by weight water content.

III. Embodiments of the invention.

A. Process for the preparation of a compound of formula IX.

In one aspect, the present invention provides an improved process for the preparation of a compound of formula IX

including

a) contacting a compound of formula XIII

R ¹ (III), with a compound of formula III

R ¹ with the first base and the first organic solvent to obtain a compound of formula IV O but

(IV) where each R ¹ is independently halogen or sulfonic acid ester;

(b) contacting a compound of formula IV with hydrazine and a second organic solvent to obtain a compound of formula V

(c) contacting a compound of formula V with a sulphonating agent or a halogenating agent to obtain a compound of formula VI

- 6 041474

N

R ² (VI), where R ² represents a halogen or ester of sulfonic acid;

(d) contacting a compound of formula VI with a compound of formula VII

a second base and a third organic solvent to give a compound of formula

VIII

COOR ³

F /=N/ (VIII), where R ³ is selected from the group consisting of C _1-2 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _3-8 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, each of which is optionally substituted;

(e) contacting a compound of formula VIII with a third base to obtain a compound of formula IX.

Step (a) involves contacting a first base and an organic solvent, with a compound of formula II

and a compound of formula III (sh) to give a compound of formula IV

One skilled in the art will appreciate that a compound of formula IV has both electrophilic and nucleophilic sites and that intramolecular transformations are possible depending on the reaction conditions. For example, under certain conditions, an alcohol group of formula IV can be added to one of the carbonyl carbons to form a six-membered ring (formula IVa). When the above addition is combined with a subsequent elimination reaction, the compound of formula IV has the structure of formula IVb. The person skilled in the art will also appreciate that the compound of formula IV may also exist as the enol tautomer A of formula IVc.

Compounds of formula IV, formula IVa, formula IVb, formula IVc are interconvertible, and depending on the reaction conditions, different concentrations of these substances are present. In some embodiments, only one variety is present.

There are many suitable bases that can be used in this conversion. For example, in some embodiments, the first base is an alkali metal carbonate, an alkali metal bicarbonate, or a combination thereof. Alkali metal carbonates may include, but are not limited to, Li ₂ CO ₃ , Na ₂ CO ₃ , and K ₂ CO ₃ , and alkali metal bicarbonates may include, but are not limited to, LiHCO ₃ , NaHCO ₃ and KHCO 3 . In some embodiments, the alkali metal carbonate is K ₂ CO ₃ .

The organic solvent in step (a) is one in which both compounds of formula II and formula III will be properly dissolved and which will be miscible with the base used. One skilled in the art will appreciate that there are a number of organic solvents that meet these characteristics. In some embodiments, the first organic solvent is a polar organic solvent. In some embodiments, polar

- 7 041474 the organic solvent is selected from the group consisting of acetone, ethyl acetate, dichloromethane, tetrahydrofuran, dimethylformamide and acetonitrile. In some embodiments, the organic solvent is acetone.

The transformation in step (a) also includes at least 1 equivalent of water to obtain a hydroxyl-containing compound of formula IV. Often this equivalent of water is provided by the reactants or solvents in the reaction, such as the first base or organic solvent, rather than by the direct addition of water. It has been found that the use of a hydrated base in the chemical conversion in step (a) provides an extremely efficient conversion. As such, in some embodiments, the first base in the conversion in step (a) is a hydrated base. In some embodiments, the first base is a hydrated alkali metal carbonate. In some embodiments, the first base is hydrated K2CO ₃ .

The compound of formula III has two R ¹ groups each independently selected from the group consisting of chloride, bromide, tosylate and mesylate. In some embodiments, each R ¹ is bromide.

It can be seen that each R ¹ group acts as a leaving group in the conversion in step (a); thus, one skilled in the art will appreciate that other leaving groups are useful in the present invention and do not deviate from the teachings herein.

In some embodiments of the present invention, the compound of formula IV obtained in step (a) is used directly in the conversion in step (b) without further purification.

Returning to step (b), the chemical conversion described involves the reaction of a compound of formula IV

O(IV) with hydrazine (N2H4) and a second organic solvent to give a compound of formula V

One skilled in the art will appreciate that a variety of different solvents can be used as the second organic solvent in this conversion. In some embodiments, the second organic solvent is a polar protic organic solvent. In some embodiments, the polar protic organic solvent is C _1-8 -OH. In some embodiments, the polar protic organic solvent is ethanol.

In some embodiments of the present invention, the compound of formula V obtained in step (b) is used directly in the conversion of step (c) without purification.

The chemical conversion of step (c) involves replacing the hydroxyl moiety in the compound of formula (V) with a halogen or converting the hydroxyl group to a sulfonic acid ester to give a compound of formula (VI)

While sulfonating or halogenating agents are specifically provided as starting materials in this conversion, one of ordinary skill in the art will recognize that many different leaving groups outside of halogens and sulfate esters are suitable substituents for R2. Thus, any starting material that will provide a suitable leaving group at the R2 position is within the scope of this invention.

There are a number of solvents that are suitable for this conversion; however, one of ordinary skill in the art will appreciate that the solvent selected for this chemical conversion will depend on the sulfonating agent or halogenating agent selected, as particular solvents may not be suitable for all starting materials. For example, when a compound of formula V is contacted with a halogenating agent, polar organic solvents are particularly useful. In some embodiments, the polar organic solvent is 1,2-dichloroethane.

Suitable halogenating agents for the conversion in step (c) include, but are not limited to, PBr ₃ , PCl ₃ , PCl5, SOBr ₂ , PBr5 and SoCl ₂ . Sulfonating conversion agents in step (c) include, but are not limited to, mesyl chloride (MsCl) and tosyl chloride (TsCl). In some embodiments, the halogenating agent is PBr ₃ .

It should be understood that the identity of R ² depends on the starting material selected for the chemical conversion in step (c). For example, if a sulfonating agent is selected, the R ² identity corresponds to the corresponding sulfate. In some embodiments, R ² is chloride, bromide, tosylate, and mesylate. In some embodiments, R ² is Br.

Focusing on step (d), compounds of formula VI । R ² \ (VI) and formulas VII

COOR ³ %

^F (VII) is brought into contact in the presence of a base and a third organic solvent to give a compound of formula VIII

The chemical conversion of step (d) can be performed with a variety of different bases. For example, in some embodiments, the second base is an alkali metal carbonate, an alkali metal bicarbonate, or a combination thereof. Alkali metal carbonates may include, without limitation, Li ₂ CO ₃ , Na ₂ CO _{3 ,} and K ₂ CO ₃ , and alkali metal bicarbonates may include, but are not limited to, LiHCO ₃ , NaHCO _{3 ,} and KHCO ₃ . In some embodiments, the alkali metal carbonate is K2CO3.

The organic solvent in step (d) is one that will suitably dissolve both compounds of formula VI and formula VII in solution, and miscible with the base used. One skilled in the art will appreciate that there are a number of organic solvents that meet these characteristics. In some embodiments, the third organic solvent is a polar aprotic organic solvent. In some embodiments, the polar organic solvent is selected from the group consisting of acetone, ethyl acetate, dichloromethane, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, and acetonitrile. In some embodiments, the third organic solvent is dimethylformamide. In some embodiments, the third organic solvent is dimethyl sulfoxide.

Suitable substituents for the R ³ group include substituents which will not interfere with the chemical conversion in step (e), discussed in more detail below. Such substituents include, but are not limited to, C1-8alkyl, C3-8cycloalkyl, C3-12heterocycloalkyl, aryl, heteroaryl, and the like. One of skill in the art will appreciate that many other R ³ ester substituents are suitable without departing from the teachings herein. In some embodiments, R ³ is 61-8alkyl. In some embodiments, R ³ is methyl.

In some embodiments, the process of step (d) provides a compound of formula VIII with at least a 70% (mol/mol) yield based on the amount of formula VII.

With respect to step (e), the compound of formula VIII

brought into contact with a third base, to obtain a compound of formula IX

The third base in the chemical conversion in step (e) may be some of a number of different bases. For example, in some embodiments, the third base is a metal hydroxide. In some embodiments, the metal hydroxide is an alkali metal hydroxide. In some embodiments, the alkali metal hydroxide is selected from the group consisting of LiOH,

- 9 041474

NaOH, KOH, RbOH and CsOH. In some embodiments, the alkali metal hydroxide is LiOH. In some embodiments, the alkali metal hydroxide is NaOH.

One skilled in the art will appreciate that a variety of different solvents can be used as the solvent in the conversion in step (e). For example, in some embodiments, the second organic solvent is a polar protic organic solvent or water. In some embodiments, the polar protic organic solvent is C1-8-OH. In some embodiments, the polar protic organic solvent is methanol. In some embodiments, the solvent is water. In some embodiments, the solvent is a combination of methanol and water.

In some embodiments, the process in step (e) further comprises:

(e-i) removing the solvent to obtain a residue;

(e-ii) dissolving the residue in water to form a solution;

(e-iii) acidifying the solution to form a precipitate; and (e-iv) filtering the solution to obtain the isolated formula IX, wherein steps (e-i)-(e-iv) are performed after step (e).

Step (e) may be performed using any suitable removal step such as reduced pressure, increased temperature, or a combination of both. In some embodiments, the solvent was removed under reduced pressure. In some embodiments, a solid is obtained in step (e) and the solvent is removed by filtration. In addition, the addition of water in step (e-ii) may be performed before step (e-i). In such a case, removal of the solvent under reduced pressure provides a concentrated water component (ie, no water is removed). It is understood that the permutation of steps (e-i) and (e-ii) does not deviate from the processes described herein.

Step (e-iii) may be acidified with any suitable acid. In some embodiments, the suitable acid is HCl. In some embodiments, the solution is acidified to a pH below 3, 0-3, or 23. In some embodiments, the solution is acidified to a pH of about 2. In some embodiments, the solution is acidified to a pH below 2, 0-2, or 1-2. In some embodiments, the solution is acidified to a pH of about 1.4-1.6.

The pH value in the acidification step (e-iii) determines which species are predominantly produced. In some embodiments, the pH of the acidification step is in the range of 5-6 and the zwitterionic form of formula IX is obtained. In some embodiments, the pH is acidified with HCl to less than about 2, or in the range of 1.4 to 1.6, to give the Formula IX HCl salt (ie, Formula Ia compound).

The process described in step (e) can provide a compound of formula IX in high yield and purity. In some embodiments, the yield in step (e) is greater than 85%, 90%, 93%, or 95% (mol/mol) relative to formula VIII. In some embodiments, the purity of the compound of formula IX obtained in step (e) is greater than 80%, 85%, 90%, 95%, or 97% (mol/mol).

In another aspect, provided herein is a process for preparing a compound of formula IX

comprising (a) contacting a compound of formula II

with a compound of formula III(w), a first base and a first organic solvent to form an adduct, wherein each R ¹ is independently a halogen or a sulfonic acid ester;

(b) contacting the adduct with hydrazine and a second organic solvent to obtain a compound of formula V

IhnA \ (V) (c) contacting a compound of formula V with a sulphonating agent or a halogenating agent to obtain a compound of formula VI

- 10 041474

where R ² represents a halogen or ester of sulfonic acid;

(d) contacting a compound of formula VI with a compound of formula VII

a second base and a third organic solvent to give a compound of formula

VIII

where R ³ is selected from the group consisting of C _1-2 alkyl, C _2-12 alkenyl, C _1-12 alkynyl, C _3-8 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, each of which is optionally substituted;

(e) contacting a compound of formula VIII with a third base to obtain a compound of formula IX.

In some embodiments, the adduct obtained in step (a) is a compound of formulas IV, IVa, IVb and/or IVc.

One skilled in the art will appreciate that the compounds shown above are interconvertible and that the relative amounts of each compound depend on the conditions of the experiment.

As noted above, one skilled in the art will appreciate that selected steps in the process may be carried out regardless of the origin of the starting material or intermediates.

B. Pharmaceutically acceptable salts of formula I.

In a second aspect, the present disclosure relates to a pharmaceutically acceptable salt represented by formula I coon

where X is a pharmaceutically acceptable protic acid anion.

Various protic acids are suitable for preparing the pharmaceutically acceptable salt of formula I. It can be seen that the pharmaceutically acceptable protic acid anion depends on the protic acid used. For example, protic acids used herein include hydrochloric acid, hydrobromic acid, sulfonic acid, tosylic acid (p-toluenesulfonic acid), methanesulfonic acid, nitric acid, or acetic acid. Thus, pharmaceutically acceptable protic acid anions include chloride (Cl ^- ), bromide (Br ^- ), sulfonate (HS(O)2O ^- ), tosylate (TsO ^- ), mesylate (MsO ^- ), besylate (BeO ^- ) ethanesulfonate ( EtSO3 ^- ), nitrate (NO3 ^- ), acetate (CH3C(O)O ^- ), glycolate (HO-CH2C(O)O ^- ) or combinations thereof.

In some embodiments, the pharmaceutically acceptable protic acid anion is mesylate. In some embodiments, the mesylate salt of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 4.

In some embodiments, the pharmaceutically acceptable protic acid anion is a besylate. In some embodiments, the mesylate salt of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 8.

In some embodiments, the pharmaceutically acceptable protic acid anion is

- 11 041474 is a tosylate. In some embodiments, the tosylate salt of formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 10.

In some embodiments, the pharmaceutically acceptable protic acid anion is esylate. In some embodiments, the esylate salt of Formula IX has an X-ray powder diffraction pattern substantially consistent with FIG. 12.

In some embodiments, the pharmaceutically acceptable protic acid anion is bromide. In some embodiments, the bromide salt of formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 14.

In some embodiments, the pharmaceutically acceptable protic acid anion is a nitrate. In some embodiments, the nitrate salt is characterized by an x-ray pattern substantially in accordance with FIG. 16. In some embodiments, the nitrate salt has an XRPD pattern substantially in accordance with FIG. 18.

In some embodiments, the pharmaceutically acceptable protic acid anion is chloride and the pharmaceutically acceptable salt of formula I is represented by formula (Ia)

In some embodiments, the salt of formula Ia is crystalline form A. In some embodiments, crystalline form A of formula Ia is characterized by an X-ray powder diffraction pattern including peaks at 12.0, 21.8, 25.9, 26.7, and 27.9 degrees 2Θ (±0.2 degrees 2θ). In some embodiments, the X-ray powder diffraction pattern further includes one or more peaks at 7.0, 10.3, 13.9, 15.6, and/or 17. Crystal form A of Formula Ia, has an X-ray powder diffraction pattern of substantially in accordance with FIG. 31.

In some embodiments, multiprotic acids, such as diprotic or tripronic acids, are used to prepare pharmaceutically acceptable salts of formula IX. In such embodiments, the pharmaceutically acceptable salt is represented by formula Ib

where Y is a multiprotic acid.

In some embodiments, Y is selected from the group consisting of ethane-1,2-disulfonic acid, sulfuric acid, citric acid, maleic acid, malic acid, tartaric acid, and oxalic acid. In some embodiments, Y is L-malic acid or L-tartaric acid.

In some embodiments, Y is ethane-1,2,-disulfonic acid. In some embodiments, the edisylate salt of formula IX has an XRPD pattern substantially in accordance with FIG. 6.

In some embodiments, Y is sulfuric acid. In some embodiments, the sulfate salt of formula IX has an XRPD pattern substantially in accordance with FIG. 19.

In some embodiments, Y is oxalic acid. In some embodiments, the oxalate salt of formula IX has an XRPD pattern substantially in accordance with FIG. 21.

In some embodiments, Y is maleic acid. In some embodiments, the maleate salt of formula IX has an XRPD pattern substantially in accordance with FIG. 23. In some embodiments, the maleate salt of formula IX has an XRPD pattern substantially in accordance with FIG. 25.

In some embodiments, Y is acetic acid. In some embodiments, the acetic acid salt of formula IX has an XRPD pattern substantially in accordance with FIG. 27.

In some embodiments, Y is L-malic acid. In some embodiments, the L-malic acid salt of Formula IX is characterized by the XRPD pattern substantially as shown in FIG. 29.

The molar ratios of AG-10 and Y in formula Ib may vary depending on the multiprotonic acid used. For example, when Y is maleic acid, the molar ratio of AG-10 to Y is 1:1; when Y is edisylate, the molar ratio of AG-10 to Y is 2:1; and when Y is malic acid, the molar ratio of AG-10 to Y is 1.8:1.

Pharmaceutically acceptable salts of formula I can be obtained using a number of conventional means in the art. For example, the free acid form of a compound of formula I is mo

- 12 041474 can be contacted with a stoichiometric amount of a suitable acid in water, an organic solvent or a mixture thereof. In some embodiments, the pharmaceutically acceptable salts of Formula I are prepared in non-aqueous vehicles such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. In some embodiments, pharmaceutically acceptable salts of Formula I are prepared by dissolving a compound of Formula IX in water, adding an appropriate amount of HX to form a mixture, and adding a non-aqueous solvent, such as the non-aqueous medium described above, to crystallize the salt. In some embodiments, a suitable amount of HX is a stoichiometric amount. It is understood that HX includes hydrogen and X is the pharmaceutically acceptable protic acid anion as defined above.

As with the pharmaceutically acceptable salts of formula I, pharmaceutically acceptable salts of formula Ib can also be prepared using a number of conventional means in the art. As a non-limiting example, the free acid form of a compound of formula Ib may be contacted with a stoichiometric or substoichiometric amount of a suitable multiprotonic acid in water, an organic solvent, or a mixture thereof to form a pharmaceutically acceptable salt of formula Ib.

C. Crystal Forms of Formula IX.

In yet another aspect, this document provides crystalline forms of formula IX

The disclosure currently describes eleven crystalline forms of Formula IX, six HCl salt forms (Form A, Form B, Form E, Form H, Form I, and Form J), three free base forms (Form K, Form C, and Form G) and two unidentified forms (form D and form F). A brief description of the properties of the identified forms is given in Table. 1 and table. 2.

In some embodiments of the present invention, the crystalline forms of formula IX provided herein are substantially free of other crystalline forms. The term substantially free refers to an amount of 10% or less of the other form, preferably 8, 5, 4, 3, 2, 1, 0.5% or less of the other form.

Table 1. Characterization results for crystalline forms of HCl salts

Crystal Form Batch No. Mass loss % Endotherm (peak, °C) HPLC purity (% area) Stoichiometry (acid: FB)* Intended form form A 1.3 1P,7, 212.6, 237.3 98.76 0.91 Anhydrate form B 1.2 161.4, 232.2, 262.3 97.86 0.86 form E 1.5 182.0, 242.7 98.60 0.91 form I 3.0 62.0, 158.4, 215.7 97.94 0.86 Hydrate form H 4.6 90.4, 200.5, 232.3 98.47 0.91 Solvate MeOH form J 21.5 120.8, 197.8, 221.5 91.69 0.90 DMAc solvate

* stoichiometry was determined by HPLC/IC using free base form A (TRM-01658-2) as standard. We used a routine method and did not conduct system development and validation.

Table 2. Results of characterization of forms of free bases

Crystal Form Batch No. Mass loss % Endotherm (peak, °C) HPLC Purity (Area %) SG content (%) * Form K (Monohydrate) 6D 159.3, 176.2, 278.4 99.12 -- form C 3.1 91.2, 173.0 — 0.17 form G 3.7 231.1 99.46 0.14

-- data not available.

* The theoretical content of Cl ^- mono-HCl salt is 10.8%. Based on the Cl- content results, the C and G forms were determined to be the free base forms.

- 13 041474

In some embodiments, what is provided herein is crystalline form A of formula IX. In some embodiments, crystalline Form A of formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 7.0, 10.4, 12.0, 13.0, and 13.9 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form A of Formula IX has an X-ray powder diffraction pattern including peaks at 12.0, 21.8, 25.9, 26.7, and 27.9 degrees 2θ (±0.2 degrees 2θ). In some embodiments, the X-ray powder diffraction pattern further includes one or more peaks at 7.0, 10.3, 13.9, 15.6, and/or 17. In some embodiments, crystalline form A of formula IX is characterized by an X-ray powder diffraction pattern, essentially, in accordance with FIG. 31. In some embodiments, crystalline form A of formula IX is essentially excluding other crystalline forms.

In some embodiments of the invention, the crystalline form A of formula IX is characterized by a weight loss in the range from about 0.7 to about 1.9% when heated to about 150°C, as measured by thermogravimetric analysis. In some embodiments, the weight loss is about 1.3% as measured by thermogravimetric analysis.

In some embodiments, crystalline form A of formula IX has a water uptake of about 1.6% at 25° C./80% relative humidity (RH) after undergoing a dynamic vapor sorption cycle that includes pre-equilibration at 0% RH. In some embodiments, the crystalline form A of formula IX has an increase of less than 2.5 wt.% after passing through the cycle of dynamic vapor sorption at relative humidity (RH) from about 0 to about 90% RH. In some embodiments, crystalline form A of Formula IX has a dynamic vapor sorption profile substantially as shown in FIG. 37.

In some embodiments of the invention, crystalline form A of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks around 211-214 and 237-239°C. In some embodiments, the differential scanning calorimetry thermogram includes endothermic peaks around 11.7, 212.6, and 237.3°C.

In some embodiments, what is provided herein is crystalline form B of formula IX. In some embodiments, crystalline form B of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 12.0, 13.8, 17.2, 17.7, and 19.8 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline form B of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 12.1, 13.9, 19.8, 23.3, and 24.4 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form B of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 40. In some embodiments, crystalline form B of formula IX is essentially excluding other crystalline forms.

In some embodiments of the invention, crystalline form B of formula IX is characterized by a weight loss of from about 0.6 to about 2.0% when heated to about 150°C, as measured by thermogravimetric analysis. In some embodiments, crystalline form B of formula IX has a weight loss of about 1.2% when heated to about 150° C., as measured by thermogravimetric analysis.

In some embodiments of the invention, crystalline form B of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks at about 161.4, 232.2 and 262.3°C.

In some embodiments, what is provided herein is crystalline form E of formula IX. In some embodiments, crystalline form E of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 11.8, 14.0, 15.1, 19.9, and 24.0 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline form E of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 11.9, 14.0, 15.1, and 25.8 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form E of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 46. In some embodiments, crystalline form E of Formula IX is substantially excluding other crystalline forms.

In some embodiments of the invention, the crystalline form E of formula IX is characterized by a weight loss of from about 0.5 to about 2.5% when heated to about 150°C, as measured by thermogravimetric analysis. In some embodiments, crystalline form E of formula IX has a weight loss of about 1.5% when heated to about 150°C, as measured by thermogravimetric analysis.

In some embodiments of the invention, crystalline form E of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks around 182.0 and 242.7°C.

In some embodiments, what is provided herein is crystalline form I of formula IX. In some embodiments, crystalline form I of formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 11.4, 12.1, 12.4, 13.6, and 13.9 deg.

- 14041474 dusa 2θ (±0.2 degrees 2θ). In some embodiments, crystalline form I of formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 12.5, 17.3, 23.4, 25.0, and 25.4 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline form I of formula IX has an X-ray powder diffraction pattern substantially corresponding to FIG. 55. In some embodiments, crystalline form I of formula IX is essentially excluding other crystalline forms.

In some embodiments of the invention, the crystalline form I of formula IX is characterized by a weight loss in the range from about 2.5 to 3.5% when heated to about 120°C, as measured by thermogravimetric analysis. In some embodiments, the crystalline form I of formula IX is characterized by a weight loss of about 3.0% when heated to about 120°C, as measured by thermogravimetric analysis.

In some embodiments of the invention, crystalline form I of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks around 62.0 and 158.4, 215.7°C.

In some embodiments, what is provided herein is a crystalline Form H of Formula IX. In some embodiments, crystalline form H of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 11.8, 12.3, 13.8, 15.7, and 16.9 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline form H of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 11.9, 12.3, 21.7, 23.3, and 25.8 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline form H of Formula IX has an X-ray powder diffraction pattern substantially corresponding to FIG. 51. In some embodiments, crystalline form H of Formula IX is essentially excluding other crystalline forms.

In some embodiments, crystalline form H of formula IX has a weight loss in the range of about 3.5 to about 5.5% when heated to about 150° C., as measured by thermogravimetric analysis. In some embodiments, crystalline form H of formula IX has a weight loss of about 4.6% when heated to about 150° C., as measured by thermogravimetric analysis.

In some embodiments of the invention, the crystalline form H of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks of about 90.4 and 200.5, 232.3°C.

In some embodiments, what is provided herein is crystalline form J of formula IX. In some embodiments, crystalline form J of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 4.6, 11.8, 12.8, 13.8, and 14.6 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form J of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 13.8, 14.7, 22.9, 26.2, and 27.7 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form J of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 59. In some embodiments, crystalline form J of Formula IX is substantially excluding other crystalline forms.

In some embodiments of the invention, the crystalline form J of formula IX is characterized by a weight loss in the range from about 17.5 to about 24% when heated to about 120°C, as measured by thermogravimetric analysis. In some embodiments, crystalline form J of Formula IX has a weight loss of about 21.5% when heated to about 120° C. as measured by thermogravimetric analysis.

In some embodiments of the invention, crystalline form J of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks at about 120.8 and 197.8, 221.5°C.

In some embodiments, what is provided herein is crystalline form K of formula IX. In some embodiments, crystalline form K of formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 7.5, 9.8, 13.9, 15.9, and 19.3 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form K of Formula IX has an X-ray powder diffraction pattern that peaks at 7.2, 7.6, 9.9, 14.0, and 19.3 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form K of formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 59. In some embodiments, crystalline form K of formula IX is essentially excluding other crystalline forms.

In some embodiments of the invention, the crystalline form K of formula IX is characterized by a weight loss in the range from about 5.0 to about 7.0% when heated to about 120°C, as measured by thermogravimetric analysis. In some embodiments, crystalline form K of formula IX has a weight loss of about 6.1% when heated to about 120° C. as measured by thermogravimetric analysis.

- 15 041474

In some embodiments of the invention, crystalline form K of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks at about 159.3 and 176.2, 278.4°C.

In some embodiments, what is provided herein is crystalline form C of formula IX. In some embodiments, the crystalline form C of formula IX is characterized by an x-ray powder diffraction pattern containing peaks at 9.5, 11.7, 12.3, 13.4, and 14.6 degrees 2Θ (±0.2 degrees 2Θ). In some embodiments, crystalline Form C of formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 14.6, 16.8, 19.5, 20.7, and 22.5 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline Form C of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 43. In some embodiments, crystalline form C of formula IX is essentially excluding other crystalline forms.

In some embodiments of the invention, the crystalline form C of formula IX is characterized by a weight loss in the range from about 2.0 to about 4.0% when heated to about 150°C, as measured by thermogravimetric analysis. In some embodiments, crystalline form C of formula IX has a weight loss of about 3.1% when heated to about 150° C., as measured by thermogravimetric analysis.

In some embodiments of the invention, crystalline form C of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks around 91.2 and 173.0°C.

In some embodiments, what is provided herein is a crystalline form G of formula IX. In some embodiments, crystalline form G of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 9.8, 12.2, 13.1, 13.4, and 14.6 degrees 2Θ (±0.2 degrees 2θ). In some embodiments, crystalline form G of Formula IX is characterized by an X-ray powder diffraction pattern containing peaks at 12.3, 13.2, 13.4, 17.8, and 26.6 degrees 2θ (±0.2 degrees 2θ). In some embodiments, crystalline form G of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 43. In some embodiments, crystalline form G of Formula IX is substantially excluding other crystalline forms.

In some embodiments, crystalline form G of formula IX has a weight loss in the range of about 1.7 to about 2.7% when heated to about 200° C., as measured by thermogravimetric analysis. In some embodiments, crystalline Form G of formula IX has a weight loss of about 3.7% when heated to about 200° C. as measured by thermogravimetric analysis.

In some embodiments of the invention, the crystalline form G of formula IX is characterized by a differential scanning calorimetry thermogram containing endothermic peaks around 231.1°C.

In some embodiments, what is provided herein is crystalline form D of formula IX. In some embodiments, crystalline form D of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 45 (upper section). In some embodiments, crystalline form D of formula IX is essentially excluding other crystalline forms.

In some embodiments, what is provided herein is a crystalline form F of formula IX. In some embodiments, crystalline form F of Formula IX has an X-ray powder diffraction pattern substantially in accordance with FIG. 45 (bottom graph). In some embodiments, crystalline form F of formula IX is essentially excluding other crystalline forms.

Methods for obtaining the described crystalline forms are further detailed in the Examples of this application. Crystallization conditions used to make forms A-K include addition of an antisolvent, slow evaporation, slow cooling, slurry conversion at room temperature (RT), slurry conversion at 50°C, solid-vapor diffusion, liquid-vapor diffusion.

IV. Examples.

Example 1 Preparation of 3-(3-hydroxypropyl)pentan-2,4-dione (compound of formula IV)

II Illa IV

The compound of formula IIIa (100 g, 495 mmol 1.0 equiv.) was dissolved in acetone (1 L). To the above solution was added a compound of formula II (49.59 g, 495 mmol, 1.0 equiv.) followed by

- 16 041474 K2CO ₃ (82.14 g, 594.38 mmol, 1.2 equiv.) and KI (41.11 g, 247 mmol, 0.5 equiv.) at room temperature with stirring. The reaction mixture was heated to 60±5°C and stirred for 40 h at this temperature. The reaction mixture was filtered and then concentrated under reduced pressure to give the compound of formula IV (102 g) as a viscous orange liquid.

Example 2 Preparation of 3 (3,5-dimethyl-1H-pyrazol-4-yl) propan-1-ol (compound of formula V)

IV V

The compound of formula IV (100 g, 632 mmol, 1.0 equiv.) was dissolved in ethanol (1 L). Hydrazine hydrate (87 g, 1738 mmol, 2.75 equiv) and conc. HCl (4.6 ml, 0.2 equiv.) was added to the above solution at room temperature. The reaction mixture was heated to 75±5°C and stirred for 3 hours at this temperature. After completion of the reaction, determined by TLC (70% ethyl acetate:nhexane, visible in iodine) and observing the peak of the product in the mass spectrum, the reaction mixture was concentrated under reduced pressure to obtain the compound of formula V (70 g) as a colorless liquid syrup, which used as such for the next step.

Example 3 Preparation of 4-(3-bromopropyl)-3,5-dimethyl-1H-pyrazole (compound of formula VIa)

V

Via

The compound of formula V (35 g, 227 mmol, 1.0 equiv.) was dissolved in 1,2-dichloroethane (525 ml). PBR ³ (64.67 ml, 681 mmol, 3 equiv.) was added in small portions at room temperature over 30 minutes. The reaction mixture was heated to 75±5°C and stirred for 3 hours at this temperature. After completion of the reaction, determined by TLC (50% ethyl acetate: n-hexane, visible in iodine) and observing the peak of the product in the mass spectrum, the reaction mixture was diluted with dichloromethane (350 ml) and quenched with saturated NaHCO ₃ solution to pH 7 to 8 Both the organic and aqueous layers were separated and collected. The organic layer was dried over MgSO ₄ and filtered. The filtrate was concentrated under reduced pressure to give the compound of formula VIa (38 g) as a viscous orange liquid.

Example 4 Preparation of 3-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy]-4fluorobenzoic acid methyl ester (compound of formula VIIIa)

The compound of formula VIIa (19 g, 111 mmol, 1.0 equiv.) was dissolved in DMF (190 ml). A compound of formula VIa (31.5 g, 145.14 mmol, 1.3 equiv.) was added followed by K ₂ CO ₃ (38.6 g, 279.18 mmol, 2.5 equiv.) at room temperature under conditions mixing. The reaction mixture was stirred for 1618 hours at room temperature. After completion of the reaction by TLC (50% ethyl acetate: n-hexane), the reaction mixture was diluted with water (190 ml) and ethyl acetate (95 ml). Both the organic layer and the aqueous layer were separated and collected. The aqueous layer was extracted with ethyl acetate (190 ml). The combined organic extract was washed with water (95 ml), brine (95 ml), dried over Na ₂ SO ₄ and filtered. The filtered organic layer was concentrated under reduced pressure to give a crude viscous orange liquid (40 g). The crude product was further purified by column chromatography using silica gel (285 g) and eluted with various amounts of ethyl acetate in hexane to give the pure product, the compound of formula VIIIa (25 g), as an off-white solid.

Example 5 Preparation of 3-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy]-4fluorobenzoic acid methyl ester (compound of formula VIIIa)

- 17 041474

4-(3-Bromopropyl)-3,5-dimethyl-1H-pyrazole hydrobromide (VIa) and DMSO were charged into a vessel and stirred at 20±10°C for 10 min. The mixture was then heated to 55±5° C. with stirring. To this mixture was transferred a stirred solution containing 4-fluoro-3-hydroxybenzoic acid methyl ester (Vila), potassium carbonate and anhydrous DMSO. A solution of alkyl bromide in DMSO was slowly transferred to maintain an internal temperature of 55.0±5°C. The addition was completed after 6 hours and the mixture was stirred at 55.0±5°C for an additional hour at 55.0±5°C. The mixture was cooled to 25±5°C over 30 min and water was added keeping the temperature below 25°C. The mixture was extracted with ethyl acetate and the aqueous layer was back extracted with ethyl acetate. The combined ethyl acetate solutions were washed with brine. The combined ethyl acetate washes were concentrated in vacuo to minimum volume and heptane was added, which precipitated Villa. The mixture was heated to 75±5°C and kept under stirring for 1 hour. The mixture was cooled to 25±5°C for two hours and the resulting solids were collected by filtration. The filter cake was washed with ethyl acetate in heptane (30%). The isolated solids were dried under nitrogen flow. The solids were loaded into a vessel and combined with ethyl acetate and heptane. The resulting mixture was heated to 75±5°C to dissolve the solids. The solution was cooled to 25±5°C for two hours and the resulting solids were collected by filtration. The solids were washed with a 30% ethyl acetate/heptane solvent mixture and dried in a vacuum oven at 55° C. to give Villa >99.5% pure.

Example 6 Preparation of 3-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy]-4-fluorobenzoic acid (compound of formula IX)

Villa IX

The compound of the formula Villa (19 g, 62 mmol, 1 equiv.) was dissolved in methanol (95 ml, 5 vol.) at room temperature. A solution of LiOH.H ₂ O (6.5 g, 155 mmol, 2.5 equiv.) in water (57 ml) was added in small portions at room temperature over 10 to 15 minutes. The reaction mixture was stirred for 2 hours at room temperature. After determining the completion of the reaction using TLC (70% ethyl acetate: n-hexane), the reaction mixture was concentrated at a temperature below 45°C under reduced pressure, obtaining a solid residue of formula IX.

Example 7 Preparation of a Pharmaceutically Acceptable Salt of Formula I

The solid residue of formula IX was dissolved in water (57 ml) and stirred for 10 min and cooled to 0±5°C. The aqueous solution was acidified with conc. HCl (20-25 ml) to pH 2 and stirred for 30 min at 0±5°C. A precipitate was observed which was filtered off and dried at room temperature to give the pure product, a compound of formula Ia (17.5 g) as an off-white solid.

Example 8 Additional Preparation of a Pharmaceutically Acceptable Salt of Formula I

Water and concentrated HCl were loaded into a vessel and cooled with stirring to 10±5°C. The compound of formula IX and water were loaded into the second vessel and cooled with stirring to 10±5°C. The HCl solution in vessel 1 was transferred to the vessel containing the compound of formula IX for at least 15 minutes while maintaining the temperature <25°C. The resulting suspension was kept under stirring at 20±5°C for 44 hours. The solids were collected by filtration, washed with 0.2 N HCl (3 times) and dried in vacuum at >55°C to give Ia as a white solid, with >99.8% purity.

Example 9 Preparation of 3-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy]-4fluorobenzoic acid hydrochloride salt (compound Ia) from VIIIa

A jacketed glass vessel was charged with a compound of formula VIIIa (1.0 equiv.) and methanol.

- 18 041474

The mixture was cooled with stirring to 10±5° C. and an aqueous sodium hydroxide solution (3 equiv.) was charged over 20 minutes. The mixture was kept under stirring at 20±5°C for 2 h, after which the reaction was completed. Stirring was stopped and water was added. Then the methanol was removed by vacuum distillation at an internal temperature of not more than 35°C. The resulting concentrated clear aqueous solution was cooled to 10° C. and concentrated HCl was added until the pH dropped to 1.4-1.6 (pH meter) to precipitate the HCl salt. The solids were collected by filtration, washed with 0.2 N HCl and dried in vacuo at 50° C. to give a compound of formula Ia with a purity of at least 99.5%.

Example 10 Preparation of 3-[3-(3,5-dimethyl-1H-n-pyrazol-4-yl)propoxy]-4-fluorobenzoic acid (compound of formula IX) from VIIIa COOMe

NaOH Me OH/water

UNSD

Villa IX

3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic acid methyl ester (compound of formula VIIIa) and methanol was charged into a vessel and the resulting mixture was stirred at 20±5°C until complete dissolution. The solution was cooled to 10±5°C and sodium hydroxide solution was added over 20 min, maintaining the temperature <25°C. The temperature of the mixture was brought to 25±5°C and kept under stirring for 18 hours, the Reaction mixture was filtered. Water was added to the filtrate and the resulting mixture was concentrated in vacuo until the volume of the mixture was reduced to the minimum volume. Water was added again and the resulting mixture was concentrated in vacuo until the volume of the mixture was reduced to the minimum volume. The pH of the aqueous mixture was adjusted to 5.5±0.5 by adding concentrated hydrochloric acid, then 0.5 N HCl. The temperature of the mixture was brought to 7±5°C and kept under stirring for an additional hour. The solids were collected with the filtrate, washed with water and partially dried in vacuo at >55° C. to give the compound of formula IX as white solids with >99.5% HPLC purity.

Example 11 Conversion of hydrochloride salt to free base.

3-[3-(3,5-Dimethyl-1H-pyrazol-4-yl)pronoxy]-4-fluorobenzoic acid hydrochloride (10.0 g, 30.4 mmol, 1.0 equiv.) was sampled in deionized water ( 30.0 ml) at room temperature and cooled to 10±5°C. To this mixture was added saturated sodium bicarbonate to pH=6-7 and stirred for 30 minutes at this temperature. The resulting off-white precipitate was filtered off and washed with deionized water (20 ml). The solid was dried at room temperature to give 3-[3(3,5-dimethyl-1H-pyrazol-4-yl)propoxy]-4-fluorobenzoic acid (compound of formula IX) (7.40 g, 83.2%) as an off-white solid.

Example 12 Oral Administration Using a Pharmaceutically Acceptable Salt of Formula I

The following examples describe pharmacokinetic measurements of a compound of formula IX in various salt and zwitterion forms. The results presented herein show that the compounds of formula I have an unexpectedly high pharmacokinetic profile.

Rats or dogs were orally administered zwitterion, Na-salt, or AG-10 HCl-salt. The AG-10 form used and dosage are shown in Table 1. 3. Plasma samples from each rat/dog, as appropriate, were measured between 0 and 96 hours post-dose of AG-10 in the indicated form. After isolation from the animal, each sample (50 μl) was precipitated with protein by adding 500 μl of 0.1% formamide in acetonitrile to the sample. After adding the formamide solution, the sample was shaken and centrifuged at 1400 rpm for 15 min at 4°C. 100 µl of the supernatant was removed and diluted with 100 µl of water. 5 µl of the diluted sample was injected for LC-MS/MS analysis. Pharmacokinetic data were calculated using the available data C _max and exposure (0-24 ng.h/ml) shown in table. 3.

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Table 3. Oral dosing of the pharmaceutically acceptable salt of formula I for several varieties

Variety form Dose (mg/kg) Stach (ng/ml) (µM) Exposure (0-24 h) ng.h/ml 1 rat Zwitterion 200 26373 (90.3 µM) .... 2 rat Na-salt 50 mg/kg daily for 28 days 12322 (42.2 µM) .... 3 rat HCl salt 200 216333 (741 µM) 395503 4 dog zwitterion 25 17533 (60 µM) 89786 5 dog HC1-salt 20 16075 (55.1 µM) 69088 6 dog HCl salt 25 25067 (85.8 µM) 128374 7 dog HCl salt 60 41800 (143.2 µM) 155814 8 dog HCl salt 200 154333 (528.5 µM) 1020640

As can be seen in Table. 3, the HCl salt of formula I showed an unexpected and significant improvement in C _max values in dogs and rats compared to zwitterion and Na salt. Compare line 3 with line 1 and line 6 with line 4. Thus, to achieve the same levels of bioavailability, a significantly lower dose of the HCl salt of formula I is required.

Example 13 Intravenous Dosing of a Pharmaceutically Acceptable Salt of Formula I

The following examples describe pharmacokinetic measurements of compounds of formula IX in various salt and zwitterion forms when administered intravenously to rats and dogs. The results presented herein show that the compounds of formula I have an unexpectedly high pharmacokinetic profile when administered both orally and intravenously.

Mice, rats and dogs were intravenously injected with zwitterion, Na-salt, or HCl-salt AG-10. Form AG-10 and dosage are listed in table. 4. Plasma samples from each mouse/rat/dog, as appropriate, were measured between 0 and 24 hours post-dose in the indicated AG-10 form. After isolation from the animal, proteins were precipitated in each sample (50 μl) by adding 500 μl of 0.1% formamide in acetonitrile to the sample. After adding the formamide solution, the sample was shaken and centrifuged at 1400 rpm for 15 min at 4°C. 100 µl of the supernatant was removed and diluted with 100 µl of water. 5 µl of diluted samples were injected for LC-MS/MS analysis. Pharmacokinetic data were calculated using the available Cmax and exposure (0-24 ng.h/ml) data shown in Table 1. 4.

Table 4. Intravenous Dosing of the Pharmaceutically Acceptable Salt of Formula I for Several Varieties

Variety Form Dose (mg/kg) Stach (ng/ml) (µM) 1 mouse zwitterion 3 4485 (15.4 µM) 2 rat zwitterion 3.43 3093 (10.6 µM) 3 rat Na-salt 10 3150 (10 μM) 4 rat HCl salt 1 4275 (14.6 μM) * 5 dog HCl salt 1 5.959 (20.4 μM) *

* derived C _max - extrapolated C ₀ .

Example 14 High bioavailability of several varieties of AG-10.

In FIG. 2 shows pharmacokinetic results demonstrating high bioavailability of AG-10 in dogs, rats and mice. The mean plasma concentration of AG-10 was measured 0-24 hours after administration of 1 mg/kg AG-10 intravenously and 5 mg/kg AG-10 orally. The calculated pharmacokinetic data are shown in FIG. 2.

Example 15 High bioavailability of AG-10 in dogs.

In FIG. 3 shows pharmacokinetic results demonstrating high bioavailability of AG-10 in female and male dogs at various dosage levels. The mean plasma concentration of AG-10 was measured 2-24 hours after oral administration of 5 mg/kg or 20 mg/kg AG-10. The calculated pharmacokinetic data are shown in FIG. 3.

Example 16 Screening of salts and co-crystals.

Numerous salt and co-crystal conditions have been tested targeting various pharmaceutically acceptable salts. Details of the experiment can be found in Table. 5 and 6. Experiments were carried out using various crystallization methods, including cooling, evaporation, slurry, and solvent milling. Solids from the salt and co-crystal screening experiments were observed by polarized light microscopy (PLM) and analyzed by XRPD. The XRPD patterns of isolated solids were compared to data from known forms of AG10 and counterion/coformer.

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Confirmed salts of AG10 have been identified from experiments aimed at salt formation with strong acids, in particular methanesulfonic acid, ethane-1,2disulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, ethanesulfonic acid, sulfuric acid, hydrogen bromide and nitric acid.

Salts or co-crystals from AG10 have also been isolated from experiments aimed at the formation of salts/co-crystals with weaker organic acids such as citric acid, acetic acid, maleic acid, oxalic acid and malic acid.

Experiments have also been carried out using L-tartaric acid, glycolic acid and fumaric acid; however, experiments conducted to isolate these materials as single-crystal phases were not successful and resulted in starting materials or a physical mixture of unique additional peaks with starting materials.

An attempt to prepare salts/co-crystals from AG10 with L-aspartic acid, benzoic acid, gentisic acid and succinic acid was unsuccessful.

Table 5. Preparation of pharmaceutically acceptable salts of formula I and formula Ib

Counterion (X:Y) ^a Conditions ^b Observations results Benzenesulfonic acid (1:1) 1. Add 2.0 ml THF to 94.7 mg AG-10 with stirring at 60°C 2. Hot Filtration 3. Add 52.3 mg acid to purify solution, leave at ET for ~15 min. 4. Cooled to room temperature, then stirred in within ~ 3 days 1. Cloudy solution 2. Transparent solution 3. Transparent solution 4. Remains solution with a solid, the solid was collected Fines, W/E (birefringence and extinction), agglomerates (PLM) Crystalline, besylate AGIO (XRPD) NVG (1:1) 1. Add 2.0 ml warmed MIBK to 94.2 mg AG-10 at 1. Remains solid 2. Transparent Fines, W/E, agglomerates (PLM) Crystal,

- 21 041474

stirring at 60°C. 2. Add 0.4 ml of heated DMSO to the sample at stirring at 60°C 3. Add 55 µl of HBr to the sample with stirring at 60°C 4. Cooled to room temperature, then placed in VO at room temperature for 3 5. Add 1.0 ml of MEK to the sample under sonication and heated to 60°C, cooled x 2 solution 3. Yellow solution and oil formed 4. Oil with solid particles 5. Remains solution with a solid, the solid was collected bromide AGIO (XRPD) Ethanesulfonic acid (1:1) 1. Add 3.0 ml THF to 110.9 mg AG-10 at 50°C at mixing 2. Hot filtration 3. Add 31 µl of acid to the sample with stirring at 50°C 4. Cooled to RT 1. Whitish solution 2. Transparent solution 3. Solid substances precipitate after ~ 1 min mixing at ET 4. Remains solution with a solid, the solid was collected Crystalline, esilate AGIO (XRPD) 1,2- ethanedisulfonic acid (eleven) 1. Add 2.0 ml of heated acetone to 101.8 mg of AG-10 with stirring at 50°C. 2. Add 0.5 ml of heated DMA to the sample at mixing 3. Add 70 mg of acid to the sample while stirring at 50°C 4. Hold at ET for ~10 min, cool to room temperature 1. Remains solid 2. Transparent solution 3. Precipitated solid 4. Remains solution with a solid, the solid was collected small fraction, agglomerates (PLM) Crystalline, AGIO edisylate + ethanedisulfonic acid (XRPD) 1,2- 1. 36.7 mg acid 1. Transparent Fine fraction, W/E,

- 22 041474

ethanedisulphonic acid (2:1) dissolved in 400 μl of acetone at 60°C 2. 106.5 mg AG-10 was dissolved in 2.0 ml DMA at 60°C 3. The acid solution was added to the AG-10 solution at ET with stirring and then cooled to room temperature. 4. Heated to 60°C, then cooled solution 2. Transparent solution 3. Cloudy solution 4. Remains solution with a solid, the solid was collected agglomerates (PLM) Crystalline, edisylate AGIO (XRPD) Methansulfonic acid (1:1) 1. Add 2.0 ml of heated MEK to 100.7 mg of AG-10 with stirring at 50°C 2. Add 0.3 ml of heated DMF to the sample at mixing 3. Add 23 µl of acid to the sample with stirring at 50°C 4. Hold at ET for ~20 min, cool 1. Remains solid 2. Transparent solution 3. Precipitated solid 4. Remains solution with a solid, the solid was collected small fraction, agglomerates (PLM) Crystalline, AGIO Mesylate (XRPD) P- toluenesulphate (eleven) 1. Add 3.5 ml ACN to 112,OmgAO-Iu with stirring at 60°C 2. Add 74.5 mg of acid to suspension at ET 3. Increased to 70°C at mixing 4. Mixed solids back into solution, cooled to room temperature 1. Present solid 2. Solution became transparent to short time, then dropped out sediment 3. Transparent solution with a solid walls 4. Remains solution with a solid, the solid was collected small fraction, agglomerates (PLM) Crystalline, Tosi Lat AGIO (XRPD) Nitric acid (1:1) 1. Add 0.7 ml DMSO to 95.8 mg AGIO at stirring at room temperature. 2. Add 330 µl of acid to the solution at room temperature 1. Transparent solution 2. Transparent solution 3. Remains solution with a solid, the solid was collected Crystalline, Form A Nitrate AGIO (XRPD)

- 23 041474

3. Left at room temperature at mixing for 2 days Nitric acid (1:1) 1. Add 3.0 ml THF to 97.2 mg AG-10 with stirring at room temperature temperature. 2. Add 330 µl of acid to the solution at room temperature 3. Left at room temperature at mixing for 2 days 4. Placed in VO at RT in RT 1. Remains solid 2. Transparent solution 3. Transparent solution 4. Remains solid Crystalline, Form B Nitrate AGIO (XRPD) Sulfuric acid (1:1) 1. Add 2.0 ml warmed EtOH to 108.5 mg AG-10 with stirring at 60°C 2. Hot filtration 3. Add 22 µl of acid to the sample while sitting at 60°C 4. Cooled to room temperature temperature and mixed in within 2 days 5. Placed at 28°C for 5 days 6. Evaporation at room temperature temperature for 11 days 7. Evaporation under nitrogen atmosphere for 2 days 1. Opaque solution 2. Transparent solution 3. Whitish solution 4. Cloudy solution 5. Remains cloudy solution 6. Transparent solution 7. Remains solid fan agglomerates, B/E (PLM) Crystalline, sulfate AGIO (XRPD)

a. X: Y=AG-10: mole ratio of counterions, b. Temperature and time are approximate.

Table 6. Preparation of pharmaceutically acceptable salts of formula I and formula Ib

Coformer (X:Y) ^a Conditions ^b Observations results Acetic acid (1:2.2) 1. Add 2.0 ml warmed EtOH to 85.0 mg AG-10 with stirring at 70°C 1. Clear solution 2. Present solid 3. Clear solution Fines, W/E, agglomerates (PLM) Crystalline AGIO (Form G [2])

- 24 041474

2. Add 37 µl of acetic acid to the sample with stirring at 70°C 3. Cooled to room temperature, kept overnight and heated to 60°C for ~ 3 hours. 4. Cooled to room temperature and stirred for ~2 days 4. Remains a solution with a solid, a solid collected (XRPD) Acetic acid (1:1) 1. Grind 102.7 mg AG- IO with 40 µl acetic acid 1. Solids are turned into a paste, then dried and collected white solid. Crystalline, Acetic Acid Salt Form A AGIO (XRPD) Citric acid (excess) 1. Added 3.0 ml of saturated solution citric acid in IP A to 78.3 mg AG-10 with stirring at room temperature. 2. Stirred at room temperature for 1 day 3. Placed at 2-8°C for 1 day 4. Evaporated at room temperature after 10 days 5. Returned for evaporation at room temperature for 7 days and then placed in VO for ~1 day. 6. Placed for evaporation in a nitrogen atmosphere for 1 d 1. Present solid 2. Clear solution 3. Clear solution 4. Collected single crystal 5. Remains a solid with a liquid 6. The remaining solid was used for XRPD Citrate AGIO (SCXRD) Citric Acid + Citrate AGIO (XRPD) Citric acid (1:1) 1. Add 1.6 ml of IP A to 72.9 mg of acid at 50°C 2. Add acid solution to 110.1 mg AG-10 with stirring at 50°C overnight 3. Cooled to room temperature at stirring, left overnight 1. Clear solution 2. Remains solid 3. Remains a solution with a solid, a solid collected small fraction, agglomerates, W/E (PLM) Crystalline AGIO (XRPD) L-malic acid (1:1) 1. Add 6.0 ml anhydrous ACN to 81.1 mg AG-10 with stirring at 50°C 2. Add 2.0 ml DCM to the sample with stirring at 50°C 3. Hot filtration 1. Solid remains 2. Whitish solution 3. Clear solution 4. Clear solution 5. Clear solution 6. Solution remains Starburst, agglomerates, W/E with gel (PLM)

- 25 041474

L-Malic Acid (Excess) Maleic acid (1:2.2) Maleic acid Maleic acid (1:1) Maleic acid (1:1) 4. Acid was added to the solution with stirring at 50°C 5. Cooled to room temperature and allowed to stir at room temperature. 6. Evaporation at room temperature 7. Placed in VO at RT 1. Add 1.0 ml saturated solution of L-malic acid in ACN to 89.3 mg AG-10 with stirring at 60°C 2. Cooled to room temperature and stirred for ~3 days 3. Placed at 2-8°C for 3 days. 1. Dissolve 85.7 mg of maleic acid in 1.5 ml of nitromethane at stirring at 70°C 2. Add acid solution to 87.8 mg AG-10 with stirring at ET 3. Cooled to room temperature, kept overnight and heated to 60°C for ~ 3 hours. 4. Cooled to room temperature and stirred for ~2 days 1. LIMS 471312 was dried at 110°C for ~7 min. 2. Cooled down to RT 1. 2.0 ml p-dioxane was added to 49.6 mg of acid and 122.9 mg of AG-10 at room temperature with stirring 2. The suspension was heated to 50°C and with stirring was added 4.0 ml of p-dioxane at 50°C. 3. Stirred at 50°C for ~2 days 1. The filtrate of the suspension LIMS 474152 was left evaporate at 7. Remains a solid with a gel 1. Clear solution 2. Clear solution 3. Thick slurry, collected solid substance 1. Clear solution 2. Solid remains 3. Solid remains 4. Remains a solution with a solid, a chalky solid substance is going 1. The solid has become gel-like with a yellow tint 2. Remains solid 1. Solid remains 2. Remains solid 3. Remains a solution with a solid, a solid collected 1. Solid remains Fines, W/E, agglomerates (PLM) Crystalline AGIO, L-malic acid (XRPD) Fines, W/E, agglomerates (PLM) Form B AGIO Maleate + Material A (XRPD) Agglomerates with minor B/E (PLM) Random form B maleate AGIO + amorphous halo (XRPD) Form B of AGIO maleate (XRPD) Form B of AGIO maleate + additional peak 16.4° + amorphous halo room temperature. (XRPD) Oxalic acid (1:1.1) 1. Add 4.0 ml anhydrous acetone and 1.0 ml DMA to 96.2 mg AG-10 with stirring at 50°C. 2. Hot filtration 3. 34.5 mg acid added to the sample at 50°C 4. Cooled to RT 1. Whitish solution 2. Clear solution 3. Transparent solution, then precipitation of a solid 4. Remains a solution with a solid, a solid collected Crystalline Oxalate AGIO (XRPD)

a. X: Y=AG-10: molar ratio of counterions, b. Temperature and time are approximate.

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Example 17 Preparation of the mesylate salt of formula IX.

The formula IX mesylate salt was prepared by adding 1 molar equivalent of methanesulfonic acid to a solution of AG-10 MEK: DMF 2: 0.3 v/v at elevated temperature. The suspension was kept at an elevated temperature for ~20 min, cooled to room temperature and a solid was separated.

The XRPD pattern is shown in Fig. 4 and shows the resolution of peaks indicative of crystalline material. An attempt was made to index the XRPD pattern; however, no indexing solution was found, possibly due to the sample containing a mixture of crystalline phases or poor peak resolution.

1H NMR spectrum is consistent with AG-10 mesylate salt at a ratio of 1:1 mol based on a peak at 2.37 ppm. Also trace amounts of DMF and additional unknown peaks were observed in the spectrum.

The DSC thermogram (Fig. 5) shows one endotherm at approx. 233°C (maximum value), probably related to melting. No significant weight loss is observed in TGA (FIG. 5) up to ~200°C, indicating that the material is rather unsolvated/anhydrous.

Example 18 Preparation of the edisylate salt of formula IX.

The edisylate salt of formula IX was prepared by adding 1 molar equivalent of 1,2ethanedisulfonic acid to a solution of AG10 in acetone:DMA at elevated temperature. The suspension was cooled to ambient temperature and a solid was isolated.

The edisylate salt of formula IX was determined by XRPD to consist of crystalline material ( ^FIG . 6). Approximately one mole of DMA was also observed, indicating DMA edisylate solvate AG-10 (2:1:1).

The DSC thermogram (FIG. 7) shows a broad signature at ~139° C. associated with an 11% weight loss based on TGA (FIG. 7) data, which is likely related to desolvation. A sharper endotherm is observed at 313°C (maximum peak), which is probably related to the melting/decomposition of the desolvated material. Hot stage microscopy has been proposed to further understand the behavior of the material when heated.

A sample of the edisylate salt of formula IX was dried at 180° C. for 10 minutes and no change in physical form was noted by XRPD.

Example 19 Preparation of the besylate salt of formula IX.

The formula IX besylate salt was prepared by cooling a THF solution containing equimolar equivalents of AG-10 and benzenesulfonic acid.

The formula IX besylate salt consists of crystalline material and the XRPD pattern is shown in FIG. 8. The 1H NMR spectrum generally corresponds to besylate AG10 in an approximate ratio of 1:1. Trace amounts of THF were also observed in the spectrum based on the peak at 3.6 ppm.

The DSC thermogram shows two endotherms peaking at ~158°C and 177°C (FIG. 9). A weight loss of 0.2% is observed between 42 and 127°C (FIG. 9).

Example 20 Preparation of the tosylate salt of formula IX.

The formula IX tosylate salt was prepared by adding 1 molar equivalent of ptoluenesulfonic acid to a solution of AG-10 in acetonitrile at elevated temperature.

Based on the XRPD results, it is clear that the tosylate salt of formula IX is composed of a crystalline material (FIG. 10). The pattern has been successfully indexed and indicates that this material consists primarily or exclusively of a single crystalline phase. The unit cell volume obtained from the indexing solution corresponds to AG10 tosylate salt 1:1 based on molecular volume.

The 1H NMR spectrum was broadly consistent with the tosylate salt of AG10 at an approximate ratio of 1:1 mol based on the peak at 2.28 ppm.

The DSC thermogram shows one endotherm at approx. 205°C (maximum peak), probably due to melting (Fig. 11). At TGA up to ~160°C, no significant weight loss is observed, suggesting that the material is rather unsolvated/anhydrous (FIG. 11).

Example 21 Preparation of the esylate salt of formula IX.

The esylate salt of formula IX was precipitated from a solution in THF containing AG-10 and ethanesulfonic acid (ratio 1:1 mol) at a temperature of 50°C. The suspension was cooled and a solid was isolated.

The esylate salt of formula IX is composed of crystalline materials as confirmed by ^XRPD (FIG. 12). THF was observed in the spectrum, approximately 0.1 mol.

A single endotherm at 199° C. (peak maximum) is observed on the DSC thermogram, probably due to melting (FIG. 13). No significant weight loss on heating to melt, which may indicate that the material is unsolvated or anhydrous (FIG. 13).

- 27 041474

Example 22 Preparation of the bromide salt of formula IX.

The bromide salt of formula IX was prepared by adding an equimolar amount of hydrogen bromide to a solution of MIBK: DMSO 2: 0.4 v/v AG-10 at ~60°C. This gave a yellow solution and an oil. The sample was placed in a vacuum oven at room temperature for 3 days and an oil with solids was obtained. MEK was added to the sample using ultrasound and heated to 60°C, then cooled twice. The solid remaining in the resulting suspension was isolated and analyzed.

The bromide salt of formula IX consists of a crystalline material (FIG. 14). 1H-NMR spectrum corresponds to the chemical structure of AG-10. Also observed DMSO based on the peak at 2.54 ppm, approximately 1 mol.

Based on the IC, the bromide content was found to be 17.7% by weight, which is consistent with the calculated bromide content (17.7%) in solvate AG10, bromide, DMSO 1:1:1.

The DSC data show an endotherm at ~105°C (peak maximum), followed by an exotherm at 155°C (peak maximum) and an endotherm at ~214°C (FIG. 15). A weight loss of 19.9% is observed upon heating to ~182°C, which is probably due to the loss of solvent and possible recrystallization to the non-solvated form (Fig. 15).

Example 23 Preparation of the nitrate salt of formula IX.

Two forms of the nitrate salt of formula IX have been identified. These two forms are referred to as form a and form b.

Nitrate salt form a.

Form a of the nitrate salt of formula IX precipitated from a DMSO solution containing AG10 and nitric acid in equimolar ratios. Material A of nitrate AG-10 consists of a single crystalline phase, as evidenced by the successful indexing of the XRPD pattern (FIG. 16).

The ¹ H-NMR spectrum of a solution of form A of AG10 nitrate corresponds to the chemical structure of AG10. DMSO, approximately 0.8 mol, is present based on the peak at 2.54 ppm. Water and minor additional peaks were also observed.

The DSC thermogram shows a wide endotherm at ~117° C. associated with a 2.5% weight loss, likely due to loss of volatiles (FIG. 17). A broad endotherm is followed by an exotherm peaking at ~173° C. associated with a ~16% weight loss, likely due to melt/decomposition (FIG. 17).

The nitrate content was found to be 7.5% by weight based on IC, which is not consistent with the calculated nitrate content expected for a non-solvated 1:1 nitrate salt (theoretical nitrate content: 17.5%) or even a DMSO solvate, nitrate, AG10 1:1:1 (theoretical nitrate content: 14.3%).

Nitrate salt form b.

Form b of the nitrate salt of formula IX was obtained by evaporating a solution of THF containing AG-10 and nitric acid in an equimolar ratio. The XRPD pattern of this solid is shown in FIG. 18.

The ¹ H-NMR spectrum of a material B solution of nitrate AG10 corresponds to the chemical structure of AG10.

The nitrate content was found to be 16.9% by weight, based on the IC, which is generally consistent with about 1:1 AG-10 nitrate salt.

Example 24 Preparation of the sulfate salt of formula IX.

The sulfate salt of formula IX was prepared by evaporating an ethanolic solution containing equimolar amounts of AG-10 and sulfuric acid, which was obtained on cooling (60 to 2-8°C). The sulfate salt of formula IX consists of a crystalline material (FIG. 19).

The ¹ H-NMR spectrum confirms the presence of AG10 and includes about 1 mole of ethanol based on peaks at 1.06 and 3.4 ppm. Additional unknown peaks were also observed in the spectrum.

The sulfate content was found to be 15.9% by weight, based on the IC, corresponding to an AG10:sulfate ratio of 1:0.58.

In the TGA thermogram between ~30°C and 96°C there is a weight loss of 6.4%, which is equivalent to 1 mole of ethanol, provided that the salt AG10 sulfate is 2:1 (Fig. 20). Broad features were observed by DSC (FIG. 20).

Example 25 Preparation of the citrate salt of formula IX.

Single crystals of the citrate salt of formula IX were obtained after the IPA solution saturated with citric acid and containing AG-10 was evaporated at room temperature. After recovery of a suitable single crystal for SCXRD, the sample was further evaporated and the collected solid consisted of a mixture of AG-10 citrate and citric acid based on XRPD.

The structure of AG10 citrate was successfully determined. The crystal system is triclinic and the space group is PI. Cell parameters and calculated volume: a=7.64605(12) A, b=8.37443(13) A, c=17.8097(3) A, α=87.9509(14)°, β=79, 7770(14)°, γ=88.3139(13)°, V= ^1121.24 (3) A cm ^-3 .

The second experiment was carried out with the aim of obtaining a set of solid particles of AG10 citrate in

- 28 041474 as one crystalline phase for further characterization. The experiment also produced a physical mixture of AG10 citrate and citric acid.

Example 26 Preparation of the oxalate salt of formula IX.

The oxalate salt of formula IX is precipitated from a DMA solution containing AG10 and oxalic acid (molar ratio 1:1) at 50°C. The sample was cooled to room temperature and the solid was isolated for characterization.

According to the results of XRPD, the oxalate salt of formula IX consists of a crystalline material (Fig. 21). The XRPD pattern of the sample was successfully indexed, indicating that the sample consists mainly or exclusively of a single crystalline phase. Indexed volume corresponds to AG10 hemisoxalate based on molecular volume.

The ¹ H-NMR spectrum corresponds to the chemical structure of AG-10. Also present in the spectrum were DMA, approximately 0.1 mol, and water.

According to IC, the oxalate content of the sample was 13.7%, confirming the ~2:1 stoichiometry of the AG10 hemisoxalate salt.

The DSC data shows a single endotherm at ~225°C (maximum peak), which is likely related to melt/decomposition based on the TGA data (FIG. 22). The TGA thermogram shows an initial weight loss of 0.9% on heating between 33 and 169° C., likely due to the loss of residual surface solvent such as DMA, which was observed by 1 H NMR (FIG. 22).

Example 27 Preparation of the maleate salt of formula IX.

Two forms of the maleate salt of Formula IX have been identified. These two forms are referred to as form a and form b.

Maleate salt form a.

Addition of a nitromethane solution of maleic acid (2.2 molar equivalents) to AG-10 at 70° C. gave a slurry. The suspension was cooled to room temperature and heated twice to 60° C. before solids were isolated.

Form a of the maleate salt of formula IX consists of crystalline material as determined by XRPD (FIG. 23). The XRPD pattern could not be indexed, which indicates that the material does not consist of a single crystalline phase and is a possible mixture of forms. XRPD analysis suggests that form a of the maleate salt of formula IX was isolated as a mixture with form b of the maleate salt of formula IX.

The 1H NMR spectrum of the sample contained AG-10:maleic acid in an approximate ratio of 1:1 mol based on a peak at 6.23 ppm. Approximately 1.3 moles of nitromethane is observed for every mole of AG10 based on the presence of a peak at 4.42 ppm. Additional unknown peaks were also observed in the spectrum.

An endotherm was observed in the DSC data at ~160° C. (peak maximum) (FIG. 24). An 8.4% weight loss was observed upon heating to 110° C., which may be due to loss of solvent (FIG. 24) , consistent with the form b of the maleate salt of formula IX according to the results of XRPD.

According to XRPD and 1H NMR, the sample most likely consists of a mixture of form b of the maleate salt of formula IX and a possible nitromethane solvate.

Maleate salt form b.

Form b of the maleate salt of formula IX was prepared in an elevated temperature suspension experiment containing AG-10 and maleic acid (1:1) in p-dioxane. The XRPD pattern of form b of the maleate salt of formula IX (FIG. 25) was successfully indexed, indicating that the pattern consists predominantly or exclusively of a single crystalline phase. The indexed volume corresponds to the maleate salt AG10 1:1.

The 1H NMR spectrum of the sample was consistent with AG10 and maleic acid in a 1:1 molar ratio. Approximately 0.3 mol of p-dioxane was observed in the spectrum.

A single endotherm is observed at ~171°C (peak maximum) in the DSC thermogram (Fig. 26). No significant weight loss was observed when the sample was heated between 33 and 120° C. (FIG. 26).

Example 28 Preparation of Acetic Acid Salt of Formula IX

Salts of acetic acid of formula IX were obtained by direct grinding of AG-10 with acetic acid in a ratio of 1: 1 mol.

According to XRPD, the acetic acid salt of formula IX consists of a crystalline material and is shown in FIG. 27. The X-ray pattern has been successfully indexed, indicating that the sample consists mainly or exclusively of one crystalline phase.

The ¹ H-NMR spectrum is consistent with the chemical structure of AG-10, with approximately 0.9 moles of acetic acid present.

The DSC thermogram showed a wide endotherm at ~113°C, which is associated with a weight loss of ~16%, probably due to the loss of acetic acid (Fig. 28). This is followed by endotherms at 186 and 192°C (peak maximum), which are likely associated with the melting of the free form of AG10 (Fig. 28).

- 29 041474

Example 29 Preparation of the L-malic acid salt of formula IX.

Cooling the solution obtained by adding a saturated solution of L-malic acid in nitromethane to AG-10 at 60° C. gave a solid below ambient temperature. The XRPD pattern consists of a unique crystalline material indicative of the L-malic acid salt of formula IX (Fig. 29).

The α-NMR spectrum contained 1.8 moles of malic acid per mole of AG-10 based on a peak at 4.2 ppm. Insignificant amounts of ACN and water were observed in the spectrum.

In the DSC data, two broad endotherms are observed with peak maxima at ~89°C and 199°C (Fig. 30). A weight loss of 0.2% is observed between 33 and 107°C (FIG. 30).

Example 30 Crystal Form A of formula IX.

The material of formula Ia (HCl salt of formula IX) obtained in example 7 was characterized by powder x-ray diffraction (XRPD) (Fig. 31), thermogravimetric analysis (TGA) (Fig. 32-34), differential scanning calorimetry (DSC) (Fig. 32-34) and polarized light microscopy (PLM) (FIG. 35) This material was named crystalline form A of formula IX. In FIG. 31 overlays three different XRPD plots showing three different formulations according to Example 7.

Typical peak values of the XRPD plots shown in FIG. 31 are presented in table. 7 below.

Table 7. Typical XRPD peak values ____ for crystalline form A of formula IX

Position [° 20] Relative intensity [%] 7.0 23.50 10.4 21.74 12.0 51.77 13.0 6.45 13.9 34.17 15.6 31.64 17.0 31.28 18.6 20.8 21.8 23.3 24.2 24.7 25.3 25.9 26.7 27.9 15.02 19.23 100.00 12.57 14.33 14.29 17.29 63.60 55.94 44.84 28.2 12.96 28.6 10.35 29.1 13.20 30.9 13.10 31.6 10.76 33.8 2.95 34.5 4.62 37.9 4.55

Three separate TGA/DSC plots of crystalline form A of formula IX are shown in FIG. 32-35. A weight loss of about 0.7 to 1.9% when heated to a temperature of about 150°C was measured by thermogravimetric analysis, and further characterization using differential scanning calorimetry showed at least two endothermic peaks at about 211-214°C and 237-239°C. The HPLC purity of crystalline form A of formula IX was determined to be 98.76 area%.

The asymmetric unit of crystalline form A of formula IX is shown in FIG. 36. It includes one AG 10 free base compound cation, and one chloride ion (the HCl molecule has transferred a proton to the N1 atom of the free base), indicating that form A is the anhydrous form of the mono-HCl salt.

In order to evaluate the hygroscopicity and physical stability of crystalline form A of formula IX at various humidity levels, dynamic vapor sorption (DVS) data were collected at 25° C. after the sample had been pre-equilibrated at 0% RH to remove free water. The DVS result (FIG. 37) showed a water uptake of 1.6% at 25° C./80% RH, indicating that crystalline form A of formula IX is slightly hygroscopic. In addition, the XRPD results (FIG. 38) showed no shape change before and after the DVS test.

Example 31 AG-10 Polymorphic Screening.

Using crystalline form A of formula IX as starting material, experiments

- 30 041474 polymorph screening was performed under 98 conditions using vapor diffusion, antisolvent addition, suspension conversion, slow evaporation and slow cooling methods. A total of ten additional crystalline forms resulted from polymorph screening and subsequent investigation, including six HCl salt forms (A/B/E/H/I/J form), two free base forms (C/G form), and two currently unidentified forms (form D/F). Forms A/B/E were identified as anhydrous. Form I was identified as a hydrate. Forms H and J were identified as MeOH solvate and DMAc solvate, respectively. Used methods and identified crystalline forms are given in table. 8.

Table 8 Summary of AG 10 polymorph screening

Way Number of experiments Isolated forms of the solid Addition of anti-solvent 24 form A, C, D, E, form A + additional peaks slow evaporation 7 form A, H slow cooling 8 shape A, E, J Slurry conversion 25 shape A, C, F, G Diffusion solid substance-vapour 13 form A, form A + additional peaks Diffusion liquid-vapor 21 form A Total 98 form A, C, D, E, F, G, H, J, form A + additional peaks

Antisolvent additions.

A total of 24 antisolvent experiments were performed. For each experiment, about 15 mg of crystalline form A of formula IX was weighed into a 20 ml glass vial followed by the addition of 0.125-0.63 ml of the appropriate solvent. The mixture was then stirred with a magnetic stirrer at 750 rpm to obtain a clear solution at room temperature. An appropriate anti-solvent was then added to the solution to cause precipitation or until the total amount of anti-solvent reached 10.0 ml. Clear solutions were transferred into suspension at 5°C. If precipitation did not occur, the solution was then transferred to rapid evaporation at room temperature or vacuum drying at room temperature. Solids were isolated for XRPD analysis. The results summarized in table. 9 showed that forms A, C, D, E and form A with additional peaks were obtained.

Table 9. Summary of Antisolvent Addition Experiments

Solvent Antisolvent Final Results DMSO IPA Form A + one peak ** Acetone Form A + one peak * EtOAc Form A THF Form A* * ACN Form A* Toluene Form E DCM Form A H ₂ O Form C Meon MEK Form A IPAc Form D 2-MeTHF Form A* SRME Form A ACN Form A H ₂ O Form C CHCE Form A + additional peaks ** Toluene Form A + additional peaks EtOH MIBK Form A EtOAc Form A* * 1,4-dioxane Gel anisole Form A* * ACN Form E* DXM Form A* * n-heptane Form A H ₂ O Form C

* solids were obtained by stirring at 5°C, ** solids were obtained by rapid evaporation or vacuum drying at room temperature.

Slow evaporation.

Slow evaporation experiments were carried out under 7 conditions. For every experiment

- 31 041474 about 15 mg of crystalline form A of formula IX was weighed into a 3 ml glass vial followed by the addition of an appropriate solvent or mixture of solvents to obtain a clear solution. The vial was then covered with 3~4 pinhole parafilm and kept at room temperature to slowly evaporate the solution. The isolated solids were verified by XRPD. As shown in Table. 10, forms A and H were obtained.

Table 10. Summary of _______________ Slow Evaporation Experiments _______________

Solvent (v/v) Final Results Meon Form A EtOH Form A DCM/MeOH 1:1 Form A Acetone/MeOH, 4:1 Form H EtOAc/EtOH, 4:1 Gel THF/MeOH, 4:1 Form A ACN/ethanol, 4:1 Form A

Slow cooling.

Slow cooling experiments were carried out in 8 solvent systems. For each experiment, about 15-35 mg of crystalline form A of formula IX was suspended in 0.8-2.0 ml of the appropriate solvent in a 3 ml glass vial at room temperature. The suspension was transferred into suspension at 50°C using a magnetic stirrer at 750 rpm. The sample was equilibrated at 50° C. for 1 hour and filtered using a 0.45 µm PTFE membrane. The filtrate was then slowly cooled from 50 to 5° C. at a rate of 0.1° C./min. If precipitation did not occur, then the solution was then transferred to rapid evaporation at room temperature or vacuum drying at room temperature. The results are given in table. 11 showed that forms A, E and J were obtained.

Table 11. Summary of experiments ________ with slow cooling ________

Solvent, v/v Final results * EtOH Form E IPA Form A** MEK/DMAc, 3:1 Form J** IP Ac/EtOH, 3:1 Form A Anisole/MeOH, 3:1 Form A ACN/NMP, 3:1 Clear solution H2O/DMAc, 3:1 Form J** SNS1 s/EUN, 3: 1 Form E

* all samples were converted to slow evaporation at room temperature, ** solids were obtained after vacuum drying at 50°C, carried out on clear solutions.

Slurry conversion at room temperature.

Suspension conversion experiments were carried out at room temperature in various solvent systems. For each experiment, about 15-35 mg of crystalline form A of formula IX was suspended in 0.3-2.0 ml of the appropriate solvent in a 1.5 ml glass vial. After the suspension was stirred with a magnetic stirrer for 4 days at room temperature, the remaining solid was isolated for XRPD analysis. The results summarized in table. 12 showed that forms A, C and G were obtained.

Table 12. Summary of experiments ____________ on suspension conversion at RT____________

Solvent, v/v Final Results EtOH Form A H ₂ O Form C EtOH/H ₂ O, 97:3, a _w = 0.2 Form A EtOH/H ₂ O, 927: 73, a _w = 0.4 Form A EtOH/H ₂ O, 86:14, a _w = 0.6 Form A EtOH/H ₂ O, 71:29, a _w = 0.8 Form A _H2O /DMAc, 3:1 Form G MIBK/MeOH, 1:1 Form A THF/ _H2O , 9:1 Form A ACN/EtOH 3:1 Form A DCM/DMSO, 3:1 Form A EtOAc/DMF, 3:1 Form A

- 32 041474

Slurry conversion at 50°C.

Suspension conversion experiments were carried out at 50°C in various solvent systems. For each experiment, about 15 mg of crystalline form A of formula IX was suspended in 1.0 ml of the appropriate solvent in a 1.5 ml glass vial. After the suspension was stirred with a magnetic stirrer for 4 days at 50°C, the remaining solid was isolated for XRPD analysis. The results summarized in table. 13 indicate that forms A and F were obtained.

Table 13 Summary of Slurry Conversion Experiments at 50°C

Solvent Temperature, °C Final Results IPA 50 Form A SNS1 ₃ Form A Acetone Form A MEK Form A IP Ac Form A EtOAc Form A anisole Form A THF Form A 2-MeTHF Form A 1,4-Dioxane Form A ACN Form A Toluene Form F 1-Butanol Form A

Diffusion solid-vapour.

Solid-vapor diffusion experiments were carried out using 13 solvents. For each experiment, about 15 mg of crystalline form A of formula IX was weighed into a 3 ml vial, which was placed in a 20 ml vial with 4 ml of the appropriate solvent. The 20 ml tube was capped and kept at room temperature for 39 days to allow solvent vapors to interact with the solid sample. The isolated solid was verified by XRPD. The results are given in table. 14 showed that form A and form A with additional peaks were obtained.

Table 14. Summary of solid-vapor diffusion experiments

Solvent Final Results H ₂ O Form A DCM Form A EtOH Form A Meon Form A ACN Form A THF Form A SNS1z Form A Acetone Form A DMF Form A EtOAc Form A 1,4-dioxane Form A IPA Form A DMSO Form A + additional peaks

Diffusion liquid-vapor.

Twenty-one liquid-vapor diffusion experiments were carried out. For each experiment, about 15 mg of crystalline form A of formula IX was dissolved in 0.125-0.6 ml of the appropriate solvent to obtain a clear solution in a 3 ml vial. The solution was then placed in a 20 ml vial containing 4 ml of the appropriate antisolvent. A 20 ml tube was capped and kept at room temperature, leaving enough time for the solvent vapors to interact with the solution. If no red precipitate formed, the solution was transferred to rapid evaporation at room temperature. Solids were isolated for XRPD analysis. The results summarized in table. 15 showed that form A was obtained.

- 33 041474

Table 15 Summary of Liquid-Vapor Diffusion Experiments

Solvent, v/v Anti solvent Final Results DCM/MeOH 1:1 Toluene Form A MIBK Form A EtOAc Form A ACN Form A anisole Form A EtOH MEK Form A IP Ac Form A 2-MeTHF Form A ACN Form A H ₂ O Form A SNS1 ₃ Form A n-heptane Form A Toluene Form A DMF IPA Clear solution Acetone Clear solution EtOAc Clear solution THF Clear solution ACN Clear solution Toluene Clear solution H ₂ O Limited Solid DCM Clear solution

A diagram summarizing the interconversions between the identified crystalline forms is shown in FIG. 39.

Abbreviation for solvents.

Table 16. List of solvent abbreviations

Reduction Solvent Reduction Solvent Meon methanol THF Tetrahydrofuran EtOH ethanol 2-MeTHF 2-Methyltetrahydrofuran IPA Isopropyl alcohol ACN Acetonitrile MIBK 4-Methyl-2-pentanone DMSO Dimethyl sulfoxide EtOAc ethyl acetate DCM dichloromethane IP Ac Isopropyl acetate DMAc N,N-dimethylformamide MTBE methyl tert-butyl ether MEK Methyl ethyl ketone

XRPD Tools and Methods.

For XRPD analysis, PANalytical X-ray powder diffractometers were used. The XRPD parameters used are listed in Table 1. 17.

Table 17. Parameters for the XRPD test

Options PANalytical (reflective mode) PANalytical (reflective mode) Model Empyrean X' Pert ³ X-ray wavelength Si, ka, Καί (A): 1.540598, Ka2 (A): 1.544426 Intensity factor Ka2/Ka1: 0.50 Si, ka, Καί (A): 1.540598, Ka2 (A): 1.544426 Intensity factor Ka2/Ka1: 0.50 X-ray tube settings 45 kV, 40 mA 45 kV, 40 mA divergence gap automatic 1/8° Scan Mode continuous continuous Scan Range (2TH) 3°-40° 3°-40° Scan stage time (s) 17.8 46.7 Stage size (°2TH) 0.0167 0.0263 Test time 5 min 30 s 5 min 04 s

TGA and DSC.

TGA data was collected using a TA Q500/Q5000 TGA from TA Instruments. DSC was performed using TA Q200/Q2000 DSC from TA Instruments. The detailed parameters used are listed in Table. 18.

-34041474

Table 18. Parameters for TGA and DSC test

Options TGA DSC Way Ramp Ramp sample cuvette Platinum, open Aluminium, corrugated Temperature RT-300°C 25- target temperature Heating rate 10°C/min 10°C/min Purge gas n ₂ n ₂

1H NMR.

1H-NMR data were obtained on a Bruker 400M NMR spectrometer using DMSO-d6.

DVS.

DVS was measured using SMS (Surface Measurement System) from DVS Intrinsic. The parameters for testing DVS are given in Table. 19.

____________________Table 19. Parameters for the DVS test____________________

Options Meaning temperature 25°С Sample size 10-20 mg Gas and flow rate N2, 200 ml/min dm/dt 0.002%/min Min. stability duration dm/dt 10 min. Max, equilibrium time 180 min. RH range 0%RH-95%RH-0%RH step size RH 10% (0%RH-90%RH-0%RH) 5% (90%RH-95%RH and 95%RH-90%RH)

HPLC.

An Agilent 1100/1260 HPLC was used for purity and solubility analysis and the detailed method is shown in Table 1. 20.

_________Table 20. HPLC method for testing purity and solubility_______

Chapter Purity Solubility Column Phenomenex Gemini C18 110A, 4.6x250 mm, 5.0 pm Mobile phase A: 0.1% _FAbH2O B: 0.1% FA in acetonitrile gradient table Time(min) %IN Time(min) %V 0.0 5 0.0 5 20.0 100 7.0 100 20.1 5 7.1 5 25.0 5 10.0 5 lead time 25.0 min. 10.0 min. fasting time 0.0 min. 0.0 min. Flow rate 1.0 ml/min injected volume 10 µl Detector wavelength UV at 254 nm Column temperature 30°C Sample temperature RT Diluent ACN: _H2O (1:1)

IC.

The IC method for measuring Cl' was listed in Table 1. 21.

___________Table 21. IC method for measuring Cl-

Chapter Meaning Column Analytical column lonPac AS 18 (4 χ 250 mm) Mobile phase 25MMNaOH injected volume 25 µl Flow rate 1.0 ml/min injected volume - 50 µl Cell temperature 35°C Column temperature 35°C Current 80 mA lead time 6.0 min

Example 32 Preparation of crystalline form B of formula IX.

Crystal Form B of formula IX was prepared by heating a sample of Crystal Form A to 212° C., cooling to 30° C. under nitrogen protection and exposure to air. HPLC purity and stoichiometry (acid: FB) of crystalline Form B were determined to be 97.86 area % and 0.86, respectively. The XRPD pattern is shown in Fig. 40 and the TGA/DSC curves are shown in FIG. 41. The results showed that form B was crystalline with a 1.2% weight loss to reach 150°C in TGA and three endothermic peaks at 161.4, 232.2 and 262.3°C (peak) in DSC.

- 35 041474

Due to the limited weight loss of TGA and pure DSC up to 150°C, Form B was presumably anhydrous. To study the thermal signal, a heating experiment was carried out. As shown in FIG. 42 Form B is converted to Form I (Form I is discussed in more detail in Example 38) upon heating to 100 or 170°C, cooling to 30°C under nitrogen protection and subsequent exposure to air.

The peak values of the XRPD plot shown in FIG. 40 are presented in table. 22 below.

Table 22. XRPD Peak Values for _________Crystal Form B of Formula IX_________

Position [° 20] Relative intensity [%] 9.8 2.40 12.0 100.00 13.8 25.37 17.2 12.82 17.7 13.85 18.8 2.52 19.8 38.56 20.7 3.48 21.1 6.60 21.9 2.68 22.6 7.05 23.3 92.78 24.4 21.44 24.8 23.23 26.3 13.18 27.0 3.20 27.7 6.15 28.3 2.26 28.9 14.47 29.9 8.96 30.2 2.79 31.5 1.89 36.1 h, p 37.8 1.64

Example 33 Preparation of crystalline form C of formula IX.

Crystalline Form C of Formula IX was prepared by adding an anti-solvent in DMSO/H ₂ O at room temperature and its XRPD is shown in FIG. 43. TGA and DSC results in FIG. 44 showed a 3.1% weight loss to 150°C and two endothermic peaks at 91.2 and 173.0°C. Since the Cl content of the Form C sample was 0.17% (the theoretical Cl content ^of the mono-HCl salt is 10.8%), Form C was confirmed as the free base form.

The peak values of the XRPD plot shown in FIG. 43 are presented in table. 23 below.

- 36 041474

Table 23. XRPD Peak Values for Crystal Form C of Formula IX

Position [° 20] Relative intensity [%] 9.5 10.95 I,7 19.10 12.3 38.53 13.4 19.21 14.6 99.67 15.8 37.90 16.7 79.33 17.2 16.59 17.8 8.43 18.4 6.88 19.5 41.44 20.3 9.55 20.7 21.39 21.4 42.38 21.7 18.03 22.5 37.96 22.9 26.38 24.0 4.72 24.7 20.49 26.1 100.00 26.7 18.16 28.8 8.11 29.6 5.55 30.4 5.96 31.1 5.56 34.4 5.29 35.3 4.44 36.3 2.70 38.1 4.10

Example 34 Preparation of Crystal Form D and Form F of formula IX.

Crystal form D of formula IX was obtained by adding an anti-solvent in the MeOH/IPAc system at room temperature. Form F of formula IX was obtained using a suspension of form A in toluene at 50°C. Their XRPD patterns are shown in Fig. 45 (Form D, top plot; Form F, bottom plot).

The peak values of the XRPD plot shown in FIG. 45 (forms D and F) are presented in table. 24 and tab. 25 below.

Table 24 XRPD Peak Values for Crystalline ______Form D of Formula IX______

Position [° 20] Relative intensity [%] 6.8 19.59 10.2 16.65 12.0 71.32 12.3 43.00 13.8 100.00 15.4 26.66 16.6 12.89 18.2 48.41 19.8 13.71 21.7 78.63 22.2 64.23 24.0 26.05 26.0 72.52 26.5 51.29 27.8 21.59 30.8 23.74 34.6 4.15

- 37 041474

Table 25. XRPD Peak Values for Crystal Form F of Formula IX

Position [° 20] Relative intensity [%] 4.6 6.80 12.4 5.23 12.9 5.29 13.3 22.78 13.8 17.01 14.7 5.10 15.5 22.78 16.3 16.83 18.1 8.86 18.6 18.97 19.0 7.62 20.1 10.89 21.6 5.75 22.3 14.39 22.5 16.27 24.1 100.00 25.2 17.28 25.7 35.11 26.0 34.41 26.4 15.15 27.2 4.40 28.2 6.46 28.9 8.86 29.4 7.40 30.0 15.47 30.6 6.43 31.0 7.51 31.3 5.94 32.0 3.26 34.4 1.81 35.1 1.46 36.3 2.36 37.0 2.00 37.8 3.29

Example 35 Preparation of crystalline form E of formula IX.

Crystal form E of formula IX was obtained by slow evaporation in a mixture of CHCl ₃ /EtOH at room temperature. HPLC purity and stoichiometry (acid: FB) of crystalline form E of formula IX were determined to be 98.60 area% and 0.91, respectively. The XRPD pattern is shown in Fig. 46 and the TGA/DSC curves are shown in FIG. 47. The results showed that crystalline form E has a mass loss of 1.5% to 130°C in TGA and two endothermic peaks at 182.0 and 242.7°C in DSC (peak). Due to the limited weight loss of TGA and pure DSC up to 170°C, form E was presumably anhydrous. To study the thermal signal at 182.0°C (peak) in DSC, a heating experiment was performed. As shown in FIG. 48 Form E converts to Form I hydrate upon heating to 195°C, cooling to 30°C under nitrogen protection and subsequent exposure to air. Based on the thermal data and the heating experiment, Form E anhydride can be converted to a new anhydrous form (an endothermic DSC ~180°C signal may be a shape transition signal), and then the anhydride form is converted to Form I hydrate due to interaction with moisture when exposed to conditions environment.

The peak values of the XRPD plot shown in FIG. 46 are presented in table. 26 below.

- 38 041474

Table 26. XRPD Peak Values for Crystal Form E of Formula IX

Position [° 2Θ] Relative intensity [%] 11.8 29.29 14.0 18.45 15.1 12.00 17.2 2.51 18.2 0.97 19.9 3.73 20.6 2.90 21.4 1.43 24.0 3.14 25.8 100.00 27.8 2.34 29.1 2.06 33.2 1.16

Example 36 Preparation of crystalline form G of formula IX.

Crystal form G of formula IX was obtained via suspension in DMAc/H ₂ O (v:v, 1:3) at room temperature and its XRPD is shown in FIG. 49. TGA and DSC results in FIG. 50 showed a 3.7% weight loss up to 200°C and one sharp endothermic signal at 231.1°C (peak). Since the content of Cl ^- in the form G sample was 0.14% (the theoretical content of Cl ^- mono-HCl salt is 10.8%), it was confirmed that form G is the free base form.

The peak values of the XRPD plot shown in FIG. 49 are presented in table. 27 below.

Table 27. XRPD Peak Values for Crystal Form G of Formula IX

Position [° 2Θ] Relative intensity [%] 6.6 1.33 9.8 3.80 12.2 47.25 13.1 66.55 13.4 46.87 14.6 8.71 15.1 8.08 16.4 3.87 17.1 5.54 17.8 46.61 18.4 14.28 18.7 13.76 19.4 16.96 19.9 8.02 20.3 4.92 21.0 5.62 21.7 32.86 22.8 5.34 23.2 13.61 23.7 8.42 24.3 29.93 24.7 23.96 24.9 25.98 25.4 8.92 26.0 13.52 26.6 100.00 28.3 24.14 29.2 2.64 30.5 8.98 30.9 7.92 33.1 1.62 35.0 1.54 35.7 2.29

Example 37 Preparation of crystalline form H of formula IX.

The crystalline form H of formula IX was obtained by slow evaporation in acetone/MeOH at room temperature and its XRPD is shown in FIG. 51. HPLC purity and stoichiometry (acid: FB) of Form H (810119-11-A4) were determined to be 98.47 area% and 0.91, respectively

- 39 041474 no. Curves TGA and DSC (Fig. 52) showed a weight loss of 4.6% up to 120°C and three endothermic peaks at 90.4, 200.5 and 232.3°C (peak). As shown in the 1H NMR spectrum (FIG. 53), 0.36 equivalents of MeOH (~3.40% by weight) were detected. Combined with the fact that shape change to shape I was observed after shape H was heated to 120°C, cooled to 30°C under nitrogen protection (FIG. 54), and exposed to ambient conditions, it was hypothesized that the H form is a MeOH solvate.

The peak values of the XRPD plot shown in FIG. 51 are presented in table. 28 below.

Table 28. XRPD Peak Values for Crystal Form H of Formula IX

Position [° 20] Relative intensity [%] 11.8 28.73 12.3 21.19 13.8 14.54 15.7 5.38 16.9 16.03 20.6 4.73 21.7 100.00 23.2 17.57 24.7 12.94 25.7 58.09 26.7 6.83 27.8 9.32 28.1 9.00

Example 38 Preparation of crystalline form I of formula IX.

Crystal form I of formula IX was prepared by heating crystal form B of formula IX to 100° C., cooling to 30° C. under nitrogen protection and exposure to air. His XRPD is shown in Fig. 55. HPLC purity and stoichiometry (acid: FB) of crystalline form I of formula IX were determined to be 97.94 area% and 0.86, respectively. Since crystalline form I was obtained by solid phase transfer of anhydrous form B and a mass loss of 3.0% (equivalent to 0.5 molar water) was observed with an endothermic peak at 62.0°C (peak, Fig. 56), then form I, presumably is a hydrate.

The peak values of the XRPD plot shown in FIG. 55 are presented in table. 29 below.

Table 29. XRPD Peak Values for _________Crystal Form I Formula IX________

Position [° 20] Relative intensity [%] I,4 4.60 12.1 35.57 12.4 89.18 13.6 8.36 13.9 14.40 14.2 10.59 14.8 6.38 16.8 9.64 17.2 41.23 17.8 21.69 18.7 10.82 19.1 9.61 19.9 15.55 20.7 27.63 21.2 8.33 21.7 12.83 22.2 10.01 22.8 18.37 23.3 100.00 24.1 9.53 24.9 41.18 25.4 52.09 25.9 10.04 27.0 26.02 28.3 14.09 29.0 11.22

- 40 041474

29.6 8.99 30.0 8.40 30.8 5.50 32.8 6.13 33.3 6.37 33.8 4.22 36.1 1.88 36.9 2.91 37.8 2.89

To further identify crystalline Form I and investigate its dehydration behavior, in-situ XRPD was performed with N ₂ flow to observe dehydrated Form I, and a KF test was performed to confirm whether the TGA weight loss was due to water content or not. As shown in FIG. 57, shape change to anhydrous crystalline Form B was observed for crystalline Form I with N ₂ purge for approximately 1.5 hours (30°C/16% RH). As shown in FIG. 58 in crystalline Form I, a weight loss of 2.6% to 120°C was observed. Based on the KF result, a water content of about 3.48% was observed in the Form I crystalline sample. Combined with the change to anhydrous Form B in the N ₂ stream, Form I was identified as a hydrate.

Example 39 Preparation of crystalline form J of formula IX.

Crystal Form J of formula IX was obtained by slow evaporation followed by vacuum drying at 50° C. in the MEK/DMAc system and its XRPD is shown in FIG. 59. HPLC purity and stoichiometry (acid: FB) of crystalline form J of formula IX were determined to be 91.69 area% and 0.90, respectively. The TGA and DSC results in FIG. 60 showed a weight loss of 21.5% to 120°C and three endothermic peaks at 120.8, 197.8 and 221.5°C (peak). As shown in the 'H NMR spectrum (FIG. 61), 4.9 DMAc equivalents (-56.51% by weight) were detected. Combined with the fact that a change in shape of the mixture of crystalline form I (isolated) and crystalline form A was observed after crystalline form J was heated to 130°C, cooled to 30°C under nitrogen protection, and exposed to ambient conditions ( Fig. 62), form J is presumed to be a solvate of DMAc.

The peak values of the XRPD plot shown in FIG. 59 are presented in table. 30 below.

Table 30. XRPD Peak Values for Crystal Form J of Formula IX

Position [° 20] Relative intensity [%] 4.6 18.19 11.8 8.94 12.8 9.10 13.8 87.83 14.6 29.55 18.4 26.65 20.5 18.63 21.1 8.10 21.9 8.04 22.8 100.00 23.7 18.41 26.2 33.09 27.7 35.16 28.8 29.02 30.4 12.86 30.8 8.86 31.3 6.46 32.6 8.32 36.0 3.83

Example 40 Preparation of crystalline form K of formula IX.

The free base material prepared in Example 11 was characterized by XRPD (FIG. 63), TGA (FIG. 64) and DSC (FIG. 64). This material has been termed crystalline form K of formula IX. With TGA, a weight loss of 6.1% to 150°C was observed, and the DSC result showed endothermic peaks at 159.3, 176.2 and 278.4°C (peak). HPLC purity of crystalline form K of formula IX was determined to be 99.12 area%.

The peak values of the XRPD plot shown in FIG. 63 are presented in table. 31 below.

Claims (11)

Table 31. XRPD Peak Values for Crystal Form K of Formula IX Position [°2Θ] Relative intensity [%] 7.1 21.72 7.5 20.44 9.8 11.27 13.9 100.00 15.9 6.59 19.3 7.18 20.9 2.30 21.9 7.51 22.7 5.79 23.0 3.40 24.0 2.73 24.9 3.09 25.5 1.35 27.3 2.70 28.1 1.86 29.1 1.70 35.0 1.79 While the foregoing has been described in more detail for purposes of illustration and example for clarity and understanding, one skilled in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each source presented in this document is incorporated by reference in its entirety to the same extent as if each source were individually incorporated by reference. CLAIM 1. Crystalline form A of formula IX as its HCl salt COOH characterized by an X-ray powder diffraction pattern including peaks at 12.0, 21.8, 25.9, 26.7, and 27.9 degrees 2Θ (±0.2 degrees 2θ). 2. Crystalline form A of formula IX as its HCl salt according to claim 1, further characterized by an X-ray powder diffraction pattern including peaks at 7.0, 10.4, 13.0 and 13.9 degrees 2θ (±0.2 degrees 2θ). 3. Crystalline Form A of formula IX as its HCl salt according to claim 1 or 2, further characterized by an X-ray powder diffraction pattern including peaks at 15.6 and 17.0 degrees 2θ (±0.2 degrees 2θ). 4. Crystalline form A of formula IX as its HCl salt according to claim 1, characterized by an X-ray powder diffraction pattern corresponding to FIG. 31. 5. Crystalline form A of formula IX as its HCl salt according to any one of claims 1 to 4, which contains less than 2% of other crystalline or amorphous forms of formula IX. 6. Crystalline form A of formula IX as its HCl salt according to any one of claims 1 to 4, further characterized by a weight loss in the range of from about 0.7 to about 1.9% when heated to about 150°C, as measured by thermogravimetric analysis . 7. Crystalline form A of formula IX as its HCl salt according to claim 6, in which the weight loss is about 1.3%. 8. Crystalline form A of formula IX as its HCl salt according to any one of claims 1 to 7, characterized by a water uptake of about 1.6% at 25° C./80% relative humidity (RH) after undergoing a dynamic vapor sorption cycle that includes pre-equilibration at 0% RH. 9. Crystalline form A of formula IX as its HCl salt according to any one of claims 1 to 7, characterized by an increase of less than 2.5% by weight after going through a dynamic vapor sorption cycle from about 0% relative humidity (RH) to about 90% RH. 10. Crystalline form A of formula IX as its HCl salt according to any one of claims 1 to 9, having a dynamic vapor sorption profile as shown in FIG. 37. 11. Crystalline form A of formula IX as its HCl salt according to any one of claims 1-10, further characterized by a differential scanning calorimetry thermogram including endothermic peaks at a temperature of about 211-214 and 237-239°C. -