Solid crystalline forms of PPAR-gamma modulators and methods of use thereof

Abstract

The present disclosure provides novel solid crystalline forms A of Compound I, compositions comprising the same, and methods of using the same, including their use in the treatment of cancer, particularly cancer in which agents known to target the RXR [alpha] and / or PPAR [gamma] pathway are useful. # imgabs0 #

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 364,402, filed on May 9, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure provides solid forms of compounds that can alter, modulate or inhibit the RXRα and / or PPARγ pathways, and methods of treating cancer, particularly cancers in which agents targeting the RXRα and / or PPARγ pathways are known to be useful.

[0003] Muscle-invasive bladder cancer (MIBC) is an aggressive, potentially fatal disease with limited treatment options. Although chemotherapy and immunotherapy are approved for the treatment of locally advanced or metastatic bladder cancer, most patients either do not respond or have only a short-lived response (Seiler et al., 2017 Eur Urology; Robertson et al., 2017 Cell). This suggests that additional efforts are needed to identify novel therapies that could benefit patients who currently do not respond to existing standard of care (SoC) therapies.

[0004] Recent studies have shown that not all advanced bladder cancers are created equal. Deep gene expression and genomic analyses have revealed distinct molecular subtypes of MIBC, including basal and luminal subtypes (Choi et al., 2014 Cancer Cell; Kardos et al., 2016 JCI Insight; Kamoun et al., 2020 Eur Urology), with unique tumor intrinsic and microenvironmental characteristics. Much like luminal breast and prostate cancers, luminal bladder cancers tend to grow slower, have less immune infiltration, and respond poorly to chemotherapy and immunotherapy (Robertson et al., 2017 Cell). As only suboptimal therapies are currently available for luminal disease, concerted efforts are needed to identify and develop novel luminal lineage-specific therapeutic nodes.

[0005] Genomic alterations in the RXRα / PPARγ pathway are evident in most luminal bladder cancers, including recurrent mutations of RXRα at serine 427 (S427F / Y), hotspot mutations of PPARγ at threonine 475 (T475M), and amplification / overexpression of PPARγ (Guo et al., 2013 Nature Genetics; Van Allen et al., 2014 Cancer Discovery). These genomic alterations enhance the PPARγ / RXRα-dependent transcriptional program in MIBC (Halstead et al., 2017 eLife; Korpal et al., 2017 Nat Communications; Goldstein et al., 2017 Cancer Research). In addition, only PPARγ 活性 Luminal bladder cancer cell lines showed reduced proliferation following genetic / pharmacological inhibition of PPARγ (Halstead et al., 2017 eLife; Goldstein et al., 2017 Cancer Research). Thus, activation of this pathway is associated with dependence on RXRα / PPARγ for growth. Subsequent analysis of the functional role of PPARγ in luminal cells revealed a key role in promoting energy production by enhancing glucose and lipid metabolism (Liu et al., 2019 Nat Communications), which may contribute to the observed dependence on PPARγ in genomic altered luminal cells.

[0006] In addition to this tumor-intrinsic role of PPARγ in luminal bladder cancers with PPARγ alterations, recent studies have shown that activated PPARγ / RXRα inhibits inflammatory cytokine expression and immune cell infiltration (Korpal et al., 2017 Nat Communications; Kardos et al., 2016 JCI Insight). Several clinical data sets and in vivo tumor models suggest that PPARγ 高 / RXRα S427F / Y Damage to CD8 +T cell infiltration and confer partial resistance to immune checkpoint inhibitors. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression and may suggest therapeutic approaches to restore immune surveillance and sensitivity to immunotherapy (Korpal et al., 2017 Nat Communications). Collectively, these studies suggest that PPARγ acts as an intrinsic “immune oncogene” of tumor cells, acting by promoting tumor cell growth, enhancing energy production, and increasing tumor cell survival through immunosuppression.

[0007] As used herein, "Compound I" can be described using a name according to ACD / naming software, which is (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide, and / or (S)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-N-((1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide. The structure of Compound I can be depicted as:

[0008]

[0009] Compound I and its preparation method are disclosed as follows: Compound 169B in U.S. Provisional Application No. 63 / 11,354 filed on November 9, 2020, U.S. Application No. 17 / 521,666 and PCT International Application No. PCT / US2021 / 058473 (both applications were filed on November 8, 2021); the entire contents of each of these applications are incorporated herein by reference.

[0010] One aspect of the present disclosure provides a new solid crystalline form of Compound I, Form A, which is useful for treating cancers in which agents targeting the RXRα and / or PPARγ pathways are known to be useful.

[0011] Another aspect of the present disclosure provides methods for treating cancer, comprising administering Form A of Compound I or a pharmaceutical composition comprising the same to a subject in need thereof.

[0012] In some embodiments, these treatment methods comprise administering to a subject in need thereof at least one additional active agent, either in the same pharmaceutical composition as Form A of Compound I or in a separate composition.

[0013] Also provided are methods of modulating PPARγ, which methods comprise administering Form A of Compound I or a pharmaceutical composition comprising the same to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 An XRPD diffraction pattern of Form A of Compound 1 is depicted.

[0015] Figure 2 Depicted is a solid of Form A of Compound I 13 C SSNMR spectrum.

[0016] Figure 3 The TGA / DSC thermogram of Form A of Compound 1 is depicted.

[0017] Figure 4 The Raman spectrum of Form A of Compound 1 is depicted.

[0018] Figure 5 The adsorption and desorption isotherms of Form A of Compound I are depicted.

[0019] Figure 6 The crystal structure of Form A of Compound 1 is depicted.

[0020] definition

[0021] When referring to the compounds of the present disclosure, the term "compound" refers to a collection of molecules having the same chemical structure, unless otherwise indicated as a collection of stereoisomers (e.g., a collection of racemates, a collection of cis / trans stereoisomers, or a collection of (E) and (Z) stereoisomers), except where there may be isotopic variation between the constituent atoms of the molecule. Therefore, it will be clear to those skilled in the art that a compound represented by a specific chemical structure containing the deuterium atom shown will also contain a smaller number of isotopologues having hydrogen atoms at one or more designated deuterium positions in the structure. The relative amount of such isotopologues in the compounds of the present disclosure will depend on a number of factors, including the isotopic purity of the reagents used to make the compound and the efficiency of incorporating isotopes in the various synthetic steps used to prepare the compound. However, as described above, the total relative amount of such isotopologues will be less than 49.9% of the compound. In other embodiments, the total relative amount of such isotopes will be less than 47.5%, less than 40%, less than 32.5%, less than 25%, less than 17.5%, less than 10%, less than 5%, less than 3%, less than 1% or less than 0.5% of the compound.

[0022] Non-limiting examples of suitable solvents that can be used in the present disclosure include, but are not limited to, water, methanol (MeOH), ethanol (EtOH), dichloromethane (or methylene chloride, CH2Cl2), toluene, acetonitrile (MeCN), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methyl acetate (MeOAc), ethyl acetate (EtOAc), heptane, isopropyl acetate (IPAc), tert-butyl acetate (t-BuOAc), isopropanol (IPA), tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-MeTHF), methyl ethyl ketone (MEK), tert-butanol, diethyl ether (Et2O), methyl-tert-butyl ether (MTBE), 1,4-dioxane, and N-methylpyrrolidone (NMP).

[0023] Non-limiting examples of suitable bases that can be used in the present disclosure include, but are not limited to, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), potassium tert-butoxide (KOtBu), potassium carbonate (K2CO3), N-methylmorpholine (NMM), triethylamine (Et3N; TEA), diisopropyl-ethylamine (i-Pr2EtN; DIPEA), pyridine, potassium hydroxide (KOH), sodium hydroxide (NaOH), lithium hydroxide (LiOH), and sodium methoxide (NaOMe; NaOCH3).

[0024] The terms "about" and "approximately" when used in conjunction with a dose, amount, or weight percentage of an ingredient of a composition or dosage form include values ​​of the specified dose, amount, or weight percentage, or ranges of doses, amounts, or weight percentages recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percentage.

[0025] "Treatment," "treat," or "treating" cancer refers to reversing, alleviating, and / or slowing the progression of a cancer as described herein.

[0026] As used herein, "subject" means an animal subject, such as a mammalian subject, and particularly a human.

[0027] As used herein, "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated therewith. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present disclosure include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates, glycine, sorbic acid, potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, cyclodextrin, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

[0028] The terms "patient" and "subject" are used interchangeably and refer to animals, including humans.

[0029] The terms "effective dose" and "effective amount" are used interchangeably herein and refer to the amount of a compound that produces the desired effect resulting from the administration of the compound. The exact amount of an effective dose will depend on the purpose of the treatment and will be determined by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0030] Form A of Compound 1 can be administered once a day, twice a day, or three times a day, for example, for the treatment of FSGS. In some embodiments, Form A of Compound 1 is administered once a day. In some embodiments, Form A of Compound 1 is administered twice a day. In some embodiments, Form A of Compound 1 is administered three times a day.

[0031] As used herein, the term "ambient conditions" means room temperature, open air conditions, and uncontrolled humidity conditions.

[0032] As used herein, the terms "crystalline form" and "Form" interchangeably refer to a crystal structure (or polymorph) having a specific molecular packing arrangement in the crystal lattice. A crystalline form can be identified and distinguished by one or more characterization techniques, including, for example, X-ray powder diffraction (XRPD), single crystal X-ray diffraction (SXRD), solid state nuclear magnetic resonance (SSNMR), Raman spectroscopy (Raman), differential scanning calorimetry (DSC), and / or thermogravimetric analysis (TGA). Thus, as used herein, the term "Form A of Compound I" refers to a unique crystalline form that can be identified and distinguished from other crystalline forms of Compound I by one or more characterization techniques, including, for example, XRPD, SXRD, SSNMR, Raman, DSC, and / or TGA. In some embodiments, the new crystalline Form A is characterized by an X-ray powder diffraction pattern having one or more signals at one or more specified 2-theta values ​​(°2θ).

[0033] As used herein, the term "SSNMR" refers to the analytical characterization method of solid-state nuclear magnetic resonance. SSNMR spectroscopy can record any magnetically activated isotopes present in a sample under ambient conditions. Typical examples of active isotopes for small molecule active pharmaceutical ingredients include 1 H. 2 H. 13 C. 19 F. 31 P. 15 N. 14 N. 35 Cl, 11 B. 7 Li, 17 O. 23 Na, 79 Br and 195 Pt.

[0034] As used herein, the term "XRPD" refers to the analytical characterization method of X-ray powder diffraction. An XRPD pattern can be recorded using a diffractometer under ambient conditions in transmission or reflection geometry.

[0035] As used herein, the terms "X-ray powder diffractogram", "X-ray powder diffraction pattern", "XRPD pattern" refer interchangeably to an experimentally obtained graph that plots signal positions (on the abscissa) versus signal intensities (on the ordinate). For amorphous materials, an X-ray powder diffraction pattern may include one or more broad signals; and for crystalline materials, an X-ray powder diffraction pattern may include one or more signals, each identified by an angle value as measured in 2θ degrees (°2θ) depicted on the abscissa of the X-ray powder diffraction pattern, which may be expressed as "a signal at 2-θ degrees ...", "a signal at one or more 2-θ values ​​of ...", and / or "a signal at at least ... 2-θ values ​​selected from ...".

[0036] As used herein, a "signal" or "peak" refers to a point in an XRPD plot where the intensity, measured in counts, is at a local maximum. One of ordinary skill in the art will recognize that one or more signals (or peaks) in an XRPD plot may overlap and may not be discernible, for example, to the naked eye. Indeed, one of ordinary skill in the art will recognize that several art-recognized methods are capable of and are applicable to determining whether a signal is present in a plot, such as Rietveld refinement.

[0037] As used herein, "a signal at 2-theta degrees of ...," "a signal at one or more 2-theta values ​​of ...," and / or "a signal at at least ... 2-theta values ​​selected from ..." refers to the X-ray reflection positions (° 2θ) as measured and observed in an X-ray powder diffraction experiment.

[0038] The repeatability of the angle value is within the range of ±0.2° 2θ, that is, the angle value can be the angle value +0.2 2-θ degree, the angle value -0.2 2-θ degree, or any value between these two endpoints (angle value +0.2 2-θ degree and angle value -0.2 2-θ degree).

[0039] The terms "signal intensity" and "peak intensity" refer interchangeably to the relative signal intensity within a given X-ray powder diffraction pattern. Factors that can affect the relative signal or peak intensity include sample thickness and preferred orientation (eg, crystal particles are not randomly distributed).

[0040] As used herein, the term "X-ray powder diffraction pattern having signals at 2-theta values" refers to an XRPD pattern containing X-ray reflection positions (° 2θ) as measured and observed in an X-ray powder diffraction experiment.

[0041] As used herein, an X-ray powder diffraction pattern is "substantially similar to a [specific] pattern" when at least 90% (e.g., at least 95%, at least 98%, or at least 99%) of the signals in the two diffraction patterns overlap. In determining "substantially similar," one of ordinary skill in the art will understand that even for the same crystalline form, there may be variations in the intensity and / or signal positions in an XRPD diffraction pattern. Thus, one of ordinary skill in the art will understand that the signal position in an XRPD diffraction pattern (referenced herein as 2-θ degrees (°2θ)) generally means that the reported value (that value reported) is ±0.2 2θ degrees of the reported value (the reported value), which is a variance recognized in the art.

[0042] As used herein, an SSNMR spectrum is "substantially similar to a [specific] pattern" when at least 90% (e.g., at least 95%, at least 98%, or at least 99%) of the signals in the two spectra overlap. In determining "substantially similar," one of ordinary skill in the art will understand that even for the same crystalline form, there may be variations in the intensity and / or signal positions in the SSNMR spectrum. Thus, one of ordinary skill in the art will understand that the signal positions (expressed in ppm) in the SSNMR diffraction patterns referred to herein generally mean that the reported value (that value reported) is ±0.2 ppm of the reported value (the reported value), which is a variance recognized in the art.

[0043] As used herein, a crystalline form is "substantially pure" when it represents an amount equal to or greater than 90% (by weight, as determined according to methods of the art, such as quantitative XRPD) of the sum of all solid forms in a sample. In some embodiments, a solid form is "substantially pure" when it represents an amount equal to or greater than 95% (by weight) of the sum of all solid forms in a sample. In some embodiments, a solid form is "substantially pure" when it represents an amount equal to or greater than 99% (by weight) of the sum of all solid forms in a sample.

[0044] As used herein, the term "DSC" refers to the analytical method of Differential Scanning Calorimetry.

[0045] As used herein, the term "TGA" refers to the analytical method of Thermo Gravimetric (or thermogravimetric) analysis.

[0046] As used herein, the term "room temperature" refers to a temperature in the range of 15°C to 30°C, or in some embodiments, a temperature in the range of 20°C to 25°C.

[0047] Disclosed herein is a novel crystalline Form A of Compound 1. In some embodiments, Form A of Compound 1 is substantially pure. In some embodiments, Form A is characterized by Figure 1 Substantially similar X-ray powder diffraction patterns. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having a signal at at least one 2-theta value selected from 14.8 ± 0.2, 15.9 ± 0.2, 17.5 ± 0.2, 18.5 ± 0.2, 19.4 ± 0.2, 20.0 ± 0.2, 20.7 ± 0.2, 21.0 ± 0.2, 22.6 ± 0.2, and 27.2 ± 0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least one 2-theta value selected from 14.8±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 21.0±0.2, and 22.6±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 21.0±0.2, and 22.6±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least one 2-theta value selected from 14.8±0.2, 18.5±0.2, 19.4±0.2, 21.0±0.2, and 22.6±0.2. In some embodiments, Form A of Compound I is characterized by an X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 18.5±0.2, 19.4±0.2, 21.0±0.2, and 22.6±0.2. In some embodiments, Form A of Compound I is characterized by an X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 18.5±0.2, and 19.4±0.2. In some embodiments, Form A of Compound I is characterized by an X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 18.5±0.2, and 19.4±0.2.

[0048] In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least three 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least four 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least five 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least six 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least seven 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least eight 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at at least nine 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2.

[0049] In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2, and 27.2±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at 14.8±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 21.0±0.2, and 22.6±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at 14.8±0.2, 18.5±0.2, 19.4±0.2, 21.0±0.2, and 22.6±0.2. In some embodiments, Form A of Compound 1 is characterized by an X-ray powder diffraction pattern having signals at 14.8±0.2, 18.5±0.2, and 19.4±0.2.

[0050] Disclosed herein, in some embodiments, are compositions comprising Form A of Compound 1. Disclosed herein, in some embodiments, are compositions comprising substantially pure Form A of Compound 1. Disclosed herein, in some embodiments, are compositions comprising at least one active compound consisting essentially of Compound 1 in Form A.

[0051] In some embodiments, Form A of Compound 1 is characterized by Figure 3 Substantially Similar TGA / DSC. In some embodiments, Form A of Compound I is characterized by a DSC with an onset temperature of about 240°C.

[0052] In some embodiments, Form A of Compound 1 is characterized by the following 13 CSSNMR spectrum having a signal at at least one ppm value selected from 41.4±0.2ppm, 43.7±0.2ppm, 47.7±0.2ppm, 54.8±0.2ppm, 70.1±0.2ppm, 127.2±0.2ppm, 134.3±0.2ppm, 148.9±0.2ppm, 161.2±0.2ppm and 179.0±0.2ppm. In some embodiments, Form A of Compound I is characterized by the following 13C SSNMR spectrum having signals at at least two ppm values ​​selected from 41.4±0.2ppm, 43.7±0.2ppm, 47.7±0.2ppm, 54.8±0.2ppm, 70.1±0.2ppm, 127.2±0.2ppm, 134.3±0.2ppm, 148.9±0.2ppm, 161.2±0.2ppm and 179.0±0.2ppm. In some embodiments, Form A of Compound I is characterized by: 13 C SSNMR spectrum having signals at at least three ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm. In some embodiments, Form A of Compound I is characterized by: 13 C SSNMR spectrum having signals at at least four ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm. In some embodiments, Form A of Compound I is characterized by: 13 C SSNMR spectrum having signals at at least five ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm. In some embodiments, Form A is characterized by: 13 C SSNMR spectrum having signals at at least six ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm. In some embodiments, Form A of Compound I is characterized by: 13CSSNMR spectrum having signals at at least seven ppm values ​​selected from 41.4±0.2ppm, 43.7±0.2ppm, 47.7±0.2ppm, 54.8±0.2ppm, 70.1±0.2ppm, 127.2±0.2ppm, 134.3±0.2ppm, 148.9±0.2ppm, 161.2±0.2ppm and 179.0±0.2ppm. In some embodiments, Form A of Compound I is characterized by the following 13 C SSNMR spectrum having signals at at least eight ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm. In some embodiments, Form A of Compound I is characterized by: 13 C SSNMR spectrum having signals at at least nine ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm. In some embodiments, Form A of Compound I is characterized by: 13 C SSNMR spectrum having signals at 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm.

[0053] In some embodiments, Form A of Compound 1 is characterized by a Raman spectrum having a wavelength selected from 579.1 ± 2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm–1 At least one cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least two cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least three cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least four cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least five cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least six cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least seven cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least eight cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at a value selected from 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 At least nine cm –1 In some embodiments, Form A of Compound I is characterized by a Raman spectrum having a signal at 579.1±2 cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There is a signal.

[0054] Another aspect of the present disclosure provides a pharmaceutical composition comprising Form A of Compound 1. In some embodiments, a pharmaceutical composition comprising Form A of Compound 1 is administered to a patient in need thereof.

[0055] The pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier. In some embodiments, at least one pharmaceutically acceptable carrier is selected from a pharmaceutically acceptable vehicle and a pharmaceutically acceptable adjuvant. In some embodiments, at least one pharmaceutically acceptable carrier is selected from a pharmaceutically acceptable filler, a disintegrant, a surfactant, a binder, a lubricant.

[0056] It should also be understood that the pharmaceutical compositions of the present disclosure can be used in combination therapy; that is, the pharmaceutical compositions described herein can further include at least one additional active therapeutic agent. Alternatively, the pharmaceutical composition comprising Form A of Compound I can be administered as a separate composition simultaneously with, before, or after a composition comprising at least one other active therapeutic agent. In some embodiments, the pharmaceutical composition comprising Form A of Compound I can be administered as a separate composition simultaneously with, before, or after a composition comprising at least one other active therapeutic agent.

[0057] As described above, the pharmaceutical compositions disclosed herein may optionally further comprise at least one pharmaceutically acceptable carrier. At least one pharmaceutically acceptable carrier may be selected from adjuvants and vehicles. At least one pharmaceutically acceptable carrier as used herein includes any and all solvents, diluents, other liquid vehicles, dispersing aids, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders and lubricants suitable for the desired specific dosage form. Remington: The Science and Practice of Pharmacy [Remington: Pharmaceutical Science and Practice], 21st edition, 2005, DBTroy editor, Lippincott Williams & Wilkins [LWW Press], Philadelphia; and Encyclopedia of Pharmaceutical Technology [Encyclopedia of Preparation Technology], J.Swarbrick and JCBoylan editors, 1988 to 1999, Marcel Dekker [Marcel Dek Press], New York discloses various carriers and known preparation techniques for preparing pharmaceutical compositions. Unless any conventional carrier is incompatible with the compounds of the present disclosure, for example by producing any undesirable biological effect or otherwise interacting in a deleterious manner with one or more of any other components of the pharmaceutical composition, its use is contemplated to be within the scope of the present disclosure. Non-limiting examples of suitable pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates, glycine, sorbic acid and potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts and electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, lanolin, sugars (such as lactose, glucose and sucrose), starches (such as corn starch and potato starch), cellulose and its derivatives ( Such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients (such as cocoa butter and suppository wax), oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil), glycols (such as propylene glycol and polyethylene glycol), esters (such as ethyl oleate and ethyl laurate), agar, buffers (such as magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, phosphate buffer solution, non-toxic compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants.

[0058] In some embodiments, Compound 1 is a crystalline solid consisting of 1% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 2% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 5% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 10% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 15% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 20% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 25% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 30% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 35% to 99% Form A relative to the total weight of crystalline solid Compound 1. In some embodiments, the crystalline solid consists of 45% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 50% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 55% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 60% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 65% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 70% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 75% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 80% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 85% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 90% to 99% Form A relative to the total weight of crystalline solid Compound I. In some embodiments, the crystalline solid consists of 95% to 99% Form A relative to the total weight of crystalline solid Compound I.

[0059] In some embodiments, a method of treating cancer comprises administering Form A of Compound 1 or a pharmaceutical composition comprising the same to a subject in need thereof.

[0060] In some embodiments, these treatment methods comprise administering to a subject in need thereof at least one additional active agent, either in the same pharmaceutical composition as Form A of Compound I or in a separate composition.

[0061] In some embodiments, a method of modulating PPARγ comprises administering Form A of Compound I or a pharmaceutical composition comprising the same to a subject in need thereof.

[0062] Non-limiting exemplary embodiments

[0063] Example 1: A crystalline form A of (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide (i.e., a crystalline form A of (S)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-carbonyl)-N-((1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide) (i.e., a crystalline form A of compound I):

[0064]

[0065] Embodiment 2: According to the crystalline form A described in embodiment 1, it is characterized in that Figure 1 Substantially similar X-ray powder diffraction patterns.

[0066] Embodiment 3: Form A according to Embodiment 1 is characterized by an X-ray powder diffraction pattern having a signal at at least one 2-θ value selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.2.

[0067] Embodiment 4: Form A according to Embodiment 1 is characterized by an X-ray powder diffraction pattern having signals at at least two 2-θ values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.2.

[0068] Embodiment 5: Form A according to Embodiment 1 is characterized by an X-ray powder diffraction pattern having signals at at least two 2-θ values ​​selected from 14.8±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 21.0±0.2 and 22.6±0.2.

[0069] Embodiment 6: Form A according to Embodiment 1 is characterized by an X-ray powder diffraction pattern having signals at at least two 2-θ values ​​selected from 14.8±0.2, 18.5±0.2, 19.4±0.2, 21.0±0.2 and 22.6±0.2.

[0070] Embodiment 7: Form A according to Embodiment 1 is characterized by an X-ray powder diffraction pattern having signals at at least two 2-θ values ​​selected from 14.8±0.2, 18.5±0.2 and 19.4±0.2.

[0071] Embodiment 8: Form A according to Embodiment 1 is characterized by an X-ray powder diffraction pattern having signals at at least three 2-θ values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.2.

[0072] Example 9: Form A according to Example 1 is characterized by an X-ray powder diffraction pattern having signals at at least four 2-θ values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.2.

[0073] Embodiment 10: Form A according to Embodiment 1 is characterized by an X-ray powder diffraction pattern having signals at at least five 2-θ values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.2.

[0074] Example 11: Form A according to Example 1 is characterized by the following X-ray powder diffraction pattern, which has signals at 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.2.

[0075] Example 12: Form A according to Example 1 is characterized by the following X-ray powder diffraction pattern, which has signals at 14.8±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 21.0±0.2 and 22.6±0.2.

[0076] Example 13: Form A according to Example 1 is characterized by an X-ray powder diffraction pattern having signals at 14.8±0.2, 18.5±0.2, 19.4±0.2, 21.0±0.2 and 22.6±0.2.

[0077] Example 14: Form A according to Example 1 is characterized by an X-ray powder diffraction pattern having signals at 14.8±0.2, 18.5±0.2 and 19.4±0.2.

[0078] Embodiment 15: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13 A CSS NMR spectrum having signals at at least five ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

[0079] Embodiment 16: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13 A CSS NMR spectrum having signals at at least four ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

[0080] Embodiment 17: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13A CSS NMR spectrum having signals at at least three ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

[0081] Embodiment 18: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13 A CSS NMR spectrum having signals at at least two ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

[0082] Embodiment 19: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13 A CSS NMR spectrum having a signal at at least one ppm value selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

[0083] Embodiment 20: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13 CSS NMR spectrum having signals at 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

[0084] Embodiment 21: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13 CSS NMR spectrum having signals at 41.4±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm and 161.2±0.2 ppm.

[0085] Embodiment 22: According to the crystalline form A described in Embodiment 1, it is characterized by the following 13 CSS NMR spectrum having signals at 41.4±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 134.3±0.2 ppm and 148.9±0.2 ppm.

[0086] Example 23: Form A of Compound I according to Example 1, characterized in that 13 C SSNMR spectrum having signals at 54.8±0.2 ppm, 70.1±0.2 ppm and 148.9±0.2 ppm.

[0087] Embodiment 24: According to the crystalline form A of embodiment 1, it is characterized in that Figure 3 Basically similar TGA / DSC.

[0088] Embodiment 25: According to the crystalline form A of embodiment 1, it is characterized in that Figure 4 Basically similar Raman spectra.

[0089] Embodiment 26: Form A according to Embodiment 1 is characterized by the following Raman spectrum, which is selected from 579.1±2cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There is a signal at at least one ppm value of .

[0090] Embodiment 27: The crystalline form A according to embodiment 1 is characterized by the following Raman spectrum, which is selected from 579.1±2cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm–1 There are signals at at least two ppm values ​​of .

[0091] Embodiment 28: The crystalline form A according to embodiment 1 is characterized by the following Raman spectrum, which is selected from 579.1±2cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There are signals at at least three ppm values ​​of .

[0092] Embodiment 29: The crystalline form A according to embodiment 1 is characterized by the following Raman spectrum, which is selected from 579.1±2cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There are signals at at least four ppm values ​​of .

[0093] Embodiment 30: Form A according to Embodiment 1 is characterized by the following Raman spectrum, which is selected from 579.1±2cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There are signals at at least five ppm values ​​of .

[0094] Example 31: Form A according to Example 1, characterized by the following Raman spectrum, the Raman spectrum at 579.1±2cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There is a signal.

[0095] Example 32: Form A according to Example 1, characterized by the following Raman spectrum, which has a Raman spectrum at 741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 and 1630.2±2cm –1 There is a signal.

[0096] Example 33: Form A of Compound I according to Example 1, characterized by the following Raman spectrum: –1 、863.7±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 and 1630.2±2cm –1 There is a signal.

[0097] Example 34: Form A according to Example 1, characterized by the following Raman spectrum, which has a Raman spectrum at 863.7±2cm –1 、1405.3±2cm –1 and 1630.2±2cm –1 There is a signal.

[0098] Embodiment 35: A pharmaceutical composition comprising the crystalline form A according to any one of Embodiments 1 to 34, and a pharmaceutically acceptable carrier.

[0099] Embodiment 36: A method for treating and / or preventing cancer, comprising administering an effective amount of the crystalline form A according to any one of Embodiments 1 to 34 or the pharmaceutical composition according to Embodiment 35 to a subject in need thereof.

[0100] Embodiment 37: The method according to embodiment 36, wherein the cancer is selected from bladder cancer, breast cancer, prostate cancer and cancers with altered RXRα and / or PPARγ pathways.

[0101] Embodiment 38: The method according to any one of Embodiments 36 and 37, wherein the cancer is bladder cancer.

[0102] Embodiment 39: The method according to any one of embodiments 37 to 38, wherein the bladder cancer is selected from advanced bladder cancer, luminal bladder cancer and basal bladder cancer.

[0103] Embodiment 40: The method according to any one of Embodiments 36 to 39, wherein the cancer is a chemotherapy-resistant and / or immunotherapy-resistant cancer.

[0104] Example 41: Use of Form A of (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide according to any one of Examples 1 to 34 and the pharmaceutical composition according to Example 35 in the treatment and / or prevention of cancer.

[0105] Example 42: A method for preparing Form A of (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide, the method comprising

[0106] (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide and acetonitrile were mixed at room temperature and

[0107] The mixture was stirred at room temperature for at least 24 hours.

[0108] Examples

[0109] In order to be able to more fully understand the disclosure described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be interpreted as limiting the present disclosure in any way.

[0110] The preparation method and structure of Compound I are reported in: U.S. Provisional Application No. 63 / 11,354 filed on November 9, 2020, U.S. Application No. 17 / 521,666, and PCT International Application No. PCT / US2021 / 058473 (both applications were filed on November 8, 2021); the contents of each of these applications are incorporated herein by reference.

[0111] Synthesis of Compound I

[0112] Tert-butyl 7-(((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)carbamoyl)-4-azaspiro[2.5]octane-4-carboxylate

[0113]

[0114] HATU (447.0 mg, 1.18 mmol) and (1r,4r)-4-amino-1-(trifluoromethyl)cyclohexan-1-ol (226.0 mg, 1.23 mmol, CAS#: 1408075-09-1, Enamine) were added to a stirred mixture of 4-(tert-butoxycarbonyl)-4-azaspiro[2.5]octane-7-carboxylic acid (300.0 mg, 1.18 mmol) and DIPEA (0.62 mL, 3.53 mmol) in DMF (6.00 mL) at 0°C. The resulting mixture was stirred at 25°C for 1 h. The reaction mixture was diluted with EtOAc, water, and then extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated. The residue was purified by silica gel column chromatography (eluting with 60%-100% EtOAc / hexanes) to afford a diastereomeric mixture of the title compound as an oil (494.0 mg) containing residual solvent. LCMS (ESI): [M+Na] + :443.4.

[0115] N-((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide

[0116]

[0117] TFA (3.00 mL) was added to a solution of tert-butyl 7-(((1r, 4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)carbamoyl)-4-azaspiro[2.5]octane-4-carboxylate (494.0 mg, 1.18 mmol) in DCM (5.00 mL). The reaction mixture was stirred at 25 ° C for 8 h. The reaction mixture was concentrated under reduced pressure and purified by reverse phase column chromatography (using the following conditions: C18 column, 0%-20% MeCN / water (0.1% formic acid)) to obtain the title compound (534.0 mg) as a solid mixture of diastereomers, which contained residual solvent. LCMS (ESI): [M+H] + :322.3.

[0118] Methyl 5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate

[0119]

[0120] At 0 ° C, SEM-Cl (2.85mL, 16.10mmol) was added dropwise to a stirred solution of methyl 5-bromo-1H-pyrazole-3-formate (3.00g, 14.63mmol) and K2CO3 (2.23g, 16.10mmol) in DMF (40.00mL). The reaction mixture was then stirred for 2h at 25 ° C. The solution was diluted with EtOAc and water and then extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated. The residue was purified by silica gel column chromatography (EtOAc / hexane, 0 / 100 to 10 / 90v / v) to obtain the title compound (3.49g) in an oily state. LCMS (ESI): [M+H] + :337.0.

[0121] Methyl 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate

[0122]

[0123] Pd(dppf)Cl2 (0.44 g, 0.60 mmol) and K2CO3 (1.24 g, 8.95 mmol) were added to a stirred solution of methyl 5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate (1.0 g, 2.98 mmol) and (5-fluoro-2-methoxypyridin-4-yl)boronic acid (0.61 g, 3.58 mmol) in water (2.00 mL) and 1,4-dioxane (8.00 mL). The reaction mixture was stirred at 100 °C under nitrogen atmosphere for 15 h. The reaction mixture was then cooled to 25 °C and diluted with EtOAc and water. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated. The residue was purified by silica gel column chromatography (EtOAc / hexane, 5 / 95 to 25 / 75 v / v) to obtain the title compound (765.0 mg) as an oil. LCMS (ESI): [M+H] + :382.2.

[0124] 5-(5-Fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylic acid

[0125]

[0126] 1M lithium hydroxide aqueous solution (2.21mL, 2.21mmol) is added to a stirred solution of methyl 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate (281.0mg, 0.74mmol) in water (2.00mL) and THF (8.00mL). The reaction mixture is stirred at 25°C for 7h. The pH of the mixture is then adjusted to 1 by adding HCl aqueous solution (1M). The solution is concentrated, and the residue is purified by silica gel column chromatography (EtOAc / hexane, 30 / 70 to 50 / 50v / v) to obtain the title compound (264.0mg) as a solid. LCMS(ESI): 369.4[M+H] + .

[0127] Methyl 5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carboxylate

[0128]

[0129] TFA (10.00 mL) was added to a stirred solution of methyl 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate (10.00 g, 26.21 mmol) in DCM (10.00 mL). The resulting mixture was stirred at 25 ° C for 4 h, and the solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH, 12 / 1 v / v) to obtain the title compound (7.00 g) as an oil. LCMS (ESI): [M+H] + :252.1.

[0130] 5-(5-Fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carboxylic acid

[0131]

[0132] LiOH . H2O (1.67 g, 39.80 mmol) was added to a stirred solution of methyl 5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carboxylate (5.00 g, 19.90 mmol) in THF (8.00 mL), MeOH (8.00 mL) and H2O (8.00 mL). The resulting mixture was stirred at 60 ° C for 14 h, and the solution was then concentrated under reduced pressure. The pH of the solution was adjusted to 3 by adding an aqueous HCl solution (1 M). The precipitated solid was collected by filtration and washed with water, then dried under pressure to obtain the title compound (4.50 g) as a solid. 1 H NMR (300MHz, DMSO-d6) δ14.37(s,1H),8.28(s,1H),7.32(s,1H),7.20(s,1H),3.87(s,3H). LCMS(ESI):[M+H] + :238.0.

[0133] Alternative Synthesis of 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylic Acid

[0134] Methyl 5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate and methyl 3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-5-carboxylate

[0135]

[0136] Two batches were run in parallel. At 0°C, dichloromethane (2.50 L) was added to a 5.00 L jacketed flask equipped with an overhead stirrer, addition funnel and thermometer, N2 balloon. 3. At 0°C, methyl 5-bromo-1H-pyrazole-3-carboxylate (500.00 g, 2.44 mol) was added to the flask. At 0°C, DIPEA (630.00 g, 4.88 mol) and SEM-Cl (610.00 kg, 3.66 mol) were added to the flask. The resulting mixture was purged with N2 3 times and stirred at 0°C to 20°C under N2 for 12 h. The two batches were combined and the reaction mixture was poured into H2O (8.00 L) and extracted with dichloromethane (4.00 L x 2). The combined organic layers were washed with brine (5.00 L), dried over Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (5 / 1 v / v) to obtain the title compound (1.03 kg) as a solid. 1 H NMR(400MHz, CDCl3)δ6.92(s,1H),5.83(s,2H),3.94(s,3H),3.64(t,2H),0.91-0.95(m,2H),2.70 -1.75(t,1H),1.73-1.74(m,4H),1.48-1.55(m,6H),1.43(s,9H),1.18-1.19(m,1H),0.00(s,9H). LCMS:[M+H] + :279.

[0137] Methyl 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate

[0138]

[0139] Two batches were run in parallel. Methanol (2.50 L) was added to a 5.00 L jacketed flask equipped with an overhead stirrer, addition funnel and thermometer, N2 balloon at 25°C. Methyl 5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate and methyl 3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-5-carboxylate (517.00 g, 1.54 mol) were added to the flask at 25°C. DIPEA (399.00 g, 3.08 mol) was added to the flask at 0°C, and (5-fluoro-2-methoxypyridin-4-yl)boronic acid (277.00 g, 1.62 mol) was added to the flask at 25°C. At 25 ° C, P (tBu) Pd G (39.50g, 77.10mmol) was added to a flask, and the resulting mixture was purged 3 times with N. The reaction mixture was stirred for 2h at 65 ° C under N. Two batches were merged, and the reaction mixture was poured into H2O (3.00L), extracted with ethyl acetate (3.00L x 2). The combined organic layer was washed with brine (4.00L), dried over Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with petroleum ether / ethyl acetate (30 / 1 to 5 / 1 v / v)) to obtain the title compound (1.15kg) as a solid. 1 HNMR (400MHz, CDCl3) δ8.09(d,1H),7.40(t,2H),5.92(s,2H),3.95(d,6H),3.66(t,2H),0.92(t,2H),0.03(s,9H). LCMS:[M+H] + :382.4.

[0140] 5-(5-Fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylic acid

[0141]

[0142] Two batches were carried out in parallel. At 25°C, THF (1.80 L) and H2O (1.80 L) were added to a 5.00 L jacketed flask equipped with an overhead stirrer, an addition funnel and a thermometer, a N2 balloon, etc. At 25°C, methyl 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate (576.00 g, 1.51 mol) and LiOH.H2O (127.00 g, 3.02 mol) were added to a flask. The reaction mixture was stirred at 25°C under N2 for 2 h. The two batches were combined, and the reaction mixture was adjusted to pH=4 and extracted with ethyl acetate (500 mL x 2). The combined organic layer was washed with brine (500 mL), dried over Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was triturated with petroleum ether / ethyl acetate (10 / 1 v / v, 100 mL) at 25 °C for 0.5 h and concentrated to give the title compound as a solid (1.01 kg). 1 H NMR (400MHz, MeOD) δ8.07(d,1H),7.33(d,1H),7.23(d,1H),6.00(s,2H),3.92(s,3H),3.67(t,2H),0.88(t,2H),0.04(s,9H). LCMS:[M+H] + :368.1.

[0143] 4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-N-((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide

[0144]

[0145] HATU (103.0 mg, 0.27 mmol) and N-((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide (164.0 mg, 0.36 mmol) were added to a stirred solution of 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylic acid (100.0 mg, 0.27 mmol) and DIPEA (0.19 mL, 1.09 mmol) in DMF (6.00 mL). The resulting mixture was stirred at 25 °C for 16 h. The reaction mixture was diluted with EtOAc and water, then extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated. The residue was purified by silica gel column chromatography (eluted with 70%-100% EtOAc / hexanes) to obtain the title compound (153.0 mg) as a solid mixture of diastereomers. LCMS (ESI): [M+H] + :670.6.

[0146] (7R)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4R)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide and (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide

[0147]

[0148] TFA (3.00 mL) was added to a stirred solution of 4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-N-((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide (89.0 mg, 0.13 mmol) in DCM (3.00 mL). The resulting mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated and purified by silica gel column chromatography (eluting with 0%-10% MeOH / DCM) to afford (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide (Compound I), (7R)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4R)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide (62.0 mg) as a solid mixture of diastereomers. 1 H NMR(400MHz,CD3OD)δ8.12(d,1H),7.27(br d,1H),7.10(br s,1H),4.89–4.95(m,1H),4.39–4.71(m,1H),3.93(s,4H),2.91(br d,1H),2.21–2.61(m,1H),1.56–2.01(m,10H),1.14–1.40(m,2H),0.81–1.10(m,2H),0.73(br s,2H). LCMS(ESI):[M+H] + :540.2.

[0149] The solid mixture of diastereomers (80.0 mg) was separated using chiral preparative HPLC.

[0150] Column: CHIRALPAK IA column

[0151] Column size: 2×25 cm, 5 μm

[0152] Mobile phase: Hexane (8mM NH3·MeOH): EtOH = 50:50, maintained for 15min Flow rate: 18mL / min

[0153] Detection: 220 / 254nm

[0154] The first eluting diastereomer was obtained as a solid (26.3 mg) with a retention time of 4.59 min.

[0155] 1 H NMR (300MHz, DMSO-d6) δ14.09–13.85(m,1H),8.29–8.23(m,1H),7.82(s,1H),7.32(d,1H),7.04(s,1H),5.70(s,1H),4.56–4. 49(m,1H),3.91–3.76(m,4H),2.86–2.70(m,2H),2.22–2.16(m,1H),1.83–1.72(m,5H),1.56–1.46(m,5H),1.30–0.43(m,5H). LCMS(ESI):[M+H] + :540.2.

[0156] The second eluting diastereomer was obtained as a solid (28.3 mg) with a retention time of 8.06 min.

[0157] 1 H NMR(300MHz,DMSO-d6)δ14.00(s,1H),8.27(d,1H),7.82(d,1H),7.33(d,1H),7.03(d,1H),5.71(s,1H),4.44(br s,1H),3.91–3.80(m,4H),3.38–3.33(m,1H),2.90–2.76(m,1H),2.21(s,1H),1.83–1. 71(m,5H),1.56–1.46(m,5H),1.29–1.00(m,1H),0.99–0.80(m,2H),0.78–0.61(m,2H). LCMS(ESI):[M+H] + :540.2.

[0158] Alternative synthesis of compound I

[0159] Synthesis of (S)-4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylic acid and (R)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-4-azaspiro[2.5]octane-7-carboxylic acid

[0160]

[0161] Methyl 4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylate

[0162]

[0163] Methyl 4-azaspiro [2.5] octane-7-carboxylate hydrochloride (6.77 g, 32.9 mmol, CAS #: 2253630-26-9, Enamine), HATU (13.66 g, 35.9 mmol) and DIPEA (11.61 g, 89.8 mmol) were added to a stirred solution of 5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylic acid (11.0 g, 29.9 mmol) in DMF (30.00 mL). The resulting mixture was stirred at 25 ° C for 1 h. The solution was diluted with EtOAc and washed with water. The organic layer was concentrated under reduced pressure to give a residue, which was purified via silica gel chromatography (EtOAc / petroleum ether, 1 / 3 v / v) to obtain the title compound (14 g) as an oil. LCMS(ESI):519.2[M+H] +

[0164] Methyl 4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylate

[0165]

[0166] TFA (60.00 mL) was added to a stirred solution of methyl 4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylate (13.00 g, 25.1 mmol) in DCM (30.00 mL). The resulting mixture was stirred at 25 ° C for 14 h. The solution was concentrated under reduced pressure. The residue was purified via silica gel chromatography (EtOAc / petroleum ether, 3 / 1 v / v) to obtain the title compound (9.65 g) as a white solid. LCMS (ESI): 389.2 [M+H] +

[0167] Methyl (S)-4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylate and methyl (R)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-4-azaspiro[2.5]octane-7-carboxylate

[0168]

[0169] Methyl 4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylate (5.54 g) was purified by preparative SFC using the following conditions:

[0170] Column: CHIRALPAK IG column

[0171] Column size: 30 x 250 mm, 5 μm

[0172] Mobile phase A: CO2; Mobile phase B: IPA

[0173] Flow rate: 23mL / min

[0174] Gradient: 50% B

[0175] Detection: 220nm;

[0176] Injection volume: 4.8 mL;

[0177] Methyl (S)-4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylate. The second eluting enantiomer was obtained as a solid (2.29 g). The retention time of the second eluting enantiomer was 14.29 min. LCMS (ESI): 389.1 [M+H] + .

[0178] Methyl (R)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-4-azaspiro[2.5]octane-7-carboxylate. The first eluting enantiomer was obtained as a solid (2.42 g). The retention time of the first eluting enantiomer was 8.25 min. LCMS (ESI): 389.1 [M+H] + .

[0179] (S)-4-[5-(5-Fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylic acid

[0180]

[0181] At 0 ° C, LiOH.HO (0.56 g, 13.4 mmol) was added to a stirred solution of methyl (S)-4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylate (2.6 g, 6.69 mmol, 1.00 equiv) in a mixture of THF (10.00 mL), MeOH (10.00 mL) and water (10.00 mL). The resulting mixture was stirred at 25 ° C for 2 h. The solution was concentrated under reduced pressure to remove as much MeOH and THF as possible, then acidified to pH 3 with HCl (1 M), and the solution was extracted with EtOAc. The organic layers were combined and concentrated under reduced pressure to obtain the title compound (2.50 g) as a solid. LCMS (ESI): 375.2 [M+H] +

[0182] (R)-4-(5-(5-Fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-4-azaspiro[2.5]octane-7-carboxylic acid

[0183]

[0184] LiOH.HO (497.0 mg, 11.84 mmol) was added to a stirred solution of methyl (R)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-4-azaspiro[2.5]octane-7-carboxylate (2.30 g, 5.92 mmol) in THF (5.00 mL), MeOH (5.00 mL), water (5.00 mL). The resulting mixture was stirred at 25 °C for 1 h. The resulting mixture was concentrated under reduced pressure to remove MeOH and THF. The mixture was acidified to pH 3 with HCl (1 M). The aqueous layer was extracted with EtOAc. The organic layers were combined and concentrated under reduced pressure to obtain the title compound (2.20 g) as a solid. LCMS (ESI): 375.1 [M+H] + .

[0185] (S)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-N-((1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide (Compound I)

[0186]

[0187] HOBt (2.44 g, 18.03 mmol) and EDCI (3.46 g, 18.00 mmol) were added to a stirred solution of (S)-4-[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl]-4-azaspiro[2.5]octane-7-carboxylic acid (4.50 g, 12.02 mmol) in DMF (20.00 mL). The resulting mixture was stirred at 25 °C for 30 min. Then (1r,4r)-4-amino-1-(trifluoromethyl)cyclohexan-1-ol hydrochloride (3.17 g, 14.42 mmol, CAS#: 2137056-98-3, Enamine) and DIPEA (7.77 g, 60.10 mmol) were added to the resulting mixture. The resulting mixture was stirred at 25 °C for 14 h. The solution was diluted with EtOAc and washed with water. The organic layer was concentrated under reduced pressure. The residue was purified by reverse phase column chromatography (using the following conditions: column: C18 column; mobile phase A: water (10 mM NH4HCO3), mobile phase B: MeCN; gradient: 5% B to 40% B in 30 min; flow rate: 40 mL / min; detector, UV 220 / 254 nm) to obtain the title compound (4.91 g). 1 H NMR(400MHz,DMSO-d6)δ14.18–13.81(m,1H),8.23(d,1H),7.81(d,1H),7.30(d,1H),7.02(brs,1H),5.69(s,1H),4.64–4.32(m,1H),3.87–3.8 4(m,4H),3.36–3.34(m,1H),2.90–2.75(m,1H),2.27–2.08(m,1H),1.79 –1.70(m,5H),1.56–1.45(m,5H),1.25–0.80(m,2H),0.68–0.44(m,3H). LCMS(ESI):[M+H] + :540.45.

[0188] Alternative synthesis of compound I

[0189] 4-Benzyl-7-(((tert-butyldiphenylsilyl)oxy)methyl)-4-azaspiro[2.5]octane

[0190]

[0191] At 5 ℃ under N2 protection, THF (30.00L) is added to a 50.0L flask equipped with a stirrer, an addition funnel and a thermometer. At 5 ℃, (4-benzyl-4-azaspiro [2.5] octane-7-yl) methanol (3.00kg, 12.90mol) is added to a flask. At 5 ℃, imidazole (2.21kg, 32.40mol) is added to a flask. At 5 ℃, tert-butyl-chloro-diphenyl-silane (4.28kg, 15.60mol) is added to a flask. The reaction mixture is stirred at 5 ℃ to 25 ℃ under N2 for 12h. The reaction mixture is poured into H2O (10.00L) and extracted with ethyl acetate (10.00L x 2). The combined organic layer is washed with brine (10.00L), dried over Na2SO4, filtered and the filtrate is concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (10 / 1 v / v) to obtain the title compound (5.50 kg) as an oil, and used directly in the next step.

[0192] tert-Butyl 7-(((tert-butyldiphenylsilyl)oxy)methyl)-4-azaspiro[2.5]octane-4-carboxylate

[0193]

[0194] MeOH (20.00 L) was added to a 50.0 L flask equipped with a stirrer, an addition funnel and a thermometer at 20 to 30 ° C under N2 protection. 4-Benzyl-7-(((tert-butyldiphenylsilyl)oxy)methyl)-4-azaspiro[2.5]octane (1.00 kg, 2.13 mol) was added to the flask at once at 20 to 30 ° C, and Pd / C (100.00 g, 213.00 mmol, 10.0% purity) was added to the flask in several portions at 20 to 30 ° C under N2. HCOOH (1.02 kg, 21.30 mol) was added to the flask at 20 to 30 ° C under N2. The mixture was stirred at 50 to 55 ° C for 1 h. The mixture was filtered through diatomaceous earth, and the filter cake was washed with MeOH (5.00 L x 2). The mixture was adjusted to pH 7 to 8 with NaHCO solution (10.00 L) and extracted with ethyl acetate (10.0 L x 2), the combined organic layers were washed with brine (10.0 L x 2), and dried over NaSO, filtered and concentrated under reduced pressure to give 1.30 kg of residue. THF (6.50 L) and H2O (3.90 L) were added to a 50.0 L jacketed flask with an overhead stirrer, an addition funnel and a thermometer, N2 balloon, etc. at 20 to 30 ° C. The residue (1.30 kg) was added to the flask in one go at 20 to 30 ° C, and NaHCO (447.00 g, 5.32 mol) was added to the flask at 20 to 30 ° C. BocO (697.00 g, 3.19 mol) was added dropwise to the flask at 10 to 20 ° C. The mixture was stirred at 20°C to 30°C for 1.5h. H2O (10.0L) was added to the reaction mixture and extracted with MTBE (10.0L×2), the combined organic layers were washed with brine (10.0L×2), and dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (eluted with petroleum ether / ethyl acetate (1 / 1 v / v)) to obtain the title compound (810.00g) as a solid. 1 H NMR(400MHz, CDCl3)δ7.66(d,4H),7.29-7.45(m,6H),4.03(d,1H),3.51(d,2H),2.90(t,1H),1.98–2.07(m,1H),1.72–1.75(m, 2H),1.48(s,9H),1.26–1.29(m,2H),1.08(s,9H),1.00–1.06(m,1H),0.83–0.99(m,1H),0.50–0.52(m,1H),0.39–0.40(m,1H). LCMS:[M+H] + :380.1.

[0195] Tert-butyl 7-(hydroxymethyl)-4-azaspiro[2.5]octane-4-carboxylate

[0196]

[0197] At 20 ℃ under N2 protection, THF (24.00L) was added to a 50.0L flask with a stirrer, an addition funnel and a thermometer. At 20 ℃, tert-butyl 7-(((tert-butyldiphenylsilyl)oxy)methyl)-4-azaspiro[2.5]octane-4-carboxylate (3.10kg, 6.46mol) was added to the flask in one go. At 20 ℃, TBAF (1.00M, 6.46L) was added to the flask, and the resulting mixture was stirred at 20 ℃ for 2h. The mixture was poured into H2O (10.00L) and extracted with ethyl acetate (10.00L×2). The combined organic layers were washed with brine (5.00L), dried over Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (3 / 1 v / v) to obtain the title compound (1.45 kg) as an oil. 1 H NMR(400MHz,DMSO-d6)δ4.43(t,1H),3.83–3.86(m,1H),3.21–3.24(m,2H),1.75–1.99(m,1H),1.65–1.70(m,1H),1.3 9(t,1H),1.18(s,9H),1.00-1.06(m,1H),0.99–1.04(m,2H),0.76–0.78(m,1H),0.37–0.38(m,1H),0.33–0.35(m,1H).

[0198] 4-(tert-Butyloxycarbonyl)-4-azaspiro[2.5]octane-7-carboxylic acid

[0199]

[0200] Five batches were carried out in parallel. At 10°C, MeCN (1.10L) and H2O (1.10L) were added to a 5.00L jacketed flask with an overhead stirrer, an addition funnel and a thermometer, and a N2 balloon. At 10°C, tert-butyl 7-(hydroxymethyl)-4-azaspiro[2.5]octane-4-carboxylate (284.00g, 1.18mol) was added to the flask. At 10°C, TEMPO (74.00g, 470.00mmol) was added to the flask once, and at 10°C under N2, PhI(OAc)2 (947.00g, 2.94mol) was slowly added to the reaction in ten portions. The mixture was stirred at 25°C under N2 for 12h. Five batches were combined, and the reaction mixture was slowly added to a saturated Na2SO3 solution (8.00L) and stirred at 20°C for 0.5h. Then add saturated K2CO3 solution and adjust to pH=11.The mixture is extracted with petroleum ether (3.00L×2), 1N HCl (approximately 20.0L) is added to the aqueous solution and adjusted to pH=3.The mixture is extracted with DCM (5.00L×3), and the combined organic layers are concentrated in vacuo.The residue is crystallized from (heptane, 2.00L, 20°C), the mixture is filtered, and the filter cake is washed with heptane (300mL×2).The residue is collected and concentrated in vacuo to obtain the title compound (1.40kg, 4.47mol) as a solid. 1 H NMR(400MHz,DMSO-d6)δ12.2(br s,1H),3.81(d,1H),2.91(t,1H),2.60(t,1H),1.75-1.78(m,2H),1.23- 1.26(m,10H),0.81-0.82(m,1H),0.51-0.79(m,1H),.0.47-0.49(m,2H). LCMS:[M–100] + :156.6.

[0201] Tert-butyl 7-(((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)carbamoyl)-4-azaspiro[2.5]octane-4-carboxylate

[0202]

[0203] Four batches were carried out in parallel. DCM (3.20 L) was added to a 5.00 L jacketed flask equipped with an overhead stirrer, an addition funnel and a thermometer, and a N2 balloon. At 0 ° C, 4-(tert-butoxycarbonyl)-4-azaspiro[2.5]octane-7-carboxylic acid (317.00 g, 1.24 mol) was added to the flask. At 0 ° C, DIPEA (321.00 g, 2.48 mol), EDCI (297.00 g, 1.55 mol) and HOBt (209.00 g, 1.55 mol) were added to the flask. At 0 ° C, (1r, 4r)-4-amino-1-(trifluoromethyl)cyclohexan-1-ol (239.00 g, 1.30 mol) was added to the flask, and the resulting mixture was purged with N2 3 times. The reaction mixture was stirred at 0 ° C to 25 ° C under N2 for 2h. The four batches were combined and the reaction was poured into H2O (10.00 L) and extracted with ethyl acetate (10.0 L x 2). The combined organic layers were washed with brine (10.00 L), dried over Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 2 v / v) to obtain the title compound (1.55 kg) as a solid. 1 H NMR(400MHz,DMSO-d6)δ7.74(d,1H),5.77(s,1H),3.80-3.88(m,2H),2.78-2.88(m,1H),2.70-2.70(m,1H),2.70-1.75(t,1H), 1.73-1.74(m,4H),1.48-1.55(m,6H),1.43(s,9H),1.18-1.19(m,1H),0.80-0.81(m,1H),0.79-0.81(m,1H),0.46-0.48(m,2H).

[0204] N-((1r,4r)-4-Hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide hydrochloride

[0205]

[0206] Under N2 protection, ethyl acetate (10.00L) is added to a 30.0L flask equipped with a stirrer, an addition funnel and a thermometer. At 0 ° C, tert-butyl 7-(((1r, 4r)-4-hydroxy-4-(trifluoromethyl) cyclohexyl) carbamoyl)-4-azaspiro [2.5] octane-4-carboxylate (1.50kg, 3.57mol) is added to a flask. The reaction mixture is purged 3 times with N2. At 0 ° C, HCl / dioxanes (4.00M, 2.50L) ​​are added to a flask. The reaction is stirred for 1h at 0 ° C to 20 ° C under N2. The reaction mixture is concentrated under vacuum to obtain the title compound (1.40kg) in a solid state, and it is used directly in the next step. 1 H NMR(400MHz,DMSO-d6)δ9.43(s,2H),7.94(d,1H),5.37(br s,1H),3.81(s,1H),3.42-3.44(m,1H),2.80-2.90(m,1H),2.51-2.64(m,1H),2.10-2.13(m,1 H),1.78-1.83(s,6H),1.74-1.78(m,4H),1.49(d,1H),1.03-1.05(m,2H),0.70-0.73(m,2H).

[0207] 4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-N-((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide

[0208]

[0209] Four batches were run in parallel. At 0°C, DMF (3.00 L) was added to a 5.00 L jacketed flask equipped with an overhead stirrer, addition funnel and thermometer, N2 balloon. At 0°C, N-((1r, 4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide hydrochloride (317.00 g, 863.00 mmol) was added to the flask. At 0°C, DIPEA (502.00 g, 3.88 mol), EDCI (298.00 g, 1.55 mol) and HOBt (209.00 g, 1.55 mol) were added to the flask. The reaction mixture was stirred at 0°C and purged with N2. At 0 ° C, 5- (5-fluoro-2-methoxypyridin-4-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H- pyrazole-3-carboxylic acid (intermediate 506, 370.00 g, 1.04 mol) was added to a flask, and the resulting mixture was stirred at 0 ° C to 30 ° C under N2 for 12 h. The four batches were combined, and the reaction mixture was poured into H2O (10.00 L) and extracted with ethyl acetate (10.00 L x 2). The combined organic layers were washed with brine (10.00 L), dried over Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with petroleum ether / ethyl acetate (1 / 2 v / v)) to obtain the title compound as an oil (1.50 kg). 1 H NMR(400MHz,DMSO-d6)δ8.31(d,1H),7.86(s,1H),7.33(d,1H),7.08(s,1H),5.81-5.80(m,1H),5.49(d,1H),3.93(s,3H),3.92-3.8 9(m,1H),3.63-3.61(m,2H),2.98-2.96(m,1H),2.06-2.02(m,1H),1.85-1.25(m,11H),1.23(t,3H),1.23-0.64(m,6H),0.00(s,9H). LCMS:[M+H] + :670.2.

[0210] 4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-N-((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide

[0211]

[0212] Three batches were carried out in parallel. At 20 ° C under N2 protection, DCM (500.00 mL) was added to a 10L flask equipped with a stirrer, an addition funnel and a thermometer. At 20 ° C under N2, 4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-N-((1r, 4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide (500 g, 747 mmol, 1.00 equivalent) was added to the flask. At 20 ° C, TFA (1.28 kg, 11.20 mol) was added to the flask. The reaction mixture was stirred at 20 ° C to 25 ° C under N2 for 2h. Three batches are combined, and the reaction mixture is poured into NaHCO (10.00L), extracted with ethyl acetate (10.0L x 2). The combined organic layer is concentrated under vacuum. The 1.20kg residue obtained is dissolved in methanol (3.00L), NH is slowly added dropwise at 5°C to 10°C. H2O (1.00L) and the mixture is stirred for 1h. The mixture is poured into a saturated NaCl solution (2.00L), extracted with ethyl acetate (4.00L x 2), the combined organic layer is dried over Na2SO4, filtered and the filtrate is concentrated under vacuum. The residue is crystallized from (MTBE, 2.00L, 25°C), the mixture is filtered and the filter cake is washed with MTBE (300mL x 2). The filter cake is collected and concentrated under vacuum to obtain the title compound (970.00g) in a solid. 1 H NMR(400MHz,DMSO-d6)δ14.0(br s,1H),8.27(s,1H),7.81(d,1H),7.32(d,1H),7.03(s,1H),4.37–4.34(m,1H),3.88(s,3H),3.83 (s,1H)2.79(s,1H),2.20–2.10(m,1H),1.79–1.50(m,11H),1.08–0.98(m,3H),0.89–0.65(m,2H). LCMS:[M+H] + :540.1.

[0213] (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide (Compound I)

[0214]

[0215] 4-(5-(5-Fluoro-2-methoxypyridin-4-yl)-1H-pyrazole-3-carbonyl)-N-((1r,4r)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide (1.15 kg, 2.13 mol) was separated using preparative SFC.

[0216] Instrument: Waters SFC prep 350

[0217] Column: DAICEL CHIRALPAK IG (250 mm × 50 mm, 10 μm)

[0218] Mobile phase A: CO2

[0219] Mobile phase B: EtOH

[0220] Gradient: B 55%

[0221] Flow rate: 200mL / min

[0222] Back pressure: 100 bar

[0223] Column temperature: 35°C

[0224] Wavelength: 220nm

[0225] Cycle time: about 12.2 minutes

[0226] Sample preparation: Dissolve 1150 g of compound in 4700 ml of ethanol

[0227] Injection: 15mL each time

[0228] Compound I (411.00 g) was obtained as a solid, and its retention time was 21.69 min.

[0229] 1 H NMR(400MHz,DMSO-d6)δ14.0(br s,1H),8.26(s,1H),7.82(d,1H),7.32(d,1H),7.03(s,1H),5.68(s,1H),4.47–4.34(m,1H),3.87(s,3H) ,3.83(s,1H)2.79(s,1H),2.20–2.10(m,1H),1.78–1.50(m,11H),1.08–0.98(m,3H),0.89–0.65(m,2H). LCMS:[M+H] + 540.1.

[0230] Crystallization of Form A of Compound I

[0231] To Compound I (597 mg) was added acetonitrile (0.6 mL), and the resulting solution was stirred at room temperature for 3 days. The precipitate was harvested by filtration to obtain Compound I (Form A, 544 mg).

[0232] X-ray powder diffraction

[0233] X-ray powder diffraction pattern of Form A of Compound I ( Figure 1 ) is obtained under the following conditions:

[0234] Equipment: SmartLab (Rigaku Co., Ltd.)

[0235] X-ray source: CuKα (45kV, 200mA)

[0236] Optical system: Converging beam optics

[0237] Soller slit: 2.5°

[0238] Detector: D / teX Ultra 250 detector (1D semiconductor inspection system)

[0239] Mode:Transfer

[0240] Scanning range: from 3° to 40°

[0241] Step size: 0.01°

[0242] Scanning speed: 5° / min

[0243] Sample holder: Mylar TM membrane

[0244] The peaks are listed in Table 1 below.

[0245] Table 1. Peak list of X-ray powder diffraction pattern of Form A

[0246]

[0247]

[0248]

[0249] Solid-state NMR

[0250] The crystalline form A of compound (I) was loaded into a 7 mm rotor for 13 C SSNMR spectroscopy. Solid samples 13 CSSNMR spectra were obtained under the following conditions:

[0251] Instrument: Avance 400MHz (BRUKER) 7mm-CPMAS probe (BRUKER)

[0252] Measurement core: 13C (100.6238359MHz)

[0253] Pulse mode: CPTOSS measurement

[0254] Rotation frequency: 5000Hz

[0255] Pulse repetition time: 5 seconds

[0256] Contact time: 1 ms

[0257] Scan count: 10240

[0258] Using glycine adamantane 176.03 ppm as the reference carbonyl carbon, the crystalline form A of compound I was obtained by rotating at 5 kHz at room temperature. 13 C CPMAS( Figure 2 ). The peaks are listed in Table 2 below.

[0259] Table 2. Crystalline Form A 13 C CPMAS peak list

[0260] Chemical shift [ppm] 41.4 43.7 47.7 54.8 70.1 127.2 134.3 148.9 161.2 179.0

[0261] Thermogravimetric and differential scanning calorimetry analysis

[0262] Thermogravimetric analysis and differential scanning calorimetry analysis of Form A of Compound I were measured using a METTLER TOLEDO TGA / DSC 3+.

[0263] About 3 mg of a crystal (Form A) sample was accurately weighed into an aluminum pan and then analyzed under the following conditions.

[0264] Atmosphere: Nitrogen flow, 50 mL / min

[0265] Reference pan: empty aluminum pan

[0266] Heating speed: 10℃ / min

[0267] Sampling interval: 1 second

[0268] Temperature range: 25℃ to 300℃

[0269] DSC curve heat map ( Figure 3 ) shows an endotherm at about 240°C (onset temperature).

[0270] Raman spectroscopy

[0271] 50-100 mg of Form A of Compound I was packed into a plastic bag for Raman spectroscopy. The Raman spectrum of the sample was obtained under the following conditions.

[0272] Instrument: TRS100 Raman (Agilent)

[0273] Laser wavelength: 830nm

[0274] Laser power: 0.65W

[0275] Exposure time: 1.000 seconds

[0276] Scan times: 10

[0277] Laser spot size: 4mm

[0278] Set size: Medium

[0279] Obtain Raman spectra ( Figure 4 ), and the wave numbers are listed in Table 3 below.

[0280] Table 3. Peak list of Raman spectrum of Form A

[0281] <![CDATA[Wavenumber [cm -1 > 579.1 741.3 863.7 1066.6 1262.2 1352.7 1405.3 1509.0 1568.9 1630.2

[0282] Hygroscopicity

[0283] Form A of Compound I (10.88 mg) was weighed into a sampling cup, and the sampling cup was placed in an isothermal container at 25°C. The relative humidity (RH) was controlled from 0% to 95% using a gravimetric vapor sorption system, and the sample weight of each RH stage was measured at predetermined time intervals (e.g., every 2 minutes). The weight change of each RH stage was evaluated in a stepwise manner and then finally determined according to the following criteria. The maximum weight change of each measurement was less than 0.002% (w / w) within 1 minute. See Figure 5 .

[0284] Single crystal X-ray diffraction

[0285] Form A of Compound I (3.24 mg) was dissolved in 600 μL of MEK (methyl ethyl ketone). 200 μL of the solution was placed in another glass vial, which was then stored in a fixed container at room temperature with 2 mL of c-hexane for 2 days (MEK / c-hexane vapor diffusion method). Colorless crystals were collected.

[0286] The colorless block single crystals (0.1×0.1×0.01 mm) found in the crystallization solution were dispersed in liquid Parabar10312 and fixed on Dual-Thickness MicroMountsTM (MiTeGen). The diffraction data were collected at -160°C on XtaLAB PRO P200 MM007HF (Rigaku Co., Ltd.) using the ω axis oscillation method and multi-layer mirror monochromatic Cu-Kα radiation. The single crystal X-ray diffraction pattern of compound I was obtained under the conditions described in Table 4.

[0287] Single crystal X-ray diffraction (SXRD) was used to determine the structure of Form A, and the results are summarized in Tables 4 to 7. The results indicate that Form A is anhydrous. Figure 6 .

[0288] Table 4. SXRD conditions

[0289]

[0290]

[0291] Table 5. Atomic coordinates and B iso / B eq

[0292]

[0293]

[0294] Beq=8 / 3π 2 (U 11 (aa*) 2 +U 22 (bb*) 2 +U 33 (cc*) 2 +2U 12 (aa*bb*)cosγ

[0295] +2U 13 (aa*cc*)cosβ+2U 23 (bb*cc*)cosα)

[0296] Table 6. Atomic coordinates and B involving hydrogen atoms iso

[0297]

[0298]

[0299] Table 7. Anisotropic displacement parameters

[0300]

[0301]

[0302]

[0303] General temperature coefficient expression: exp(-2π 2 (a* 2 U 11 h 2 +b* 2 U 22 k 2 +c* 2 U 33 l 2 +2a*b*U 12 hk+2a*c*U 13 hl+2b*c*U 23 kl))

[0304] TR-FRET assay to measure the recruitment of PPARγ by Co-R peptides

[0305] The His6-TEV-PPARγ-(234-505) protein used for the assay was expressed and purified according to the following reference: Korpal, M. et al., Evasion of immunosurveillance by genomic alterations of PPARgamma / RXRalpha in bladder cancer. Nature communications, 2017. 8(1): p.103.

[0306] The assay buffer consisted of 50 mM potassium chloride (Sigma), 50 mM HEPES pH 7.4 (Teknova), 2 mM DTT (Boston Bioproducts), 0.1 mg / mL bovine gamma-globulin (Sigma), and 0.001% Pluronic F-127 (ThermoFisher). A 2x working stock of His6-PPARγ-(234-505) was diluted to a final concentration of 10 nM in assay buffer. The 2x detection solution contained an anti-6xHis-terbium antibody (CisBio) and a FITC-labeled Co-R peptide (SMRT-ID2 from New England Peptide) at final concentrations of 5 nM and 200 nM, respectively.

[0307] The acoustic dispenser delivers 4 nL of compound or DMSO from an 11-point master dose response (MDR) source plate to a 384-well assay plate (Corning, 3820) with a maximum final concentration of 20 μM. In these assay plates, the positive control in column 24 contains 500 nM (final) T0070907, and the negative control in column 23 contains the same volume of DMSO. Then 5 μL of 2x protein working stock solution or control is added to the plate and incubated at room temperature for 20 minutes. Reagent addition is performed using a Combi (Thermo) or Mantis (Formulatrix) pipetting device. 5 μL of 2x detection solution is added after incubation. The plate is covered, centrifuged and incubated for another hour. TR-FRET data are recorded using an Envision plate reader (Perkin Elmer) using the settings recommended by Thermo Fisher Scientific. TR-FRET signals recruited by FITC-Co-R peptide to positive and negative controls were used to normalize the TR-FRET signals to percent response and determine the assay Z prime. Experiments were performed in triplicate and analyzed in GraphPad Prism 7.

[0308] The assay results are reported in Table 8.

[0309] Table 8. Bioassay results

[0310]

Claims

1. A crystalline form A of (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide (i.e., crystalline form A of (S)-4-(5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-carbonyl)-N-((1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl)-4-azaspiro[2.5]octane-7-carboxamide) (i.e., crystalline form A of compound I):

2. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern substantially similar to that of Figure 1.

3. Form A according to claim 1, characterized in that An X-ray powder diffraction pattern having a signal at at least one 2-theta value selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.

2.

4. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.

2.

5. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 21.0±0.2 and 22.6±0.

2.

6. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 18.5±0.2, 19.4±0.2, 21.0±0.2 and 22.6±0.

2.

7. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern having signals at at least two 2-theta values ​​selected from 14.8±0.2, 18.5±0.2 and 19.4±0.

2.

8. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern having signals at at least three 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.

2.

9. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern having signals at at least four 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.

2.

10. The crystalline form A according to claim 1, characterized in that An X-ray powder diffraction pattern having signals at at least five 2-theta values ​​selected from 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.

2.

11. The crystalline form A according to claim 1, characterized in that The following X-ray powder diffraction pattern has signals at 14.8±0.2, 15.9±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 20.7±0.2, 21.0±0.2, 22.6±0.2 and 27.2±0.

2.

12. The crystalline form A according to claim 1, characterized in that The following X-ray powder diffraction pattern has signals at 14.8±0.2, 17.5±0.2, 18.5±0.2, 19.4±0.2, 20.0±0.2, 21.0±0.2 and 22.6±0.

2.

13. The crystalline form A according to claim 1, characterized in that The following is an X-ray powder diffraction pattern, which has signals at 14.8±0.2, 18.5±0.2, 19.4±0.2, 21.0±0.2 and 22.6±0.

2.

14. The crystalline form A according to claim 1, characterized in that The following is an X-ray powder diffraction pattern, which has signals at 14.8±0.2, 18.5±0.2 and 19.4±0.

2.

15. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at at least five ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm.

16. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at at least four ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm.

17. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at at least three ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

18. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at at least two ppm values ​​selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

19. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having a signal at at least one ppm value selected from 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm and 179.0±0.2 ppm.

20. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at 41.4±0.2 ppm, 43.7±0.2 ppm, 47.7±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm, 161.2±0.2 ppm, and 179.0±0.2 ppm.

21. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at 41.4±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 127.2±0.2 ppm, 134.3±0.2 ppm, 148.9±0.2 ppm and 161.2±0.2 ppm.

22. The crystalline form A according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at 41.4±0.2 ppm, 54.8±0.2 ppm, 70.1±0.2 ppm, 134.3±0.2 ppm and 148.9±0.2 ppm.

23. The crystalline form A of compound I according to claim 1, characterized in that as follows 13 C SSNMR spectrum having signals at 54.8±0.2 ppm, 70.1±0.2 ppm and 148.9±0.2 ppm.

24. The crystalline form A according to claim 1, characterized in that TGA / DSC substantially similar to Figure 3.

25. The crystalline form A according to claim 1, characterized in that Raman spectra similar to those in Figure 4.

26. The crystalline form A according to claim 1, characterized in that The Raman spectrum is as follows: –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There is a signal at at least one ppm value of .

27. The crystalline form A according to claim 1, characterized in that The Raman spectrum is as follows: –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There are signals at at least two ppm values ​​of .

28. The crystalline form A according to claim 1, characterized in that The Raman spectrum is as follows: –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There are signals at at least three ppm values ​​of .

29. The crystalline form A according to claim 1, characterized in that The Raman spectrum is as follows: –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There are signals at at least four ppm values ​​of .

30. The crystalline form A according to claim 1, characterized in that The Raman spectrum is as follows: –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There are signals at at least five ppm values ​​of .

31. The crystalline form A according to claim 1, characterized in that The Raman spectrum is shown below. The Raman spectrum is at 579.1±2cm –1 、741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1262.2±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 、1568.9±2cm –1 and 1630.2±2cm –1 There is a signal.

32. The crystalline form A according to claim 1, characterized in that The Raman spectrum is shown below. The Raman spectrum is at 741.3±2cm –1 、863.7±2cm –1 、1066.6±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 、1509.0±2cm –1 and 1630.2±2cm –1 There is a signal.

33. The crystalline form A of compound I according to claim 1, characterized in that The Raman spectrum is shown below. The Raman spectrum is at 741.3±2cm –1 、863.7±2cm –1 、1352.7±2cm –1 、1405.3±2cm –1 and 1630.2±2cm –1 There is a signal.

34. The crystalline form A according to claim 1, characterized in that The Raman spectrum is shown below. The Raman spectrum is at 863.7±2cm –1 、1405.3±2cm –1 and 1630.2±2cm –1 There is a signal.

35. A pharmaceutical composition comprising the crystalline form A according to any one of claims 1 to 34, and a pharmaceutically acceptable carrier.

36. A method for treating and / or preventing cancer, comprising administering an effective amount of the crystalline form A according to any one of claims 1 to 34 or the pharmaceutical composition according to claim 35 to a subject in need thereof.

37. The method of claim 36, wherein the cancer is selected from bladder cancer, breast cancer, prostate cancer, and cancers with altered RXRa and / or PPARγ pathways.

38. The method according to any one of claims 36 and 37, wherein the cancer is bladder cancer.

39. The method according to any one of claims 37 to 38, wherein the bladder cancer is selected from advanced bladder cancer, luminal bladder cancer and basal bladder cancer.

40. The method according to any one of claims 36 to 39, wherein the cancer is a chemotherapy-resistant and / or immunotherapy-resistant cancer.

41. Use of the crystalline form A of (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide according to any one of claims 1 to 34 and the pharmaceutical composition according to claim 35 in the treatment and / or prevention of cancer.

42. A method for preparing a crystalline form A of (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide, the method comprising (7S)-4-{[5-(5-fluoro-2-methoxypyridin-4-yl)-1H-pyrazol-3-yl]carbonyl}-N-[(1r,4S)-4-hydroxy-4-(trifluoromethyl)cyclohexyl]-4-azaspiro[2.5]octane-7-carboxamide and acetonitrile were mixed at room temperature and The mixture was stirred at room temperature for at least 24 hours.

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