HK40095644A - Crystalline salt of a multi-tyrosine kinase inhibitor, method of preparation, and use thereof - Google Patents

Description

Crystalline salts of multiple tyrosine kinase inhibitors, their preparation methods and uses

Technical Field

This article discloses the crystalline salt form of a multiple tyrosine kinase inhibitor. Specifically, this article discloses the crystalline form of the multiple tyrosine kinase inhibitor (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide, a pharmaceutical composition comprising the crystalline form, a method for preparing the crystalline form, and a method for using the crystalline form.

Background Technology

N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 1 (sitravatinib), free base) is a multi-tyrosine kinase inhibitor with proven potent inhibition of a series of closely related tyrosine kinases (including RET, CBL, CHR4q12, DDR, and Trk), which are important regulators of signaling pathways that drive cell growth, survival, and tumor progression.

Compound 1 induced tumor regression in multiple human xenograft tumor models in mice and is currently being used in human clinical trials as a monotherapy and in combination for the treatment of a wide range of solid tumors. Compound 1 is currently undergoing a phase 2 clinical trial in patients with advanced cancer, advanced liposarcoma, and non-small cell lung cancer (NSCLC).

Compound 1, as a free base, and the corresponding pharmaceutical product exhibit poor long-term stability at room temperature, necessitating refrigerated storage of both the drug and pharmaceutical product. Therefore, there is particular interest in identifying and developing a solid form exhibiting enhanced stability (especially at room temperature), thereby improving the commercial acceptability and viability of Compound 1 as a drug candidate.

It is also well known in the pharmaceutical industry that any improvement in bioavailability will have a positive economic impact on the company and is also beneficial to our environment, as less API will be needed and manufactured. Although the formation of crystalline salts is sometimes a possible way to improve the solubility and dissolution rate of drugs, whether salt formation will improve or decrease bioavailability is unpredictable and can only be discovered through manufacturing and testing in animals and humans.

In practice, it is difficult to predict which physical form of a particular compound will be stable and suitable for pharmaceutical processing. It is even more difficult to predict whether a specific crystalline solid form with the chemical and physical properties required for pharmaceutical formulations can be prepared.

For pharmaceutical applications, a single solid phase with well-controlled polymorphism is required to ensure product homogeneity. Compound 1 possesses a large number of carbon-carbon, carbon-oxygen, and carbon-nitrogen σ bonds (13 in total) capable of participating in rotational isomerism. In the solid state, extensive polymorphism or mixtures between energy-similar rotational isomers are possible. Overcoming rotational isomerism by defining stable, scalable methods to reliably generate single-crystal phases is a key obstacle for the development and commercialization of drug candidates.

For all the foregoing reasons, there is a strong need to discover a crystalline form of the salt of compound 1 that can impart to a pharmaceutical composition the following properties: enhanced dissolution rate, adequate bioavailability with minimal or no surfactant use, acceptable stability/shelf life during storage at room temperature, and good manufacturability. The present invention advantageously addresses one or more of these needs.

Summary of the Invention

This invention addresses the aforementioned challenges and needs by providing a stable crystalline form of the salt of compound 1. For this purpose, various salts produced by pharmaceutically acceptable salt-forming agents (including citric acid, 1,2-ethanedisulfonic acid, hydrochloric acid, sulfuric acid, maleic acid, L-malic acid, succinic acid, L-tartaric acid, malonic acid, p-toluenesulfonic acid, p-toluenecarboxylic acid, and mandelic acid) were investigated, and polymorphism screening of the salts of interest was performed. It was determined that the salts produced by these experiments readily form large quantities of solid matter, making them difficult to obtain as single-crystal, non-soluble phases.

After extensive experimentation, the inventors discovered that the crystalline form of the malate of compound 1 has excellent physical and chemical properties suitable for pharmaceutical formulations and can be manufactured on a large commercial scale with high quality and good reproducibility in both monocrystalline and polycrystalline phases.

In a first aspect, this document discloses a crystalline salt of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (hereinafter referred to as Compound 1), selected from the malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxate, and mandelate of Compound 1. In one embodiment, the salt is pharmaceutically acceptable.

The inventors have discovered that it is extremely difficult to reproducibly prepare the crystalline form of a single phase of the salts of Compound 1 (including L-malate, succinate, or L-tartrate), and most of the obtained crystalline materials are solvated, in a somewhat disordered state, and/or constitute a multiphase form. For example, the L-malate of Compound 1 can exist in various crystalline forms or materials, which are referred to below as malate form A (a single phase of crystalline form, abbreviated as malate form A), malate material B, malate material C, malate material D, malate form E, malate material F, malate material G, malate material H, malate material J, malate material K, malate material L, malate material M, malate material N, malate material O, malate material P, and malate material Q. The L-tartrate of Compound 1 can also exist in various crystalline forms or materials, which are referred to below as tartrate form A, tartrate material B, tartrate material C, tartrate material D, tartrate material E, and tartrate material F.

Despite the existence of many competing crystalline forms of the malate and tartrate of compound 1, it was found that the malate form A (abbreviated as malate form A) and the tartrate form A (abbreviated as tartrate form A) of compound 1 can be prepared reproducibly on a large scale in single-crystal phase form.

Furthermore, the inventors have discovered that the malate form A exhibits improved and unpredictable properties favorable for pharmaceutical use compared to the previously disclosed free base, including increased long-term chemical/physical stability and improved bioavailability. The improved chemical stability in practice enables the storage at ambient temperatures for pharmaceuticals prepared from L-malate form A, compared to the requirement for temperatures below ambient temperature for pharmaceuticals prepared from the free base. Improved bioavailability has been demonstrated in clinical trials, where a 20%-30% dose reduction was achieved when patients were administered the salt, resulting in bioequivalence between 100 mg of Compound 1 malate form A capsules and 120 mg of Compound 1 free base capsules.

In the second aspect, this article discloses the crystalline form of malic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), hereinafter referred to as the crystalline form of the malic acid salt of compound 1 (1:1).

In one embodiment, the crystalline form of the malate of compound 1 is (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), hereinafter referred to as (S)-2-hydroxysuccinic acid (1:1) of compound 1 or the crystalline form of L-malate of compound 1 (1:1).

In one embodiment, per mole of (S)-2-hydroxysuccinate (1:1) of compound 1, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 contains 0-3.0 moles of _H2O .

In one embodiment, the X-ray powder diffraction pattern of the crystalline form contains diffraction peaks with 2θ angle values in the range of 9.4° to 10.2°.

In one embodiment, the X-ray powder diffraction pattern (XRPD) of the crystalline form further includes diffraction peaks with a 2θ angle value of 12.6 ± 0.2°.

In one embodiment, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 is represented as malate form A.

In one embodiment, malate form A has an XRPD pattern substantially as shown in FIG1A. Furthermore, the XRPD pattern of malate form A has peak diffraction angles substantially as shown in Table 1A.

In another embodiment, the malate form A has an XRPD diagram substantially as shown in Figures 2A(a) and 2A(b).

In another embodiment, the malate form A is a variable hydrate having an XRPD plot that exhibits a significant shift in the XRPD peak when heated and cooled or when the relative humidity changes, as shown in Figures 2A(c) and 2A(d).

In one embodiment, the X-ray powder diffraction pattern of malate form A includes diffraction peaks with a 2θ angle in the range of 9.4° to 10.2°.

In one embodiment, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 is expressed as malate form E.

In another embodiment, the malate form E has an XRPD plot substantially as shown in FIG2A(e).

In another embodiment, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 exhibits XRPD patterns substantially as shown in Figures 1A, 2A(a), 2A(b), 2A(c), 2A(d) and 2A(e).

In the third aspect, this paper discloses the crystalline form of tartaric acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (hereinafter referred to as the tartrate of compound 1).

In one embodiment, the tartrate of compound 1 is crystalline in the form of tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), hereinafter referred to as the crystalline form of the tartrate of compound 1 (1:1).

In one embodiment, the crystalline form of the tartrate of compound 1 is (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), hereinafter referred to as (2R,3R)-2,3-dihydroxysuccinate (1:1) of compound 1 or the crystalline form of L-tartrate of compound 1 (1:1).

In one embodiment, the crystalline form of (2R,3R)-2,3-dihydroxysuccinate (1:1) of compound 1 is represented as tartrate form A.

In another embodiment, the tartrate form A has an XRPD plot substantially as shown in FIG2B(a).

In one embodiment, the crystalline form is at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% crystalline.

In a fourth aspect, this document discloses a pharmaceutical composition comprising a therapeutically effective amount of a salt of compound 1 and a pharmaceutically acceptable excipient. In one embodiment, the salt is selected from malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxylate, and mandelate of compound 1. In one embodiment, the salt is pharmaceutically acceptable. In a preferred embodiment, the salt is in crystalline form.

In one embodiment, the malate is malate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the malate is (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the salt is in crystalline form of L-malate and L-tartrate, particularly in malate form A, malate form E, and tartrate form A. In another embodiment, the salt is in crystalline form, namely malate form A.

In a fifth aspect, this document discloses a method for inhibiting the activity of multiple tyrosine kinases in cells, comprising contacting cells for which inhibition of multiple tyrosine kinase activity is desired with a therapeutically effective amount of a pharmaceutically acceptable salt of Compound 1 and a pharmaceutically acceptable excipient. In one embodiment, the salt is selected from malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxylate, and mandelate of Compound 1. In one embodiment, the salt is pharmaceutically acceptable. In a preferred embodiment, the salt is in crystalline form.

In one embodiment, the malate is malate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the malate is (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the salt is in crystalline form of L-malate and tartrate, particularly malate form A, malate form E, and tartrate form A. In another embodiment, the salt is in crystalline form, namely malate form A.

In the sixth aspect, this article discloses a method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective dose of a pharmaceutically acceptable salt of compound 1 and a pharmaceutically acceptable excipient.

In one embodiment, the salt is selected from malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxylate, and mandelate of compound 1. In one embodiment, the salt is pharmaceutically acceptable. In a preferred embodiment, the salt is in crystalline form.

In one embodiment, the malate is malate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the malate is (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the salt is in crystalline form of L-malate and L-tartrate, particularly in malate form A, malate form E, and tartrate form A. In another embodiment, the salt is in crystalline form, namely malate form A.

In a seventh aspect, a method for preparing the crystalline form of the salt of compound 1 is disclosed herein. In one embodiment, the crystalline form is selected from malate form A and tartrate form A. In another embodiment, the crystalline form is malate form A.

Attached Figure Description

Figure 1A illustrates the X-ray powder diffraction (XRPD) pattern of crystalline form A (malate form A) of compound 1 prepared according to Example 1B.

Figure 1B illustrates the differential scanning calorimetry (DSC) curve of crystalline form A (malate form A) of compound 1 prepared according to Example 1.

Figure 1C illustrates the thermogravimetric analysis (TGA) curves of crystalline form A (malate form A) of compound 1 prepared according to Example 1.

Figure 1D illustrates the dynamic vapor adsorption (DVS) isotherm curves of the crystalline form A of the malate of compound 1 prepared according to Example 1 (malate form A).

Figure 2A illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with L-malic acid according to Example 2.

Figure 2A(a) illustrates the pointer solution of the X-ray powder diffraction (XRPD) pattern of the crystalline form A of the malate of compound 1 prepared according to Example 2 (malate form A).

Figure 2A(b) illustrates the pointer solution of the X-ray powder diffraction (XRPD) pattern of the crystalline form A (malate form A) of compound 1 prepared according to Example 2.

Figure 2A(c) illustrates the temperature-variable-temperature X-ray powder diffraction (VT-XRPD) pattern shift of the malate form A of compound 1 during heating and cooling.

Figure 2A(d) illustrates the shift of the variable relative humidity X-ray powder diffraction (VRH-XRPD) pattern of the malate form A of compound 1 as the relative humidity changes.

Figure 2A(e) illustrates the pointer solution of the X-ray powder diffraction (XRPD) pattern of the crystalline form E (malate form E) of compound 1 prepared according to Example 2.

Figure 2B illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with L-tartaric acid according to Example 2.

Figure 2B(a) illustrates the pointer solution of the X-ray powder diffraction (XRPD) pattern of the crystalline form (tartrate form A) of compound 1 prepared according to Example 2.

Figure 2B(b) illustrates the pointer-based solution of the X-ray powder diffraction (XRPD) pattern of the crystalline form (tartrate form A) of compound 1 prepared according to Example 2, after offset.

Figure 2C illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with succinic acid according to Example 2.

Figure 2D illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with citric acid according to Example 2.

Figure 2E illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with 1,2-ethanedisulfonic acid according to Example 2.

Figure 2F illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with hydrochloric acid according to Example 2.

Figure 2G illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with sulfuric acid according to Example 2.

Figure 2H illustrates the X-ray powder diffraction (XRPD) pattern of the salt/eutectic of compound 1 prepared with maleic acid according to Example 2.

Figure 2I illustrates the X-ray powder diffraction (XRPD) pattern of the salt/cocrystal of compound 1 prepared from citric acid, maleic acid, malonic acid, D-tartaric acid, p-toluenesulfonic acid, and p-toluenecarboxylic acid.

Figure 2J illustrates ^{the 1H -} NMR spectrum of the salt/cocrystal of compound 1 prepared from L-malic acid according to Example 2.

Figure 2K illustrates ^{the 1H} -NMR spectrum of the salt/cocrystal of compound 1 prepared with L-tartaric acid according to Example 2.

Figure 2L illustrates ^{the 1H} -NMR spectrum of the salt/cocrystal of compound 1 prepared with succinic acid according to Example 2.

Figure 2M illustrates ^{the 1H -} NMR spectrum of the salt/cocrystal of compound 1 prepared with citric acid according to Example 2.

Figure 2N illustrates ^{the 1H -} NMR spectrum of the salt/cocrystal of compound 1 prepared with hydrochloric acid according to Example 2.

Figure 2O illustrates ^{the 1H} -NMR spectrum of the salt/eutectic of compound 1 prepared with sulfuric acid according to Example 2.

Figure 2P illustrates ^{the 1H -} NMR spectrum of the salt/cocrystal of compound 1 prepared with maleic acid according to Example 2.

Figure 2Q illustrates ^{the 1H} -NMR spectrum of the salt/cocrystal of compound 1 prepared with malonic acid.

Figure 2R illustrates ^{the 1H} -NMR spectrum of the salt/cocrystal of compound 1 prepared with D-tartaric acid.

Figure 2S illustrates the ^1H -NMR spectrum of the salt/cocrystal of compound 1 prepared with p ^- toluenesulfonic acid.

Figure 2T illustrates the ^1H -NMR spectrum of the salt/cocrystal of compound 1 prepared with p ^- toluic acid.

Figure 2U illustrates the X-ray powder diffraction (XRPD) patterns of malate form A, malate material D, tartrate form A, and succinate material G of compound 1 prepared according to Example 2 with proportionally increased amounts.

Figure 2V illustrates the X-ray powder diffraction (XRPD) pattern of the malate material D of compound 1 prepared according to Example 2.

Figure 2W illustrates the differential scanning calorimetry (DSC) curve of malate material D of compound 1 prepared according to Example 2.

Figure 2X illustrates the thermogravimetric analysis (TGA) curves of malate material D of compound 1 prepared according to Example 2.

Figure 2Y illustrates the dynamic vapor adsorption (DVS) analysis of malate material D of compound 1 prepared according to Example 2.

Figure 3A illustrates a comparison of the mean plasma concentration curves over time for Compound 1 administered in capsule and tablet form as the free base of Compound 1, compared to L-malate capsules of Compound 1, in beagle dogs.

Figures 3B and 3C illustrate the comparison of the mean plasma concentration profiles of Compound 1 administered in the form of 120 mg free base capsules and 100 mg L-malate capsules over time in humans, and the corresponding ratios of the geometric mean of the key parameters of the profiles and the 90% confidence intervals, to determine the bioequivalence between the two formulations.

Detailed Implementation

This invention relates to a salt of compound 1. In particular, this invention discloses a crystalline form of the salt of compound 1, a pharmaceutical composition comprising the crystalline form, a method for preparing the crystalline form, and a method for using the crystalline form.

In a first aspect, this document discloses a salt of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 1), which is in the form of malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxate, and mandelate of selected compounds 1. In one embodiment, the salt is pharmaceutically acceptable. In a preferred embodiment, the salt is in crystalline form.

Furthermore, the inventors have discovered that it is extremely difficult to reproducibly prepare single-phase crystalline forms of malate or tartrate, and most of the obtained crystalline materials are in a somewhat disordered state, composed of multiphase forms, or are fused materials. For example, the L-malate of compound 1 can exist in various crystalline forms or materials, which are referred to below as malate form A (a single-phase crystalline form, abbreviated as malate form A), malate material B, malate material C, malate material D, malate form E, malate material F, malate material G, malate material H, malate material J, malate material K, malate material L, malate material M, malate material N, malate material O, malate material P, and malate material Q. The L-tartrate of compound 1 can also exist in various crystalline forms or materials, which are referred to below as tartrate form A, tartrate material B, tartrate material C, tartrate material D, tartrate material E, and tartrate material F.

Despite the prospect of polymorphic forms of the highly complex salt of compound 1, it has been found that the malate form A can be reproducibly isolated under well-controlled crystallization conditions and is suitable for the manufacture of homogeneous pharmaceutical products. Furthermore, although other crystalline forms are obtained by solution with solvents other than water or by mixtures, it has been found that the L-malate form A can be manufactured on a large scale in a single-crystal phase substantially free of solvents other than water.

Furthermore, malate form A is a variable hydrate exhibiting unpredictable properties, such as good stability. The improved long-term chemical stability of malate form A enables room temperature storage of formulated drugs prepared with malate, compared to formulated drugs containing the free base of stratinib, which must be stored under refrigeration. It was also found that at ambient temperature, above approximately 23% RH, malate form A is the most physically stable malate form of compound 1, and is physically stable upon exposure to 40°C/75% RH and 30°C/60% RH. Although maintaining the same form under these conditions, malate form A appears to be a variable hydrate, with its water content depending on relative humidity and temperature. After exposure to 30% and 93% RH for approximately 4 days, malate form A was found to contain 3.7% and 5.3% water, respectively (equivalent to approximately 1.5 and 2.5 mol). Outside the specified physical stability range of RH and temperature, malate form A was found to reversibly dehydrate into a seemingly anhydrous but potentially unstable material or a low-hydrate form (referred to as malate materials B, C, and D). Among these materials, malate material D was further characterized by DVS and Karl Fischer, thus confirming its low water content (approximately 0.67%, equivalent to approximately 0.3 mol).

Furthermore, malate form A was found to possess superior properties compared to the free base, particularly improved long-term chemical/physical stability and higher bioavailability. Significantly improved bioavailability of malate form A in dogs was confirmed compared to the free base. Improved bioavailability was also demonstrated in clinical trials, where a 20%–30% dose reduction was achieved in RP2D cases when administering the salt-based drug to patients.

In the second aspect, this article discloses the crystalline form of malic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), hereinafter referred to as the crystalline form of the malic acid salt of compound 1 (1:1).

In one embodiment, the crystalline form of the malate of compound 1 is (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), hereinafter referred to as (S)-2-hydroxysuccinic acid (1:1) of compound 1 or the crystalline form of L-malate of compound 1 (1:1).

In one embodiment, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 contains 0-3 moles of _H₂O .

In one embodiment, the X-ray powder diffraction pattern of the crystalline form contains diffraction peaks with 2θ angle values in the range of 9.4° to 10.2°.

In one embodiment, the X-ray powder diffraction pattern of the crystalline form contains diffraction peaks with 2θ angle values in the range of 9.6 ± 0.2°.

In another embodiment, the X-ray powder diffraction pattern (XRPD) of the crystalline form further includes diffraction peaks with a 2θ angle value of 12.6 ± 0.2°.

In another embodiment, the X-ray powder diffraction pattern (XRPD) in crystalline form further comprises diffraction peaks with 2θ angle values independently selected from the group consisting of: 15.3±0.2°, 20.3±0.2°, 21.0±0.2°, and 24.1±0.2°.

In another embodiment, the X-ray powder diffraction pattern (XRPD) in crystalline form further comprises diffraction peaks with 2θ angle values independently selected from the group consisting of: 12.0±0.2°, 15.3±0.2°, 20.3±0.2°, 21.0±0.2°, 22.0±0.2°, 23.2±0.2°, 24.1±0.2°, and 24.2±0.2°.

In another embodiment, the X-ray powder diffraction pattern (XRPD) in crystalline form further comprises diffraction peaks with 2θ angle values independently selected from the group consisting of: 6.1±0.2°, 8.6±0.2°, 12.0±0.2°, 13.5±0.2°, 15.3±0.2°, 16.9±0.2°, 17.4±0.2°, 17.7±0.2°, 18.1±0.2°, 18.4±0.2°, 20.0±0.2°, 20.3±0.2°, 21.0±0.2°, 22.0±0.2°, 22.9±0.2°, 23.2±0.2°, 24.1±0.2°, 24.2±0.2°, 24.8±0.2°, and 25.4±0.2°.

In another embodiment, the X-ray powder diffraction pattern (XRPD) of the crystalline form further comprises diffraction peaks with 2θ angle values independently selected from the group consisting of: 6.1±0.2°, 8.6±0.2°, 12.0±0.2°, 13.5±0.2°, 15.3±0.2°, 16.9±0.2°, 17.4±0.2°, 17.7±0.2°, 18.1±0.2°, and 18.4±0.2°. 2°, 20.0±0.2°, 20.3±0.2°, 21.0±0.2°, 22.0±0.2°, 22.9±0.2°, 23.2±0.2°, 24.1±0.2°, 24.2±0.2°, 24.8±0.2°, 25.4±0.2°, 25.6±0.2°, 26.2±0.2°, 27.8±0.2°, 28.2±0.2° and 29.1±0.2°.

In one embodiment, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 is represented as malate form A.

In one embodiment, malate form A has an XRPD pattern substantially as shown in FIG1A, and malate form A typically has an XRPD pattern with the following peak diffraction angles shown in Table 1A below.

In another embodiment, by differential scanning calorimetry (“DSC”), malate form A is characterized by having a wide range of endothermic events and overlapping endothermic events between approximately 50°C and 125°C, with a peak maximum at approximately 171°C. In another embodiment, malate form A has a DSC thermal analysis plot substantially as shown in Figure 1B.

In one embodiment, malate form A is characterized by a weight reduction at 120°C by thermogravimetric analysis (“TGA”). In another embodiment, malate form A has a TGA curve substantially as shown in Figure 1C.

In one embodiment, malate form A is characterized by a weight loss of about 1.2 wt% at equilibrium at 5% relative humidity (RH), a weight gain of about 2.1 wt% between 5-25% RH, and an additional weight gain of 1.1 wt% between 25%-95% RH during the adsorption phase. During desorption cycles, the material loses about 1.3 wt% and 1.7% at 95-15% RH and 15-5% RH, respectively, with a very small hysteresis between about 25% and 5% RH, as measured by dynamic vapor adsorption (“DVS”). In another embodiment, malate form A has a DVS isotherm substantially as shown in Figure 1D.

The malate form A was measured as a variable hydrate, and its X-ray powder diffraction pattern contained shifted diffraction peaks with 2θ angle values ranging from 9.4° to 10.2°.

In another embodiment, the malate form A has an XRPD diagram and pointerization solution substantially as shown in Figures 2A(a) and 2A(b).

In one embodiment, malate form A is substantially free of residual organic solvents.

In one embodiment, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 is expressed as malate form E.

In another embodiment, the malate form E has essentially the XRPD diagram and pointerization solution shown in Figure 2A(e).

In another embodiment, the crystalline form of (S)-2-hydroxysuccinate (1:1) of compound 1 has substantially the XRPD diagrams shown in Figures 1A, 2A(a), 2A(b), 2A(c), 2A(d) and 2A(e).

In the third aspect, this paper discloses the crystalline form of tartaric acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (hereinafter referred to as the tartrate of compound 1).

In one embodiment, the tartrate of compound 1 is crystalline in the form of tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), referred herein to as the tartrate (1:1) crystalline form of compound 1.

In one embodiment, the crystalline form of the tartrate of compound 1 is (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), hereinafter referred to as (2R,3R)-2,3-dihydroxysuccinate (1:1) of compound 1 or the crystalline form of L-tartrate of compound 1 (1:1).

In one embodiment, the crystalline form of (2R,3R)-2,3-dihydroxysuccinate (1:1) of compound 1 is represented as tartrate form A.

In another embodiment, the tartrate form A has an XRPD plot substantially as shown in FIG2B(a).

In one embodiment, the crystalline form is at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% crystalline.

In a fourth aspect, this document discloses a pharmaceutical composition comprising a therapeutically effective amount of a salt of compound 1 and a pharmaceutically acceptable excipient. In one embodiment, the salt is selected from malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxylate, and mandelate of compound 1. In one embodiment, the salt is pharmaceutically acceptable. In a preferred embodiment, the salt is in crystalline form.

In one embodiment, the malate is malate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the malate is in the crystalline form of (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the salt is in a crystalline form selected from malate form A, malate form E, and tartrate form A. In another embodiment, the salt is in a crystalline form, namely malate form A.

The crystalline form of the salt of compound 1 can be formulated by any method well known in the art and can be prepared for administration via any route, including (but not limited to) parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or rectal administration. In some embodiments, the crystalline form of the salt of compound 1 is administered intravenously in a hospital setting. In one embodiment, it can be administered orally.

The characteristics of the carrier will depend on the route of administration. As used herein, the term “pharmaceuticalally acceptable” means a non-toxic substance that is compatible with biological systems (such as cells, cell cultures, tissues, or organisms) and does not interfere with the effectiveness of the bioactivity of the active ingredient. Therefore, in addition to inhibitors, compositions may contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Preparation of pharmaceutically acceptable formulations is described, for example, in Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing, Easton, Pennsylvania, 1990.

The active compound is included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver a therapeutically effective amount to the patient without causing serious toxicity in the treated patient.

In one embodiment, the pharmaceutical composition contains 95% of the crystalline form of a salt of compound 1. In another embodiment, the pharmaceutical composition of the present invention contains at least 95% of the crystalline form of a salt of compound 1. In another embodiment, the pharmaceutical composition contains at least 90% of the crystalline form of a salt of compound 1. In another embodiment, the pharmaceutical composition contains at least 80% of the crystalline form of a salt of compound 1. In another embodiment, the pharmaceutical composition contains at least 70% of the crystalline form of a salt of compound 1. In another embodiment, the pharmaceutical composition contains at least 60% of the crystalline form of a salt of compound 1. In another embodiment, the pharmaceutical composition contains at least 50% of the crystalline form of a salt of compound 1.

The pharmaceutical composition comprising the salt of compound 1 in crystalline form may be used as described herein.

In a fifth aspect, this document discloses a method for inhibiting the activity of multiple tyrosine kinases in cells, comprising contacting cells for which inhibition of multiple tyrosine kinase activity is desired with a therapeutically effective amount of a pharmaceutically acceptable salt of Compound 1 and a pharmaceutically acceptable excipient. In one embodiment, the salt is selected from malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxylate, and mandelate of Compound 1. In a preferred embodiment, the salt is in crystalline form.

In one embodiment, the malate is malate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the malate is (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate salt of compound 1 is in the crystalline form of (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the salt is in a crystalline form selected from malate form A, malate form E, and tartrate form A. In another embodiment, the salt is in a crystalline form, namely malate form A.

In the sixth aspect, this article discloses a method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective dose of a pharmaceutically acceptable salt of compound 1 and a pharmaceutically acceptable excipient.

In one embodiment, the cancer is a multiple tyrosine kinase-associated cancer.

In one embodiment, the multiple tyrosine kinase-associated cancer is lung cancer, including non-small cell lung cancer (NSCLC).

The active compound is included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver a therapeutically effective dose to a patient without causing serious toxicity in the treated patient. In one embodiment, the dose of the active compound for all the above-described conditions is in the range of about 0.01 to 300 mg/kg, for example, 0.1 to 100 mg/kg daily, and as another embodiment, 0.5 to about 25 mg per kilogram of the recipient's body weight daily. In suitable carriers, the typical local dose will be in the range of 0.01-3% wt/wt. The effective dose range of a pharmaceutically acceptable derivative can be calculated based on the weight of the parent compound to be delivered. If the derivative itself is active, the effective dose can be approximated using the weight of the derivative as described above or by other means known to those skilled in the art.

In one embodiment, a salt of compound 1, a multiple tyrosine kinase inhibitor, is administered orally once daily (QD). In another embodiment, a salt of compound 1 is administered orally twice daily (BID).

In one embodiment, the salt is selected from malate, tartrate, succinate, citrate, 1,2-ethanedisulfonate, hydrochloride, sulfate, maleate, malonate, p-toluenesulfonate, p-toluenecarboxylate, and mandelate of compound 1. In one embodiment, the salt is pharmaceutically acceptable. In a preferred embodiment, the salt is in crystalline form.

In one embodiment, the malate is malate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the malate is (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is tartrate N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the tartrate is (2R,3R)-2,3-dihydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

In one embodiment, the salt is in crystalline form of L-malate and L-tartrate, particularly in malate form A, malate form E, and tartrate form A. In another embodiment, the salt is in crystalline form, namely malate form A.

In some embodiments of any of the methods described herein, prior to treatment with the compositions or methods of the present invention, the patient had received one or more of the following treatments: chemotherapy, targeted anticancer agents, radiation therapy, and surgery, and optionally, the prior treatment was unsuccessful; and/or the patient had undergone surgery, and optionally, the surgery was unsuccessful; and/or the patient had been treated with platinum-based chemotherapy agents, and optionally, it had been previously determined that the patient did not respond to treatment with platinum-based chemotherapy agents; and/or the patient had been treated with kinase inhibitors, and optionally, the prior treatment with kinase inhibitors was unsuccessful; and/or the patient had been treated with one or more other therapeutic agents.

Those skilled in the art will recognize that in vivo and in vitro tests using suitable, known and recognized cell and/or animal models can predict the ability of a combination of test compounds or combinations to treat or prevent a given disorder.

Those skilled in the art will further recognize that human clinical trials, including first-in-human dose range and efficacy trials in healthy patients and/or patients with a given disability, can be conducted using methods well known in clinical and medical techniques.

In a seventh aspect, this document discloses a method for preparing the crystalline form of the salt of compound 1. In one embodiment, the crystalline form is selected from malate form A, malate form E, and tartrate form A. In another embodiment, the crystalline form is malate form A. Furthermore, malate form A appears to be a variable hydrate, the water content of which depends on relative humidity and temperature.

In another embodiment, this type of method describes a crystallization procedure for preparing malate form A from another form or mixture of forms of malate from compound 1.

In one embodiment, malate form A is obtained by a method comprising any of the following procedures:

a) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) is suspended in a water-immiscible solvent selected from methanol, ethanol, acetone or mixtures thereof, and heated, cooled or further removed from the solvent to obtain malate form A;

b) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) and malate form A as a seed crystal were suspended in acetone, a solvent immiscible with water, heated, methanol was added, and cooled to obtain malate form A;

c) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) was suspended in acetone, a solvent immiscible with water, and slurried to obtain malate form A;

d) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) was suspended in water-immiscible ethanol, heated, slurried, cooled, and stirred to obtain malate form A (offset).

In one embodiment, malate form A is obtained by a method including the following steps:

1) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) was suspended in a mixture of acetone, an immiscible solvent in water, and methanol, an organic solvent miscible in water;

2) Heat the resulting suspension to approximately 70°C to produce a clear solution;

3) Cooling the solution to promote crystal formation; and

4) Remove the acetone-methanol mixture to obtain malate form A.

In one embodiment, the volume percentage of acetone to methanol is 59:41 (v/v).

In another embodiment, the method further includes collecting malate form A. In another embodiment, the method further includes drying malate form A in a vacuum oven before or after collection.

In one embodiment, malate form A is obtained by a method including the following steps:

1) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) and malate form A as a seed crystal are suspended in acetone, a solvent that is immiscible with water;

2) Heat the resulting suspension to approximately 70°C;

3) Add hot, water-miscible organic solvent methanol;

4) Cooling the solution to promote crystal formation; and

5) Remove the acetone-methanol mixture to obtain malate form A.

In one embodiment, the volume percentage of acetone to methanol is 59:41 (v/v).

In another embodiment, the method further includes collecting malate form A. In another embodiment, the method further includes drying malate form A in a vacuum oven before or after collection.

In one embodiment, malate form A is obtained by a method including the following steps:

1) The malate form A of compound 1 was suspended in ethanol, an organic solvent miscible with water;

2) Heat the resulting suspension to approximately or above 55°C;

3) Raise the temperature of the resulting suspension to approximately 65°C-70°C; and

4) Slowly cool the suspension to ambient temperature to obtain malate form A.

In another embodiment, the method further includes collecting malate form A. In another embodiment, the method further includes drying malate form A in a vacuum oven before or after collection.

In another embodiment, the method describes a crystallization procedure for preparing the malate of compound 1 from compound 1 and malic acid.

In one embodiment, malate form A is obtained by a method comprising any of the following procedures:

1) Compound 1 was suspended in ethanol, an organic solvent miscible with water, heated, cooled, and L-malic acid was added to form malate. The mixture was cooled to the initial crystalline form and further cooled to a lower temperature to obtain malate form A.

In one embodiment, malate form A is obtained by a large-scale method including the following steps:

2) Compound 1 was suspended in ethanol, an organic solvent miscible with water;

3) Heat the resulting suspension to approximately 75°C;

4) Cool the resulting suspension to approximately 62°C;

5) Add L-malic acid to form malate;

6) Slowly cool the suspension to approximately 25°C in its initial crystallization state; and

7) Cool the suspension to approximately 0°C to obtain malate form A.

In another embodiment, the method further includes collecting malate form A. In another embodiment, the method further includes drying malate form A in a vacuum oven before or after collection.

In one embodiment, the malate form A obtained by the method disclosed herein is at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% crystalline.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, patent applications, and publications mentioned herein are incorporated herein by reference.

As used herein, “Compound 1” refers to N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide, which is a free base.

As used herein, “salt of compound 1,” “salt of compound 1,” “salt of multiple compounds 1,” or “salt of multiple compounds 1” refers to the salt of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide. For example, “malate of compound 1” or “malate of compound 1” refers to the malate of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide.

As used herein, “the crystalline form of the salt of compound 1”, “the crystalline form of the salt of compound 1”, “the crystalline form of the salt of multiple compounds 1” or “the crystalline form of the salt of multiple compounds 1” refers to the crystalline form of the salt of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide”.

If the compound does not involve other characteristic types, a continuous Roman letter characteristic is assigned as the default name for materials exhibiting a unique crystallographic XRPD plot. The name is experimentally associated with the term 'material' until the phase purity obtained via pointerization of the XRPD plot and the chemical consistency obtained via proton nuclear magnetic resonance spectroscopy ( ^1H NMR) are determined. When the characteristic definition data is consistent with a unique crystalline form consisting of a single phase, the material is further represented as a "form" (i.e., material C becomes form C) using the same letter name. In this invention, the XRPD plot of a compound's "form" can be successfully pointerized. However, the XRPD plot of a "material" cannot be pointerized because it exists as a crystalline material with some degree of disorder or as a mixture.

As used herein, when used alone, the term "malate form A" or "malate form A of compound 1" refers to crystalline form A of (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1). The terms "malate form A" or "malate form A of compound 1", "malate form E" or "malate form E of compound 1", and "tartrate form A" or "tartrate form A of compound 1" have similar meanings to "malate form A" or "malate form A of compound 1".

As used herein, the term "solvent" refers to the crystalline form of compound 1 or its salt containing a solvent.

As used herein, the term "hydrate" refers to a solvate in which the solvent contains water. Due to the specific crystal structure of the malate form A disclosed herein, it can be a variable hydrate containing or not containing water, which does not affect the properties of the malate form A.

As variable hydrates, the chosen malate form A and modes or materials C and D represent a series of related crystal types, differing only in the water content within the crystal lattice. The relationships between them are demonstrated by the following observations:

1.) Complete reversibility of transformation from form A to modes C and D, and back to form A, is achieved through dehydration and rehydration without loss of crystallinity. This characteristic is typical of variable hydrates, where the XRPD reflection angle shifts slightly due to changes in the unit cell space size caused by the inflow or outflow of water. However, the lattice remains intact throughout the RH cycle, showing no signs of degradation.

2.) A strong correlation was observed between weight gain/weight loss from changes in DVS and KF varying with RH; and

3.) DVS does not exhibit significant hysteresis, confirming the complete reversibility of the hydration state.

As used herein, the term “residual organic solvent” refers to volatile organic chemicals used or generated during crystallization/manufacturing processes that were not completely removed during the manufacturing process.

As used herein, the term “substantially free of residual organic solvents” means that the manufactured pharmaceutical preparation, such as the crystalline form of the salt of compound 1, contains less than 0.5% by weight of residual organic solvents, less than 0.4% by weight of residual organic solvents, less than 0.3% by weight of residual organic solvents, less than 0.2% by weight of residual organic solvents, or less than 0.1% by weight of residual organic solvents.

As used in this article, “multiple tyrosine kinase-related disease or disorder” refers to a disease or disorder associated with or mediated by oncogenic driver mutations in RET, CBL, CHR4q12, DDR and/or Trk.

As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, a patient is a human. In some embodiments, the subject has experienced and/or presents at least one symptom of the disease or disorder being treated and/or prevented. In some embodiments, the subject is suspected of having multiple tyrosine kinase-related cancer.

In one embodiment, the multiple tyrosine kinase-associated cancer is lung cancer, including non-small cell lung cancer (NSCLC).

As used herein, a “therapeuticly effective amount” of the crystalline form of the salt of compound 1 is an amount sufficient to improve or reduce symptoms in some way, or to stop or reverse the progression of the disease, or to negatively regulate or inhibit the activity of multiple tyrosine kinases. Such an amount may be administered as a single dose or according to a regimen in which it is effective.

As used herein, treatment means any means of improving or otherwise beneficially altering the symptoms or pathology of a condition, disorder, or disease. Treatment also covers any medical use of the compositions described herein.

As used herein, improvement of symptoms of a particular disorder by administration of a particular pharmaceutical composition means any relief attributable to or related to the administration of the composition, whether permanent or temporary, persistent or transient.

As used herein, when used to modify parameters defined numerically (e.g., the dose of the crystalline form of a salt of compound 1 described in detail herein, or the length of treatment time described herein), the term “about” means that the parameter may be up to 10% smaller or larger than the numerical value stated with respect to that parameter. For example, a dose of about 5 mg/kg may vary between 4.5 mg/kg and 5.5 mg/kg. When “about” is used at the beginning of a list of parameters, it means to modify each parameter. For example, about 0.5 mg, 0.75 mg, or 1.0 mg means about 0.5 mg, about 0.75 mg, or about 1.0 mg. Similarly, about 5% or more, 10% or more, 15% or more, 20% or more, and 25% or more mean about 5% or more, about 10% or more, about 15% or more, about 20% or more, and about 25% or more.

As used herein, when referring to XRPD peak positions, the term "about" refers to the inherent variability of the peak, which depends on instrument calibration, the method used to prepare the crystalline form of the invention, the aging of the crystalline form, and the type of instrument used in the analysis. The variability of the instrument used for XRPD analysis is about ±0.2°2θ.

As used herein, when referring to the onset of the DSC endothermic peak, the term "about" refers to the inherent variability of the peak, which depends on the instrument calibration, the method used to prepare the sample of this invention, and the type of instrument used in the analysis. The variability of the instrument used for DSC analysis is about ±1°C.

General methods

Unless otherwise stated, the illustrative examples use the general methods outlined below.

I. Crystallization Technology

The crystalline forms disclosed herein can be prepared using a variety of methods well known to those skilled in the art, including crystallization or recrystallization from a suitable solvent or by sublimation. A variety of techniques (including those exemplified in the examples) can be used for crystallization or recrystallization, including evaporation of water-miscible or water-immiscible solvents or solvent mixtures, seeding crystals in a supersaturated solution, lowering the temperature of a solvent mixture, or freeze-drying a solvent mixture.

The crystallization disclosed herein can be carried out with or without a seed crystal. The seed crystal can be derived from any previous batch in the desired crystal form.

Abbreviations and abbreviations

The following examples are intended to illustrate certain other embodiments of the present invention and are not intended to limit the scope of the present invention.

Example 1

Preparation of (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) in crystalline form A

This example illustrates the preparation of crystalline form A of (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1).

Methods for preparing the multiple tyrosine kinase inhibitor disclosed herein, namely N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 1), are known. For example, commonly owned international application publications WO2009/026717A and WO 2009/026720 A describe suitable intermediates and general reaction procedures for preparing multiple tyrosine kinase inhibitor compounds, and also provide detailed synthetic routes for the free bases used to prepare the multiple tyrosine kinase inhibitors disclosed herein. Furthermore, the malate of compound 1 can be prepared, for example, according to the method described in Example 1.

Example 1A: Preparation of crystalline form A of (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) (malate form A of compound 1, or malate form A)

N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 1, free base) was placed in pure ethanol (30 volumes) and heated to 75±5°C to obtain a clear solution, which was then cooled to 62±5°C. L-(-)malic acid solution (1.0 equivalent in 3.8 volumes of pure ethanol) was added dropwise while stirring for approximately 24 hours. The reaction mixture was slowly cooled to 25±5°C over a period of approximately 8 hours, followed by stirring at 25±5°C for approximately 8 hours. The reaction mixture was cooled to 0±5°C, stirred for approximately 1 hour, filtered, and the solid was washed with pure ethanol (2.0 volumes). The XRPD and purity of the sample were analyzed by HPLC. The sample was dried in a vacuum at 45±5℃ to obtain a grayish-white solid malate of compound 1 (malate form A of compound 1).

The malate form A of compound 1: ^¹H NMR (500 MHz, DMSO) δ = 10.41 (s, 1H), 10.00 (s, 1H), 8.64 (d, J = 1.5 Hz, 1H), 8.53 (d, J = 5.5 Hz, 1H), 8.37 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.98 (dd, J = 2.0, 6.0 Hz, 1H), 7.89 (dd, J = 2.0, 11.5 Hz, 1H), 7.62 (td,J＝5.0,2.0Hz,2H),7.45(m,2H),7.12(tq,J＝3.5Hz,2H),6.65(d,J＝5.5Hz,1H),4.03(s,4H),3 .50(t,J＝5.5Hz,7H),2.92(m,3H),2.51(m,1H),2.34(dd,5.5,10.5Hz,1H),1.47(d,J＝5.0Hz,4H).

Example 1B: Preparation of malate form A of compound 1 (salt formation and crystallization)

3.0 kg of Compound 1 (free base) was suspended in a flask containing 91.25 L of pure ethanol, and the suspension was heated to 75 ± 5 °C to form a clear solution, which was then cooled to approximately 62 ± 5 °C. 0.69 kg of L-(-)malic acid was added dropwise while stirring for up to 24 hours. The solution was then slowly cooled to 25 ± 5 °C over an 8-hour period and stirred for another 8 hours at approximately 25 ± 5 °C. The solution was cooled to approximately 0 ± 5 °C, stirred for 1 hour, filtered under vacuum, and washed with 7.6 L of chilled pure ethanol. The final solid (malate of Compound 1) was dried at approximately 45 ± 5 °C and collected. The weight of the malate of Compound 1 was 3.38 kg, with a yield of 92.86% and a purity of 98.7%, as measured by HPLC analysis.

X-ray powder diffraction (XRPD)

In one instance, an X-ray powder diffraction pattern (diffraction pattern) was collected using a PANalytical X'Pert PRO MPD diffractometer with an incident beam of Cu radiation generated using an Optix long-focus focal length source. The Cu Kα X-rays were focused through the sample and onto the detector using an elliptical multilayer mirror. Prior to analysis, a silicon sample (NIST SRM 640e) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position.

Each sample was sandwiched between two 3 μm thick films and analyzed using transmission geometry. A beam trap, short antiscattering extension, and antiscattering blade were used to minimize background from air. A Soller slit was applied to both the incident and diffracted beams to minimize the widening of axial divergence. Diffraction patterns were collected using Data Collector software v2.2b with a scan position-sensitive detector (X'Celerator) located 240 mm from the sample.

In another example, XRPD patterns were collected using a PANalytical X'Pert PRO MPD diffractometer with an incident beam of Cu radiation generated using an Optex long-focus light source and a nickel filter. The diffractometer was configured with symmetric Bragg-Brentano geometry. Prior to analysis, a silicon sample (NIST SRM640e) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position.

Each sample was prepared into a thin circular layer and placed in the center of a zero-background silicon substrate. An anti-scattering slit (SS) was used to minimize the background caused by air. Soler slits for the incident and diffracted beams were used to minimize the widening of axial divergence. Diffraction patterns were collected using a scan position sensitive detector (X'Celerator) located 240 mm from the sample, using Data Collector software v2.2b.

The characteristics of the L-malate of compound 1 obtained were defined using X-ray powder diffraction (XRPD) patterns, which confirmed that the L-malate of compound 1 is in a crystalline form, referred to as crystalline form A of the L-malate of compound 1 (malate form A of compound 1, or malate form A), see Figure 1A. The characteristic peaks and peak intensity percentages obtained by XRPD analysis are listed in Table 1A.

Table 1A. XRPD diagram of crystalline form A (malate form A) of compound 1

Example 1C: Preparation of malate form A of compound 1 (recrystallization)

Weigh 34 mg of a mixture of the malate form A of compound 1 and another crystalline material of the malate of compound 1, place it in a test tube, and mix it with 2 mL of acetone added in four 500 μL aliquots. Heat the suspension at 70 °C on a hot plate, and continuously add 1.4 mL of methanol at 70 °C in five separate aliquots (500 μL × 2; 200 μL; then 100 μL × 2) until a clear solution is obtained. The final acetone to methanol ratio of the clear solution is 59:41 (volume percentage).

After the solution cleared, the heating plate was turned off and the sample was slowly cooled to ambient temperature. After three days at ambient temperature, aggregates of small fine particles and needle-like structures were observed in the solution by birefringence and extinction. The sample was then transferred to a temperature below ambient temperature (e.g., 2°C–8°C). After 11 days, the solvent was decanted and the remaining solids were simply dried with a gentle stream of nitrogen, followed by XRPD analysis, producing an XRPD plot substantially the same as shown in Figure 1A.

Example 1D: Preparation of malate form A of compound 1 (recrystallization)

Weigh 48 mg of a mixture of the malate form A of compound 1 and another crystalline material of the malate of compound 1, place it in a test tube, and mix it with 1.4 mL of ethanol in the form of seven 200 μL aliquots. Heat the suspension to approximately 55 °C. Continuously add an additional twenty-six 200 μL aliquots of ethanol at 55 °C and raise the temperature to approximately 65 °C–70 °C.

After heating at approximately 65°C–70°C, solids remained. Twenty aliquots of 200 μL each of ethanol at approximately 65°C were then added sequentially. The resulting solution was stirred at approximately 65°C–70°C for one hour, and then ten aliquots of 200 μL each of ethanol at approximately 65°C were added sequentially. A slightly turbid solution was obtained, and the heating plate was turned off, allowing the turbid solution to slowly cool to ambient temperature. After one day at ambient temperature, the resulting solids were collected by vacuum filtration. The XRPD plot of the alternative formulation of crystalline malate is substantially the same as shown in Figure 1A.

Example 1E: Differential scanning calorimetry (DSC) analysis of malate form A of compound 1

Differential scanning calorimetry (DSC)

DSC data were collected using a TA Instruments Q2000 differential scanning calorimeter. Temperature calibration was performed using NIST-monitorable indium. The sample was placed in the DSC aluminum disk, covered, and its weight was accurately recorded. The weighed aluminum disk, configured as the sample disk, was placed on the reference side of the unit cell.

Alternatively, DSC analysis was performed using a Mettler-Toledo TGA/DSC3+ analyzer. Temperature calibration was performed using adamantane, phenyl salicylate, indium, tin, and zinc. The sample was placed in the DSC aluminum disk, covered, and its weight was accurately recorded. The weighed aluminum disk, configured as the sample disk, was placed on the reference side of the unit cell. The lid of the sample disk was punctured before heating and analysis.

Typically, the sample is heated from a temperature of about -30°C to a temperature of about 250°C at a rate of 10°C/min for analysis.

DSC analysis of the malate form A of compound 1 was performed using a TA Instruments Q2000 differential scanning calorimetry module according to the manufacturer's instructions.

In short, approximately 3 mg of malate form A was placed in a balanced aluminum dish, covered with a rolled-up aluminum cap, and heated at a base heating rate of 10 °C/min, with temperature adjustments from 25 °C to 300 °C. The sample was maintained under dry nitrogen purging at 50 ml/min. The weighed aluminum dish, configured as the sample dish, was placed on the reference side of the unit cell. The instrument control software used was Advantage for Q Series and Thermal Advantage, and the data were analyzed using Universal Analysis.

The DSC thermal analysis chromatogram of the sample of malate form A of compound 1 is shown in Figure 1B. As shown in Figure 1B, malate form A of compound 1 exhibits extensive endothermic events and overlapping endothermic events between approximately 50 °C and 125 °C, with a maximum peak temperature of approximately 171 °C and degradation above approximately 185 °C.

Example 1F: Thermogravimetric analysis (TGA) of malate form A of compound 1

Thermogravimetric analysis (TGA)

TGA data were collected using a TA Instruments Q5000 IR thermogravimetric analyzer. Each sample was placed in an aluminum pan with a rolled-up lid and inserted into the TG furnace. The furnace was heated under nitrogen.

In one embodiment, the sample is heated from ambient temperature to approximately 300°C at a rate of 10°C/min. The percentage of weight loss is calculated at a temperature of approximately 120°C.

TGA analysis was also performed using a calibrated TAInstruments Q500 TGA.

In short, approximately 5 mg of compound 1 in malate form A was loaded onto a pre-balanced 100 μL platinum TGA dish and heated to 300 °C from ambient temperature at 10 °C/min. The sample was maintained under nitrogen purging at 25 mL/min. The instrument control software used was Advantage and Thermal Advantage for the Q series, and the data were analyzed using Universal Analysis.

The TGA thermal analysis chromatogram of malate form A is shown in Figure 1C. As shown in Figure 1C, malate form A exhibits a very small, gradual weight loss (approximately 2%) at 120°C until the degradation begins at approximately 175°C.

Example 1G: Dynamic vapor adsorption (DVS) analysis of malate form A of compound 1

Dynamic vapor adsorption (DVS)

Moisture adsorption/desorption data were collected according to the manufacturer's instructions using a Surface Measurement System (SMS) DVS Intrinsic instrument. Samples were not dried prior to analysis. Adsorption/desorption data were collected under nitrogen purging in 10% RH increments, ranging from 5% to 95% relative humidity (RH). The equilibrium criterion used for analysis was a weight change of less than 0.0100% over 5 minutes, with a maximum equilibrium time of three hours. Data were not corrected for the initial moisture content of the samples.

The sample was recovered after isotherm analysis and XRPD reanalysis.

Adsorption isotherms for the malate form A of compound 1 were obtained using an SMSDVS intrinsic moisture adsorption analyzer controlled by DVS intrinsic control software. The sample temperature was maintained at 25°C under instrument control. Humidity was controlled by a mixed flow of drying and humidifying nitrogen gas at a total flow rate of 200 mL/min. Relative humidity was measured using a calibrated Rotronic probe (dynamic range 1.0–100% RH) located near the sample. The weight change (mass relaxation) of the sample with % RH was continuously monitored using a microbalance (accuracy ±0.005 mg).

In short, under ambient conditions, malate form A was placed in a balun-equipped stainless steel basket. Samples were loaded and unloaded at 40% RH and 25°C (typical indoor conditions) and equilibrated against 5% RH. Moisture adsorption isotherms were obtained as outlined below (one scan per complete cycle). Standard isotherms were obtained at 25°C in 10% RH intervals over the range of 5–95% RH. Data analysis was performed in Microsoft Excel using the DVS Analysis Suite.

The DVS isotherm curves for malate form A are shown in Figure 1D. As shown in Figure 1D, malate form A is characterized by a weight loss of approximately 1.2 wt% at equilibrium at 5% relative humidity (RH), a weight gain of approximately 2.1 wt% between 5-25% RH, and an additional weight gain of 1.1 wt% between 25%-95% RH during the adsorption phase. During desorption cycles, the material loses approximately 1.3 wt% and 1.7% wt% at 95-15% RH and 15-5% RH, respectively, with a very small hysteresis between approximately 25% and 5% RH, indicating that the sample exists in a variable hydrate form.

Example 1H: Variable temperature (VT) and variable relative humidity (VRH) XRPD of malate form A of compound 1

Variable Temperature X-ray Powder Diffraction (VT-XRPD)

Field XRPD images varying with temperature were collected using an Anton Paar TTK 450 stage. Samples were heated by resistance heaters located directly beneath the sample holder, with temperature monitored by platinum-100 resistance sensors located within the sample holder. The heaters were powered and controlled by an Anton Paar TCU 100 connected to the data collector.

The VT-XRPD data for malate form A shown in Figure 2A(c) illustrates the XRPD peak shifts during the dehydration of malate form A at gradually heated to 55°C and during the rehydration synthesis of malate form A at cooled back to ambient temperature.

Variable relative humidity X-ray powder diffraction (VRH-XRPD)

Field XRPD maps of humidity variations were collected using an Anton Paar Temperature-Humidity Chamber (THC). Sample temperature was varied via a Peltier thermoelectric device located directly beneath the sample rack and monitored by a platinum-100 resistance sensor located within the rack. The thermoelectric device was powered and controlled by an Anton Paar TCU 50 connected to the data collector. Humidity was generated by a VTI-manufactured RH-200 and transported via a nitrogen flow. Humidity and temperature were monitored by a Rotronic-manufactured HygroClip sensor located adjacent to the samples within the THC.

VRH-XRPD data for malate form A indicate that it is stable between approximately 23% and approximately 78% RH (the highest relative humidity tested) and appears to reversibly dehydrate to malate material D (or C) at relative humidities below approximately 15%–23%. This is consistent with VT-XRPD data. In this respect, malate form A, material C, and material D can be considered as a series of related crystal types, differing only in the water content within the crystal lattice, which causes XRPD peak shifts.

Example 2

Salt/cocrystal of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 1)

Salts of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 1) with eight salt-forming agents were prepared and further studies of salt/cocrystals were conducted. These salt-forming agents include L-malic acid, L- and D-tartaric acid, succinic acid, citric acid, 1,2-ethanedisulfonic acid, hydrochloric acid, sulfuric acid, maleic acid, malonic acid, p-toluenesulfonic acid, p-toluenecarboxylic acid, and mandelic acid.

X-ray powder diffraction (XRPD)

X-ray powder diffraction pattern overlays were created using PatternMatch versions 2.3.6 and 3.0.4. Images labeled "Images obtained by PatternMatch v3.0.4" were generated using unverified software and are not representative of cGMP.

PANalytical, Transmission Mode

XRPD patterns were collected using a PANalytical X'Pert PROMPD diffractometer with an incident beam of Cu radiation generated by an Optex long-focus light source. Cu Kα X-rays were focused through the sample and onto the detector using an elliptical multilayer mirror. Prior to analysis, a silicon sample (NIST SRM 640d) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position. The sample was sandwiched between 3 μm thick films and analyzed using transmission geometry. A beam trap, short antiscattering extension, and antiscattering kerf were used to minimize background from air. Soler slits for the incident and diffracted beams were used to minimize the widening of axial divergence. Diffraction patterns were collected using a scan position-sensitive detector (X'Celerator) located 240 mm from the sample and Data Collector software version 2.2b.

PANalytical, Reflection Mode

XRPD patterns were collected using a PANalytical X'Pert PRO MPD diffractometer with an incident beam of Cu Kα radiation generated by an Optix long-focus light source and a nickel filter. The diffractometer used a symmetric Bragg-Brentano geometry configuration. Data were collected and analyzed using data collection software version 2.2b. Prior to analysis, a silicon sample (NIST SRM640d) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position. The sample was filled into nickel-coated copper vias. Anti-scattering slits (SS) were used to minimize background caused by air scattering. Soler slits for both the incident and diffracted beams were used to minimize the broadening of axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) located at a distance of 240 mm from the sample.

Indicators

XRPD plots were pointerized using X'Pert High Score Plus [X'Pert High Score Plus 2.2a (2.2.1)] or the dedicated SSCI software TRIADS. The selected pattern was pointerized using previously obtained unit cell parameters and optimized using CheckCell [CheckCell 11/01/04; http://www.ccp14.ac.uk/tutorial/lmgp/]. Molecular alignment was not attempted to confirm the experimental pointerization scheme.

Polarizing microscope (PLM)

Samples were observed using a Leica MZ12.5 stereo microscope and a first-order red compensator. Samples were observed using a variety of objectives typically in the 0.8–10x range and under orthogonally polarized light. Selected samples were observed in mineral oil.

Thermogravimetric analysis (TGA)

TG analysis was performed using a TA Instruments 2950 thermogravimetric analyzer. Temperature calibration was performed using nickel and Alumel™. Each sample was placed in an aluminum pan and inserted into the TG furnace. The furnace was heated under nitrogen purging. Data acquisition parameters are displayed above the individual TG plots in the data section of this report. The method codes on the TG plots are abbreviations for the start and end temperatures and heating rates; for example, 25-350-10 means "from 25°C to 350°C at 10°C/min".

Differential scanning calorimetry (DSC)

DSC analysis was performed using a TA Instruments 2920 and Q2000 differential scanning calorimeter. Temperature calibration was performed using NIST-monitored indium. The sample was placed in the DSC aluminum disk, covered, and its weight was accurately recorded. A weighed aluminum disk configured as the sample disk ( ^T0C -Tzero coiled disk, T0CMP-Tzero coiled disk, manually perforated, T0HSLP-hermetic-sealed Tzero cap with laser-perforated pinhole, or T0HSMP-hermetic-sealed Tzero cap with manual perforation) was placed on the reference side of the unit cell. The data acquisition parameters and disk configuration for each thermal analysis plot are shown in the images in the data section of this report. The method codes on the thermal analysis plots are abbreviations for the start and end temperatures and heating rates; for example, -30-250-10 means "from -30°C to 250°C at 10°C/min".

Solution proton nuclear magnetic resonance spectroscopy ( ^¹H NMR)

Deuterated DMSO was used to acquire solution proton NMR spectra using a 400MHz instrument.

Karl Fischer titration

Water measurements were performed using a Mettler Toledo DL39 Karl Fischer titrator connected to a Strombololi oven. Prior to analysis, the samples were exposed to a specified relative humidity for approximately 2 hours. Two replicates of the samples were placed in a drying oven set to approximately 140°C or 145°C. The drying oven was purged into the titrator container with dry nitrogen. The samples were then titrated using a generating electrode that produces iodine via electrochemical oxidation: 2I⁻ → I₂ + 2e⁻. Analysis was performed against the NIST Hydranal Water Standard 10.0 to verify the operation of the coulometric titrator.

Example 2A: Salt/eutectic of Compound 1

Experiments are conducted with the aim of producing crystalline materials containing salts of several compounds 1, typically involving crystallization techniques such as cooling of compound 1 based on its solubility and stability under various conditions, slurrying at high or ambient temperatures, evaporation, or combination, and observing properties under selected conditions. Experiments are typically conducted on a medium scale (40-100 mg). Equimolar amounts of free base and salt-forming agent are used in most experiments. Selected experiments are conducted with a 2:1 excess of free base or salt-forming agent.

The resulting solids were initially analyzed typically by polarized light microscopy (PLM) as a preliminary assessment of crystallinity. Next, X-ray powder diffraction (XRPD) was used as the primary analytical technique for initial identification of the new crystalline phase. XRPD patterns were compared with each other and with patterns of free bases and available salt-forming agents. Where possible, ^1H -NMR spectroscopy was used to confirm salt formation, and the stoichiometric properties of the resulting candidates were measured to ensure no degradation occurred.

Crystalline materials of compound 1 were prepared using several salt-forming agents, including L-malic acid, L-tartaric acid, succinic acid, citric acid, 1,2-ethanedisulfonic acid, hydrochloric acid, sulfuric acid, maleic acid, malonic acid, p-toluenesulfonic acid, p-toluenecarboxylic acid, and mandelic acid. The conditions and results of salt screening for malate, tartrate, and succinate are summarized in Table 2A.

Characterization of at least one of the materials produced with each salt-forming agent was defined by ^1H -NMR. The ^1H -NMR spectra of the salt/eutectic of the corresponding compound 1 are shown in Figures 2J, 2K, 2L, 2M, 2N, 2O, 2P, 2Q, 2R, 2S, and 2T. In various cases, the data are consistent with salt or eutectic formation based on peaks in the range of 4.4–2.6 ppm (possibly related to non-aromatic protons near secondary amino groups), peaks observed due to the corresponding relative ions (where appropriate), or shifts in other peaks near 9–10 ppm that may be attributed to the protonation of compound 1.

XRPD was used to define the characteristics of the salt/eutectic of compound 1 prepared. Materials exhibiting unique XRPD plots are illustrated in Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I. Most of the prepared materials were either crystalline solids showing various degrees of disorder or solvated. The material prepared with 1,2-ethanedisulfonic acid exhibited significant disorder. One crystalline form of malate and one tartrate can be successfully prepared separately in the single-crystal phase, as demonstrated by the pointer-based XRPD plots. These two forms are referred to as malate form A and tartrate form A. The pointer-based XRPD plots of malate form A, malate form A (offset), tartrate form A, and tartrate form A (offset) are further shown in Figures 2A(a), 2A(b), 2B(a), and 2B(b). When prepared under different conditions, these peak shifts and slightly different indexing results indicate that the two crystal structures have a similar ability to expand and contract to some level depending on the formation conditions or water content. Furthermore, malate materials B and C have essentially the same XRPD plots as malate form A, but with some shifted peaks.

Table 2A. Salts/Cocrystals of Compound 1

(a) The ratio of free base to acid is 1/1. The temperature and duration of the experiment are approximate.

(b) Unless otherwise instructed, observations shall be made in mineral oil.

Example 2B: Scale-up of the salt/eutectic of compound 1

Crystalline materials of the salt/eutectic of compound 1 (including malate form A, tartrate form A, and succinate form A of compound 1) were selected for scale-up to generate additional quantities for further characterization and stabilization of the form's activity. Due to the large number of different materials prepared from the same salt-forming agent (most of which are not indexable by XRPD), the selection of the target form for scale-up was primarily based on the apparent crystallinity obtained by XRPD and the absence of organic solvents confirmed by ^1H -NMR.

The conditions and results of the scale-up experiments are summarized in Table 2B. The characteristics of the resulting salt/eutectic of compound 1 were defined using XRPD plots, illustrated in Figure 2U. Based on the XRPD data, a new material, termed malate material D, was obtained at a larger scale; a tartrate form A with a slightly shifted plot was obtained; and a new material, termed succinate material G (possibly containing succinate material D), but not the target succinate material A, was obtained. ^1H NMR and TGA/DSC data of the samples were acquired to further define the characteristics of the obtained salt/eutectic.

The obtained malate material D

Based on other peaks in the range of approximately 4.0 ppm and 2.6–2.2 ppm (which can be attributed to relative ions and overlap with protons from the analytical solvent), the ^1H NMR spectrum is consistent with the process solvent containing approximately 0.8 mol of malic acid and not detected by ^1H NMR.

The XRPD, DSC, and TGA plots of malate material D are further shown in Figures 2V, 2W, and 2X. The TGA thermal analysis plot shows the loss of volatiles in the temperature range of 26°C–63°C, which is likely related to water loss, and indicates that the sample is a sesquihydrate with an approximate 3.4 wt% water loss. The sample exhibits a small weight reduction (approximately 1.2 wt%) that may be attributed to partial dehydration during storage or to the use of dry nitrogen during analysis. Upon further heating, the sample shows a sharp loss at approximately 197°C (the starting point), which may be attributed to degradation. The DSC thermal analysis plot indicates extensive endothermic activity between approximately 25°C and 122°C (with a peak maximum at approximately 84°C), which corresponds to the volatile loss observed during TG analysis and is therefore likely related to water release. Overlapping endothermic events starting at approximately 122°C are also observed. Furthermore, the XRPD plot of malate material D is substantially the same as that of malate form A, but with some shifted peaks.

Furthermore, based on the DVS shown in Figure 2Y, malate material D exhibits approximately 5.4 wt% (equivalent to approximately 2.5 mol) of water uptake across the entire RH range (0-95% RH), which may be related to its rehydration to synthesize malate form A. Following desorption, the material was observed to lose all the acquired water with a small hysteresis. Based on XRPD data, the solids after the desorption cycle appear to be consistent with malate material D. This is consistent with the dehydration expected during desorption.

The obtained tartrate form A

1) Based on other peaks around 4.1 ppm (attributable to the tartaric acid relative ion), the ^1H NMR spectrum is consistent with that of compound 1 containing about 1 mol/mol of tartaric acid, and the sample does not show residual organic solvents; 2) The TGA thermal analysis plot shows a loss of about 1.9 wt% associated with the release of volatiles around 25 °C–56 °C, and a sharp weight loss is found at about 203 °C (the starting point), typically observed due to degradation; 3) The DSC thermal analysis plot shows a wide range of endothermic events between about 25 °C and about 123 °C, with multiple endothermic events observed above 123 °C, with peak maximums at about 129 °C and about 189 °C.

The obtained succinate material G

1) Based on other peaks around 2.4 ppm attributable to the relative ion, the ^1H NMR spectrum of the sample is generally consistent with that of compound 1 containing about 1 mol/mol of succinic acid, and the sample also contains residual chloroform; 2) The TGA spectrum of the sample shows volatile losses (about 1.1 and about 0.7 wt%) in the temperature range of 24 °C–116 °C, and a sharp weight loss possibly attributable to degradation was observed at about 166 °C (starting point); 3) The DSC thermal analysis plot indicates extensive endothermic activity between about 26 °C and about 102 °C (peak maximum at about 71 °C), corresponding to the volatile losses observed during TG analysis, and above 102 °C, the sample exhibits multiple endothermic activities, with peak maximums at about 132 °C and about 150 °C.

Table 2B. Proportional scaling of salt/eutectic of compound 1

(a) The temperature, duration, recovery and yield of the experiment were approximate.

(b) Unless otherwise stated, microscopic observation shall be performed in mineral oil.

(c) No mineral oil was used.

Example 2C: Screening for stable forms of the salt/eutectic of compound 1

To attempt to evaluate the thermodynamically most stable forms of compound 1, namely L-malate, L-tartrate, and succinate/eutectic, the material was scaled up by wet milling for up to a week in a solvent system with suitable solubility. Experiments targeting potential hydrates were also conducted, mostly under conditions of high water activity.

XRPD was used as the primary analytical technique for the initial identification of materials obtained from L-malic acid, L-tartaric acid, and succinic acid, as illustrated in Figures 2A, 2B, and 2C, respectively. The pointer XRPD plot for malate form E is further shown in Figure 2A(e). The conditions and results for stable form screening are summarized in Table 2C.

Table 2C. Stable Form Screening Experiment

Example 3

Evaluation of the malate form A of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (malate form A of compound 1)

Example 3A: Critical Relative Humidity

Critical relative humidity (RH) experiments were designed to confirm the physical stability of solid malate form A within the RH range. Mixtures of malate form A/malate material H, as well as samples of malate form A, malate material B, malate material C, and malate material D, were tested under selected RH conditions (see Table 3A(1)). The water content characteristics of malate form A and material D were defined by Karl Fischer analysis (see Table 3A(2)).

Experiments were conducted by exposing the solid material to various RH conditions. Samples for XRPD analysis of the post-stress solid were prepared with minimal exposure to environmental conditions. For selected samples, Karl Fischer analysis and XRPD testing were performed simultaneously. Therefore, these samples were held in glove bags equilibrated at RH conditions similar to the exposure conditions, and XRPD samples were prepared inside the glove bags.

The mixture of malate form A/malate material H was subjected to 0%, 11%, 23%, 69%, and 93% RH for approximately 5 days each. It should be noted that stress RH was not maintained during XRPD sample preparation and analysis. Therefore, solids from all these stress conditions were simply exposed to ambient RH, which was approximately 21%–23% at the time of analysis.

The solids under stress at 23% RH still consisted of a mixture of malate form A and malate material H. However, based on the relative intensities of the XRPD peaks, the amount of malate material H in the stressed samples appeared to be reduced compared to the original samples. The solids under stress at 69% and 93% RH were consistent with malate form A with improved crystallinity. Malate material H was no longer observed in these samples, indicating that it transformed into malate form A at high RH.

XRPD analysis of solids under stress equal to or below 11% RH indicates dehydration of malate form A. XRPD plots after exposure to 0% and 11% RH show peaks attributable to malate materials C and D, respectively, containing malate form A.

Malate materials C and D were suspected to be lower-level hydrates. Both materials exhibited similar XRPD plots to malate material B, which is another potential lower-level hydrate or an unstable anhydrous material. After re-exposure of malate materials C and D to 75% RH for approximately 5 days, both materials were observed to transform into malate form A. This indicates that the dehydration of malate form A was reversible under the test conditions.

Based on these results, it is confirmed that malate form A is physically stable above 23% RH, and reversible dehydration was observed below 23% RH. Although maintaining the same form within the specified RH range, malate form A from various samples exhibited a shift in the XRPD peak. This suggests that malate form A may be a variable hydrate. For variable hydrates, the amount of water contained within the lattice can vary without a change in form.

To further investigate this, stress experiments were conducted at 30% and 93% RH with minimal exposure to ambient RH, allowing XRPD and moisture content analysis to be representative of the stress conditions. For this purpose, RH exposure and XRPD sample preparation were performed in glove bags under the following assumptions: 1) the glove bags remained sealed throughout the duration of each experiment; 2) the entire glove bag maintained the same humidity without any "bags" with higher or lower humidity; 3) the polymer film used for the XRPD samples remained sealed throughout the XRPD analysis; and 4) the sample exposure to ambient conditions during preparation for Karl Fischer analysis was transient and did not significantly affect the moisture content. In these experiments, a mixture of malate form A/malate material H was initially exposed to 93% RH for approximately 8 days. XRPD and Karl Fischer analyses were then performed immediately. The same samples were subsequently exposed to 30% RH.

As expected by the critical RH research institute, both samples are consistent with malate form A based on XRPD data. It should be noted that the peaks in the XRPD plots of the sample exposed to 93% RH are consistently shifted towards a smaller angle compared to the peaks of the sample exposed to 30% RH. This shift indicates lattice expansion due to the containment of a larger amount of water, consistent with the higher water content of malate form A after exposure to high humidity. Specifically, malate form A was found to contain 5.3% water (equivalent to approximately 2.5 mol) after stress at 93% RH, while the measured water content of the sample after stress at 30% RH was 3.7% (equivalent to approximately 1.5 mol). The observed peak shift in the XRPD of malate form A is consistent with the fact that malate form A is a variable hydrate. Furthermore, malate form A may contain approximately 1.5–2.5 mol of _H₂O .

Table 3A(1) Relative humidity and thermal stress of L-malate of compound 1

(a) The temperature, RH percentage and duration of the experiment are approximate.

(b) Prepare samples for XRPD analysis in a glove bag equilibrated at the specified RH level. Prepare samples for Karl Fischer analysis with minimal exposure to ambient RH.

(c) The peak shift of malate form A is consistent with the material being a variable hydrate.

Table 3A(2). Physical characteristics of L-malate of compound 1

Example 3B: Thermal and High Temperature/Relative Humidity Stress

Thermal and high-temperature/RH stresses were designed to evaluate the physical stability of the malate form A of compound 1 at high temperatures. Tests were conducted using a mixture of malate form A of compound 1 and malate material H. Experiments were performed by heating the solid of the test material at 40°C, 80°C, 120°C, and 140°C–145°C. The solid was also subjected to combinations of high temperature and RH, namely, 40°C/75% RH and 30°C/60% RH (Table 3B).

As previously observed at lower RH, heating the malate form A of compound 1 to 40°C or higher causes partial dehydration. Solid XRPD patterns obtained under stresses of 40°C and 80°C for 7 days and under stresses of 120°C or higher for approximately 1 hour primarily consist of either malate material C or malate material D of compound 1, both suspected to be lower-level hydrates of the salt. Peaks attributable to malate form A are still observed in the patterns.

The samples held for one day under stress at 40°C/75% RH and 30°C/60% RH consisted of malate form A and no longer exhibited malate material H.

Table 3B shows the relative humidity and thermal stress of malate for compound 1.

(a) The temperature, RH percentage and duration of the experiment are approximate.

(b) The peak shift of malate form A is expected to be a variable hydrate.

Example 3C: Key Water-Active/Solvate Formation

The physical stability of malate form A under solvent-mediated conditions and the potential competition between solvate and hydrate formation were assessed by slurrying malate form A in various organic solvent/water systems (Table 3C).

The acetone/water system was chosen for stability studies at water activity levels between 0.4 and 0.9. Aqueous ACN, dioxane, DMF, DMSO, and NMP were also investigated. A mixture of malate form A and malate material H was primarily used as the starting material. Samples of malate form A were combined and used as the starting material in a single experiment (acetone/water at low water activity).

No change in the form of malate form A was observed after slurrying in acetone/water at water activities of approximately 0.8 and 0.9. Based on XRPD, the solids from both experiments appear to consist of malate form A and no longer contain malate material H. This indicates the physical stability of malate form A in the highly water-active acetone/water system and the transformation of malate material H under these conditions.

Slurrying in acetone/water at a water activity of 0.7 or less produces a mixture of malate material B and malate form A. It should be noted that the amount of malate material B in the slurry solid may depend on the presence of malate material H in the starting material. Specifically, based on experiments at a water activity of 0.3, the amount of malate material B appears to be higher in samples using a mixture of malate form A/malate material H and lower in samples using malate form A. In this study, based on XRPD data, it was found that malate material B was rehydrated to malate form A after 75% RH stress.

All other aqueous mixtures containing organic solvents other than acetone produce materials that appear to be unique and potentially soluble. These are novel crystalline materials and are referred to as malate materials L to P. These results are consistent with previously proposed competition between solvates and malate form A, depending on solvent and water activity. Indeed, slurrying in NMP/water at a water activity of 0.8 causes complete conversion of malate form A into a unique material (referred to as malate material O), while increasing the water activity to 0.9 in the same solvent system only causes partial conversion, and the sample still contains malate form A.

Table 3C. Aqueous active slurries of malate of compound 1

(a) The organic solvent was dehydrated using a molecular sieve before use. The temperature, solvent ratio (v/v), and duration of the experiment were approximate.

(c) The peak shift of malate form A is expected to be a variable hydrate.

Example 3D: Crystallization Experiment - Malate Form A of Compound 1

Preliminary crystallization experiments were conducted targeting the pure phase of the malate form A of compound 1 to search for more suitable and controllable crystallization conditions. Several of these experiments used acetone/water, a solvent system in which the malate form A of compound 1 exhibits physical stability under certain aqueous reactivity. Techniques such as cooling, evaporation, and slurrying were used in the experiments. Details are provided in Table 3D.

In these experiments, the malate form A of compound 1 was produced by slow cooling in acetone, slow cooling in EtOH, and slow cooling in acetone/MeOH, followed by sub-ambient crystallization. The disordered malate form A was produced by slurrying in acetone/water or water. Other solvent systems produced unique materials typically containing the malate form A. Based on XRPD, a predominantly crystalline sample of the malate form A was produced by slow cooling in acetone/MeOH followed by sub-ambient crystallization.

Table 3D. Crystallization experiments of malate form A of compound 1

(a) The temperature, solvent ratio (v/v), and duration of the experiment are approximate.

(b) The peak shift of malate form A is expected to be a variable hydrate.

The peak shift of malate material G is relative to the peak shift in Example 2. The peak shift of malate material L is relative to the peak shift in the previous sample of malate material L (disordered) in Table 3C.

Example 4

Stability and in vivo studies of the malate of N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (malate form A of compound 1)

Example 4A: Stability of malate form A of compound 1

Long-term stability studies of the malate form A of compound 1 were conducted at 2℃–8℃ and 25℃/60% RH. For each test interval, samples were individually sealed in double-layered polyethylene bags, tied with cable ties, and stored in 30mL white screw-cap HDPE bottles.

Stability data up to the 24th month are provided in Table 4A. The results indicate that the malate form A of compound 1 is stable after 24 months of storage at 2℃-8℃ or 25℃/60% RH, after 6 months of storage at 40℃/75% RH, and after 12 months of storage at 40℃/75% RH.

Table 4A. Stability of malate form A of compound 1

Example 4B: In the presence of a surfactant, the malate form A of compound 1 exhibits improved PK parameters compared to the free base amorphous form of compound 1.

The following examples illustrate that, compared with formulations of compound 1 containing Tween-20, the malate form A of compound 1 exhibits twice the improvement in pharmacokinetic (PK) parameters and in vivo bioavailability.

method

(1) Animals

Pharmacokinetic studies were conducted using three male and three female purebred beagles (non-untreated) in each group to test the formulations. Animals were acclimatized to study conditions for 3 days prior to initial dose administration and housed in captivity in accordance with all applicable laws, regulations, and guidelines of the Institutional Animal Care and Use Committee (IACUC). At initial administration, animals weighed 6.6 to 10.8 kg and were juvenile/adult.

(2) Administration

For Phase 1, individual doses are administered based on a fixed dose of 20 mg/day. The dose is administered orally, followed by rinsing with 10 mL of water to help ensure swallowing.

For Phase 2, individual doses are administered based on a fixed intravenous dose of 2 mg/day (single dose). The single dose is administered via intravenous bolus injection followed by flushing with approximately 2 mL of normal saline.

On each day of administration (in all groups), animals were kept fasting for approximately 1 hour prior to treatment. Food was resumed approximately 1 hour after administration.

(3) Sample collection and analysis

For each stage, blood (approximately 1 mL) was collected from the jugular vein into test tubes containing _K₂EDTA before administration and at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours after administration.

Keep the blood in chilled cryoracks before centrifugation to obtain plasma. Begin centrifugation within 1 hour of collection. Place the plasma in 96-well tubes with barcode labels. Keep the plasma on dry ice before storing at approximately -70°C.

Samples were analyzed using recognized liquid chromatography/mass spectrometry (LC-MS/MS) methods.

result

The results comparing the results of a single oral dose of 20 mg/kg or intravenous administration of a solution of 2 mg/kg for compound 1 in malate form A and Tween-20 containing the free base of compound 1 are shown in Table 4B and Figure 3A. The absolute bioavailability of compound 1 in malate form A and Tween-20 containing the amorphous form of compound 1 is shown in Table 4C.

Table 4B. Comparison of oral and intravenous exposures of compound 1 in its malate form A and Tween-20 containing the amorphous form of compound 1.

Table 4C. Absolute bioavailability of oral compound 1 in malate form A and Tween-20 containing compound 1 amorphous form.

These data indicate that the malate form A of compound 1 exhibits a 2-fold improvement in oral bioavailability in dogs in the absence of a surfactant, compared to the amorphous form of the free base of compound 1 with a surfactant. However, IV administration of both formulations resulted in similar in vivo exposures, suggesting that the malate form A of compound 1 presents a significantly improved oral exposure to the compound, thus avoiding the need to include a surfactant in oral formulations containing the malate form A of compound 1.

Example 4C: In healthy patients, the malate of compound 1 showed improved bioavailability compared to the free base of compound 1.

A phase 1, part 2, open-label, single-dose, crossover study was conducted to evaluate the relevant bioavailability of the malate and free base formulations of compound 1 in healthy subjects after oral administration.

The mean plasma concentration curves and associated bioavailability of the two formulations over time are shown in Figures 3B and 3C, demonstrating bioequivalence between 100 mg of compound 1 malate and 120 mg of compound 1 free base capsule formulation after oral administration in healthy subjects. The results also indicate that, after oral administration, compound 1 malate increased absorption and bioavailability compared to compound 1 free base.

Although the invention has been described in conjunction with specific embodiments thereof, it should be understood that further modifications are possible, and this application is intended to cover any variations, uses or modifications of the invention that generally follow the principles of the invention and include deviations from the invention within the scope of known or customary practice in the art to which the invention pertains, and that can be applied to the basic features set forth above and follow the scope of the claims.

Claims (31)

1. A crystalline form comprising malic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1). 2. A crystalline form comprising (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1). 3. A crystalline form comprising (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1), wherein each mole contains 0-3.0 moles of _H₂O . 4. The crystalline form as described in claim 2 or 3, wherein the X-ray powder diffraction pattern of the crystalline form comprises diffraction peaks having 2θ angle values in the range of 9.4° to 10.2°. 5. The crystalline form as described in any one of claims 2 to 4, wherein the X-ray powder diffraction pattern of the crystalline form contains diffraction peaks having a 2θ angle value of 9.6 ± 0.2. 6. The crystalline form as claimed in claim 4 or 5, wherein the X-ray powder diffraction pattern of the crystalline form further comprises diffraction peaks having a 2θ angle value of 12.6 ± 0.2. 7. The crystalline form as claimed in claim 6, wherein the X-ray powder diffraction pattern of the crystalline form further comprises diffraction peaks having °2θ angle values independently selected from the group consisting of: °2θ values at 15.3±0.2°, 20.3±0.2°, 21.0±0.2° and 24.1±0.2°. 8. The crystalline form as claimed in claim 6, wherein the X-ray powder diffraction pattern of the crystalline form further comprises diffraction peaks having angle values of °2θ independently selected from the group consisting of: 12.0±0.2°, 15.3±0.2°, 20.3±0.2°, 21.0±0.2°, 22.0±0.2°, 23.2±0.2°, 24.1±0.2°, and 24.2±0.2°. 9. The crystalline form as claimed in claim 6, wherein the X-ray powder diffraction pattern of the crystalline form further comprises diffraction peaks having angle values of °2θ independently selected from the group consisting of: 6.1±0.2°, 8.6±0.2°, 12.0±0.2°, 13.5±0.2°, 15.3±0.2°, 16.9±0.2°, 17.4±0.2°, 17.7±0.2°, 18.1±0.2°, 18.4±0.2°, 20.0±0.2°, 20.3±0.2°, 21.0±0.2°, 22.0±0.2°, 22.9±0.2°, 23.2±0.2°, 24.1±0.2°, 24.2±0.2°, 24.8±0.2°, and 25.4±0.2°. 10. The crystalline form as claimed in claim 6, wherein the X-ray powder diffraction pattern of the crystalline form further comprises diffraction peaks having angle values of °2θ independently selected from the group consisting of: 6.1±0.2°, 8.6±0.2°, 12.0±0.2°, 13.5±0.2°, 15.3±0.2°, 16.9±0.2°, 17.4±0.2°, 17.7±0.2°, 18.1±0.2°, 18.4± 0.2°, 20.0±0.2°, 20.3±0.2°, 21.0±0.2°, 22.0±0.2°, 22.9±0.2°, 23.2±0.2°, 24.1±0.2°, 24.2±0.2°, 24.8±0.2°, 25.4±0.2°, 25.6±0.2°, 26.2±0.2°, 27.8±0.2°, 28.2±0.2° and 29.1±0.2°. 11. The crystalline form as claimed in any one of claims 4 to 10, wherein the crystalline form is referred to as malate form A. 12. The crystalline form of claim 11, wherein the malate form A has a substantially XRPD pattern as shown in FIG1A. 13. The crystalline form of claim 11, wherein the malate form A, as determined by differential scanning calorimetry (DSC), is characterized by having a broad endothermic peak and overlapping endothermic events in the range of 50°C to 125°C, wherein the peak maximum is located at approximately 171°C. 14. The crystalline form of claim 11, wherein the malate form A has a substantially DSC thermal analysis pattern as shown in FIG1B. 15. The crystalline form as described in any one of claims 4 or 5, wherein the crystalline form has a substantially XRPD pattern as shown in FIG2A(a). 16. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline form as described in claim 2 or 3. 17. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline form as described in any one of claims 4 to 15. 18. The pharmaceutical composition of any one of claims 16 to 17, further comprising at least one pharmaceutically acceptable excipient. 19. A method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the crystalline form as described in claim 2 or 3. 20. A method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the crystalline form as described in any one of claims 4 to 15. 21. The method of claim 20, wherein the therapeutically effective amount of the crystalline form is between about 0.01 and 100 mg/kg per day. 22. The method of claim 20, wherein the therapeutically effective amount of the crystalline form is between about 0.1 and 50 mg/kg per day. 23. The method of claim 19 or 20, wherein the cancer is a multiple tyrosine kinase-associated cancer. 24. The method of claim 23, wherein the multiple tyrosine kinase-associated cancer is lung cancer, including non-small cell lung cancer. 25. The method of claim 19 or 20, wherein the subject is a human. 26. The crystalline form as described in any one of claims 4 to 15, obtained by a method comprising any one of the following procedures: a) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) is suspended in a water-miscible solvent selected from methanol, ethanol, acetone or an aqueous mixture thereof, and heated, cooled or further removed from the solvent to obtain the desired crystalline form; b) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) and the desired crystalline form as a seed crystal are suspended in acetone, a solvent miscible with water. The mixture is heated, methanol is added, and the mixture is cooled to obtain the desired crystalline form. c) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) was suspended in acetone, a solvent miscible with water, and slurried to obtain the desired crystalline form; d) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) was suspended in water-miscible ethanol, heated, slurried, cooled, and stirred to obtain the desired crystalline form (offset). 27. The crystalline form as described in any one of claims 4 to 15, obtained by a method comprising the following steps: 1) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) was suspended in a mixture of acetone and methanol; 2) Heat the resulting suspension to approximately 70°C to produce a clear solution; 3) Cool the solution to promote crystal formation; and 4) Remove the organic solvent mixture to obtain the desired crystalline form. 28. The crystalline form as described in any one of claims 4 to 15, obtained by a method comprising the following steps: 1) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) and the desired crystalline form as seed crystals are suspended in acetone, a water-miscible solvent; 2) Heat the resulting suspension to approximately 70°C; 3) Add hot, water-miscible organic solvent methanol; 4) Cool the solution to promote crystal formation; and 5) Remove the acetone-methanol mixture to obtain the desired crystalline form. 29. A crystalline form obtained by the method of any one of claims 26 to 28, wherein the volume percentage of acetone to methanol is 59:41 (v/v). 30. The crystalline form as described in any one of claims 4 to 15, obtained by a method comprising the following steps: 1) (S)-2-hydroxysuccinic acid N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1:1) was suspended in ethanol; 2) Heat the resulting suspension to approximately or above 55°C; 3) Raise the temperature of the resulting suspension to approximately 65°C-70°C; and 4) Slowly cool the suspension to ambient temperature to obtain the desired crystalline form. 31. The crystalline form as described in claims 4 to 15, obtained by a method comprising the following steps: 1) N-(3-fluoro-4-((2-(5-((((2-methoxyethyl)amino)methyl)pyridin-2-yl)thiopheno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide was suspended in ethanol; 2) Heat the resulting suspension to approximately 75°C; 3) Cool the resulting suspension to approximately 62°C; 4) Add L-malic acid to form malate; 5) Slowly cool the suspension to approximately 25°C in its initial crystallization state; and 6) Cool the suspension to approximately 0°C to obtain the desired crystalline form.