Solid forms of rxfp1 modulators

Abstract

The present specification relates generally to solid forms, such as crystalline and amorphous forms, of (1S, 4s)-4-(2-fluoro-4-methoxy-5-(((1S, 2R, 3S, 4R)-3-(((1-methylcyclobutyl) methyl) carbamoyl) bicyclo [2.2. 1] hept-2-yl) carbamoyl) phenoxy)-1-methylcyclohexane-1-carboxylic acid (Compound (I)). In particular, amorphous forms of Compound (I), solid dispersions and pharmaceutical compositions comprising such amorphous forms, and crystalline Form G of Compound (I). # imgabs0 #

Description

Technical Field

[0001] The present specification describes solid forms (e.g., crystalline and amorphous forms) of the RXFP1 modulator (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (also referred to herein as Compound (I)), pharmaceutical compositions thereof, and methods for preparing such solid forms. Background Art

[0002] Relaxin is a pleiotropic hormone known to mediate systemic hemodynamic changes and renal adaptive changes during pregnancy. Relaxin has also been shown to have anti-fibrotic properties and has beneficial effects in heart failure, such as acute decompensated heart failure (ADHF). Heart failure is associated with significant morbidity and mortality. It is characterized by complex tissue remodeling involving increased cardiomyocyte death and interstitial fibrosis. Relaxin activates a number of signaling cascades that have been shown to be beneficial in the setting of ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and activation of the nitric oxide signaling pathway (Bathgate RA et al. (2013) Physiol. Rev. 93(1):405-480; Mentz RJ et al. (2013) Am. Heart J. 165(2):193-199; Tietjens J et al. (2016) Heart 102:95-99; Wilson SS et al. (2015) Pharmacology 35:315-327).

[0003] A large proportion of heart failure patients also suffer from pulmonary hypertension (HF+PH patients). It is estimated that approximately 50% of heart failure patients with preserved ejection fraction also suffer from pulmonary hypertension, and this proportion increases to 60% in heart failure patients with reduced ejection fraction (Guazzi, (2014) Circ Heart Fail. [Circulation: Heart Failure], 7:367-377; Miller et al., (2013) JACC Heart Fail. [Journal of the American College of Cardiology: Heart Failure], 1(4):290-299). Patients with heart failure and pulmonary hypertension have a decreased survival rate compared with heart failure patients without pulmonary hypertension (Barnett and DeMarco, (2012) Heart Fail. Clin. [Heart Failure Clinical] 8:447-459). In patients with heart failure, an increase or decrease of 3 mmHg in estimated pulmonary artery diastolic pressure (ePAD) (equivalent to an increase or decrease of approximately 4 mmHg in mean pulmonary artery pressure (mPAP)) is associated with a 24% increase or 19% decrease in cardiovascular mortality, respectively (Zile MR, et al. (2017) Circ Heart Fail. 10:e003594). A 4 mmHg decrease in mPAP is also associated with improved dyspnea in patients with heart failure and pulmonary hypertension (Solomonica A, et al. (2013) Circ Heart Fail. 6:53-60).

[0004] Resistant hypertension (rHT) is defined as a hypertensive patient whose blood pressure remains above target despite concurrent use of optimized doses of 3 different classes of antihypertensive agents, one of which is a diuretic. The current SoCs for initial treatment of hypertension are calcium channel blockers (CCBs), renin-angiotensin system blockers (angiotensin-converting enzyme [ACE] inhibitors or angiotensin receptor blockers [ARBs]), and diuretics. For patients with rHT, there are multiple options for the next medication to add (such as mineralocorticoid receptor antagonists (MRAs), β-blockers, or α-blockers), and current guidelines recommend MRAs as the preferred choice for rHT treatment. rHT also includes patients whose blood pressure is adequately controlled while receiving 4 or more antihypertensive drugs simultaneously (Carey et al., Hypertension, 2018, 72, e53-e90). Patients with rHT typically have a history of long-term, severe elevations in blood pressure, placing them at higher risk for cardiovascular disease than hypertensive patients whose blood pressure is controlled under treatment (Acelajado et al., Circulation Research, 2019, 124, 1061-1070). It has been suggested that relaxin may have therapeutic potential for hypertensive disorders (Lekgabe et al., Hypertension, 2005, 46, 412-8).

[0005] Clinical trials have been conducted using unmodified recombinant human relaxin-2 (serelaxin). Continuous intravenous administration of serelaxin to hospitalized patients improved markers of cardiac, renal, and liver damage and congestion (Felker GM et al. (2014) J. Am. Coll. Cardiol. [Journal of the American College of Cardiology] 64(15): 1591-1598; Metra M et al. (2013) J. Am. Coll. Cardiol. [Journal of the American College of Cardiology] 61(2): 196-206; Teerlink JR et al. (2013) Lancet [Lancet] 381(9860): 29-39). However, since serelaxin is rapidly cleared from the patient's circulation, the therapeutic effect is limited, and once the intravenous injection is stopped, the positive effect disappears rapidly. In addition, approximately one-third of patients experience a significant decrease in blood pressure (>40 mmHg) after receiving intravenous serelaxin, and the dose must be reduced by half or even more.

[0006] The cognate receptor for human relaxin is RXFP1 and is a well-validated pharmacologically important member of GPCR family 1c, whose activation by the hormone relaxin is associated with hemodynamic, antifibrotic and anti-inflammatory properties (Halls ML et al., (2015), Pharmacol Rev. 67(2):389-440).

[0007] Small molecule modulators of RXFP1 have been sought as relaxin mimetics. For example, Marugan, JJ, et al., WO2013 / 165606 A1; Xiao J et al. (2013) Nat. Commun. [Nature Communications] 4: 1953; and McBride A et al. (2017) Scientific Reports [Science Report] 7: 10806 discuss small molecule modulators of RXFP1.

[0008] Despite the foregoing, there is still a need for additional compounds (such as compound (I)) that are modulators of RXFP1, which may make these compounds particularly promising for development as therapeutic agents. Such compounds may also exhibit improved RXFP1 regulation compared to other known RXFP1 modulators. Such compounds may also exhibit favorable pharmacokinetic profiles (e.g., lower intrinsic clearance) and / or favorable physical properties (e.g., higher water solubility) compared to other known RXFP1 modulators. Therefore, such compounds may be particularly useful for treating disease states in which RXFP1 regulation is beneficial. There is also a need for stable solid forms of such compounds having favorable physical properties (such as water solubility at low pH) that may be used for oral administration of such compounds. Summary of the invention

[0009] The present specification relates to a solid form of compound (I), which has the following structure:

[0010]

[0011] and has the chemical name: (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid.

[0012] Briefly, this specification describes in part the amorphous compound (I).

[0013] The specification also describes, in part, a pharmaceutical composition comprising amorphous Compound (I) and a pharmaceutically acceptable excipient.

[0014] This specification also describes, in part, a solid dispersion comprising amorphous Compound (I).

[0015] This specification also describes, in part, a pharmaceutical composition comprising a solid dispersion comprising amorphous Compound (I).

[0016] This specification also describes, in part, an amorphous compound (I) or a solid dispersion or a pharmaceutical composition described herein for use in therapy.

[0017] The present specification also partially describes the amorphous compound (I) or solid dispersion or pharmaceutical composition described herein for use in treating a subject suffering from a disease or condition selected from the following: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension and resistant hypertension.

[0018] The present specification also describes, in part, a crystalline form of Compound (I), wherein the crystalline form has a melting onset of about 217.5°C, and a method for preparing such a crystalline form, the method comprising heating crystalline Form A of Compound (I) to a temperature greater than about 43.9°C.

[0019] The present specification also describes, in part, a crystalline form of Compound (I) which can be obtained by heating the crystalline Form A of Compound (I) to a temperature greater than about 43.9°C, and a method for preparing such a crystalline form, the method comprising heating the crystalline Form A of Compound (I) to a temperature greater than about 43.9°C.

[0020] This specification also describes, in part, pharmaceutical compositions comprising a crystalline form as disclosed herein.

[0021] The present specification also describes, in part, a crystalline form as disclosed herein for use in the manufacture of a medicament for treating a disease or condition in a human patient selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension.

[0022] Additional aspects of the present disclosure will be apparent to those skilled in the art from reading this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the X-ray powder diffraction pattern of Form A of Compound (I).

[0024] Figure 2 is the X-ray powder diffraction pattern of amorphous compound (I).

[0025] Figure 3 Is the X-ray powder diffraction pattern of an amorphous solid dispersion of Compound (I) (20%, 30% and 40% loading of Compound (I)) after formation, after storage at 40°C / 75% relative humidity for 4 weeks, and after storage at 50°C at ambient relative humidity for 4 weeks.

[0026] Figure 4 Shown are the DSC outputs for Form A / Form G analysis of Compound (I).

[0027] Figure 5 TGA analysis of Form A / Form G of Compound (I) is shown.

[0028] Figure 6 Shown is the light scattering intensity as a function of the total concentration of amorphous Compound (I) in simulated gastric fluid at pH 1.2. DETAILED DESCRIPTION

[0029] Many embodiments are described in detail throughout the specification and will be apparent to the skilled reader.This specification is not to be construed as limited to any particular embodiment or embodiments described herein.

[0030] Terms not expressly defined herein should be understood to have the meaning that one skilled in the art would give them based on this disclosure and context.

[0031] "About" can generally mean an acceptable degree of error for a measured amount given the nature or precision of the measurements. Exemplary degrees of error are within a percentage (%) of a given value or range of values, typically within 10%, and more typically within 5%.

[0032] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of corresponding embodiments “consisting of such features”.

[0033] Concentrations, amounts, volumes, percentages and other numerical values ​​can be presented in a range format. It should also be understood that such a range format is used only for convenience and brevity, and should be flexibly interpreted to include not only the numerical values ​​explicitly listed as the limits of the range, but also all individual numerical values ​​or sub-ranges contained within the range, just as each numerical value and sub-range is explicitly listed.

[0034] The chemical names of the compounds described in this specification are derived from of Produced by Professional Edition 19.0.0.22. The skilled person will appreciate that different chemical naming software may generate different chemical names for a particular compound. Where the compounds described herein are described in the form of a chemical name and a chemical formula, in the event of any inconsistency, the chemical formula shall prevail.

[0035] The compounds and salts described in this specification may exist in solvated and non-solvated forms. For example, the solvated form may be a hydrated form, such as a hemihydrate, a monohydrate, a dihydrate, a trihydrate or an alternative amount thereof. All such solvated and non-solvated forms of the compounds described herein are encompassed herein.

[0036] The atoms of the compounds and salts described in this specification may exist as their isotopes. This document covers all compounds described herein in which atoms are replaced by one or more of their isotopes (e.g., compounds described herein in which one or more carbon atoms are 11 C or 13 C carbon isotope, or one or more hydrogen atoms are 2 H or 3 H isotope).

[0037] The compounds described herein may exist in one or more geometric forms, optical forms, enantiomeric forms, and diastereomeric forms (including but not limited to cis and trans, E- and Z-forms, and R-, S- and meso forms). Unless otherwise indicated, reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Where appropriate, such isomers may be separated from their mixtures by application or modification of known methods (e.g., chromatography techniques and recrystallization techniques). Where appropriate, such isomers may be prepared by application or modification of known methods.

[0038] The compounds described herein may include one or more chiral centers. Where a structure or chemical name in this specification does not indicate chirality, the structure or name is intended to encompass any single stereoisomer corresponding to the structure or name, as well as any mixture of stereoisomers (e.g., racemates). Structures in this specification include bonds drawn as solid and dashed wedges (i.e., ), the real and imaginary wedges are intended to represent the absolute configuration of the chiral center.

[0039] It is well known in the art how such optically active forms are separated. For example, a single stereoisomer can be obtained by separating it from a mixture of isomers (e.g., a racemate) using, for example, chiral chromatographic separation. In other embodiments, a single stereoisomer is obtained by, for example, direct synthesis of chiral starting materials.

[0040] According to one embodiment, the compounds described herein are provided as single enantiomers in an enantiomeric excess (%ee) ≥ 95%, ≥ 98% or ≥ 99%. Conveniently, the single enantiomer is present in an enantiomeric excess ≥ 99%.

[0041] According to one embodiment, the compounds described herein are provided as single enantiomers in an enantiomeric excess (%ee) ranging from 95% to 100%.

[0042] The compounds described herein can exist in one or more tautomeric forms, including but not limited to keto- and enol-forms. Reference to a particular compound includes all tautomeric forms, including mixtures thereof. Therefore, structures depicted herein as one tautomer are also intended to include other tautomers.

[0043] The pharmaceutical compositions described herein may include one or more pharmaceutically acceptable excipients. The one or more excipients selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art and are described, for example, in Handbook of Pharmaceutical Excipients, 6th edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmaceutically acceptable excipients may be used, for example, as adjuvants, diluents, carriers, stabilizers, flavoring agents, colorants, fillers, adhesives, disintegrants, lubricants, glidants, thickeners, and coating agents. As will be appreciated by those skilled in the art, certain pharmaceutically acceptable excipients may be used for more than one function and may be used for alternative effects, depending on how many excipients are present in the composition and which other excipients are present in the composition.

[0044] The pharmaceutical composition may be in a form suitable for oral use (e.g. as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, or dispersible powders or granules), topical use (e.g. as creams, ointments, or aqueous or oily suspensions), administration by inhalation (e.g. as finely divided powders), administration by insufflation (e.g. as finely divided powders), or as suppositories for rectal administration. The composition may be obtained by conventional procedures well known in the art. Compositions intended for oral use may contain additional components, for example, one or more coloring agents, sweeteners, flavoring agents and / or preservatives.

[0045] Amorphous compound (I)

[0046] In an embodiment, an amorphous compound (I) is provided. In an embodiment, the amorphous compound (I) has substantially the same Figure 2 The X-ray powder diffraction pattern shown in the same X-ray powder diffraction pattern (CuK α In one embodiment, the amorphous compound (I) is in the range of 3 to 40° 2-θ (CuK α No reflection is shown within the range of radiation).

[0047] Pharmaceutical composition comprising amorphous compound (I)

[0048] In an embodiment, a pharmaceutical composition is provided, comprising an amorphous compound (I) and a pharmaceutically acceptable excipient. In one embodiment, at least 80% of the compound (I) by weight in the pharmaceutical composition is amorphous. In one embodiment, at least 90% of the compound (I) by weight in the pharmaceutical composition is amorphous. In one embodiment, at least 95% of the compound (I) by weight in the pharmaceutical composition is amorphous. In one embodiment, at least 99% of the compound (I) by weight in the pharmaceutical composition is amorphous. In one embodiment, substantially all of the compound (I) in the pharmaceutical composition is amorphous. In one embodiment, all of the compound (I) in the pharmaceutical composition is amorphous.

[0049] In one embodiment, less than 20% by weight of Compound (I) in the pharmaceutical composition is crystalline. In one embodiment, less than 10% by weight of Compound (I) in the pharmaceutical composition is crystalline. In one embodiment, less than 5% by weight of Compound (I) in the pharmaceutical composition is crystalline. In one embodiment, less than 1% by weight of Compound (I) in the pharmaceutical composition is crystalline. In one embodiment, substantially no Compound (I) in the pharmaceutical composition is crystalline. In one embodiment, no Compound (I) in the pharmaceutical composition is crystalline.

[0050] Solid dispersion containing amorphous compound (I)

[0051] In another embodiment, a solid dispersion comprising amorphous compound (I) is provided. Solid dispersions typically include compounds dispersed in a suitable carrier medium, such as one or more polymers. In one embodiment, the solid dispersion further comprises one or more polymers. In one embodiment, one or more polymers are water soluble. It should be understood that water soluble polymers can be dissolved in aqueous media (such as water or gastric juice).

[0052] In one embodiment, at least 90% by weight of Compound (I) in the solid dispersion is amorphous. In one embodiment, at least 95% by weight of Compound (I) in the solid dispersion is amorphous. In one embodiment, at least 99% by weight of Compound (I) in the solid dispersion is amorphous. In one embodiment, substantially all of Compound (I) in the solid dispersion is amorphous. In one embodiment, all of Compound (I) in the solid dispersion is amorphous.

[0053] In one embodiment, less than 20% by weight of Compound (I) in the solid dispersion is crystalline. In one embodiment, less than 10% by weight of Compound (I) in the solid dispersion is crystalline. In one embodiment, less than 5% by weight of Compound (I) in the solid dispersion is crystalline. In one embodiment, less than 1% by weight of Compound (I) in the solid dispersion is crystalline. In one embodiment, substantially no Compound (I) in the solid dispersion is crystalline. In one embodiment, no Compound (I) in the solid dispersion is crystalline.

[0054] In one embodiment, compound (I) is present in the solid dispersion in an amount of 5% by weight to 95% by weight. In one embodiment, compound (I) is present in the solid dispersion in an amount of 5% by weight to 80% by weight. In one embodiment, compound (I) is present in the solid dispersion in an amount of 5% by weight to 60% by weight. In one embodiment, compound (I) is present in the solid dispersion in an amount of 10% by weight to 40% by weight. In one embodiment, compound (I) is present in the solid dispersion in an amount of 20% by weight to 40% by weight.

[0055] In one embodiment, the one or more polymers are selected from poly(1-vinyl-2-pyrrolidone), copolymers of 1-vinyl-2-pyrrolidone and vinyl acetate, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymers, methacrylic acid-ethyl acrylate copolymers, polyacrylic acid, hypromellose acetate succinate, cellulose acetate phthalate, and polyvinyl acetate phthalate.

[0056] In one embodiment, one or more polymers are selected from 30. VA 64, L100-55S, polyacrylic acid, HPMC AS-LF, HPMCAS-HF, cellulose acetate phthalate, and polyvinyl acetate phthalate.

[0057] In one embodiment, the one or more polymers are copolymers of 1-vinyl-2-pyrrolidone and vinyl acetate. In one embodiment, the one or more polymers are copolymers of 1-vinyl-2-pyrrolidone and vinyl acetate in a ratio of 6:4 by weight. In one embodiment, the one or more polymers are VA 64.

[0058] In one embodiment, one or more polymers are present in the solid dispersion in an amount of 5% by weight to 95% by weight. In one embodiment, one or more polymers are present in the solid dispersion in an amount of 20% by weight to 90% by weight. In one embodiment, one or more polymers are present in the solid dispersion in an amount of 40% by weight to 90% by weight. In one embodiment, one or more polymers are present in the solid dispersion in an amount of 50% by weight to 90% by weight. In one embodiment, one or more polymers are present in the solid dispersion in an amount of 60% by weight to 80% by weight.

[0059] Pharmaceutical composition comprising a solid dispersion containing an amorphous compound (I)

[0060] In an embodiment, a pharmaceutical composition is provided, comprising a solid dispersion comprising amorphous Compound (I) as described herein.

[0061] Crystalline Form G of Compound (I)

[0062] Differential scanning calorimetry (DSC) analysis of crystalline Form A of Compound (I) shows an endothermic transition, which is observed to start at around 43.9° C. This endothermic peak is assigned to a solid-solid phase transition, and the phase produced at a temperature above the completion of the endothermic peak observed at around 43.9° C. is referred to as Form G. This new anhydrous phase Form G exists until the last endothermic peak in DSC, which corresponds to the onset of melting at 217.5° C.

[0063] Thus, in one embodiment, a crystalline form of Compound (I) (Form G) is provided, wherein the crystalline form has a melting onset of about 217.5°C. In one embodiment, the melting onset is 217.5±5°C. In one embodiment, the melting onset is 217.5±4°C. In one embodiment, the melting onset is 217.5±3°C. In one embodiment, the melting onset is 217.5±2°C. In one embodiment, the melting onset is 217.5±1°C.

[0064] In one embodiment, the crystalline form (Form G) of Compound (I) can be obtained by heating the crystalline Form A of Compound (I) to a temperature greater than about 43.9°C. In one embodiment, the crystalline form can be obtained by heating the crystalline Form A of Compound (I) to a temperature greater than about 45°C. In one embodiment, the crystalline form can be obtained by heating the crystalline Form A of Compound (I) to a temperature greater than about 50°C. In one embodiment, the crystalline form can be obtained by heating the crystalline Form A of Compound (I) to a temperature greater than about 55°C. In one embodiment, the crystalline form can be obtained by heating the crystalline Form A of Compound (I) to a temperature greater than about 60°C. In one embodiment, the crystalline form can be obtained by heating the crystalline Form A of Compound (I) to a temperature greater than about 65°C.

[0065] In one embodiment, a method for preparing a crystalline form of compound (I) (Form G) is provided, the method comprising heating the crystalline Form A of compound (I) to a temperature greater than about 43.9°C. In one embodiment, the crystalline Form A of compound (I) is heated to a temperature greater than about 45°C. In one embodiment, the crystalline Form A of compound (I) is heated to a temperature greater than about 50°C. In one embodiment, the crystalline Form A of compound (I) is heated to a temperature greater than about 55°C. In one embodiment, the crystalline Form A of compound (I) is heated to a temperature greater than about 60°C. In one embodiment, the crystalline Form A of compound (I) is heated to a temperature greater than about 65°C.

[0066] The ten most significant X-ray powder diffraction peaks [angle 2-θ (2θ), intensity] of Form A of Compound (I) are: 7.5 (s), 10.3 (m), 10.7 (s), 12.8 (s), 14.5 (vs), 15.3 (s), 15.8 (s), 17.5 (m), 19.6 (s) and 21.3 (s). In one embodiment, the X-ray powder diffraction pattern (CuK αRadiation) has at least five specific peaks at about 2-θ = 7.5, 10.7, 12.8, 14.5 and 15.8 °. In one embodiment, the X-ray powder diffraction pattern of Form A of Compound (I) has specific peaks at about 2-θ = 7.5, 10.3, 10.7, 12.8, 14.5, 15.3, 15.8, 17.5, 19.6 and 21.3 °. In one embodiment, the X-ray powder diffraction pattern of Form A of Compound (I) has at least five specific peaks at 2-θ = 7.5, 10.7, 12.8, 14.5 and 15.8 °, wherein the value may be plus or minus 0.2 ° 2-θ. In one embodiment, the X-ray powder diffraction pattern of Form A of Compound (I) has specific peaks at 2-θ=7.5, 10.3, 10.7, 12.8, 14.5, 15.3, 15.8, 17.5, 19.6 and 21.3°, wherein the value may be plus or minus 0.2° 2-θ. In one embodiment, Form A of Compound (I) has substantially the same Figure 1 The X-ray powder diffraction pattern is the same as the X-ray powder diffraction pattern shown in .

[0067] The specific solid forms described herein provide X-ray powder diffraction patterns substantially identical to those shown in the accompanying drawings, and have different 2-θ values ​​as described herein. It should be understood that the 2-θ values ​​of the X-ray powder diffraction patterns may vary slightly from machine to machine or sample to sample, and therefore the quoted values ​​are not to be interpreted as absolute.

[0068] It is known that, depending on the measurement conditions (such as the equipment or machine used), an X-ray powder diffraction pattern with one or more measurement errors may be obtained. In particular, it is generally known that the intensity in an X-ray powder diffraction pattern may fluctuate depending on the measurement conditions. Therefore, it should be understood that the solid forms described herein are not limited to crystals that provide an X-ray powder diffraction pattern identical to the X-ray powder diffraction pattern shown in the accompanying drawings, and any crystals that provide an X-ray powder diffraction pattern that is substantially identical to those shown in the accompanying drawings fall within the scope of the embodiments described herein. A person of ordinary skill in the art of X-ray powder diffraction is able to judge the substantial identity of an X-ray powder diffraction pattern.

[0069] Those skilled in the art of X-ray powder diffraction will recognize that the relative intensity of the peaks can be affected by, for example, grains with a size greater than 30 μm and non-single aspect ratios, which may affect the analysis of the sample. The skilled person will also recognize that the position of the reflections may be affected by the exact height at which the sample is located in the diffractometer and the zero point correction of the diffractometer. The surface planarity of the sample may also have a subtle effect. Therefore, the diffraction pattern data presented cannot be taken as absolute values. (Jenkins, R and Snyder, RL 'Introduction to X-Ray Powder Diffractometry' John Wiley & Sons 1996; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; Klug, HP and Alexander, LE (1974), X-Ray Diffraction Procedures). Peak intensities are described herein as vs (very strong), s (strong), m (moderate) and w (weak), and correspond to % relative intensities (based on the most intense peak) of 25%-100%, 10%-25%, 3%-10% and 1%-3%, respectively. Relative intensities are derived from diffraction patterns measured with a fixed slit.

[0070] Typically, the measurement error of the diffraction angle in the X-ray powder diffraction pattern is about 5% or less, particularly plus or minus 0.5° 2-θ, and when reading the data described herein, when considering the X-ray powder diffraction patterns in the figures herein, such a degree of measurement error should be taken into account. In addition, it should be understood that the intensity may fluctuate depending on the experimental conditions and sample preparation (preferred orientation).

[0071] Pharmaceutical compositions comprising Form G of Compound (I)

[0072] In an embodiment, a pharmaceutical composition is provided, which comprises Form G of Compound (I) described herein and a pharmaceutically acceptable excipient.

[0073] Use of solid forms in therapy

[0074] Compound (I) is expected to be useful in therapy (due to its modulation of RXFP1).

[0075] The term "therapy" is intended to have its normal meaning: treating a disease or condition so as to fully or partially relieve one, some or all of its symptoms, or so as to correct or compensate for an underlying pathology. The term "therapy" also includes "prophylaxis," unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted accordingly.

[0076] The term "prophylaxis" is intended to have its normal meaning and includes primary prevention, which prevents the development of a disease or condition, and secondary prevention, wherein the disease or condition has already developed and the patient is temporarily or permanently protected against an exacerbation or worsening of the disease or condition or against the development of new symptoms associated with the disease or condition.

[0077] The term "treatment" is used synonymously with "therapy". Similarly, the term "treat" may be viewed as "applying therapy", where "therapy" is as defined herein.

[0078] The term "therapeutically effective amount" refers to the amount of a compound that effectively provides "therapy" in a subject, or effectively "treats" a disease or condition in a subject. As described in the definitions of "therapy", "treatment" and "prophylaxis" above, a therapeutically effective amount can cause any observable or measurable change in a subject. As recognized by those skilled in the art, the effective amount can vary according to the route of administration, the use of excipients, and the use in conjunction with other agents. For example, in the case of using a combination therapy, the amount of one or more pharmaceutically active agents and the amount of one or more other pharmaceutically active agents are combined to effectively treat the subject's targeted disorder or condition. In this context, if they are combined to reduce the symptoms of a disease or condition that is responsive to the regulation and / or excitement of RXFP1 as described above, the combined amount is a "therapeutically effective amount". Typically, such an amount can be determined by those skilled in the art.

[0079] As used herein, the terms "subject" and "patient" are used interchangeably. "Subject" includes, for example, mammals, such as humans. In some embodiments, the subject is a human.

[0080] Therefore, the amorphous compound (I), Form G of compound (I), a solid dispersion comprising amorphous compound (I), or a pharmaceutical composition comprising compound (I) described herein can be used in therapy, for example, for treating a disease or disorder. Also provided is a method for treating a disease or disorder, the method comprising administering a therapeutically effective amount of the amorphous compound (I), Form G of compound (I), a solid dispersion comprising amorphous compound (I), or a pharmaceutical composition comprising compound (I) described herein to a subject or patient in need thereof.

[0081] It should be understood that the amorphous Compound (I), Form G of Compound (I), a solid dispersion comprising amorphous Compound (I) or a pharmaceutical composition comprising Compound (I) described herein can be used to treat cardiovascular diseases, such as for the treatment of heart failure and hypertension.

[0082] As used herein, the term "heart failure" includes acute heart failure, chronic heart failure (CHF), and acute decompensated heart failure (ADHF). The term "heart failure" may also include more specific diagnoses such as heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate ejection fraction (HFmrEF; also known as heart failure with mildly reduced ejection fraction), or heart failure with reduced ejection fraction (HFrEF).

[0083] As used herein, the term "resistant hypertension" is defined as the blood pressure of a hypertensive patient that is above target despite the simultaneous use of optimized doses of 3 different classes of antihypertensive agents (one of which is a diuretic), or the blood pressure of a patient whose blood pressure is adequately controlled when receiving 4 or more antihypertensive drugs simultaneously (Carey et al., Hypertension, 2018, 72, e53-e90). The initial treatment of hypertension can be a calcium channel blocker (CCB), a renin-angiotensin system blocker (angiotensin converting enzyme [ACE] inhibitor or angiotensin receptor blocker [ARB]), and a diuretic. For rHT patients, additional treatment may include mineralocorticoid receptor antagonists (MRAs), beta-blockers, and / or alpha-blockers. Typically, subjects with resistant hypertension have a systolic blood pressure ≥140 mmHg, and / or a diastolic blood pressure ≥90 mmHg when the subject is at rest. Alternatively, a subject with resistant hypertension typically has a systolic blood pressure of ≥130 mmHg, and / or a diastolic blood pressure of ≥80 mmHg when the subject is at rest. Alternatively, a subject with resistant hypertension typically has a systolic blood pressure of ≥150 mmHg, and / or a diastolic blood pressure of ≥90 mmHg when the subject is at rest. In some cases, resistant hypertension may be resistant essential hypertension. Essential hypertension (essential hypertension / primaryhypertension) is hypertension without a known definite secondary cause.

[0084] The amorphous compound (I), Form G of compound (I), a solid dispersion comprising amorphous compound (I), or a pharmaceutical composition comprising compound (I) described herein can also be used to treat kidney disease (including chronic kidney disease), acute kidney injury, lung disease, and fibrotic disorders, such as fibrotic disorders of the kidney, heart, lung, and liver; wound healing (Sherwood OD (2004) Endocrine Reviews 25(2):205-234); reversal of insulin resistance in diabetic patients (Bonner JS et al. (2013) Diabetes 62(9):3251-3260); various forms of pulmonary hypertension, a disorder caused by arterial stiffness, reduced arterial elasticity, reduced arterial compliance and distensibility, including hypertension, kidney disease, peripheral arterial disease, carotid and cerebrovascular disease (i.e., stroke and dementia), diabetes, microvascular disease leading to end-organ damage, coronary artery disease and heart failure, and preeclampsia.

[0085] In one embodiment, an amorphous form of Compound (I) is provided for use in therapy.

[0086] In one embodiment, an amorphous compound (I) is provided for use in treating a disease or condition selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and refractory hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is refractory hypertension.

[0087] In one embodiment, an amorphous compound (I) is provided for use in the manufacture of a medicament for treating a disease or condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and refractory hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is refractory hypertension.

[0088] In one embodiment, a method of treating a disease or condition selected from the following in a human patient is provided: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, the human patient being in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of amorphous compound (I). In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0089] In one embodiment, Form G of Compound (I) is provided for use in treating a disease or condition selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and refractory hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is refractory hypertension.

[0090] In one embodiment, Form G of Compound (I) is provided for use in the manufacture of a medicament for treating a disease or condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0091] In one embodiment, a method of treating a disease or condition selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension in a human patient in need of such treatment is provided, the method comprising administering to the human patient a therapeutically effective amount of Form G of Compound (I). In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0092] In one embodiment, a solid dispersion comprising amorphous compound (I) as described herein is provided for use in treating a disease or condition selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and refractory hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is refractory hypertension.

[0093] In one embodiment, a solid dispersion comprising amorphous compound (I) as described herein is provided for use in the manufacture of a medicament for treating a disease or condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and refractory hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is refractory hypertension.

[0094] In one embodiment, a method of treating a disease or condition selected from the following in a human patient is provided: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, the human patient being in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of a solid dispersion comprising amorphous compound (I) as described herein. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0095] In one embodiment, a pharmaceutical composition comprising a compound (I) as described herein is provided for use in the treatment of a disease or condition selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0096] In one embodiment, a pharmaceutical composition comprising compound (I) as described herein is provided for use in the manufacture of a medicament for treating a disease or condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0097] In one embodiment, a method of treating a disease or condition selected from the following in a human patient is provided: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, the human patient being in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of a pharmaceutical composition comprising a compound (I) as described herein. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0098] Examples

[0099] In general, all solvents used were commercially available and of analytical grade. Anhydrous solvents were routinely used for the reactions. The phase separator used in these examples was Phase separation column. The compounds described below were named using ChemDraw Professional version 19.0.0.22 from PerkinElmer.

[0100] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements were performed using TG Discovery 550 (TA Instruments, Germany) and DSC Discovery 2500 (TA Instruments, Germany), respectively. 5.152 mg (for TGA) of sample and 2.474 mg (for DSC) of sample were weighed into an aluminum pan. The sample was then heated from room temperature to 350°C (for TGA) and from 0°C to 230°C (for DSC) under a nitrogen purge of 100 mL / min, with a heating rate of 10°C / min. An empty aluminum pan was used as a reference for DSC.

[0101] X-ray powder diffraction analysis is performed according to standard methods, which can be found in, for example, Kitaigorodsky, AI (1973), Molecular Crystals and Molecules, Academic Press, New York; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; or Klug, HP and Alexander, LE (1974), X-ray Diffraction Procedures, John Wiley & Sons, New York.

[0102] XRPD Method A

[0103] X-ray powder diffraction (referred to herein as XRPD) patterns were determined by mounting the sample on a zero-background holder single crystal silicon and developing the sample into a thin layer.

[0104] The X-ray wavelength was 400 nm and the wavelength was 200 nm. Nickel-filtered CuK α Powder X-ray diffraction was recorded with a θ-2θ scanning axis and one-dimensional scanning (radiation, 40 kV, 15 mA). Automatic variable divergence and anti-scattering slits were used, and the sample was rotated at 80 revolutions per minute during the measurement. The sample was scanned using a step size and scanning speed of 0.01° and 1° / min, respectively.

[0105] XRPD Method B

[0106] The samples were mounted on a silicon wafer holder and analyzed using a Bruker D4 Endeavor diffractometer (Cu anode, ) analysis. The samples were measured in reflection geometry in a θ-2θ configuration over a 2° to 40° 2θ scan range with 0.12 sec exposure / 0.02° increment (continuous scan mode). X-rays were generated by a copper long fine focus tube operated at 40 kV and 40 mA.

[0107] The following abbreviations are used

[0108] Aq water based

[0109] B 2 Pin 2 4,4,5,5-Tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1,3,2-dioxaborolane

[0110] Calcd

[0111] DCM Dichloromethane

[0112] DIA Diisopropylamine

[0113] DIAD (E)-Diisopropyl diazene-1,2-dicarboxylate

[0114] DIPEA N-ethyl-N-isopropyl-propan-2-amine

[0115] DMA N,N-Dimethylacetamide

[0116] DMSO Dimethyl sulfoxide

[0117] DPPA Diphenylphosphorylazide

[0118] DTBBPY 4,4'-di-tert-butyl-2,2'-bipyridine

[0119] ESI Electrospray ionization

[0120] Et Ethyl

[0121] EtOAc Ethyl acetate

[0122] EtOH

[0123] h / hr hour

[0124] HRMS High Resolution Mass Spectrometry

[0125] IPA Isopropyl Alcohol

[0126] IPAC Isopropyl Acetate

[0127] [Ir(COD)OMe] 2 Bis(1,5-cyclooctadiene)di-μ-methoxyiridium(I)

[0128] L Liter

[0129] Me Methyl

[0130] MeCN Acetonitrile

[0131] mL milliliters

[0132] MeOH Methanol

[0133] 2-Me-THF 2-Methyltetrahydrofuran

[0134] Min Minutes

[0135] MS mass spectrometry

[0136] MTBE Methyl tert-butyl ether

[0137] NMR Nuclear Magnetic Resonance

[0138] PE Petroleum Ether

[0139] Rt Room temperature

[0140] Sat

[0141] TEA Triethylamine

[0142] TFA Trifluoroacetic acid

[0143] THF Tetrahydrofuran

[0144] TLC Thin layer chromatography

[0145] UHPLC Ultra-High Performance Liquid Chromatography

[0146] Preparation of compound (I)

[0147] Intermediate 1: 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid ethyl ester

[0148]

[0149] A solution of DIA (576 mL, 413 g, 4.08 mol) in THF (3.50 L) was cooled to -50 to -40 °C and a solution of n-BuLi (2.5 M in hexanes, 1.09 kg, 3.92 mol) was added over 3 h, with the temperature maintained between -50 to -40 °C. The solution was stirred at -50 to -40 °C for 3 h, followed by the addition of a solution of ethyl 1,4-dioxaspiro[4.5]decane-8-carboxylate (700 g, 3.27 mol, 2.34 M in THF) over 2 h, with the temperature maintained between -50 to -40 °C. The reaction mixture was stirred at -50 to -40 °C for 4 h, and then iodomethane (603 g, 4.25 mol, 3.04 M in THF) was added over 3 h, with the temperature maintained between -50 to -30 °C. The reaction mixture was further stirred at -50°C to -30°C for 2 h, followed by the addition of aqueous NH 4 Cl (3.50 L, 20% w / w, in H 2 O) with the temperature maintained at <0°C. The solution was warmed to between 15°C and 25°C for 0.5 h, then the layers were separated and the organic layer was washed with aqueous NH 4 Cl (2x3.50 L, 20% w / w, in H 2 The organic reaction solvent was exchanged from THF to EtOH under reduced pressure (with the temperature maintained at <45°C) to give the title compound (2.51 kg, 2.91 mol, 89%) as a 27% w / w solution in EtOH. 1 HNMR (400MHz CDCl 3 )δ1.18(3H,s),1.27-1.22(3H,m),1.47-1.35(2H,m),1.70-1.56(4H,m),2.13(2H,d),3.92(4H,s),4.14(2H,q),MS(ESI):m / z[M+H] + 229.2.

[0150] Intermediate 2: 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid

[0151]

[0152] To a solution of Intermediate 1 (1.13 kg, 1.31 mol, 27% w / w in EtOH) was added EtOH (900 mL) followed by aqueous NaOH (2.63 L, 5.26 mol, 2 M in H 2O), wherein the temperature is maintained between 15°C and 30°C. The solution is heated to between 50°C and 60°C and then maintained for 6 h, then cooled to 15°C to 30°C and the solution is concentrated to 1.8 to 2.4 L under reduced pressure. Hexane (1.50 L) is added and the layers are separated. The aqueous layer is collected and separated by adding aqueous HCl (1.30 L, 5.2 mol, 4 M, in H 2 The pH was adjusted to between 3 and 4 in DCM (2x1.50 L) with the temperature maintained at <20°C. The aqueous solution was extracted with DCM (2x1.50 L) and the combined organic phases were concentrated under reduced pressure (with the temperature maintained at <30°C) to give the title compound (1.43 kg, 1.24 mol, 94%) as a 17% w / w solution in DCM. 1 H NMR (500MHzCDCl 3 )δ1.25(3H,s),1.53(2H,dt),1.62-1.72(4H,m),2.05-2.19(2H,m),3.93(4H,s). MS(ESI):m / z[M+Na] + 223.1.

[0153] Intermediate 3: 1-Methyl-4-oxocyclohexane-1-carboxylic acid

[0154] Method A

[0155]

[0156] To a solution of intermediate 2 (367 g, 250 mmol, 14% w / w in DCM) was added TFA (95.3 mL, 142 g, 1.25 mol). The reaction temperature was maintained between 25°C and 35°C for 20 h and then cooled to between 0°C and 10°C. H 2 O (250 mL) and by adding aqueous NaOH (440 mL, 1.76 mol, 4 M, in H 2 The pH of the aqueous phase was adjusted to between 9 and 10 (in H 2 O). The layers were separated and the aqueous layer was retained and cooled to between 0° C. and 10° C. The reaction mixture was stirred for 2 hours by adding aqueous HCl (73.5 mL, 294 mmol, 4 M in H 2 The pH was adjusted to between 2 and 3 with 1% 4% iodine (in 0.5% ethanol) and then extracted with DCM (3 x 250 mL), and the combined DCM solutions were concentrated to between 150 and 200 mL under reduced pressure. The organic reaction solvent was exchanged from DCM to MeCN under reduced pressure (wherein the temperature was maintained at <40° C.), giving the title compound (119 g, 227 mmol, 91%) as a 30% w / w solution in MeCN. The purified compound 1H NMR (400MHz CDCl3) δ1.39(3H,s),1.73(2H,td),2.43(6H,m). MS(ESI):m / z[M+H] + 157.1.

[0157] Method B

[0158] To a solution of intermediate 2 (6.17 kg, 3.83 mol, 12.4% in DCM) was added TFA (1.42 L, 2.18 kg, 19.13 mol). The reaction temperature was maintained between 25°C and 35°C for 20 h, then cooled to between 0°C and 10°C. Aqueous NaOH (918 g, 22.96 mol, dissolved in 7.66 L H 2 O) was added to the reaction solution and the pH of the aqueous phase was adjusted to between 9 and 11. The layers were separated and the aqueous layer was retained and cooled to between 0°C and 10°C. DCM (3.83 L) was added followed by aqueous HCl (1.52 L, 6.08 mol, 4 M in H 2 The pH was adjusted to between 3 and 4 by adding 4% 4-nitropropene (2.85 kg, 3.32 mol, 87%) of DCM (2x 3.83 L). The organic layer was retained and the aqueous layer was extracted with DCM (2 x 3.83 L) and the combined organic phases were washed with brine (2.3 L, 15% w / w NaCl). The organic phase was concentrated to 2.3 to 3.1 L under reduced pressure. The organic reaction solvent was exchanged from DCM to MeCN under reduced pressure (wherein the temperature was maintained at <45° C.) to give the title compound (2.85 kg, 3.32 mol, 87%) as a 18% solution in MeCN. The purified compound 1 HNMR(400MHz CDCl3)δ1.39(3H,s),1.73(2H,td),2.43(6H,m). MS(ESI):m / z[M+H] + 157.1.

[0159] Intermediate 4: 1-Methyl-4-oxocyclohexane-1-carboxylic acid naphth-1-ylmethyl ester

[0160] Method A

[0161]

[0162] To a solution of intermediate 3 (119 g, 192 mmol, 25% w / w in MeCN) was added 1-chloromethylnaphthalene (32.2 g, 183 mmol), followed by DIPEA (70.0 mL, 49.7 g, 384 mmol) and NaI (2.88 g, 19.2 mmol). The solution was heated to between 50°C and 60°C for 8 h, then cooled to between 0°C and 10°C. H2 O (240 mL) and by adding aqueous HCl (55.0 mL, 220 mmol, 4 M in H 2 The pH of the reaction mixture was adjusted to between 3 and 4 (in 0.5% CO). The reaction mixture was extracted with MTBE (2 x 150 mL), and the combined organic phases were washed with aqueous NaHCO 3 (150 mL, 144 mmol, 8% w / w, in H 2 The organic reaction solvent was exchanged from MTBE to IPA under reduced pressure, with the temperature maintained at <40°C. The temperature of the reaction solution was lowered to between -10°C and 3°C, and the solution was stirred for 2 h, at which time a solid precipitate formed. The solid was filtered and heated to N 2 Dry at 400 °C for 15 h to give the title compound (42.8 g, 144 mmol, 74%) as a white solid; 1 HNMR (500MHz, CDCl 3 )1.30(3H,s),1.65(2H,td),2.16-2.47(6H,m),5.66(2H,s),7.46(1H,dd),7.51-7.63(3H,m),7.78-7.93(2H,m),7.93-8.05(1H,m). MS(ESI):m / z[M+Na] + 319.1.

[0163] Method B

[0164] To a solution of intermediate 3 (2.66 kg, 3.09 mol, 18.2% in MeCN) was added 1-chloromethylnaphthalene (535 g, 2.94 mol), followed by potassium carbonate (513 g, 3.71 mol) and another portion of fresh MeCN (714 mL). The suspension was heated to between 50°C and 60°C for 17 h, then cooled to between 25°C and 30°C. The solid was removed by filtration through a Celite pad and washed with MeCN (2x967 mL). The filtrate was concentrated to 1.45 to 1.93 L under reduced pressure. MeCN was exchanged into isopropanol under reduced pressure, wherein the temperature was maintained at <50°C. The temperature of the mixture was reduced to between 20°C and 25°C, at which time a solid precipitate was formed. The mixture was further cooled to -10°C to 0°C, then the solid was filtered, washed with isopropanol and stirred at N 2 Dry at 400 °C to give the title compound as a white solid (752.6 g, 2.49 mol, 80.5%); 1 H NMR (500 MHz, CDCl 3)1.30(3H,s),1.65(2H,td),2.16-2.47(6H,m),5.66(2H,s),7.46(1H,dd),7.51-7.63(3H,m),7.78-7.93(2H,m),7.93-8.05(1H,m). MS(ESI):m / z[M+Na] + 319.1.

[0165] Intermediate 5: 5-(1,3,6,2-dioxaboroctan-2-yl)-4-fluoro-2-methoxybenzoic acid methyl ester

[0166] Method A

[0167]

[0168] B 2 Pin 2 (362 g, 1.43 mol) was added to the 2 Degas to <1% oxygen in 2-Me-THF (1.75 L). Maintain the solution between 20°C and 30°C and add methyl 4-fluoro-2-methoxybenzoate (250 g, 1.36 mol). Add DTBBPY (1.09 g, 4.10 mmol) and evacuate the reaction vessel and use N 2 Refill until oxygen level < 0.5%. Add [Ir(COD)OMe] 2 (1.35 g, 2.04 mmol), and the reaction vessel was evacuated and heated with N 2 Refill until oxygen level <0.5%. Heat the reaction mixture to between 80°C and 85°C and maintain at this temperature for another 2 h. Cool the reaction mixture to between 0°C and 5°C, then slowly add diethanolamine (428 g, 4.07 mol, 10.9 M in IPA) over a period of 2.5 h while generating H 2 The reaction mixture was stirred at 0°C to 5°C for 2.5 h, then filtered and the solid was washed with 2-Me-THF (3 x 750 mL). 2 Dry at 400 °C for 10 h to give the title compound (356 g, 1.20 mol, 88%) as a white solid; 1 H NMR(500MHz,DMSO-d6)δ2.81-2.89(2H,m),3.14(2H,dq),3.71(2H,ddd),3.7 4(3H,s),3.78(3H,s),3.84(2H,td),6.77(1H,d),7.10(1H,s),7.83(1H,d). MS(ESI):m / z[M+H] +297.1.

[0169] Method B

[0170] B 2 Pin 2 (29.0 g, 114 mmol) and methyl 4-fluoro-2-methoxybenzoate (20.6 g, 109 mmol) were added to the mixture which had been treated with N 2 Degas to <1% oxygen in 2-Me-THF (140 mL). Maintain the solution between 20°C and 30°C before adding DTBBPY (88 mg, 0.33 mmol) and [Ir(COD)OMe] 2 (108 mg, 0.16 mmol), and the reaction vessel was evacuated and heated with N 2 Refill until the oxygen level is <0.5%. The reaction mixture is heated to between 80°C and 85°C and maintained at this temperature for another 3 h. The reaction mixture is cooled to between 0°C and 10°C, and then isopropanol (12.4 mL, 218 mmol) is slowly added while generating H 2 Gas. Seed crystals (100 mg of intermediate 5) were added, followed by diethanolamine (22.84 g, 218 mmol) dissolved in IPA (20 mL) to give a mobile slurry. The slurry was warmed to 20°C to 30°C and the solid was collected by filtration. It was then washed with 2-Me-THF (160 ml) and the solid was concentrated under N 2 Dry at 400 °C for 10 h to give the title compound (29.1 g, 96 mol, 88%) as a white solid; 1 H NMR(500MHz,DMSO-d6)δ2.81-2.89(2H,m),3.14(2H,dq),3.71(2H,ddd),3.7 4(3H,s),3.78(3H,s),3.84(2H,td),6.77(1H,d),7.10(1H,s),7.83(1H,d). MS(ESI):m / z[M+H] + 297.1.

[0171] Intermediate 6: 4-Fluoro-5-hydroxy-2-methoxybenzoic acid methyl ester

[0172] Method A

[0173]

[0174] Intermediate 5 (350 g, 1.18 mol) was reacted with H 2 To the suspension in 2% CO (1.05 L) was added THF (1.75 L) and the reaction mixture was stirred until a clear solution was obtained. (NH4 ) 2 CO 3 (136 g, 1.41 mol), and the heterogeneous mixture was cooled to between 0°C and 10°C. NaBO was added in 10 equal portions over a period of 2 h. 3 .4H 2 O (217 g, 1.41 mol), wherein the reaction temperature is maintained between 0°C and 30°C. The reaction temperature is adjusted to between 20°C and 30°C and maintained for 1 h. NaHSO is added over 3 h 3 An aqueous solution (1.96 L, 942 mmol, 0.48 M, in H 2 O) and the reaction mixture was stirred for an additional 0.5 h. The reaction mixture was filtered, the solids were washed with ethyl acetate (700 mL), and the filtrate and washings were combined to give a two-phase solution. The solution was separated and the remaining organic phase solvent was exchanged from THF / ethyl acetate to MeOH under reduced pressure, with the temperature maintained at <40°C. H was added dropwise over a period of 4 h. 2 O (3.50 L), and the reaction mixture was cooled to between 0°C and 5°C and maintained for 2 h. The reaction mixture was filtered and the collected solid was washed with H 2 O (3 x 350 mL) and dried under hot air at <40 °C to give the title compound (195 g, 974 mmol, 83% yield) as a white solid; 1 HNMR (500MHz, CDCl 3 )δ3.82(3H,s),3.86(3H,s),6.72(1H,d),7.54(1H,d). MS(ESI):m / z[M+H] + 201.0.

[0175] Method B

[0176] Intermediate 5 (32.41 g, 67.3 mmol) and acetic acid (12.13 g, 202 mmol) were dissolved in 2-Me-THF (100 mL) and cooled to between 0°C and 10°C. Hydrogen peroxide solution (30% w / w, 9.16 g, 80.8 mmol) was added over 2 hours, and then the reaction temperature was adjusted to between 20°C and 30°C and maintained for 18 hours. 2 S 2 O 3 .5H 2 The mixture was quenched with an aqueous solution of O (20% w / w, 50 mL) and the phases were separated. The aqueous phase was discarded and the organic phase was washed with Na 2 S 2 O 3 .5H 2O aqueous solution (5% w / w, 100mL) washed twice. The organic phase was concentrated to 60mL under reduced pressure, followed by 2-Me-THF (100mL) for another 2 vacuum distillations to give a dissolved solution (at 35 ° C to 45 ° C). Nucleation was controlled by adding seed crystals (100mg of intermediate 6), followed by slowly adding 300mL of n-heptane over 5 hours. The resulting slurry was adjusted to between 20 ° C and 30 ° C, stirred overnight, and then filtered. The collected solid was washed with n-heptane (2x60mL) and dried to give the title compound (12.5g, 62.5mmol, 93% yield) as a white solid; 1 HNMR (500MHz, CDCl 3 )δ3.82(3H,s),3.86(3H,s),6.72(1H,d),7.54(1H,d). MS(ESI):m / z[M+H] + 201.0.

[0177] Intermediate 7: (1R,2R,3S,4S)-3-(Methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

[0178]

[0179] To a solution of (3aR,4R,7S,7aS)-3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran-1,3-dione (387 g, 2.36 mol) in toluene (4.64 L) was added quinidine (843 g, 2.60 mol) followed by toluene (774 mL). The reaction mixture was cooled to between -10°C and -5°C and MeOH (227 g, 286 mL, 7.08 mol) was added dropwise over 1.5 h and then maintained between -10°C and -5°C for 14 h. The reaction mixture was warmed to between -5°C and 5°C, maintained for 2 h, and then filtered. The solid was washed with toluene (3 x 387 mL), and the filtrate and washings were combined and cooled to between 0°C and 10°C. In a separate container, an aqueous solution of HCl (590 mL, 7.08 mol, 12 M, in H 2 O) and NaCl (1.24 kg, 21.2 mol) were added to H 2 O (6.39 L) and the resulting solution was added dropwise to the main reaction vessel, where the reaction solution was maintained at <10°C. The reaction mixture was warmed to between 10°C and 20°C for 0.5 h and then filtered. The solid was washed with toluene (1.94 L), the filtrate and washings were combined, and the two phases were separated. The organic phase was washed with aqueous NaCl (3.87 L, 20% w / w, in H 2O) and stored at <5°C to give the title compound (6.19 kg, 1.83 mol, 78%) as a 5.9% w / w solution in toluene; the purified compound 1 HNMR (400MHzDMSO-d 6 )δ1.25-1.32(1H,m),1.95(1H,d),2.48-2.50(2H,m),2.93(2H,s),3.51(3H,s),6.15-6.22(2H,m),12.21(1H,s). MS(ESI):m / z[M+Na]+219.1.

[0180] Intermediate 8: (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester hydrochloride

[0181]

[0182] To a solution of intermediate 7 in toluene (6.19 kg, 5.9% w / w, 1.85 mol) between -5°C and 5°C was added TEA (307 mL, 223 g, 2.22 mol) followed by DPPA (538 g, 1.94 mol), wherein the reaction solution was maintained at <5°C. The reaction mixture was stirred between -5°C and 5°C for 4 h, then TEA (767 mL, 557 g, 5.55 mol) was added, followed by citric acid (352 g, 1.85 mol). The reaction mixture was stirred between -5°C and 5°C for 6 h, then H 2 O (3.6 L), wherein the reaction solution was maintained at <10° C. The two-phase reaction solution was stirred for 0.5 h, the phases were separated and the organic phase was washed with H 2 O (3.6 L) and aqueous NaCl (3.6 L, 15% w / w in H 2O) and then stored between 2°C and 8°C to give (1S,2S,3R,4R)-3-(azidocarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester (Intermediate 9) as a solution in toluene, which was used directly in the next step. Intermediate 9 as a solution in toluene between 2°C and 8°C was added to a reactor containing toluene (1.80 L) between 70°C and 80°C over 2h, maintaining the reaction temperature at <80°C. The resulting solution was stirred for 1h and then cooled to between 20°C and 30°C. The organic reaction solvent was exchanged from toluene to 1,4-dioxane under reduced pressure, maintaining the temperature at <50°C, giving (1S,2S,3R,4R)-3-isocyanatobicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester (Intermediate 10) as a solution in 1,4-dioxane, which was used directly in the next step. To a solution of intermediate 10 in 1,4-dioxane between 10°C and 20°C was added HCl (420 mL, 1.68 mol, 4 M in 1,4-dioxane), followed by H 2 O (360mL, 1.68mol, 4.67M in 1,4-dioxane). The reaction mixture was warmed to between 25°C and 35°C and maintained for 16h. MTBE (1.65L) was added dropwise, and the reaction mixture was filtered, the solid was washed with MTBE / 1,4-dioxane (1:1, 660mL) and MTBE (660mL), and then dried under vacuum between 30°C and 40°C to give the title compound (258g, 1.27mol,>99%ee, 75%) as a white solid; 1 HNMR(400MHz,DMSO-d6)δ1.45(1H,d),2.04(1H,d),2.52-2.67(1H,m),2.94-3 .10(2H,m),3.19(1H,d),3.65(3H,s),6.21(1H,m),6.30(1H,m),8.34(3H,s). MS(ESI):m / z[M+H] + 168.1.

[0183] Intermediate 11: (1r,4r)-4-hydroxy-1-methylcyclohexane-1-carboxylic acid naphth-1-ylmethyl ester

[0184]

[0185] To 20℃ to 30℃ Na 2 HPO 4 .12H 2 O (8.25 g, 23.0 mmol), NaH 2 PO 4 (0.55 g, 4.48 mmol) and MgCl2 (0.11 g, 1.10 mmol) in H 2 To a solution in 450 mL of 1% ethanol (550 mL), intermediate 4 (50.0 g, 169 mmol) was added (in solution in IPA (450 mL)). The pH of the reaction solution was adjusted to between 7.3 and 7.8 using 6 M HCl, and NAD+ (0.66 g, 1.00 mmol) was added, followed by ADH-230 (7.50 g, 0.15 wt%). ADH-230 is an alcohol dehydrogenase (catalog number ADH-230) obtained from Johnson Matthey PLC, UK. The reaction mixture was then maintained at 33° C. to 37° C. for 18 h, and then concentrated under reduced pressure to between 300 and 400 mL, with the temperature maintained at <45° C. NaCl (150 g), (20.0 g, 0.4 wt%) and MTBE (500 mL) were added and the reaction was maintained for 0.5 h. The mixture was filtered and the filter cake was washed with MTBE (250 mL). The combined filtrate was separated and the aqueous phase was extracted with MTBE (500 mL). The organic phases were combined and washed with H 2 O (250 mL), then the solvent was exchanged to THF under reduced pressure (wherein the temperature was maintained at <45° C.) to give the title compound as a solution in THF (138 g, 33% w / w%, >99:1 trans:cis, <0.1% IPA, 92% yield), which was used directly in the next step. The purified compound 1 H NMR (500 MHz, CDCl 3 )δ1.21(3H,s),1.48-1.58(2H,m),1.62-1.77(4H,m),1.82-1.93(2H,m),3.74-3.77(1H,m),5. 57(2H,s),7.41-7.48(1H,m),7.48-7.57(3H,m),7.85(1H,d),7.87-7.91(1H,m),7.98(1H,d). MS(ESI):m / z[M+Na]+321.1.

[0186] Route B

[0187] Under nitrogen, a solution of lithium tri-sec-butyl borohydride (1.06 g, 5.6 mmol) in THF (5 mL) was added dropwise over a 1 min period to a stirred solution of intermediate 4 (1.00 g, 3.37 mmol) in THF (10 mL) cooled to -78 ° C. The resulting solution was stirred at -78 ° C for 2 h. The reaction mixture was quenched with 0.1 M HCl (10 mL) (at -78 ° C) and then extracted with EtOAc (3x50 mL). The organic layers were combined and purified by Na 2 SO 4 Dried, filtered and evaporated. The residue was purified by preparative TLC (EtOAc / PE, 1:3) to give the title compound (0.488 g, 48.5%) as a light yellow gum. The isolated material had a cis / trans ratio of 3:100. 1 HNMR (400MHz, CDCl 3 )δ1.21-1.25(s,3H),1.37-1.49(m,1H),1.49-1.61(m,2H),1.61-1.74(m,4H),1.83-1.95(m,2H),3.74-3.83 (dq,1H),5.57-5.61(s,2H),7.43-7.54(dd,1H),7.50-7.61(m,3H),7.84-7.94(m,2H),7.97-8.04(m,1H).). MS(ESI):m / z[M+Na] + 321.

[0188] Intermediate 12: methyl 4-fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate

[0189]

[0190] To a solution of intermediate 11 in THF (736 g, 34% w / w, 839 mmol) was added THF (156 mL), PPh 3 (248 g, 944 mmol) and intermediate 6 (140 g, 699 mmol). The solution was heated to 30°C and DIAD (184 g, 909 mmol) was added dropwise over 1 h, with the reaction temperature maintained at <40°C. The solution was kept between 30°C and 40°C for 1 h, then cooled to between 20°C and 30°C, followed by the addition of an aqueous solution of NaCl (700 mL, 20% w / w, in H 2 O). The layers were separated and the crude solution of the title compound in THF was used directly in the next step. 1 H NMR (500 MHz, CDCl3 )δ1.17(3H,s),1.20-1.30(2H,m),1.58(2H,qd),1.88-1.98(2H,m),2.29(2H,d),3.84(3H,s),3.88 (3H,s),4.05(1H,tq),5.61(2H,s),6.72(1H,d),7.43-7.58(5H,m),7.82-7.94(2H,m),8.00(1H,d). MS(ESI):m / z[M+Na]+503.2.

[0191] Intermediate 13: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid

[0192]

[0193] To a crude solution of intermediate 12 used directly from the previous step was added LiOH.2H 2 O in water (88.0 g, 2.10 mol, in 525 mL of H 2 O), wherein the reaction temperature is maintained at <10°C. The solution is warmed to between 15°C and 30°C and stirred vigorously for 16h. IPAC (1.68L) is added, and the solution is cooled to between 0°C and 10°C, followed by dropwise addition of H 3 PO 4 (1.26L, 2.52M, 2M, in H 2 O) (wherein the reaction temperature was maintained at <10°C) to give a solution with a pH between 4.0 and 5.0. The organic layer was separated and washed with an aqueous solution of NaCl (700 mL, 20% w / w, in H 2 The THF was removed under reduced pressure with the temperature maintained at <50°C and IPAC (4.20 L) was added to give the title compound in IPAC which was used directly in the next step. 1 H NMR (500 MHz, CDCl 3 )δ1.18(3H,s),1.22-1.36(2H,m),1.58(2H,qd),1.95(2H,dt),2.29(2H,d),4.02(3H,s),4.19(1H,td), 5.60(2H,s),6.82(1H,d),7.46(1H,dd),7.49-7.62(3H,m),7.78(1H,d),7.82-7.94(2H,m),7.99(1H,d).

[0194] Intermediate 14: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid cyclohexyl ammonium salt

[0195]

[0196] To a crude solution of intermediate 13 from the previous step, used directly, in IPAC between 50 and 55 °C was added a solution of cyclohexylamine (280 mL, 699 mmol, 2.5 M in IPAC) dropwise over 3 h. The heterogeneous slurry was stirred between 50 and 55 °C for 0.5 h, then between 40 and 45 °C for another 1 h. The reaction mixture was filtered and the solids were washed with IPAC (3 x 0.98 L) preheated to between 40 and 45 °C and stirred at 45 °C under N 2 The mixture was dried under reduced pressure for 16 h. MeOH (3.64 L) was added to the dried collected solid and the mixture was heated to between 55° C. and 56° C. H was added dropwise over 1 h. 2 O (1.58 L), and the mixture was stirred for 1 h before being cooled to between 0°C and 5°C over 3 h. The heterogeneous slurry was maintained for an additional 1 h before being filtered and washed with 5:3 MeOH:H 2 O (2 x 750 mL), and the solid was stirred under N 2 and dried at 45 °C for 16 h to give the title compound as a white solid (332 g, 85% from methyl 4-fluoro-5-hydroxy-2-methoxybenzoate); 1 HNMR (500MHz, CDCl 3 )δ0.96(1H,ddt),1.03-1.36(6H,m), overlap 1.14(3H,S),1.46-1.7(5H,m),1.91(4H,dt),2.26(2H,d),2.81(1H,t),3.76( 3H,s),4.03(1H,tt),5.59(2H,s),6.65(1H,d),7.37-7.49(2H,m),7.49-7.6(3H,m),7.81-7.93(2H,m),7.98(1H,d). MS(ESI):m / z[M+Na]+489.2.

[0197] Intermediate 15: (1S,2S,3R,4R)-3-(4-fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamido)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester

[0198]

[0199] To a solution of intermediate 14 (149 g, 264 mmol) in DCM (750 mL) between 15°C and 30°C was added H 2 O (450 mL), followed by slow addition of HCl (300 mL, 1 M in H 2 The two-phase solution was stirred for 0.5 h, then separated, and the organic phase was washed with HCl (750 mL, 0.2 M in H 2 O) and then washed with H 2 The organic solution was concentrated under reduced pressure (with the temperature maintained below 30° C.) to dryness to <0.1% H 2 O. The solution was diluted with DCM (450 mL) to bring the total volume to 750 mL, then intermediate 8 (59.3 g, 291 mmol) was added to give a heterogeneous slurry. To this mixture was added DIPEA (137 g, 1.06 mol), followed by T3P (252 g, 397 mmol, 50% w / w in EtAOc), and the solution was stirred for 1 h. The solution was cooled to between 0°C and 10°C, then H 2 O (750 mL), and then stirred for another 0.5 h. The two phase solution was separated, and the organic phase was washed with H 2 O (2 x 750 mL), then the solvent was exchanged to THF under reduced pressure to give the title compound in THF, which was used directly in the next step. 1 H NMR (500 MHz, CDCl 3 )δ1.16(3H,s),1.25(2H,td),1.49-1.69(3H,m),1.92-2.01(2H,m),2.04-2.1(1 H,m),2.28(2H,d),2.71(1H,dd),2.83(1H,s),2.92-3.05(1H,m),3.61(3H,s),3 .93(3H,s),4.17(1H,td),4.46(1H,td),5.60(2H,s),6.25(2H,ddd),6.72(1H,d ),7.46(1H,dd),7.48-7.6(3H,m),7.81-7.95(3H,m),7.99(1H,d),8.60(1H,d). MS (ESI): m / z [M+H] + 616.3.

[0200] Intermediate 16: (1S,2S,3R,4R)-3-(4-fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamido)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

[0201]

[0202] A crude solution of intermediate 15 from the previous step in THF (750 mL) was cooled to between 0°C and 5°C. LiOH.2H 2 O in water (27.7 g, 661 mmol, in 150 mL of H 2 O) and the solution was maintained for 36 h. The pH of the solution was adjusted to 2 with slow portionwise addition of HCl (0.5 M, 1.45 L, 2.90 mol) and maintained between 0°C and 5°C for 1 h. The heterogeneous slurry was filtered and the solid was washed with 1:3 MeOH:H at 0°C. 2 O (600 mL), and the solid was heated at 45 °C under N 2 The crude product (150 g) was slurried in IPAC (1.13 L) at 60°C to 65°C for 0.5 h. The heterogeneous mixture was cooled to between 0°C and 5°C over 3 h, then stirred for another 1 h, and then filtered. The collected solid was washed with IPAC at 0 and 5°C (2 x 300 mL), then stirred at N 2 Dry at 45 °C for 12 h to give the title compound as a white solid (127 g, 82% from Intermediate 14); 1 HNMR (500MHz, CDCl 3 )δ1.16(3H,s),1.2-1.35(2H,m),1.50-1.69(3H,m),1.89-2.08(3H,m),2. 27(2H,ddd),2.72(1H,dd),2.80(1H,s),3.06(1H,s),3.75(3H,s),4.15(1H ,tt),4.43-4.54(1H,m),5.59(2H,s),6.24(2H,ddd),6.53(1H,d),7.45(1 H,dd),7.47-7.58(3H,m),7.8-7.9(3H,m),7.94-8.05(1H,m),8.59(1H,d). MS(ESI):m / z[M+H] + 602.3.

[0203] Intermediate 17: (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1R,2R,3S,4S)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-5-en-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid naphth-1-ylmethyl ester

[0204]

[0205] To a solution of DIPEA (6.45 g, 49.9 mmol) in DCM (300 mL) between 0°C and 5°C was added intermediate 16 (30.6 g, 49.9 mmol) followed by (1-methylcyclobutyl)methanamine hydrochloride (8.63 g, 62.4 mmol). DIPEA (25.8 g, 200 mmol) was added dropwise with the temperature maintained between 0°C and 5°C followed by T3P (50.8 g, 79.8 mmol, 50% w / w in EtAOc) over 0.5 h. The solution was warmed to between 15°C and 25°C and stirred for 1 h before H was added dropwise. 2 O (150 mL), wherein the temperature was maintained below 30°C. The two phases were separated and the organic phase was washed with H 2 O (2 x 150 mL), then the solvent was exchanged to EtOH under reduced pressure to give the title compound as a crude solution in EtOH (128 g, 26% w / w, 96% yield), which was used directly in the next step. 1 HNMR (500MHz, CDCl 3 )δ0.98(3H,s),1.16(3H,s),1.21-1.29(2H,m),1.51-1.66(5H,m),1.66-1.76(3H,m),1.76-1.82 (1H,m),1.88-2.02(2H,m),2.26(3H,dd),2.40(1H,dd),2.80(1H,s),3.00(1H,s),3.05(1H,dd),3 .21(1H,dd),3.93(3H,s),4.06-4.2(1H,m),4.39(1H,td),5.60(2H,s),5.64(1H,t),6.19-6.38(2 H,m),6.70(1H,d),7.46(1H,dd),7.49-7.62(3H,m),7.75-7.93(3H,m),8.00(1H,d),8.66(1H,d). MS(ESI):m / z[M+H] + 683.3.

[0206] Compound (I): (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0207]

[0208] To a solution of intermediate 17 in EtOH (206 g, 19% w / w, 57.9 mmol) was added EtOH (385 mL) followed by 10 wt% Pd / C (3.96 g, 5% w / w). The vessel was heated with N 2 Purge six times, followed by H 2 Purge six more times. 2 The pressure was increased to 0.4 MPa, and the reaction solution was stirred between 20°C and 30°C for 20 h. 2 Completely replace H 2 Atmosphere, then the reaction mixture is filtered and the solid is washed with EtOH (3x 80mL).Carry out the same operation of the second batch, and merge the EtOH solution collected, to give a single solution.Under reduced pressure, the solvent is exchanged into EtOAc, wherein the temperature is maintained at less than 45 ° C.EtOAc solution (280mL) is heated to 70 ° C and 75 ° C for 0.5h, then cooled to between 40 ° C and 45 ° C, and n-heptane (475mL) is added dropwise through 0.5h. The mixture is stirred for 0.5h and then cooled to between 20 ° C and 25 ° C through 2h, and then kept for 2h.The heterogeneous slurry is filtered, then the solid is washed twice with 1:2EtOAc / normal heptane (160mL), then dried 20h at less than 45 ° C, to give the thick title compound (55.7g, 87%) as a white solid.

[0209] Part 1: The crude title compound (2.50 g, 4.59 mmol) was dissolved in EtOH (15.0 mL). The temperature of the solution was maintained at 25.0°C ± 2.0°C during the dropwise addition of water (7.50 mL), during which time a precipitate formed. The heterogeneous slurry was stirred for an additional 1.0 h and then collected by filtration. The solid was washed with a (2:3) mixture of EtOH / water (2 x 5.00 mL), collected and precipitated under N 2 Drying at 400 °C gave the title compound as a white solid (1.80 g, 72%). This material was characterized as Form A and used as seed crystals following the procedure described in Section 2.

[0210] Part 2: The crude title compound (50.0 g, 91.8 mmol) was dissolved in EtOH (350 mL) and then passed through a filter. EtOH (100 mL) was added to a vessel and then passed through a filter to give a combined EtOH solution. H was added slowly over 0.5 h. 2 O (150 mL), the temperature of the solution was maintained at 25.0°C ± 2.0°C. The solution was stirred for another 0.5 h, then the seed material from Part 1 (0.005 g, 0.1% w / w) was added. The solution was maintained for 6 h, then cooled to 20.0°C ± 0.5°C over 2 h, then maintained for another 6 h. H2 O (150 mL), and the mixture was kept for another 2 h before filtering. EtOH (45 mL) and H 2 O (30 mL) was added to the container and then used to wash the filter cake. The solid was collected and heated at below 45 °C under N 2 Dry at 400 °C for 12 h to give the title compound Form A (42.2 g, 85%) as a white solid; 1 H NMR (500 MHz, CDCl 3 )δ0.97(3H,s),1.12-1.42(5H,m),overlapping1.25(3H,s),1.43-1.82(10H,m),1.92-2.1(3H,m),2.25(3H,dd),2.51(2H,dd),2.96(1H,dd),3.18(1H,dd),3.92(3H,s),4.12-4.28(1H,m),4.41(1H,t),5.81(1H,t),6.70(1H,d),7.86(1H,d),8.60(1H,d). For C30H42FN2O6, HRMS(ESI)m / z[M+H] + Calculated value: 545.3022, found value: 545.3019. The XRPD pattern (XRPD method A) is shown in Figure 1 middle.

[0211] Example 1

[0212] Amorphous (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0213] A solution of (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (3915 mg, 7.19 mmol) in 1,4-dioxane / water 80:20 (67 mL) was frozen in liquid nitrogen and freeze-dried. The mixture was dried overnight in a desiccator (ChristAlpha2-4LD) to yield amorphous (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (3915 mg, 7.19 mmol) as a white solid. The X-ray powder diffraction pattern (XRPD Method A) of this amorphous form is shown in Figure 2 middle.

[0214] Example 2

[0215] Solid dispersion of amorphous (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0216] Polymers compatible with compound (I) were screened to select suitable polymers for solid dispersion. The following polymers were found to be stable at 50°C and 40°C / 75% relative humidity (RH) for more than 4 weeks with no signs of crystallization and were found to be suitable for 10%, 20% and 40% drug loading;

[0217] · 30 (poly(1-vinyl-2-pyrrolidone), obtained from BASF Pharma)

[0218] · VA 64 (copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate in a mass ratio of 6:4, obtained from BASF Pharmaceuticals)

[0219] · (Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, obtained from BASF Pharmaceuticals)

[0220] · L100-55S (methacrylic acid-ethyl acrylate copolymer (1:1), obtained from Evonik Industries AG)

[0221] Polyacrylic acid

[0222] · HPMC AS-LF (hydroxypropyl methylcellulose acetate succinate, obtained from Shin-Etsu Chemical Co., Ltd.)

[0223] · HPMC AS-HF (hydroxypropyl methylcellulose acetate succinate, obtained from Shin-Etsu Chemical Co., Ltd.)

[0224] Cellulose acetate phthalate

[0225] Polyvinyl acetate phthalate

[0226] Three amorphous solid dispersions of Compound (I) with drug loadings of 20%, 30% and 40% w / w were prepared according to the following method:

[0227] 1. Weigh compound (I) in a glass container and add VA 64 (copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate in a mass ratio of 6:4, obtained from BASF Pharmaceuticals). The powders were mixed in a blender at 32 rpm for 5 minutes.

[0228] 2. First clean the hot melt extruder with molten beads, then extrude the mixture. Extrude the solid dispersion with 20% drug loading at 200°C, and collect the filaments in a clean glass container. Extrude the solid dispersion with 30% drug loading at 200°C, and the solid dispersion with 40% drug loading at 210°C.

[0229] 3. The collected filaments were then manually ground using a laboratory grinder.

[0230] The samples were stabilized at 40°C / 75% relative humidity (RH) (open container) and 50°C (closed container, ambient RH) for 4 weeks and XRPD of the samples was recorded weekly. Figure 3XRPD diffractograms of each sample after formation and after storage for 4 weeks at 40°C / 75% relative humidity (RH) (open container) and 4 weeks at 50°C (closed container, ambient RH) are shown (XRPD Method B). The XRPD patterns indicate that no crystallization of compound (I) occurred after storage of the amorphous solid dispersion for 4 weeks at 40°C / 75% relative humidity (RH) (open container) and 4 weeks at 50°C (closed container, ambient RH). Dissolution experiments of solid dispersions with 30% drug loading showed improved dissolution in simulated gastric fluid at pH 1.8 and fasting state simulated intestinal fluid (FaSSIF) at pH 6.5 compared to particles based on crystalline Form A material of compound (I).

[0231] Example 3

[0232] Form G of (1S,4S)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0233] Upon heating Form A of (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid, DSC exhibited an endothermic transition with an observed onset at around 43.9°C. This endothermic peak was assigned as a solid-solid phase transition, and the phase produced at temperatures above the completion of the endothermic peak observed at around 43.9°C was designated Form G. This new anhydrous phase Form G persisted until the last endothermic peak in the DSC, which corresponded to a melting onset of 217.5°C. The DSC output for Form A / Form G is shown in Figure 4 The TGA analysis of Form A / Form G is shown in Figure 5 middle.

[0234] Biological and physicochemical data of compound (I)

[0235] RXFP1 Hu cAMP (Test A)

[0236] To screen for modulators of hRXFP1, an assay to identify compounds that stimulate cAMP production via the Gs-coupled hRXFP1 receptor was used. cAMP was detected using a large number of cAMP HiRange HTRF kits (obtained from CisBio Bioassays, France; catalog number 62AM6PEJ) according to the manufacturer's recommendations. The HTRF method is a competitive immunoassay between native cAMP produced by cells and cAMP labeled with the dye d2. Tracer binding is visualized by antibodies labeled with cryptates against cAMP, and therefore the signal is inversely proportional to the amount of cAMP produced.

[0237] Preparation of assay reagents:

[0238] Assay buffer: HBSS (ThermoFisher, 14065) with 5 mM Hepes (ThermoFisher, 15630), pH 7.4, containing 0.1% BSA (Sigma, A8806)

[0239] Cells: Jump-In cells stably transfected with human RXFP1 TM T-REx TM CHO-K1 cells (Thermo Fisher Scientific). Cells were induced to express human RXFP1 by treating with 10 ng / ml doxycycline for 24 h. They were then cryopreserved for long-term storage. At the beginning of each experiment, cells were thawed, washed with PBS and resuspended in assay buffer to 1.875*10^5 cells / ml

[0240] cAMP Standard: Dilute the stock standard cAMP provided in the CisBio kit in assay buffer to a maximum final concentration of 2.8 μM in the assay.

[0241] HTRF detection reagents: cAMP-d2 and anti-cAMP cryptate reconstituted according to the CisBio instructions were diluted 1:40 in the lysis buffer provided in the HTRF-kit.

[0242] Step-by-step procedure for running the assay:

[0243] 1. 40 nL of test compound dissolved in DMSO was acoustically dispensed (Labcyte Echo) into a white 384-well plate (Greiner; 784075), sealed and stored at room temperature until assayed.

[0244] 2. On the day of the assay, add 40 nL of 200 nM Relaxin-2 in DMSO (1 nM final concentration) to the 100% control wells and 40 nL of DMSO to the 0% wells using an Echo acoustic dispenser.

[0245] 3. Using a Multidrop Combi (Thermo Fisher Scientific), add 4 μL of assay buffer containing 1 mM phosphodiesterase-blocking IBMX (0.5 mM final concentration).

[0246] 4. Add 4 μL of cell solution (1.875*10^5 cells / ml) using Multidrop Combi to obtain 750 cells / well.

[0247] 5. Incubate at room temperature for 45 minutes.

[0248] 6. Add 4 μL of cAMP-d2 in lysis buffer using the Multidrop Combi.

[0249] 7. Add 4 μL of anti-cAMP cryptate in lysis buffer using Multidrop Combi

[0250] 8. Incubate at room temperature for 2 hours

[0251] 9. Homogeneous time-resolved fluorescence (HTRF) signals were detected using an Envision (PerkinElmer) or Pherastar (BMG Labtech) reader (λex = 340 nm, λem = 665 and 615 nm).

[0252] Using a cAMP standard curve, the HTRF data were converted to the amount of cAMP produced in the sample, which was then used to calculate the concentration response. The concentration response data were fit using a four-parameter logistic fit, the Hill equation. The results from the assay are reported in Table 1 as EC 50 (μM) and S inf (%).

[0253] EC 50 It is defined as the concentration at which the stimulatory activity reaches 50% of its maximal level. When multiple assays were run for the same compound, the geometric mean is reported.

[0254] S inf is the activity level, potency, fitted at infinite concentration of the test compound. To facilitate comparison of potency data, potency was normalized to the % effect of the response stimulated by a saturating concentration of relaxin (1 nM). When multiple assays were run for the same compound, the arithmetic mean was reported.

[0255] Human plasma protein binding (Test B)

[0256] The assay was performed according to the human plasma protein binding assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), 157-179, pp. 167-170. The data are reported in Table 1 as the unbound fraction (f u ) (% free). When multiple assays were run for the same compound, the arithmetic mean is reported.

[0257] Human liver microsome stability (Test C)

[0258] The assay was performed according to the human liver microsomal stability assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), 157-179, pp. 170-174. Data are reported in Table 1 as CL int (μl / min / mg protein). When multiple assays were run for the same compound, the arithmetic mean is reported.

[0259] Human hepatocyte stability (Test D)

[0260] The metabolic stability of compounds in human hepatocytes was assessed using the following protocol:

[0261] 1. Prepare 10 mM stock solutions of compound and control compounds in appropriate solvent (DMSO). Place incubation medium (L-15 medium) in a 37°C water bath and allow to warm for at least 15 minutes before use.

[0262] 2. Add 80 μL of acetonitrile to each well of a 96-well deep well plate ("Quench Plate").

[0263] 3. In a new 96-well plate, dilute the 10 mM test and control compounds to 100 μM by combining 198 μL of acetonitrile and 2 μL of the 10 mM stock solution.

[0264] 4. A vial of cryopreserved (below -150°C) human hepatocytes (LiverPool from Bioreclamation IVT) was placed in the TM 10-Donor human hepatocytes (Product No. S01205)) were removed from storage, ensuring that the vials remained cold until thawing. As quickly as possible, the cells were thawed by placing the vials in a 37°C water bath and gently shaking the vials. The vials were kept in the water bath until all ice crystals were dissolved and no longer visible. After thawing was complete, the vials were sprayed with 70% ethanol and transferred to a biosafety cabinet.

[0265] 5. Open the vial and pour the contents into a 50 mL conical tube containing lysis medium. Place the 50 mL conical tube in a centrifuge and spin at 100 g for 10 minutes (room temperature). After the spin is complete, aspirate the lysis medium and resuspend hepatocytes in enough incubation medium to produce approximately 1.5 × 10 6 cells / mL.

[0266] 6. Use Vision, count cells and determine viable cell density. Cells with poor viability (<80% viability) are unacceptable for use. Dilute cells with incubation medium to reach 1.0 × 10 6 The working cell density was 10 viable cells / mL.

[0267] 7. Transfer 247.5 μL of hepatocytes to each well of a 96-well cell incubation plate. Place the plate on an Eppendorf Thermomixer Comfort plate shaker to allow the hepatocytes to warm for 10 minutes.

[0268] 8. Add 2.5 μL of 100 μM test compound or control compound to the incubation wells containing cells to initiate the reaction.

[0269] 9. Incubate the plate at 37°C and 900 rpm on an Eppendorf Thermomixer Comfort plate shaker. At 0.5, 5, 15, 30, 45, 60, 80, 100 and 120 min, transfer 20 μL of the incubated mixture to a separate "quench plate" and mix the samples by vortexing for 2 min.

[0270] 10. Centrifuge the quenched plates at 4,000 rpm for 20 minutes. Transfer 30 μL of supernatant of each compound to a 96-well assay plate. Combine the 4 compounds in one box. Then dilute the combined sample by adding 180 μL of purified water. All incubations were performed in duplicate.

[0271] All calculations were performed using Microsoft Excel. Peak areas were determined from the extracted ion chromatograms. In vitro intrinsic clearance (IVCl) of the parent compound was determined by regression analysis of the Ln parent percent disappearance versus time curve. int , in μL / min / 10 6 In vitro intrinsic clearance (in vitro Cl int , in μL / min / 10 6 The cell counts (per cell) are reported in Table 1 and determined from the slope values ​​using the following equation:

[0272] In vitro Cl int =kV / N

[0273] V = incubation volume (0.25 mL);

[0274] N = number of hepatocytes per well (0.25×10 6 cells)

[0275] When multiple assays were run for the same compound, the geometric mean was reported.

[0276] Rat hepatocyte stability (Test E)

[0277] The assay was performed according to the rat hepatocyte stability assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), pp. 170-174, 157-179. The data are reported in Table 1 as mean Cl int (μl / min / 10 6 When multiple assays were run for the same compound, the geometric mean is reported.

[0278] Solubility (pH 7.4) (Test F)

[0279] The assay was performed according to the solubility assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), pp. 164-167, 157-179. The data are reported as solubility (μM) in Table 1. When multiple assays were run for the same compound, the arithmetic mean was reported.

[0280] Table 1 - Determination data of compound (I)

[0281]

[0282] Human RXFP1 cGMP Production Assay (Test G)

[0283] To profile the RXFP1 agonist activity of compounds with respect to cGMP production, the Green GENIecGMP assay (Montana Molecular; Cat. No. D800G) was used. The assay is based on a mNeonGreen fusion protein fluorescent biosensor delivered to mammalian cells in a BacMam vector. When cGMP binds to the biosensor, fluorescence decreases.

[0284] Preparation of assay reagents:

[0285] Assay buffer: DPBS (Gibco; 14040133) containing 0.1% BSA (Sigma; A8806)

[0286] Cells: HEK293s cells stably transfected with human RXFP1 in pIRESneo3 were used. The cells were cultured in DMEM medium (Gibco; 31966) supplemented with 10% FBS at 0.8 mg / mL to maintain RXFP1 expression.

[0287] Step-by-step protocol for running the assay:

[0288] Day 1

[0289] 1. The cells were split one day before transduction and seeded at 63,000 cells / cm2 in DMEM medium containing 10% FBS but without antibiotics in tissue culture flasks.

[0290] Day 2

[0291] 2. After washing with PBS, the cells were detached using accutase (Gibco; 1737341), resuspended in culture medium and collected in a 50 mL tube.

[0292] 3. The cells were counted using CEDEX (Innovatis) and diluted to 267,000 cells / mL with culture medium.

[0293] 4. Prepare viral transduction mastermix by mixing reagents in the following ratios (for a single well):

[0294] 6 μL GENIe BacMAM vector

[0295] 0.2 μL 500 mM sodium butyrate

[0296] 13.8 μL DMEM medium containing 10% FBS

[0297] 20 μL total volume

[0298] 5. Combine cells and transduction mastermix at a ratio of 30 μL cells to 20 μL mastermix for a single well.

[0299] 6. In a black poly-D-lysine-coated μclear 384-well plate (Greiner; 781946), dispense 50 μL of the cell transduction mixture from above into each well.

[0300] 7. Incubate the plate in the dark at 37°C, 5% CO2 for 24 h.

[0301] Day 3

[0302] 8. Remove the culture medium from the plate using Bluewasher (BluCatBio).

[0303] 9. Add 20 μL of assay buffer using a Multidrop Combi (Thermo Fisher Scientific).

[0304] 10. Incubate the plate at room temperature in the dark for 30 min prior to assay.

[0305] 11. Assay plate using FLIPR Tetra (Molecular Devices): 10 μL of compound diluted in assay buffer was added to each well by FLIPR Tetra and green fluorescence was measured over time for up to 3 h.

[0306] Data were processed using Screener software (Genedata AG). After subtracting background fluorescence (before compound addition), the area under the curve from 0 to 90 min after compound addition was used to calculate the response. The concentration response data were fitted using a four-parameter logistic fit, and the EC 50 The values ​​(nM) are reported in Table 2.

[0307] Human RXFP1 phosphorylation ERK (phospho-ERK) assay (Test H)

[0308] To analyze the RXFP1 agonist activity of compounds with respect to ERK phosphorylation, the Advanced Phospho-ERK (Thr202 / Tyr204) Cell Kit (CisBio; 64AERPEH) was used. The assay uses two antibodies. One is labeled with a donor fluorophore (Eu cryptate) and the second is labeled with an acceptor (d2). The first antibody binds specifically to phosphorylated ERK and the second antibody binds to another motif of ERK and is independent of its phosphorylation state. ERK phosphorylation allows the formation of an immune complex involving the two antibodies, thereby generating a FRET signal. Its intensity is proportional to the concentration of phosphorylated ERK in the sample. The assay was performed according to the manufacturer's recommendations.

[0309] Preparation of assay reagents:

[0310] Cells: HEK293s cells stably transfected with human RXFP1 in pIRESneo3 were used. Cells were cultured in DMEM medium (Gibco; 31966) supplemented with 10% FBS at 0.8 mg / mL to maintain RXFP1 expression. Cells maintained in continuous culture were assayed.

[0311] Dilution of test compounds: Compounds were diluted to the desired concentration using serum-free DMEM without phenol red (Gibco; 31053-038). DMSO concentration was adjusted to 0.4%.

[0312] Antibody mixture: Eu- and D2-labeled anti-ERK1 / 2 antibodies were diluted 20-fold with the detection buffer provided in the kit. Shortly before the experiment, equal volumes of each diluted antibody solution were combined into the antibody mixture.

[0313] Step-by-step protocol for running the assay:

[0314] Day 1

[0315] 1. The cells were detached from the culture flask using accutase (Gibco; 1737341), resuspended in phenol red-free DMEM medium containing 10% FBS and collected in a 50 mL tube.

[0316] 2. The cells were counted using CEDEX (Innovatis) and diluted to 320,000 cells / mL using the above culture medium.

[0317] 3. Dispense 100 μL of the cell suspension into each well of a black μclear poly-D-lysine-coated μclear 96-well plate (Greiner; 655946).

[0318] 4. Incubate the plate at 37°C, 5% CO2 for 24 h.

[0319] Day 2

[0320] 5. Serum starvation method: Remove the medium and replace with 50 μL of serum-free DMEM without phenol red. Incubate the plate at 37°C, 5% CO2 for 5 h.

[0321] 6. Add 50 μL of test compound solution to each well.

[0322] 7. Incubate the plate at room temperature for 5 min.

[0323] 8. Stop stimulation by quickly removing the medium and adding 50 μL of lysis buffer per well (diluted to 1x final concentration before addition).

[0324] 9. Transfer the plates to -80°C and freeze the lysates.

[0325] Day 3

[0326] 10. Thaw the plate and shake at room temperature for 30 min.

[0327] 11. Homogenize the cell lysate by pipetting.

[0328] 12. Transfer 16 μL of homogenate per well to a white low-volume 384-well plate (Greiner; 784075)

[0329] 13. Add 4 μL of antibody mixture to each well.

[0330] 14. Incubate the plate at room temperature in the dark for 4 h.

[0331] 15. Homogeneous time-resolved fluorescence (HTRF) signals were detected using a Pherastar (BMG Laboratory Technologies) reader (λex = 340 nm, λem = 665 and 615 nm).

[0332] HTRF ratio data were processed using Screener software (Gene Data). Concentration response data were fitted with a four-parameter logistic fit, and EC 50 The values ​​(nM) are reported in Table 2.

[0333] Table 2 - Measurement data

[0334]

[0335]

[0336] Solubility of amorphous compound (I) in SGF at pH 1.2 (Test I)

[0337] Preparation of amorphous nanosuspension of compound (I)

[0338] A 100 mM stock solution of compound (I) (9.44 mg in 140.2 μL of DMA and 23.6 μL in myglyol 812) was prepared in DMA and miglyol 812 by sonication and stirring. Under a few seconds of sonication, 10 μL of the stock solution was rapidly injected into 990 μL of a stabilizer solution containing 0.2% w / w PVPK30 and 0.2% w / w Pluronic F127 and 0.25 mM SDS in water to make a 1 mM amorphous nanosuspension. This formulation was further diluted to 0.25 mM with purified water.

[0339] Solubility experiment

[0340] At 37°C, the bulk concentration in equilibrium with the amorphous particles was determined by turbidimetry (Lindfors et al., Langmuir, 2006, 22(3), 911-916). A small amount of drug suspension was added directly to a fluorescent cuvette containing 2000 μL simulated gastric fluid (pH 1.2) (SGF) and then mixed to give the desired concentration. The light scattering intensity at 700 nm was recorded at a scattering angle of 90° as a function of the total drug concentration. As a light scattering device, a Perkin-Elmer LS 55 luminescence spectrometer was used, with both the emission and excitation wavelengths set to 700 nm. The slit excitation was set to 2.5 nm and the slit emission was set to 5 nm. Before the measurement began, the particles were allowed to dissolve for at least 30 seconds. The data was then collected for 1 minute and the average light scattering intensity was calculated. The solubility check was performed within 45 min after the amorphous nanosuspension was formed.

[0341]

[0342]

[0343] Light scattering is plotted as a function of total concentration of Compound (I). At low concentrations, the particles dissolve, while at concentrations above solubility, the particles remain suspended, resulting in an almost linear increase in light scattering intensity as a function of total concentration. The amorphous solubility of Compound (I) in SGF at pH 1.2 at 37°C was estimated using a linear extrapolation of the fitted high concentration data to zero intensity and was determined to be 8 μM. The light scattering intensity as a function of total concentration is shown in Figure 6 middle.

[0344] Solubility of crystalline form A of compound (I) in SGF at pH 1.2 (Test J)

[0345] Crystal solubility in simulated gastric fluid (pH 1.2) (SGF) was measured using a micro shake flask method (stirring at 37°C for 24 h). Excess Form A of Compound (I) was weighed into a vial, 1.2 mL of pH 1.2 SGF was added, and the sample was sonicated for 45 min.

[0346] The sample was stirred in an Eppendorf shaker at 37°C for 24 hours with a stirring speed of 1000rpm. The sample was then centrifuged at 10000RPM for 15 minutes at 37°C. Approximately 500μL of the supernatant was drawn into a new vial with an automatic pipette and centrifuged again at 10000rpm for 15min at 37°C. Then from the second centrifugation, samples were taken with an automatic pipette to the final dilution. The pH of the remaining sample solution balanced after the second centrifugation was measured. XRPD was used to confirm that no form changes occurred. UHPLC was used to quantify the concentration in the sample and it was found that the solubility of Form A of Compound (I) in SGF at pH 1.2 at 37°C was 0.4μM.

[0347] Those skilled in the art will appreciate that the assays described above may be performed using alternative equipment and minor variations in the protocol without significantly affecting the results.

[0348] Although this specification and its specific examples show certain embodiments, they are intended for illustrative purposes only. Therefore, this disclosure is not limited to the illustrative embodiments described in this specification and can be modified differently. In addition, it should be understood that, for reasons of clarity, the various embodiments described in the context of the separate embodiments can also be combined to form a single embodiment. On the contrary, for the sake of brevity, the various embodiments described in the context of a single embodiment can also be combined to form a sub-combination thereof.

[0349] Any publications disclosed within this specification are hereby incorporated by reference.

Claims

1. An amorphous compound (I) 2. A pharmaceutical composition comprising an amorphous compound (I) and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition of claim 2, wherein at least 80% by weight of Compound (I) in the pharmaceutical composition is amorphous.

4. A solid dispersion comprising an amorphous compound (I).

5. The solid dispersion of claim 4, further comprising one or more polymers.

6. The solid dispersion of claim 4 or 5, wherein at least 80% by weight of Compound (I) in the solid dispersion is amorphous.

7. The solid dispersion of any one of claims 4 to 6, wherein less than 20% by weight of Compound (I) in the solid dispersion is crystalline.

8. The solid dispersion according to any one of claims 4 to 7, wherein compound (I) is present in an amount of 5% by weight to 95% by weight.

9. The solid dispersion of any one of claims 4 to 8, wherein the one or more polymers are present in an amount of 5% by weight to 95% by weight.

10. The solid dispersion of any one of claims 4 to 9, wherein the one or more polymers are water soluble.

11. A solid dispersion as described in any one of claims 4 to 10, wherein the one or more polymers are selected from poly(1-vinyl-2-pyrrolidone), copolymers of 1-vinyl-2-pyrrolidone and vinyl acetate, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymers, methacrylic acid-ethyl acrylate copolymers, polyacrylic acid, hydroxypropyl methylcellulose acetate succinate, cellulose acetate phthalate and polyvinyl acetate phthalate.

12. The solid dispersion of any one of claims 4 to 10, wherein the one or more polymers is a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate, optionally a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate in a ratio of 6:4 by weight.

13. A pharmaceutical composition comprising the solid dispersion according to any one of claims 4 to 12.

14. The pharmaceutical composition of any one of claims 2, 3 and 13, wherein the pharmaceutical composition is in the form of a tablet or a capsule.

15. The pharmaceutical composition of any one of claims 2, 3, 13 and 14, wherein the pharmaceutical composition is suitable for oral administration.

16. A compound, solid dispersion or pharmaceutical composition as claimed in any preceding claim for use in therapy.

17. A compound, solid dispersion or pharmaceutical composition as described in any one of claims 1 to 15 for use in treating a subject suffering from a disease or condition selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension and resistant hypertension.

18. A crystalline form of Compound (I), wherein the crystalline form has a melting onset of about 217.5°C.

19. A crystalline form of compound (I), which can be obtained by heating crystalline form A of compound (I) to a temperature above about 43.9°C, optionally above about 45°C, optionally above about 50°C, optionally above about 55°C, optionally above about 60°C, optionally above about 65°C.

20. A method for preparing a crystalline form as claimed in claim 18 or 19, which comprises heating the crystalline Form A of Compound (I) to a temperature above about 43.9°C, optionally above about 45°C, optionally above about 50°C, optionally above about 55°C, optionally above about 60°C, optionally above about 65°C.

21. The crystalline form of claim 19, or the method of claim 20, wherein the X-ray powder diffraction pattern (Cu K α radiation) has at least five specific peaks at about 2-θ = 7.5, 10.7, 12.8, 14.5 and 15.8°.

22. The crystalline form of claim 19, or the method of claim 20, wherein Form A of Compound (I) has an X-ray powder diffraction pattern (CuK α radiation).

23. A pharmaceutical composition comprising the crystalline form of any one of claims 18 to 22 and a pharmaceutically acceptable excipient.

24. The crystalline form of any one of claims 18 to 22, for use in the manufacture of a medicament for treating a disease or condition in a human patient selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension.

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