Crystalline forms of amido heteroaromatic compounds

Abstract

This specification relates to crystalline forms of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methanone, pharmaceutical compositions containing the crystalline forms, and uses of the crystalline forms in the treatment of diseases such as liver disease. Formula (I). (I)

Description

Cross-referencing of related patent applications

[0001] This specification claims the priority benefit of U.S. Provisional Patent Application No. 63 / 599,658 (filed November 16, 2023). The entire text of the aforementioned patent application is incorporated herein by reference. Technical Field

[0002] This specification relates to the crystalline form of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone, which inhibits 17β-hydroxysteroid dehydrogenase 13 (17βHSD13 or HSD17B13), and the use of this crystalline form in the treatment of diseases such as liver diseases. This specification also relates to pharmaceutical compositions containing this crystalline form. Background Technology

[0003] Nonalcoholic fatty liver disease (NAFLD) represents a range of liver diseases, from simple steatosis (nonalcoholic fatty liver) to nonalcoholic steatohepatitis (NASH) with or without fibrosis, and cirrhosis. Hepatic steatosis is defined as excessive accumulation of fat in the liver, with more than 5% caused by factors other than alcohol intake. NASH is defined as hepatic steatosis with inflammation and hepatocellular damage, with or without fibrosis. It is estimated that approximately 25% of the global population has NAFLD, and mortality from NAFLD-related diseases is projected to increase significantly by 2030.

[0004] To date, there are no approved treatments for NAFLD (such as NASH), and therapeutic interventions focus on addressing comorbidities that contribute to the development of NAFLD, including treating insulin resistance, obesity, type 2 diabetes, and dyslipidemia.

[0005] Recently, variants of the 17βHSD13 gene have been associated with reduced serum aminotransferase levels and a lower risk of liver diseases, including alcoholic and non-alcoholic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) in an allele-dose-dependent manner (Abul-Husn et al., N Engl J Med. 2018, 378(12), 1096-106; Wang et al., Eur RevMed Pharmacol Sci, 2020, 24(17), 8997-9007). The 17βHSD13 splice variant (rs72613567:TA) results in a truncated, unstable, and enzyme-inactive protein and has therefore been characterized as a 17βHSD13 loss-of-function (LoF) variant (Ma et al., Hepatology 2019, 69(4), 1504-19). The association between LoF17βHSD13 (rs72613567:TA) and reduced disease severity has been replicated in an additional cohort with histologically confirmed NAFLD, and in a study of 111,612 individuals from the general population of Denmark, it was also associated with lower plasma transaminases, reduced risk of cirrhosis, HCC, and liver-related mortality (Gellert-Kristensen et al., Hepatology, 2020, 71(1), 56-66). Interestingly, the protective effect of the LoF17βHSD13 (rs72613567:TA) variant against plasma transaminase levels appears to be amplified by several key risk factors for liver disease (e.g., obesity, alcohol consumption) and identified genetic risk factors (such as, but not limited to, the (rs738409 C>G) variant in protein 3 (PNPLA3) containing a potato glycoprotein-like phospholipase domain). In addition, two additional 17βHSD13LoF variants (rs62305723) and (rs143404524) have been reported to confer protection against the progression of chronic liver disease (Kozlitina et al., N Engl J Med, 2018, 379(19), 1876-7). Generally, protective variants of LoF 17βHSD13 are more strongly associated with fibrosis and progression to advanced liver disease, but not with steatosis.

[0006] Based on genetic validation of the 17βHSD13LoF variant, which confers protection against the risk and progression of liver disease, inhibition of 17βHSD13 activity with small molecule inhibitors could be an effective treatment for liver diseases such as NAFLD (e.g., NASH, liver fibrosis, cirrhosis, and solitary steatosis), hepatitis, alcoholic steatohepatitis (ASH), hepatitis C virus (HCV), and hepatocellular carcinoma (HCC), such as in individuals with several key risk factors for liver disease (e.g., obesity, alcohol consumption) and identified genetic risk factors (e.g., the (rs738409 C>G) variant in PNPLA3).

[0007] The compounds disclosed herein provide anti-hepatic effects by acting as at least 17βHSD13 inhibitors. Furthermore, the compounds disclosed herein can selectively inhibit 17βHSD13 relative to 17βHSD4 and / or 17βHSD9.

[0008] Fifteen 17β-HSD (HSD17B) members have been identified in humans. Sequence homology among different members is fairly low, but the overall structure appears to be conserved. 17β-hydroxysteroid dehydrogenases are primarily involved in sex hormone metabolism. Some 17β-HSD enzymes also play key roles in cholesterol and fatty acid metabolism (Labrie et al., Journal of Molecular Endocrinology, 2000, 25, 1–16; Wen Su et al., Molecular and Cellular Endocrinology, 2019, 489, 119–125). The ability to eliminate off-target features is an advantage of 17β-HSD13 inhibitors in avoiding potential toxicity caused by off-target activities. This includes selectivity for other 17β-HSD members.

[0009] 17βHSD4 / D-bifunctional protein (DBP) is involved in fatty acid β-oxidation and steroid metabolism. 17βHSD4 is universally expressed and plays an important role in estrogen inactivation in a range of peripheral tissues. Mutations in 17βHSD4 are known to cause DBP deficiency, an autosomal recessive disorder of peroxisome fatty acid β-oxidation that is usually fatal within the first two years of life. Homozygous missense variants of 17βHSD4 have been identified in Perrault syndrome, a recessive disorder characterized by female ovarian hypoplasia, sensorineural hearing loss in both men and women, and neurological manifestations in some patients (Pierce et al., Am. J. Hum. Genet., 2010, 87, 282-8; and Chen et al., BMC Med Genet., 2017, 18, 91).

[0010] 17βHSD9 / RDH5 (retinol dehydrogenase 5) is involved in retinoid metabolism. This enzyme is primarily expressed in the retinal pigment epithelium. The RDH5 gene encodes 11-cis-retinol dehydrogenase, an enzyme that is part of the visual cycle and catalyzes the reduction of 11-cis-retinol to 11-cis-retinaldehyde. Mutations in the RDH5 gene cause progressive cone dystrophy or macular dystrophy and night blindness. White spot fundus is a rare congenital form of night blindness with damage to the rod system, characterized by numerous small white-yellow retinal lesions. This disorder is primarily caused by mutations in the RDH5 gene (Hotta et al., Am. J. Ophthalmol., 2003, 135, 917-9; and Skorczyk-Werner et al., J. Appl. Genet., 2015, 56, 317-27).

[0011] Compared to other known 17βHSD13 inhibitors, the compounds described herein may also exhibit favorable physical properties (e.g., lower lipophilicity, higher water solubility, higher permeability, lower plasma protein binding, and / or greater chemical stability) and / or favorable toxicological characteristics (e.g., reduced activity against hERG) and / or favorable metabolic or pharmacokinetic characteristics. Therefore, these compounds may be particularly suitable as therapeutic agents, such as for the treatment of liver diseases. Summary of the Invention

[0012] In one aspect, the crystalline form of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone was provided.

[0013]

[0014] (Compound (III)), which is in form A.

[0015] In pharmaceutical formulations, it is advantageous for the pharmaceutical substance (active compound) to be in a form that is easy to handle and process. The chemical and physical stability of the active compound can be an important factor in determining the suitability of a solid form for developing pharmaceutical formulations. It is also advantageous for the active compound and the formulation containing it to be able to be stored effectively for a considerable period of time without exhibiting any significant changes in the physicochemical properties of the active compound (e.g., chemical composition, density, hygroscopicity, and solubility).

[0016] In another aspect, a pharmaceutical composition is provided comprising compound (III) form A and a pharmaceutically acceptable excipient.

[0017] On the other hand, compound (III) form A is provided for use in therapy.

[0018] On the other hand, compound (III) form A is provided for the treatment of liver disease.

[0019] On the other hand, the use of compound (III) form A in the manufacture of pharmaceuticals is provided.

[0020] On the other hand, the use of compound (III) form A in the manufacture of a medicament for the treatment of liver disease is provided.

[0021] On the other hand, a method for treating a patient’s liver disease is provided, the method comprising administering to the patient an effective amount of compound (III) form A.

[0022] definition

[0023] To make this specification easier to understand, certain terms are explicitly defined below. Furthermore, definitions are appropriately elaborated throughout the specific implementation.

[0024] Units, prefixes, and symbols are represented in their internationally recognized (SI) form. Numerical ranges include the values ​​that define that range.

[0025] 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 disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure. Attached Figure Description

[0026] The implementation schemes and experiments illustrating the principles of this disclosure will now be discussed with reference to the accompanying drawings, in which:

[0027] Figure 1A The powder X-ray diffraction pattern of form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone is shown.

[0028] Figure 1B Differential scanning calorimetry (DSC) analysis of form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone is shown.

[0029] Figure 2A Thermogravimetric analysis (TGA) of form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone is shown.

[0030] Figure 2B Gravimetric vapor adsorption analysis of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone form A is shown. Detailed Implementation

[0031] In the first aspect, ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) methyl ketone form A is provided, which is described herein as compound (III) form A.

[0032] According to this specification, compound (III) form A is provided, which has an X-ray powder diffraction pattern with a specific peak at 2θ = 10.7° when measured using CuKα radiation, ±0.2° 2θ.

[0033] According to this specification, compound (III) form A is provided, which has an X-ray powder diffraction pattern with a specific peak at 2θ = 19.8° when measured using CuKα radiation, ±0.2° 2θ.

[0034] In the embodiments, compound (III) form A is provided, which, when measured using CuKα radiation, has an X-ray powder diffraction pattern with specific peaks at 2θ = 10.7° and 19.8°, wherein the values ​​can be ±0.2°2θ.

[0035] In the embodiments, compound (III) form A is provided, which, when measured using CuKα radiation, has an X-ray powder diffraction pattern with specific peaks at 2θ = 8.1°, 10.7°, 19.8°, 24.9° and 27.6°, wherein the values ​​can be ±0.2°2θ.

[0036] In the embodiments, compound (III) form A is provided, which, when measured using CuKα radiation, has an X-ray powder diffraction pattern having specific peaks at 2θ = 8.1°, 10.7°, 13.6°, 15.4°, 18.9°, 19.8°, 21.4°, 23.1°, 24.9°, 27.6°, and 31.2°, wherein said values ​​can be ±0.2°2θ.

[0037] According to this specification, compound (III) form A is provided, which has an X-ray powder diffraction pattern with a specific peak at about 2θ = 10.7° when measured using CuKα radiation.

[0038] According to this specification, compound (III) form A is provided, which has an X-ray powder diffraction pattern with a specific peak at approximately 2θ = 19.8° when measured using CuKα radiation.

[0039] In the implementation, compound (III) form A is provided, which has an X-ray powder diffraction pattern with specific peaks at approximately 2θ = 10.7° and 19.8° when measured using CuKα radiation.

[0040] In the embodiments, compound (III) form A is provided, which has an X-ray powder diffraction pattern with specific peaks at approximately 2θ = 8.1°, 10.7°, 19.8°, 24.9° and 27.6° when measured using CuKα radiation.

[0041] In the embodiments, compound (III) form A is provided, which, when measured using CuKα radiation, has an X-ray powder diffraction pattern with specific peaks at approximately 2θ = 8.1°, 10.7°, 13.6°, 15.4°, 18.9°, 19.8°, 21.4°, 23.1°, 24.9°, 27.6°, and 31.2°.

[0042] According to this specification, compound (III) form A is provided, which, when measured using CuKα radiation, exhibits the same properties as... Figure 1A The X-ray powder diffraction patterns shown are essentially the same.

[0043] When reference is made to compound (III) form A in this specification, the crystallinity is greater than about 60%, greater than about 80%, greater than about 90%, or greater than about 95%. In the embodiments, the crystallinity is greater than about 98%.

[0044] In the embodiments, compound (III) form A is provided as described herein, wherein the purity is greater than 99%. In the embodiments, the purity is greater than about 99.5%. In the embodiments, the purity is greater than about 99.9%.

[0045] Compound (III) form A provides with Figure 1A The X-ray powder diffraction patterns shown are substantially the same and have essentially ten most prominent peaks (2θ angle values) as shown in Table 2. It should be understood that the 2θ values ​​of X-ray powder diffraction patterns may vary slightly between machines or samples, and therefore the values ​​cited should not be interpreted as absolute.

[0046] It is known that X-ray powder diffraction patterns with one or more measurement errors can be obtained depending on the measurement conditions (such as the equipment or machine used). In particular, it is generally known that the intensity in an X-ray powder diffraction pattern can fluctuate depending on the measurement conditions. Therefore, it should be understood that compound (III) form A of this specification is not limited to the form provided with Figure 1A The crystal with the same X-ray powder diffraction pattern as shown is provided. Figure 1A Any crystal with substantially identical X-ray powder diffraction patterns shown falls within the scope of this specification. Those skilled in the art of X-ray powder diffraction can determine the substantial similarity of the X-ray powder diffraction patterns.

[0047] This specification is intended to include all isotopes of the atoms present in the compounds of this invention. Isotopes will be understood to include atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include... 13 C and 14 C. Nitrogen isotopes include 15 N. Isotopes of fluorine include 18 F.

[0048] In another aspect, a pharmaceutical composition is provided comprising compound (III) form A and a pharmaceutically acceptable excipient.

[0049] The term "pharmaceutical composition" refers to a formulation in a form that allows for the biological activity of the active ingredient and does not contain any additional components that would have unacceptable toxicity to a patient to whom the composition will be administered. Such compositions may be sterile. Pharmaceutical compositions according to this specification will comprise compound (III) form A and pharmaceutically acceptable excipients.

[0050] Pharmaceutical formulations of compound (III) form A can be conveniently administered in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field, for example, as described in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA., (1985).

[0051] Due to its 17BHSD13 inhibitory activity, compound (III) form A is expected to be used in therapies, for example for the treatment of diseases or medical conditions at least partially mediated by 17BHSD13, including liver diseases such as NASH.

[0052] In one aspect of this specification, compound (III) form A is provided for use in therapy.

[0053] In one aspect of this specification, compound (III) form A is provided for the treatment of liver diseases. In embodiments, the liver disease is selected from alcoholic liver disease, non-alcoholic liver disease, NAFLD (such as NASH, liver fibrosis, cirrhosis, and solitary steatosis), hepatitis, alcoholic steatohepatitis (ASH), hepatitis C virus (HCV), and hepatocellular carcinoma (HCC).

[0054] The term "therapy" is intended to have its normal meaning, namely, treating a disease to completely or partially alleviate one, some, or all of its symptoms, or to correct or compensate for the underlying pathological condition. Unless specifically indicated to the contrary, the term "therapy" also includes "prevention." The terms "treatment" and "in treatment" should be interpreted accordingly.

[0055] The term “prevention” is intended to have its normal meaning and includes: primary prevention, which is used to prevent the development of disease; and secondary prevention, which involves taking temporary or permanent protective measures for patients after the disease has developed to prevent the disease from worsening or deteriorating or new symptoms related to the disease from appearing.

[0056] The term “treatment” is used synonymously with “therapeutic therapy.” Similarly, the term “treatment” can be considered as “the application of a therapeutic therapy,” where “therapeutic therapy” is as defined herein.

[0057] In the implementation scheme, compound (III) form A is provided, which provides inhibition of 17βHSD13.

[0058] In the implementation scheme, compound (III) form A is provided for the treatment of diseases mediated by 17βHSD13, such as liver diseases (e.g., NASH).

[0059] In the implementation scheme, compound (III) form A is provided for the treatment of fatty liver disease.

[0060] In one embodiment, compound (III) form A is provided for the treatment of non-alcoholic fatty liver disease (NAFLD), such as solitary steatosis, non-alcoholic steatohepatitis (NASH), liver fibrosis, or cirrhosis. In another embodiment, the liver disease is end-stage liver disease.

[0061] In the implementation plan, compound (III) form A is provided for the treatment of liver diseases, such as NASH, in which the patient also has or is susceptible to one or more conditions selected from the following: obesity, dyslipidemia, insulin resistance, type 2 diabetes and renal insufficiency.

[0062] In the implementation plan, compound (III) form A is provided for the treatment of liver diseases such as NASH, in which patients have a liver condition of 27 kg / m². 2 Up to 40kg / m 2 Body mass index (BMI). In another implementation, the subject had a body mass index (BMI) of 30 kg / m². 2 Up to 39.9 kg / m 2 BMI. In another implementation, the patient has a BMI of at least 40 kg / m². 2 The patient's BMI. In another implementation, the patient is overweight. In another implementation, the patient is obese.

[0063] In the implementation plan, compound (III) form A is provided for the treatment of liver diseases, such as NASH, in which patients also have or are prone to dyslipidemia.

[0064] In the implementation plan, compound (III) form A is provided for the treatment of liver diseases such as NASH, in which patients also have or are susceptible to insulin resistance.

[0065] In the implementation plan, compound (III) form A is provided for the treatment of liver diseases such as NASH, in which patients also have or are susceptible to type 2 diabetes.

[0066] In the implementation plan, compound (III) form A is provided for the treatment of liver diseases, such as NASH, in which patients also have or are susceptible to renal insufficiency.

[0067] In one implementation, compound (III) form A is provided for the treatment of liver diseases, such as NASH, in which the patient also has or is susceptible to liver fibrosis. In another implementation, the patient (i) has or is susceptible to liver fibrosis, and (ii) has or is susceptible to one or more of the following conditions: obesity, dyslipidemia, insulin resistance, type 2 diabetes, and renal insufficiency.

[0068] In one implementation, compound (III) form A is provided for the treatment of liver diseases, such as NASH, in which the patient also has or is susceptible to cirrhosis. In another implementation, the subject (i) has or is susceptible to cirrhosis, and (ii) has or is susceptible to one or more of the following conditions: obesity, dyslipidemia, insulin resistance, type 2 diabetes, and renal insufficiency.

[0069] In one embodiment, compound (III) form A is provided for the treatment of NAFLD. In another embodiment, the NAFLD is stage 1 NAFLD. In another embodiment, the NAFLD is stage 2 NAFLD. In another embodiment, the NAFLD is stage 3 NAFLD. In another embodiment, the NAFLD is stage 4 NAFLD. See, for example, “The Diagnosis and Management of Nonalcoholic Fatty Liver Disease: Practice Guidance From the American Association for the Study of Liver Diseases,” Hepatology, Vol. 67, No. 1, 2018.

[0070] In one embodiment, compound (III) form A is provided for the treatment of NAFLD, such as NASH. In another embodiment, the patient is obese. In another embodiment, the patient has alcoholic liver disease. In another embodiment, the patient has genetic risk factors for liver disease, such as the (rs738409 C>G) variant of PNPLA3.

[0071] In one implementation, compound (III) form A is provided for the treatment of NASH. In another implementation, the NASH is stage 1 NASH. In another implementation, the NASH is stage 2 NASH. In another implementation, the NASH is stage 3 NASH. In another implementation, the NASH is stage 4 NASH. In another implementation, the subject also has or is susceptible to one or more of the following conditions: obesity, dyslipidemia, insulin resistance, type 2 diabetes, and renal insufficiency.

[0072] In one implementation, compound (III) form A is provided for the treatment of liver fibrosis. In another implementation, the liver fibrosis is stage 3 liver fibrosis. In yet another implementation, the subject also has or is susceptible to one or more of the following conditions: obesity, dyslipidemia, insulin resistance, type 2 diabetes, and renal insufficiency.

[0073] In one implementation, compound (III) form A is provided for the treatment of cirrhosis. In another implementation, the cirrhosis is stage F4 cirrhosis. In yet another implementation, the subject also has or is susceptible to one or more of the following conditions: obesity, dyslipidemia, insulin resistance, type 2 diabetes, and renal insufficiency.

[0074] In one embodiment, compound (III) form A is provided for the treatment of hepatitis. In another embodiment, the inflammation is chronic inflammation. In another embodiment, the chronic inflammation is selected from: rheumatoid arthritis, osteoarthritis, and Crohn's disease. In another embodiment, the chronic inflammation is rheumatoid arthritis.

[0075] In the implementation plan, compound (III) form A is provided for the treatment of hepatocellular carcinoma (HCC).

[0076] In the implementation scheme, compound (III) form A is provided for the treatment of alcoholic steatohepatitis (ASH).

[0077] In the implementation scheme, compound (III) form A is provided for the treatment of hepatitis C virus (HCV).

[0078] In one aspect of this specification, the use of compound (III) form A as described herein in the manufacture of medicaments, such as medicaments for treating diseases (e.g., NASH).

[0079] In one aspect of this specification, a method for treating a patient’s disease (such as NASH) is provided, the method comprising administering to the patient an effective amount of compound (III) form A.

[0080] Terms such as “treatment” refer to (1) therapeutic measures that cure, alleviate, reduce or stop the progression of a diagnosed pathological condition or disorder, and (2) preventive or preventative measures that prevent and / or slow the development of the targeted pathological condition or disorder. Therefore, those who require treatment include those who already have the disorder; those who are susceptible to the disorder; and those who need to prevent the disorder.

[0081] The term "effective amount" means an amount of active ingredient sufficient to significantly and positively alter the symptoms and / or condition to be treated (e.g., provide a positive clinical response). The effective amount of active ingredient used in a pharmaceutical composition will vary depending on the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapies, the specific active ingredient used, the specific pharmaceutically acceptable excipient / carrier utilized, and similar factors within the knowledge and expertise of the attending physician.

[0082] The term "patient" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will be the recipient of a particular treatment. Typically, the term "patient" refers to a human subject.

[0083] In the implementation plan, a method for treating a patient’s disease is provided, the method comprising administering to the patient an effective amount of compound (III) form A, wherein the disease is selected from isolated steatosis, NASH, liver fibrosis and cirrhosis.

[0084] In the implementation plan, a method for treating 17βHSD13-mediated disease in a patient is provided, the method comprising administering to the patient an effective amount of a compound (III) form A, such as NASH.

[0085] The compounds disclosed herein can be used as a single pharmacological agent or in combination with other pharmacological agents or technologies in the methods described above. Such combination therapies can be achieved by administering the individual components simultaneously, sequentially, or separately. These combination therapies (and corresponding combination products) utilize the compounds disclosed herein and other pharmacological agents.

[0086] In one embodiment, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and a sodium-glucose cotransporter 2 (SGLT2) inhibitor. In another embodiment, the SGLT2 inhibitor is selected from canagliflozin, dapagliflozin, empagliflozin, eleggliflozin, ioggliflozin, luggliflozin, and repaggliflozin.

[0087] In the implementation plan, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and metformin or a pharmaceutically acceptable salt thereof.

[0088] In one embodiment, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and a glucagon-like peptide-1 receptor (GLP1) agonist. In another embodiment, the GLP1 agonist is selected from exenatide, liraglutide, lixinatide, abiglutide, dulaglutide, and semaglutide.

[0089] In one embodiment, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and a dipeptidyl peptidase 4 (DPP4) inhibitor. In another embodiment, the DPP4 inhibitor is selected from: sitagliptin, vendalliptin, saxagliptin, linagliptin, giglitazone, angliptin, terliglitazone, alogliptin, treagliptin, ocagliptin, edagliptin, gogliptin, and dugliptin.

[0090] In one embodiment, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and a PPAR agonist. In another embodiment, the PPAR agonist is a PPARα agonist. In another embodiment, the PPAR agonist is a PPARγ agonist. In another embodiment, the PPAR agonist is a PPARα / γ agonist. In another embodiment, the PPAR agonist is selected from: clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and fenofibrate. In another embodiment, the PPAR agonist is a thiazolidinedione. In another embodiment, the thiazolidinedione is selected from: pioglitazone, rosiglitazone, lobeglitazone, and linaglitazone. In another embodiment, the PPAR agonist stimulates hepatic expression of FGF21. PPAR refers to the peroxisome proliferator-activated receptor.

[0091] In one embodiment, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and a pan-PPAR agonist. In another embodiment, the pan-PPAR agonist is lanilanol.

[0092] In one embodiment, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and a ThrB agonist. In another embodiment, the ThrB agonist is resmetiro.

[0093] In one embodiment, a combination for treating liver diseases such as NASH is provided, comprising compound (III) form A and an FXR agonist. In another embodiment, the FXR agonist is obeticholic acid.

[0094] Although compound (III) forms A are primarily valued as therapeutic agents for patients, they can also be used whenever inhibition of 17βHSD13 is required. Therefore, they can be used as pharmacological standards for the development of new biological tests and the search for novel pharmacological agents.

[0095] Example

[0096] This specification will now be illustrated by the following non-limiting embodiments, wherein, generally speaking:

[0097] (i) Operation is carried out at room temperature (rt) (i.e., in the range of 17°C to 28°C) and, if necessary, in an atmosphere of an inert gas such as N2;

[0098] (ii) In cases where the reaction involves degassing or purging, this can be done, for example, by purging the reaction solvent with a constant stream of nitrogen for a suitable period of time (e.g., 5 to 10 minutes) or by repeatedly evacuating the container and backfilling it with a suitable inert atmosphere (e.g., nitrogen (g) or argon (g)).

[0099] (iii) In cases where the reaction involves the use of a microwave reactor, use one of the following microwave reactors: Biotage Initiator, Personal Chemistry Emrys Optimizer, Personal Chemistry Smith Creator, or CEM Explorer;

[0100] (iv) Generally, reaction progress is tracked by thin-layer chromatography (TLC) and / or analytical high-performance liquid chromatography (HPLC or UPLC), which is usually coupled with a mass spectrometer (LCMS).

[0101] (v) If necessary, the organic solution may be dried with anhydrous MgSO4 or Na2SO4, or by using an ISOLUTE phase separator, and post-processed using conventional phase separation techniques. When using a desiccant such as MgSO4 or Na2SO4 to dry the organic layer, it should be understood that the organic layer should be filtered before concentration.

[0102] (vi) Evaporation by vacuum rotary evaporation or by Genevac HT-4 / EZ-2 or Biotage V10;

[0103] (vii) Unless otherwise specified, rapid column chromatography shall be performed on straight-phase silica using Merck silica gel (Art. 9385) or pre-packed columns such as BIOTAGE SNAP columns (40µm-63µm silica, 4g-330g), BIOTAGE Sfär silica HC D columns (20µm, 10g-100g), INTERCHIM PURIFLASH columns (25µm, 4g-120g), INTERCHIM PURIFLASH columns (50µm, 25g-330g), GRACE GRACERESOLVE silica rapid columns (4g-120g), or Agela rapid column silica-CS columns (80g-330g), or on reversed-phase silica using Agela Technologies C-18 spherical columns (20µm-35µm, 100A, 80g–330g) with Grace REVELERIS. The X2 Fast System or similar systems can be operated manually or automatically.

[0104] (viii) Preparative TLC was performed in a glass chamber on a silica plate (20cm × 20cm) with a glass backing covered with 1mm thick silica gel (particle size 10μm–40μm) as the eluent.

[0105] (ix) Preparative reversed-phase HPLC and preparative reversed-phase SFC were performed using isocratic or gradient mobile phases as described in the Experimental Section and using standard HPLC and SFC instruments equipped with MS and / or UV-triggered fractionation collection instruments, respectively: Preparation method F: The compound was purified by preparative HPLC on a Kromasil C8 column (10 µm, 250 mm × 50 mm ID) using a gradient of MeCN in H2O / MeCN / FA (95 / 5 / 0.2) as the mobile phase.

[0106] In some cases, the compound can be dissolved in a solvent (such as DMSO), filtered through a syringe filter, and then purified on a preparative HPLC.

[0107] Collect the relevant fractions, combine them, and freeze-dry or evaporate them to obtain the purified compound; or collect the relevant fractions, combine them, concentrate them under reduced pressure, extract them with DCM or EtOAc, and dry the organic phase with Na2SO4 or by using a phase separator, and then concentrate it under reduced pressure to obtain the purified compound.

[0108] (x) Preparative chromatography was performed using HPLC or SFC on a standard HPLC or SFC instrument and with isocratic or gradient operation using the mobile phase as described in the experimental section;

[0109] (xi) The yield (in the presence of) is not necessarily the maximum obtainable value, and the reaction can be repeated if a larger amount of reaction product is required.

[0110] (xii) In cases where certain compounds are obtained as acid addition salts (e.g., monohydrochloride or dihydrochloride), the stoichiometry of the salt is based on the number and nature of the basic groups in the compound, and the precise stoichiometry of the salt cannot usually be determined, for example, by elemental analysis data;

[0111] (xiii) Generally, the structure of the final product is confirmed by nuclear magnetic resonance (NMR) and / or mass spectrometry; a Bruker Avance III 300, 400, 500, and 600 spectrometer (at 300 MHz, 400 MHz, 500 MHz, and 600 MHz, respectively) is used. 1Proton NMR chemical shifts are measured on the delta scale (operated at H frequencies). Experiments are typically recorded at 25 °C. Chemical shifts are given in ppm, with the solvent used as an internal standard. Protons on heteroatoms, such as NH and OH protons, are only reported if detected in the NMR and may therefore be lost. In some cases, protons may be masked or partially masked by solvent peaks and thus either lost and not reported or reported as multiplets overlapping with the solvent. The following abbreviations (and their derivatives, such as dd, doublet, etc.) have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad peak; qn, quintet; p, pentet. In some cases, the structure of the final product may appear as a rotational isomer in the NMR spectrum; in such cases, only the peak of the dominant rotational isomer is reported. In certain cases, the structures of intermediates and / or end products may appear as rotational isomers in NMR spectra with more equal relationships. In such cases, if the signals of the rotational isomers partially overlap, the peaks of such rotational isomers are reported as multiplets; or if the signals of the rotational isomers are well separated and only the total number of protons is reported, the peaks of such rotational isomers are reported as separate peaks. If known, the ratio of the major rotational isomer to the minor rotational isomer is reported.

[0112] (xiv) Electrospray mass spectrometry data were acquired using a WatersAcquity UPLC coupled to a Waters single quadrupole mass spectrometer or similar device, acquiring both cation and anion data, and typically only reporting ions relevant to the parent structure; High-resolution electrospray mass spectrometry data were acquired using a Waters XEVO qToF mass spectrometer coupled to a Waters Acquity UPLC or similar device, acquiring both cation and anion data, and typically only reporting ions relevant to the parent structure.

[0113] (xv) Intermediates may not be completely purified, but their structure and purity are assessed by TLC, analytical HPLC / UPLC and / or NMR analysis and / or mass spectrometry.

[0114] (xvi) Unless otherwise stated, compounds containing asymmetric carbon and / or sulfur atoms are not resolved;

[0115] (xvii) Generally, the examples and intermediate compounds are named using ChemDraw Professional version 22.2.0 from PerkinElmer. ChemDraw Professional version 22.2.0 uses the Cahn-Ingold-Prelog (CIP) rules for stereochemistry to generate names of chemical structures, and adheres as strictly as possible to the IUPAC rules when generating chemical names. Stereoisomers are distinguished from each other by the stereo descriptors referenced in their names and are assigned according to the CIP rules.

[0116] ChemDraw optionally uses notations such as “&” and “or” in the illustration of stereocenters to describe the configuration of the stereochemical centers present in the structure. The numbers following the “&” and “or” notations are assigned to each stereocenter present in the structure. The numbers increment automatically to indicate that the stereocenters can vary independently of each other.

[0117] Generally, for embodiments and intermediates containing more than one stereocenter with a fixed relative configuration, the same number is used after the symbols “&” and “or” to indicate that the stereocenter forms a group. A third stereocenter existing in the same chemical structure, independent of the preceding stereocenter, is indicated by a unique new number after the symbols “&” and “or”.

[0118] Generally, the chemical structure of an embodiment or intermediate containing the symbol "&" at the stereocenter indicates that the configuration of such an embodiment or intermediate at that stereocenter is a mixture of both (R) and (S); and the symbol "or" indicates that the configuration of such an embodiment or intermediate at that stereocenter is (S) or (R). Absolute, unspecified, "&", and "or" stereocenters can all exist in a single structure.

[0119] Generally, for chemical structures of embodiments and intermediates in which there is only one stereocenter and the stereocenter is racemic, no label is specified for the stereocenter, and the structure is drawn with straight bonds at the stereocenter.

[0120] Generally, for embodiments and intermediates containing more than one stereocenter with a fixed relative configuration, the same number is used after the symbols “&” and “or” to indicate that the stereocenter forms a group. A third stereocenter existing in the same chemical structure, independent of the preceding stereocenter, is indicated by a unique new number after the symbols “&” and “or”.

[0121] Generally, for embodiments and intermediates where all stereocenters are designated as "&", the structure is named with the "rac-" prefix. For embodiments and intermediates where all stereocenters are designated as "or", the structure is named with the "rel-" prefix.

[0122] (xviii) In addition to those mentioned above, the following abbreviations and units are also used:

[0123]

[0124] unit

[0125]

[0126] In vitro 17bHSD13 enzyme assay

[0127] Ten concentrations (0.2 μl) of the compound in DMSO solution were added to each well of a Greiner PP 384-well plate (781280) using an ECHO dispenser (BECKMAN COULTER), followed by the addition of 20 μl of recombinant 17bHSD13 (N2-K300). The enzyme reaction was initiated by adding 20 μl of substrate solution containing NAD (SIGMA, N1511) and estradiol (SIGMA, E8875) using a CERTUS-FLEX dispenser (GYGER). After each addition, the plate was centrifuged at 150 x g for 1 min (EPPENDORF, 5810R, A-4-81). The final assay conditions were as follows: 80 nM 17bHSD13, 0.5 mM NAD, 20 μM estradiol, and different concentrations of the compound in a buffer solution (5 mM EDTA (TEKNOVA E0306), 0.01% DDM (AFFYMETRIX D310) in 50 mM Tris-Cl solution, pH 7.4). After 2.5 hours, the reaction was terminated by adding 20 μl of 0.6% formic acid (MERCK 5.33002), and the samples were analyzed using LC-MS / MS.

[0128] SCIEX LC-MS / MS System: Samples were injected using a CTC analyzer and a SHIMATZU LC pump LC20, and analyzed on a SCIEX API 5000 LC-MS / MS system as follows. Samples were chromatographically separated on a WATERS (SYMMETRY, C8, 3.5 μm, 2.1 mm x 50 mm) column at a constant flow rate of 0.5 mL / min. The mobile phase consisted of solvent A (water containing 0.2% formic acid) and solvent B (acetonitrile containing 0.2% formic acid). The LC gradient distribution was as follows: 50% B from 0 min to 0.5 min, linearly increasing to 100% B from 0.5 min to 1 min, held at 100% B from 1 min to 1.6 min, and then returning to 50% B from 1.6 min to 2 min. The run time was 2 min, with retention times of approximately 0.8 min for estradiol and 1.07 min for estrone. Detection was performed on an API 5000 LC / MS / MS system with a triple quadrupole mass spectrometer, a TURBO V ion source, and an APCI probe in positive polarity multiple reaction monitoring (MRM) mode. The MRM pairs for estradiol and estrone were m / z 273.1 to m / z 107.0 and m / z 271.3 to 107.0, respectively. The residence time for each transition was 100 ms, and the depolarization and collision energies were 100 and 40, respectively. Data from the MS signal were obtained using the area under the curve (AUC). Ratio = estrone / (estrone + estradiol)

[0129] In vitro 17bHSD13 cell assay

[0130] Inhibition of 17bHSD13 was measured in cell-based assays in which HSD17β13 was overexpressed in HEK293S cells and the conversion of estradiol to estrone was measured by LCMS / MS.

[0131] Cells were seeded in 30 μl of medium (DMEM with GLUTAMAX and 10% FBS) at 10 K c / w in 384-well plates (GREINER CELL 384w black / clear poly-D-lysine). After 6 hours of cell attachment, 0.15 μl of the compound at 10 concentrations and 0.03 μl of 10 mM estradiol (SIGMA, E8875) in DMSO solution were added using an ECHO dispenser (BECKMAN COULTIER). After 18 hours of cell culture, 20 μl of medium was transferred to a GREINER PP 384-well plate (781280) using a BRAVO dispenser robot (AGILENT), and 40 μl of 50% acetonitrile was added. Samples were analyzed using LC / MS / MS.

[0132] SCIEX LC-MS / MS System: Samples were injected using a CTC analytical injector and a SHIMATZU LC pump LC20, and analyzed on a SCIEX API 5000 LC-MS / MS system as follows. Samples were chromatographically separated on a WATERS (symmetry, C8, 3.5 μm, 2.1 mm x 50 mm) column at a constant flow rate of 0.5 mL / min. The mobile phase consisted of solvent A (water containing 0.2% formic acid) and solvent B (acetonitrile containing 0.2% formic acid). The LC gradient distribution was as follows: 50% B from 0 min to 0.5 min, linearly increasing to 100% B from 0.5 min to 1 min, remaining at 100% B from 1 min to 1.6 min, and then returning to 50% B from 1.6 min to 2 min. The run time was 2 min, with retention times of approximately 0.8 min for estradiol and 1.07 min for estrone. Detection was performed on an API 5000 LC / MS / MS system with a triple quadrupole mass spectrometer, a TURBO V ion source, and an APCI probe in positive polarity multiple reaction monitoring (MRM) mode. The MRM pairs for estradiol and estrone were m / z 273.1 to m / z 107.0 and m / z 271.3 to 107.0, respectively. The residence time for each transition was 100 ms, and the depolarization and collision energies were 100 and 40, respectively. Data from the MS signal were obtained using the area under the curve (AUC). Ratio = estrone / (estrone + estradiol)

[0133] In vitro 17bHSD4 enzyme assay

[0134] Ten concentrations of the compound (0.2 μl) in DMSO solution were added to a Greineer Fluotrac 200 384-well plate (781076) using an ECHO partition (BECKMAN COULTER). 80 nl of 10 mM estradiol (SIGMA, E8875) was added using an Echo partition. The enzyme reaction was initiated by adding 40 μl of a mixture containing recombinant 17bHSD4 (M1-N311) and NAD using a MULTIDROP COMBI partition (THERMO FISHER). The final assay conditions were 40 nM 17bHSD4, 0.125 mM NAD, 15 μM estradiol, and different concentrations of the compound in a buffer solution (5 mM EDTA (TEKNOVA E0306), 0.01% DDM (AFFYMETRIX D310) in 50 mM Tris-Cl solution, pH 7.4). After each addition, the plate was centrifuged at 150 x g for 1 minute (EPPENDORF, 5810R, A-4-81). NADH formation was measured by fluorescence intensity (FI) (Ex360 / Em460) at time zero (t0) and 1.5 hours (t1) in a PHERASTAR FSX (BMG LABTECH). The FI for each sample was calculated as the FI at t1 minus the FI at t0.

[0135] In vitro 17bHSD9 cell assay

[0136] Inhibition of 17bHSD9 was measured in cell-based assays in which HSD17β9 was overexpressed in HEK293S cells and the conversion of retinol to retinaldehyde was measured by LCMS / MS.

[0137] Cells were seeded at 10 K c / w in 30 μl of medium (DMEM with GLUTAMAX and 10% FBS) in 384-well plates (GREINER CELL 384w black / clear poly-D-lysine). After 6 hours of cell attachment, 0.15 μl of the compound at 10 concentrations and 0.015 μl of 10 mM all-trans retinol (CAYMAN CHEMICAL, 20241) in DMSO solution were added using an ECHO dispenser (BECKMAN COULTIER). After 18 hours of cell culture, 20 μl of medium was transferred to a GREINER PP 384-well plate (781280) using a BRAVO dispenser robot (AGILENT), and 40 μl of 50% acetonitrile was added. Samples were analyzed using LC / MS / MS.

[0138] SCIEX LC-MS / MS System: Samples were injected using a CTC analytical injector and a SHIMATZU LC pump LC20, and analyzed on a SCIEX API 5000 LC-MS / MS system as follows. Samples were chromatographically separated on a WATERS (symmetry, C8, 3.5 μm, 2.1 mm x 50 mm) column at a constant flow rate of 0.5 mL / min. The mobile phase consisted of solvent A (water containing 0.2% formic acid) and solvent B (acetonitrile containing 0.2% formic acid). The LC gradient distribution was as follows: 50% B from 0 min to 0.1 min, linearly increasing to 100% B from 0.1 min to 0.8 min, held at 100% B from 0.8 min to 1.5 min, then returning to 50% B from 1.5 min to 1.6 min, and held for the entire run time. The run time was 2 min, with retention times of approximately 1.54 min for retinol and 1.62 min for retinal. Detection was performed on an API 5000 LC / MS / MS system with a triple quadrupole mass spectrometer, a TURBO V ion source, and an ESI probe in positive polarity in multiple reaction monitoring (MRM) mode. The MRM pairs for retinol and retinal were m / z 269.3–93.0 and m / z 285.2–161.0, respectively. The residence time for each transition was 100 ms, and the depolarization and collision energies were 50 and 25, respectively. Data from the MS signal were obtained using the area under the curve (AUC). Ratio = retinal / (retinal + retinol).

[0139] Data Analysis

[0140] Curve fitting and IC using GENEDATA SCREENER 50 Calculation of values.

[0141] The effects of compounds are calculated using the following formula:

[0142] Compound % effect = -100 x ((X-min) / (max-min))

[0143] Where X represents the effect in the presence of the test compound, min is DMSO, and max is the maximum inhibition of the enzyme using a known inhibitor as a control.

[0144] Form A ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazine) (3-Azol-3-yl)methyl ketone

[0145] This article discloses form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) methyl ketone.

[0146] ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazole-3- Synthesis of methyl ketone (compound (III))

[0147]

[0148] ((2R,6S)-2,6-dimethylmorpholine (0.520 g, 4.51 mmol) was suspended in anhydrous toluene (12 mL), and a toluene solution of Me3Al (2 M, 4.34 mL, 8.68 mmol) was added under N2 (g) atmosphere. The resulting mixture was stirred at room temperature for 1 hour. This mixture was then added to a stirred toluene (12 mL) slurry of ethyl 5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazole-3-carboxylic acid intermediate A (1 g, 3.47 mmol). The resulting solution was heated to 60 °C for 20 hours. The mixture was cooled to room temperature, tartaric acid (30%, aqueous solution, 100 mL) was added, and the mixture was extracted with EtOAc. The organic layer was concentrated, and the residue was purified by preparative HPLC method F (gradient: 20%–80%) to give the title compound (1.01 g, 81%) as a white solid; HRMS (ESI). m / z [M+H] + C 15 H 15 Calculated value of F3N3O4: 358.1008, measured value: 358.0978; 1 H NMR (500MHz, CD3OD) δ 1.14 (3H, d), 1.25 (3H, d), 2.65 (1H,dd), 2.96 (1H, dd), 3.57–3.79 (2H, m), 3.98 (1H, dt), 4.52 (1H, dt), 7.43–7.67 (1H, m).

[0149] Intermediate A: Ethyl 5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazole-3-carboxylic acid ester

[0150]

[0151] Under a nitrogen (N2) atmosphere, EDC (43.5 g, 227.07 mmol) and HOBt (15.34 g, 113.53 mmol) were added to a DMF (150 mL) solution of (Z)-2-amino-2-(hydroxyimino)ethyl acetate (15 g, 113.53 mmol), 2,4,5-trifluoro-3-hydroxybenzoic acid (21.81 g, 113.53 mmol), and NaHCO3 (28.6 g, 340.60 mmol). The resulting solution was stirred at 100 °C for 1 hour. The reaction mixture was filtered through a CELITE filter, and the filtrate was concentrated, diluted with DCM (300 mL), and washed with water (300 mL). The organic layer was dried over Na2SO4, filtered, and evaporated. The crude product was purified by rapid chromatography on a C18 column (gradient: 50%–60% MeCN aqueous solution (FA)) to give the title compound (7.0 g, 21%) as a white solid; MS (ESI) m / z [M+H] + 289; 1 H NMR (300MHz, DMSO-d6) δ 1.36 (t, 3H), 4.45 (m, 2H), 7.28 – 7.81 (m, 1H), 11.70 (s, 1H).

[0152] Table 1

[0153]

[0154] The data in Table 1 may be from a single experiment or the average of two or more experiments.

[0155] 1) Cooling crystallization

[0156] A saturated solution of the separated sample of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl-1,2,4-oxadiazol-3-yl) ketone was obtained by stirring at 50 °C in one of the following solvents: 1-propanol, 2-butanol, acetone, and ethyl acetate. The solution was slowly cooled to 5 °C. The precipitate was separated from the liquid phase, dried under ambient or vacuum (5 mbar and 50 °C), and analyzed by XRPD at room temperature.

[0157] 2) Evaporation crystallization

[0158] A high-concentration solution of the separated sample of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl-1,2,4-oxadiazol-3-yl) methyl ketone, prepared as above, was obtained by stirring at room temperature (RT) in one of the following solvents: methanol, ethanol, methyl ethyl ketone (MEK), and acetonitrile (ACN). The liquid phase was evaporated under ambient conditions, and the resulting solids were analyzed by XRPD.

[0159] Analysis of the corresponding samples prepared by cooling crystallization and evaporation crystallization revealed that the resulting powders were crystalline. Furthermore, the XRPD of each powder produced similar results to... Figure 1A The diffraction pattern shown is equivalent to the diffraction pattern. Further analysis as described herein reveals that the substance is ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) methyl ketone (compound (III)) in anhydrous solid form, form A.

[0160] Table 2: List of eleven most prominent peaks in the X-ray powder diffraction pattern of compound (III) form A .

[0161]

[0162] XRPD

[0163] X-ray powder diffraction (XRPD) was performed according to standard methods, which can be found in, for example, Kitaigorodsky, AI (1973), Molecular Crystals and Molecules, Academic Press, New York; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; or Klug, HP and Alexander, LE (1974), X-ray Diffraction Procedures, John Wiley & Sons, New York.

[0164] Those skilled in the field of X-ray powder diffraction will recognize that the relative intensity of peaks can be affected by, for example, grain sizes larger than 30 micrometers and non-uniform aspect ratios, which can influence sample analysis. They will also recognize that the position of reflections can be affected by the precise height of the sample within the diffractometer and the zero calibration of the diffractometer. The surface flatness of the sample can also have a slight effect. Therefore, the presented diffraction pattern data are not considered absolute values. (Jenkins, R & Snyder, RL'Introduction to X-Ray Powder Diffractometry' John Wiley & Sons 1996; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; Klug, HP & Alexander, LE (1974), X-Ray Diffraction Procedures).

[0165] Generally speaking, the measurement error of the diffraction angle in an X-ray powder diffraction pattern can be approximately ±0.2°2θ, and when considering... Figure 1A When reading XRPD data and Table 2, this level of measurement error should be considered. Furthermore, peak intensity can fluctuate depending on experimental conditions and sample preparation (preferably orientation).

[0166] The XRPD spectrum is determined as follows: The sample is placed on a zero-background sample holder with pits filled with abrasive material. A uniformly distributed sample with the correct sample height is obtained using a glass slide, and the peak positions are adjusted by referring to a second sample containing an internal corundum standard.

[0167] Powder X-ray diffraction patterns were recorded using a Rigaku Miniflex 600 equipped with a D / Tex Ultra detector (X-ray wavelength 1.5418 Å, nickel-filtered CuKα radiation, 40 kV, 15 mA) operating in one-dimensional scanning mode along the θ-2θ scan axis. Fixed divergence and receiving slits were used, along with an automatically variable anti-scattering screen. The sample was rotated at 30 rpm during the measurement. The sample was scanned from 3° to 50° 2θ (2θ) using a step size of 0.01° and a scan rate of 1° / min.

[0168] Definition of relative intensity

[0169]

[0170] The relative intensity is derived from the diffraction pattern measured using a fixed slit.

[0171] Differential scanning calorimetry (DSC) with ramp angle

[0172] The onset of melting point temperature (Tm) was determined using a TA Instruments DSC (Discovery 2500) via differential scanning calorimetry. The sample (approximately 2-4 mg) was weighed into an aluminum sample dish. The sample was filled to the bottom of the unsealed sample dish, and the lid was pressed down to ensure good thermal contact. The instrument was purged with nitrogen at 50 mL / min, and data were collected between 0°C and 220°C using a heating rate of 5°C / min.

[0173] Thermogravimetric analysis (TGA)

[0174] Thermogravimetric analysis was performed using a TA Instruments TGA (model TGA5500). Samples (approximately 5-10 mg) were transferred to a balanced sample holder. The instrument was purged with nitrogen, heated in an oven at 25 mL / min, and balanced at 10 mL / min. Data were collected between room temperature and 300 °C using a heating rate of 5 °C / min.

[0175] Gravimetric vapor adsorption (GVS)

[0176] Gravimetric vapor adsorption analysis was performed using a DVS Resolution instrument from Surface Measurement Systems. Samples (approximately 5-10 mg) were transferred to a balanced sample holder. Initially, the instrument was purged with dry nitrogen at 25°C, followed by data collection at different relative humidities (RH%) by mixing wet and dry nitrogen streams. The RH was increased in 10% increments from 20% to 80% RH, then gradually decreased to 0% RH, followed by a second cycle increasing to 90% RH and returning to 0% RH. The equilibrium index for moving to the next RH% was reached when the drift index (dm / dt) was below 0.002% for 10 minutes. The mass at the end of the 0% RH stage was used as a reference mass for calculating the mass change during the experiment.

[0177] Hygroscopicity can be assessed, for example, according to the European Pharmacopoeia (EP) classification: non-hygroscopic: <0.2%; slightly hygroscopic: ≥0.2% and <2%; hygroscopic: ≥2% and <15%; very hygroscopic: ≥15%; deliquescent: absorbs enough water to form a liquid; all values ​​are measured as weight gain at 80% RH and 25°C.

[0178] result

[0179] Figure 1BRepresentative DSC thermograms of form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone, recorded during heating from 0°C to 220°C, are shown. Exothermic events are plotted upwards. Figure 1B The melting endothermic reaction shown has an onset temperature of approximately 207 °C and an enthalpy of approximately 133 J / g. The obtained melting temperature can vary by up to ±5 °C, depending on the instrument used, how the sample was prepared, and batch-to-batch variability. No thermal events were detected in the DSC signal prior to the melting onset temperature, indicating that form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)methyl ketone is thermally stable in the temperature range of 0 °C to 200 °C under these experimental conditions. The observed range of thermal stability can be considered favorable for future formulation development.

[0180] Figure 2A A representative TGA thermogram of form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) ketone is shown. Form A exhibits minimal heat loss (less than about 0.1%) when heated from room temperature (RT), approximately 25 °C, to 200 °C. Therefore, form A is confirmed as the anhydrous solid form of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) ketone.

[0181] Figure 2B Representative GVS curves for form A of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) ketone, recorded during two adsorption / desorption cycles, are shown. Form A exhibits reversible water absorption of <0.05% by mass at 20% to 80% relative humidity at 25°C ± 0.1°C during the first adsorption / desorption cycle. The desorption curves show that form A loses water at a rate similar to that gained during adsorption, with limited hysteresis. No change in form was observed by XRPD after the GVS experiments. Form A is therefore identified as the nonhygroscopic (i.e., <0.2% weight increase) solid form of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) ketone according to the European Pharmacopoeia (EP) classification.

[0182] The above description of the illustrative embodiments is intended only to familiarize others skilled in the art with the applicant's specification, its principles, and its practical applications, enabling them to readily adapt and apply this specification in various forms to best suit the requirements of a particular application. This description and its specific examples are intended for illustrative purposes only when indicating embodiments of this specification. Therefore, this specification is not limited to the illustrative embodiments described herein, and various modifications are possible. Furthermore, it should be understood that, for clarity, the various features of the specification described in the case of individual embodiments may be combined to form a single embodiment. Conversely, for brevity, the various features of the specification described in the case of individual embodiments may be combined to form sub-combinations.

Claims

1. A crystalline form (form A) of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) methyl ketone, which, when measured using CuKα radiation, has an X-ray powder diffraction pattern with characteristic peaks at 2θ = 10.7° and 19.8°, wherein the values ​​are ±0.2°2θ.

2. The crystalline form according to claim 1, when measured using CuKα radiation, has an X-ray powder diffraction pattern with specific peaks at 2θ = 8.1°, 10.7°, 19.8°, 24.9° and 27.6°, wherein the values ​​can be ±0.2°2θ.

3. The crystalline form according to claim 1 or claim 2, when measured using CuKα radiation, has an X-ray powder diffraction pattern with specific peaks at 2θ = 8.1°, 10.7°, 13.6°, 15.4°, 18.9°, 19.8°, 21.4°, 23.1°, 24.9°, 27.6°, and 31.2°, wherein the values ​​can be ±0.2°2θ.

4. A crystalline form (form A) of ((2R,6S)-2,6-dimethylmorpholino)(5-(2,4,5-trifluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-3-yl) methyl ketone, which, when measured with CuKα radiation, has an X-ray powder diffraction pattern substantially as shown in FIG1A.

5. A pharmaceutical composition comprising a crystalline form according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

6. The crystalline form according to any one of claims 1 to 4, wherein the crystalline form is used in a therapeutic application.

7. The crystalline form according to any one of claims 1 to 4, wherein the crystalline form is used to treat liver disease.

8. The crystalline form according to any one of claims 1 to 4, wherein the crystalline form is used to treat liver diseases selected from: alcoholic liver disease, non-alcoholic liver disease, NAFLD, NASH, liver fibrosis, cirrhosis, solitary steatosis, hepatitis, alcoholic steatohepatitis (ASH), hepatitis C virus (HCV), and hepatocellular carcinoma (HCC).

9. The crystalline form according to any one of claims 1 to 4, wherein the crystalline form is used to treat NASH.

10. The crystalline form according to any one of claims 1 to 4, wherein the crystalline form is used to treat liver fibrosis.

11. The crystalline form according to any one of claims 1 to 4, wherein the crystalline form is used to treat cirrhosis.

12. A method of treating a patient with liver disease, the method comprising administering to the patient the crystalline form according to any one of claims 1 to 4.

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