Solid Forms of the RXFP1 Modulator

Abstract

TIFF2025523405000024.tif38170 This specification generally relates to solid forms of (1S,4s)-4-(2-fluoro-4-methoxy-5-((((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (Compound (I)), such as crystalline forms and amorphous forms. In particular, it relates to the amorphous form of Compound (I), solid dispersions and pharmaceutical compositions containing such amorphous forms, and the crystalline form G of Compound (I).

Description

Technical Field

[0001] This specification describes solid 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]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (referred to herein as Compound (I)), such as crystalline and amorphous forms, pharmaceutical compositions thereof, and processes for preparing such solid forms.

Background Art

[0002] Relaxin is a multifaceted hormone known to mediate adaptive changes in systemic hemodynamics and the kidney during pregnancy. Relaxin has also been shown to have antifibrotic properties and beneficial effects in heart failure, such as acute decompensated heart failure (ADHF). Heart failure is associated with significant morbidity and mortality. Heart failure is characterized by complex tissue remodeling involving increased cardiomyocyte death and interstitial fibrosis. Relaxin activates many signaling cascades that have been shown to be beneficial in situations such as 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] In patients with heart failure, a significant subgroup also has pulmonary hypertension (HF+PH patients). Approximately 50% of heart failure patients with preserved ejection fraction also have pulmonary hypertension, which is estimated to increase to 60% in heart failure patients with a reduced ejection fraction (Guazzi, (2014) Circ Heart Fail., 7:367-377, Miller et al., (2013) JACC Heart Fail., 1(4):290-299). Patients with heart failure complicated by pulmonary hypertension have been shown to have a lower survival rate compared to patients with heart failure without pulmonary hypertension (Barnett and De Marco, (2012) Heart Fail. Clin. 8:447-459). In heart failure patients, a 3 mmHg increase or decrease in estimated pulmonary artery diastolic pressure (ePAD) (equivalent to an approximately 4 mmHg increase or decrease in mean pulmonary artery pressure (mPAP)) was 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 was also associated with an improvement in 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 the blood pressure of hypertensive patients that remains elevated above target despite the concurrent use of optimal doses of three antihypertensive agents from different classes, one of which is a diuretic. The current standard of care (SoC) for the initial treatment of hypertension is calcium channel blockers (CCBs), renin-angiotensin system blockers (angiotensin-converting enzyme [ACE] inhibitors or angiotensin receptor blockers [ARBs]), and diuretics. In the case of rHT patients, there are multiple options for what to add next (e.g., mineralocorticoid receptor antagonists [MRAs], β-blockers or α-blockers), and the guidelines currently recommend MRAs as the preferred option for the treatment of rHT. rHT includes patients whose blood pressure is adequately controlled when four or more antihypertensive drugs are administered simultaneously (Carey et al., Hypertension, 2018, 72, e53-e90). Patients with rHT typically have a long history of severe blood pressure elevation and are at higher cardiovascular risk than treated hypertensive patients with controlled blood pressure (Acelajado et al., Circulation Research, 2019, 124, 1061-1070). Relaxin has been suggested to have therapeutic potential in hypertensive disorders (Lekgabe et al., Hypertension, 2005, 46, 412-8).

[0005] Clinical trials were conducted using selaxin, which is unmodified recombinant human relaxin 2. By continuously administering selaxin intravenously to inpatients, disorders and congestion of the heart, kidneys, and liver were improved (Felkerg M et al. (2014) J. Am. Coll. Cardiol. 64(15):1591 - 1598, Metra M et al. (2013) J. Am. Coll. Cardiol. 61(2):196 - 206, Teerlink JR et al. (2013) Lancet 381(9860):29 - 39). However, since selaxin is rapidly removed from the patient's blood circulation, its therapeutic effect is limited, and once the intravenous injection is stopped, the positive effect rapidly disappears. Furthermore, since approximately one - third of the patients experienced a significant decrease in blood pressure (more than 40 mm Hg) after intravenous administration of selaxin, it was concluded that the dose had to be reduced by half or even more.

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

[0007] Small - molecule modulators of RXFP1 are sought as relaxin mimetics. For example, Marugan, J.J., et al., International Publication No. WO 2013 / 165606 A1 pamphlet; Xiao J et al. (2013) Nat. Commun. 4:1953; and McBride A et al. (2017) Scientific Reports 7:10806 discuss small - molecule modulators of RXFP1.

[0008] Notwithstanding the above, there remains a need for additional compounds that modulate RXFP1 (e.g., compound (I), etc.), which may be particularly promising for development as therapeutic agents. Such compounds may also exhibit improved modulation of RXFP1 compared to other known RXFP1 modulators. Such compounds may also exhibit favorable pharmacokinetic profiles (e.g., lower intrinsic clearance) and / or advantageous physical properties (e.g., higher water solubility) compared to other known RXFP1 modulators. Accordingly, such compounds may be particularly useful in the treatment of disease states where modulation of RXFP1 is beneficial. Stable solid forms of such compounds having advantageous physical properties such as water solubility at low pH that may be useful for oral administration of such compounds are also needed. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0009] This specification relates to solid forms of compound (I) having the following structure:

Chemical formula

[0010] Briefly, this specification describes in part amorphous compound (I).

[0011] This specification also describes in part pharmaceutical compositions comprising amorphous compound (I) and pharmaceutically acceptable excipients.

[0012] This specification also describes in part solid dispersions comprising amorphous compound (I).

[0013] This specification also partially describes a pharmaceutical composition comprising a solid dispersion containing an amorphous compound (I).

[0014] This specification also partially describes the amorphous compound (I) or solid dispersion or pharmaceutical composition described herein for use in therapy.

[0015] This specification also partially describes the amorphous compound (I) or solid dispersion or pharmaceutical composition described herein for use in the treatment of a subject having a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension.

[0016] This specification also partially describes a crystalline form of compound (I) having a melting onset temperature of about 217.5 °C, and a method for preparing such a crystalline form comprising heating the crystalline form A of compound (I) to a temperature above about 43.9 °C.

[0017] This specification also partially describes a crystalline form of compound (I) obtainable by heating the crystalline form A of compound (I) to a temperature above about 43.9 °C, and a method for preparing such a crystalline form comprising heating the crystalline form A of compound (I) to a temperature above about 43.9 °C.

[0018] This specification also partially describes a pharmaceutical composition comprising the crystalline form disclosed herein.

[0019] This specification also partially describes the crystalline form disclosed herein for use in the manufacture of a medicament for the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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.

[0020] Further aspects of the disclosure will be apparent to those skilled in the art upon reading this specification.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0022] Numerous embodiments are detailed throughout this specification and will be apparent to those skilled in the art. This specification should not be construed as being limited to any particular embodiment described herein.

[0023] Terms not specifically defined herein are to be understood as having the meaning that would be given to them by one of ordinary skill in the art in light of the disclosure and context.

[0024] "About" may generally mean an acceptable degree of error for the quantity being measured, taking into account the nature or precision of the measurement method. Exemplary degrees of error are within a percentage (%) of the given value or range of values, typically within 10%, more typically within 5%.

[0025] Embodiments described herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" such features.

[0026] Concentrations, amounts, volumes, percentages, and other numerical values may be presented in this specification in a range format. Such range formats are used merely for convenience and brevity and should be flexibly construed to include not only the numerical values explicitly recited as the limits of the range but also all the individual numerical values or sub-ranges subsumed within that range as if each were explicitly recited.

[0027] The chemical names of the compounds described herein were generated using ChemDraw® Professional version 19.0.0.22 of PerkinElmer®. Those skilled in the art will understand that different chemical naming software may generate different chemical names for a particular compound. Where the compounds described herein are shown as formulas in the form of chemical names, the formula shall prevail in case of any contradiction.

[0028] The compounds and salts described herein may exist in solvated and unsolvated forms. For example, solvated forms may be hydrate forms such as hemihydrate, monohydrate, dihydrate, trihydrate, or alternative amounts thereof. All such solvated and unsolvated forms of the compounds described herein are included in this specification.

[0029] The atoms of the compounds and salts described herein may exist as these isotopes. All compounds described herein in which an atom is replaced by one or more of its isotopes (e.g., one or more carbon atoms are 11 C or 13 C carbon isotopes, or one or more hydrogen atoms are 2 H or 3 H isotopes) are included in this specification.

[0030] The compounds described herein may exist in one or more geometric, optical, enantiomeric and diastereomeric forms, including but not limited to cis- and trans-forms, E- and Z-forms, and R-, S- and meso-forms. Unless otherwise specified, references to a particular compound include all such isomeric forms, including racemic mixtures and other mixtures. Where appropriate, such isomers can be separated from their mixtures by the application or adaptation of known methods (e.g., chromatography techniques and recrystallization techniques). Where appropriate, such isomers can be prepared by the application or adaptation of known methods.

[0031] The compounds described herein may contain one or more chiral centers. Unless the structure or chemical name in this specification indicates chirality, the structure or name is intended to encompass any single stereoisomer corresponding to that structure or name, as well as any mixture of stereoisomers (e.g., a racemate). Where the structure herein contains solid and dashed wedges (i.e.,

Chemical formula

[0032] How such optically active forms can be separated is well known in the art. For example, a single stereoisomer can be obtained by isolating it from a mixture of isomers (e.g., a racemate) using, for example, chiral chromatography separation. In other embodiments, a single stereoisomer can be obtained, for example, by direct synthesis from a chiral starting material.

[0033] According to one embodiment, the compounds described herein are provided as a single enantiomer having an enantiomeric excess (%ee) of 95% or more, 98% or more, or 99% or more. For simplicity, the single enantiomer is present with an enantiomeric excess of 99% or more.

[0034] According to one embodiment, the compounds described herein are provided as a single enantiomer having an enantiomeric excess (%ee) in the range of 95 to 100%.

[0035] The compounds described herein may 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. Thus, the structures shown herein as one tautomer are intended to include the other tautomers as well.

[0036] The pharmaceutical compositions described herein may contain one or more pharmaceutically acceptable excipients. The excipients selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition being provided. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art and are described, for example, in Handbook of Pharmaceutical Excipients, Sixth edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmaceutically acceptable excipients can function, for example, as adjuvants, diluents, carriers, stabilizers, flavoring agents, coloring agents, fillers, binders, disintegrants, lubricants, glidants, thickening agents, and coating agents. As will be recognized by those skilled in the art, a particular pharmaceutically acceptable excipient may perform two or more functions and may perform alternative functions depending on the amount of excipient present in the composition and which other excipients are present in the composition.

[0037] The pharmaceutical composition can 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 fine powders), administration by insufflation (e.g., as fine powders), or as suppositories for rectal administration. This composition can be obtained by conventional procedures known in the art. Compositions intended for oral use can contain additional ingredients, for example, one or more coloring agents, sweetening agents, flavoring agents, and / or preservatives.

[0038] Amorphous compound (I) In one embodiment, an amorphous compound (I) is provided. In one embodiment, the amorphous compound (I) has an X-ray powder diffraction pattern that is substantially the same as the X-ray powder diffraction pattern (CuK α radiation) shown in Figure 2. In one embodiment, the amorphous compound (I) shows no reflections in the range of 2θ (CuK α radiation) from 3 to 40°.

[0039] Pharmaceutical composition comprising amorphous compound (I) In one embodiment, a pharmaceutical composition comprising an amorphous compound (I) and a pharmaceutically acceptable excipient is provided. In one embodiment, at least 80 wt% of the compound (I) in the pharmaceutical composition is amorphous. In one embodiment, at least 90 wt% of the compound (I) in the pharmaceutical composition is amorphous. In one embodiment, at least 95 wt% of the compound (I) in the pharmaceutical composition is amorphous. In one embodiment, at least 99 wt% of the compound (I) 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.

[0040] In one embodiment, less than 20 wt% of compound (I) in the pharmaceutical composition is crystalline. In one embodiment, less than 10 wt% of compound (I) in the pharmaceutical composition is crystalline. In one embodiment, less than 5 wt% of compound (I) in the pharmaceutical composition is crystalline. In one embodiment, less than 1 wt% of compound (I) in the pharmaceutical composition is crystalline. In one embodiment, substantially none of compound (I) in the pharmaceutical composition is crystalline. In one embodiment, none of compound (I) in the pharmaceutical composition is crystalline.

[0041] Solid dispersion containing amorphous compound (I) In another embodiment, a solid dispersion containing amorphous compound (I) is provided. The solid dispersion typically contains the compound 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, the one or more polymers are water-soluble. It will be understood that water-soluble polymers can dissolve in an aqueous medium such as water or gastric juice.

[0042] In one embodiment, at least 90 wt% of compound (I) in the solid dispersion is amorphous. In one embodiment, at least 95 wt% of compound (I) in the solid dispersion is amorphous. In one embodiment, at least 99 wt% 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.

[0043] In one embodiment, less than 20 wt% of compound (I) in the solid dispersion is crystalline. In one embodiment, less than 10 wt% of compound (I) in the solid dispersion is crystalline. In one embodiment, less than 5 wt% of compound (I) in the solid dispersion is crystalline. In one embodiment, less than 1 wt% of compound (I) in the solid dispersion is crystalline. In one embodiment, substantially none of compound (I) in the solid dispersion is crystalline. In one embodiment, none of compound (I) in the solid dispersion is crystalline.

[0044] In one embodiment, compound (I) is present in the solid dispersion in an amount of 5 wt% to 95 wt%. In one embodiment, compound (I) is present in the solid dispersion in an amount of 5 wt% to 80 wt%. In one embodiment, compound (I) is present in the solid dispersion in an amount of 5 wt% to 60 wt%. In one embodiment, compound (I) is present in the solid dispersion in an amount of 10 wt% to 40 wt%. In one embodiment, compound (I) is present in the solid dispersion in an amount of 20 wt% to 40 wt%.

[0045] In one embodiment, the one or more polymers are selected from poly(1-vinyl-2-pyrrolidone), a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, methacrylic acid-ethyl acrylate copolymer, polyacrylic acid, hypromellose acetate succinate, cellulose acetate phthalate, and polyvinyl acetate phthalate.

[0046] In one embodiment, the one or more polymers are selected from Kollidon® 30, Kollidon® VA 64, Soluplus®, Eudragit® L100-55S, polyacrylic acid, AQOAT® HPMC AS-LF, AQOAT® HPMC AS-HF, cellulose acetate phthalate, and polyvinyl acetate phthalate.

[0047] In one embodiment, the one or more polymers are a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate. In one embodiment, the one or more polymers are a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate with a weight ratio of 6:4. In one embodiment, the one or more polymers is Kollidon® VA 64.

[0048] In one embodiment, the one or more polymers are present in an amount of 5 wt% to 95 wt% in the solid dispersion. In one embodiment, the one or more polymers are present in an amount of 20 wt% to 90 wt% in the solid dispersion. In one embodiment, the one or more polymers are present in an amount of 40 wt% to 90 wt% in the solid dispersion. In one embodiment, the one or more polymers are present in an amount of 50 wt% to 90 wt% in the solid dispersion. In one embodiment, the one or more polymers are present in an amount of 60 wt% to 80 wt% in the solid dispersion.

[0049] A pharmaceutical composition comprising a solid dispersion comprising an amorphous compound (I) In one embodiment, there is provided a pharmaceutical composition comprising a solid dispersion comprising an amorphous compound (I) as described herein.

[0050] Crystal form G of compound (I) Differential scanning calorimetry (DSC) analysis of crystal form A of compound (I) shows an endothermic transition with an onset observed at about 43.9 °C. This endothermic peak is assigned to a solid-solid phase transition, and the phase obtained at a temperature above the completion of the endothermic peak observed at about 43.9 °C is designated as form G. This new anhydrous phase form G exists up to the last endothermic peak in the DSC corresponding to the onset of melting at 217.5 °C.

[0051] Accordingly, in one embodiment, there is provided a crystal form (form G) of compound (I) having a melting onset temperature of about 217.5 °C. In one embodiment, the melting onset temperature is 217.5 ± 5 °C. In one embodiment, the melting onset temperature is 217.5 ± 4 °C. In one embodiment, the melting onset temperature is 217.5 ± 3 °C. In one embodiment, the melting onset temperature is 217.5 ± 2 °C. In one embodiment, the melting onset temperature is 217.5 ± 1 °C.

[0052] 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 above 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 above about 45 °C. In one embodiment, the crystalline form can be obtained by heating the crystalline form A of Compound (I) to a temperature above about 50 °C. In one embodiment, the crystalline form can be obtained by heating the crystalline form A of Compound (I) to a temperature above about 55 °C. In one embodiment, the crystalline form can be obtained by heating the crystalline form A of Compound (I) to a temperature above about 60 °C. In one embodiment, the crystalline form can be obtained by heating the crystalline form A of Compound (I) to a temperature above about 65 °C.

[0053] In one embodiment, a method for preparing the crystalline form (Form G) of Compound (I) is provided, which includes heating the crystalline form A of Compound (I) to a temperature above about 43.9 °C. In one embodiment, the crystalline form A of Compound (I) is heated to a temperature above about 45 °C. In one embodiment, the crystalline form A of Compound (I) is heated to a temperature above about 50 °C. In one embodiment, the crystalline form A of Compound (I) is heated to a temperature above about 55 °C. In one embodiment, the crystalline form A of Compound (I) is heated to a temperature above about 60 °C. In one embodiment, the crystalline form A of Compound (I) is heated to a temperature above about 65 °C.

[0054] The ten most prominent X-ray powder diffraction peaks [angle 2θ (2θ), intensity] for Form A of Compound (I) are as follows: 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, Form A of Compound (I) has an X-ray powder diffraction pattern (CuK αIt has a (line). In one embodiment, the Form A compound (I) has an X-ray powder diffraction pattern with specific peaks at approximately 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, Form A of compound (I) has an X-ray powder diffraction pattern with at least five specific peaks at 2θ = 7.5, 10.7, 12.8, 14.5 and 15.8°, where the values can be ±0.2° 2θ. In one embodiment, Form A of compound (I) has an X-ray powder diffraction pattern with specific peaks at 2θ = 7.5, 10.3, 10.7, 12.8, 14.5, 15.3, 15.8, 17.5, 19.6 and 21.3°, where the values can be ±0.2° 2θ. In one embodiment, Form A of compound (I) has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 1.

[0055] The defined solid forms described herein provide an X-ray powder diffraction pattern substantially identical to the X-ray powder diffraction pattern shown in the drawings and have various 2-θ values described herein. It is understood that the 2-θ values of the X-ray powder diffraction pattern vary slightly from machine to machine or from sample to sample, and thus the cited values should not be interpreted as absolute values.

[0056] It is known that, depending on the measurement conditions (e.g., the apparatus 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 vary depending on the measurement conditions. Thus, 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 drawings, and it should be understood that any crystal that provides an X-ray powder diffraction pattern substantially the same as that shown in the drawings falls within the scope of the embodiments described herein. Those skilled in the art of X-ray powder diffraction can determine the substantial identity of X-ray powder diffraction patterns.

[0057] Those skilled in the art of X-ray powder diffraction understand that the relative intensity of peaks can be affected by, for example, particles larger than 30 μm in size and non-unitarity aspect ratios that can affect the analysis of samples. Those skilled in the art will also recognize that the position of reflections can be affected by the exact height at which the sample is positioned within the diffractometer and the zero calibration of the diffractometer. The planarity of the surface of the sample may also have a slight effect. Therefore, the presented diffraction pattern data should not be regarded as absolute values (Jenkins, R & Snyder, R.L. ‘Introduction to X-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C.W. (1948), Chemical Crystallography, Clarendon Press, London; Klug, H.P. & Alexander, L.E. (1974), X-Ray Diffraction Procedures). Peak intensities are described herein as vs (very strong), s (strong), m (moderate), and w (weak), corresponding to % relative intensities (based on the strongest peak) of 25 - 100%, 10 - 25%, 3 - 10%, and 1 - 3% respectively. The relative intensities are derived from diffractograms measured with a fixed slit.

[0058] Generally, the measurement error of the diffraction angle in an X-ray powder diffraction pattern is about 5% or less, particularly ±0.5° 2θ, and such a degree of measurement error should be considered when reading the data described herein and when considering the X-ray powder diffraction pattern in the drawings of this specification. Furthermore, of course, the intensity can vary depending on the experimental conditions and sample preparation (preferred orientation).

[0059] A pharmaceutical composition comprising Form G of compound (I) In one embodiment, there is provided a pharmaceutical composition comprising Form G of compound (I) described herein and a pharmaceutically acceptable excipient.

[0060] Use of solid forms in therapy As a result of the modulation of RXFP1, compound (I) is expected to be useful in therapy.

[0061] The term "therapy" is intended to have its ordinary meaning of treating a disease or condition in order to completely or partially relieve one, several, or all of its symptoms, or to correct or compensate for the underlying pathology. The term "therapy" also includes "prevention" unless the specific instruction to the contrary is given. The terms "therapeutic" and "therapeutically" should be construed in the corresponding manner.

[0062] The term "prevention" is intended to have its standard meaning and includes primary prevention to prevent the occurrence of a disease or condition, and secondary prevention to protect a patient temporarily or continuously against the worsening or deterioration of a disease or condition that has already occurred, or the development of new symptoms associated with the disease or condition.

[0063] The term "treatment" is used synonymously with "therapy". Similarly, the term "treat" can be considered to mean "apply therapy" (where "therapy" is as defined herein).

[0064] The term "therapeutically effective amount" refers to an amount of a compound effective to provide "therapy" to a subject or to "treat" a disease or condition in a subject. A therapeutically effective amount can cause any observable or measurable change in a subject, as described in the definitions of "therapy", "treatment", and "prevention" above. As will be recognized by those of ordinary skill in the art, the effective amount can vary depending upon the route of administration, the use of excipients, and co-administration with other agents. For example, when combination therapy is used, the amount of the pharmaceutically active agent and the amount of the other pharmaceutically active agent are effective together to treat the target disorder or condition of the subject when combined. In this context, the combined amounts are a "therapeutically effective amount" if they are sufficient to alleviate the symptoms of a disease or condition responsive to the modulation and / or agonism of RXFP1 as described above. Typically, such amounts can be determined by those of ordinary skill in the art.

[0065] As used herein, the terms "subject" and "patient" are used interchangeably. "Subjects" include, for example, mammals such as humans. In some embodiments, the subject is a human.

[0066] Accordingly, the amorphous compound (I), Form G of compound (I), solid dispersion comprising the amorphous compound (I), or pharmaceutical composition comprising compound (I) described herein can be used in therapy, for example, to treat a disease or disorder. Also provided is a method of treating a disease or disorder comprising administering to a subject or patient in need thereof a therapeutically effective amount of the amorphous compound (I), Form G of compound (I), solid dispersion comprising the amorphous compound (I), or pharmaceutical composition comprising compound (I) described herein.

[0067] It will be understood that the amorphous compound (I), Form G of compound (I), solid dispersion comprising the amorphous compound (I), or pharmaceutical composition comprising compound (I) described herein can be used in the treatment of cardiovascular diseases, for example, for the treatment of heart failure and hypertension.

[0068] 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 mid-range ejection fraction (HFmrEF; also called heart failure with mildly reduced ejection fraction), or heart failure with reduced ejection fraction (HFrEF).

[0069] As used herein, the term "resistant hypertension" is defined as the blood pressure of a hypertensive patient that remains elevated above the target despite the simultaneous use of optimized doses of three different classes of antihypertensive agents, one of which is a diuretic, or as the blood pressure of a patient whose blood pressure is well controlled when four or more antihypertensive agents are administered concomitantly (Carey et al., Hypertension, 2018, 72, e53-e90). 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. In the case of rHT patients, additional treatments can include a mineralocorticoid receptor antagonist (MRA), a β-blocker and / or an α-blocker. Subjects with resistant hypertension can typically have a systolic blood pressure of 140 mmHg or more and / or a diastolic blood pressure of 90 mmHg or more at rest. Alternatively, subjects with resistant hypertension can typically have a systolic blood pressure of 130 mmHg or more and / or a diastolic blood pressure of 80 mmHg or more at rest. Alternatively, subjects with resistant hypertension can typically have a systolic blood pressure of 150 mmHg or more and / or a diastolic blood pressure of 90 mmHg or more at rest. In some cases, resistant hypertension can be resistant essential hypertension. Essential hypertension, also known as primary hypertension, is a form of hypertension without identified known secondary causes.

[0070] The amorphous compound (I), Form G of the compound (I), a solid dispersion comprising the amorphous compound (I), or a pharmaceutical composition comprising the compound (I) described herein can also be used in the treatment of kidney diseases (including chronic kidney diseases), acute kidney injury, lung diseases 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, disorders that are the result or cause of atherosclerosis, decreased arterial elasticity, decreased arterial compliance and distensibility (i.e., stroke and dementia), diabetes, microvascular diseases that result in end-organ damage, coronary artery disease and heart failure, and preeclampsia.

[0071] In one embodiment, an amorphous compound (I) for use in treatment is provided.

[0072] In one embodiment, an amorphous compound (I) for use in the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension and resistant hypertension is provided. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0073] In one embodiment, an amorphous compound (I) for use in the manufacture of a medicament for the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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 is provided. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0074] In one embodiment, provided is a method of treating a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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, 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.

[0075] In one embodiment, provided is Form G of compound (I) for use in the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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.

[0076] In one embodiment, provided is Form G of compound (I) for use in the manufacture of a medicament for the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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 one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0077] In one embodiment, provided is a method of treating a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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, 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.

[0078] In one embodiment, a solid dispersion comprising the amorphous compound (I) described herein for use in the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension is provided. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0079] In one embodiment, a solid dispersion comprising the amorphous compound (I) described herein for use in the manufacture of a medicament for the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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 is provided. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0080] In one embodiment, a method of treating a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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, the method comprising administering to the human patient a therapeutically effective amount of a solid dispersion comprising the 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.

[0081] In one embodiment, a pharmaceutical composition comprising the compound (I) described herein for use in the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension is provided. In one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0082] In one embodiment, there is provided a pharmaceutical composition comprising the compound (I) described herein for use in the manufacture of a medicament for the treatment of a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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 one embodiment, the disease or condition is heart failure. In one embodiment, the disease or condition is resistant hypertension.

[0083] In one embodiment, there is provided a method of treating a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate 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, the method comprising administering to the human patient a therapeutically effective amount of a pharmaceutical composition comprising the 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.

Examples

[0084] Generally, all of the solvents used were commercially available for analysis. The anhydrous solvents were those commonly used for reactions. The phase separator used in the examples was an ISOLUTE® Phase Separator column. The compounds described below were named using PerkinElmer's ChemDraw Professional version 19.0.0.22.

[0085] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements were performed using a TG Discovery 550 (TA instruments, Germany) and a DSC Discovery 2500 (TA instruments, Germany), respectively. For TGA, a 5.152 mg sample and for DSC, a 2.474 mg sample were weighed into aluminum pans. Subsequently, the samples were heated from room temperature to 350 °C for TGA and from 0 °C to 230 °C for DSC at a heating rate of 10 °C / min under a nitrogen purge of 100 mL / min. An empty aluminum pan was used as a reference for DSC.

[0086] X-ray powder diffraction analysis was carried out according to standard methods that can be found, for example, in Kitaigorodsky, A.I. (1973), Molecular Crystals and Molecules, Academic Press, New York; Bunn, C.W. (1948), Chemical Crystallography, Clarendon Press, London; or Klug, H.P. & Alexander, L.E. (1974), X-ray Diffraction Procedures, John Wiley & Sons, New York.

[0087] XRPD Method A The X-ray powder diffraction (referred to herein as XRPD) pattern was determined by placing the sample on a silicon single crystal, which is a zero-background holder, and spreading the sample into a thin layer.

[0088] Powder X-ray diffraction was recorded in a one-dimensional scan using a Rigaku Miniflex 600 equipped with a D / Tex detector (X-ray wavelength 1.5418 Å, Cu K α irradiation, 40 kV, 15 mA) with a theta-2theta scanning axis. Automatic variable divergence and anti-scattering slits were used, and the sample was rotated at 80 revolutions per minute during measurement. The sample was scanned using step widths and scan rates of 0.01° and 1° / min, respectively.

[0089] XRPD Method B The sample was mounted on a silicon wafer mount and analyzed using a Bruker D4 Endeavour diffractometer (Cu anode, λ = 1.5418 Å). The sample was measured in a θ-2θ configuration reflection geometry over a scanning range of 2° to 40° 2θ with an exposure of 0.12 seconds per 0.02° increment (continuous scan mode). The X-rays were generated by a copper long fine focus tube operated at 40 kV and 40 mA.

[0090] The following abbreviations were used Aq Aqueous B2Pin2 4,4,5,5-Tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane Calcd Calculated DCM Dichloromethane DIA Diisopropylamine DIAD Diisopropyl (E)-diazene-1,2-dicarboxylate DIPEA N-Ethyl-N-isopropyl-propan-2-amine DMA N,N-Dimethylacetamide DMSO Dimethyl sulfoxide DPPA Diphenylphosphoryl azide DTBBPY 4,4’-Di-tert-butyl-2,2’-dipyridyl ESI Electrospray ionization Et Ethyl EtOAc Ethyl acetate EtOH Ethanol h / hr Hour HRMS High resolution mass spectrometry IPA Isopropyl alcohol IPAC Isopropyl acetate [Ir(COD)OMe]2 Bis(1,5-cyclooctadiene)-di-μ-methoxydiiridium(I) L Liter Me Methyl MeCN Acetonitrile mL Milliliter MeOH Methanol 2-Me-THF 2-Methyltetrahydrofuran Min Minute MS Mass spectrometry MTBE Methyl tert-butyl ether NMR Nuclear magnetic resonance PE Petroleum ether Rt Room temperature Sat Saturated TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography UHPLC Ultra-high performance liquid chromatography

[0091] Preparation of Compound (I) Intermediate 1: Ethyl 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylate

Chem.

[0092] Intermediate 2: 8-Methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid

Chemical Structure

[0093] Intermediate 3: 1-Methyl-4-oxocyclohexane-1-carboxylic acid Method A

Chemical Structure

[0094] Method B To a solution of Intermediate 2 (6.17 kg, 3.83 mol, 12.4% in DCM), TFA (1.42 L, 2.18 kg, 19.13 mol) was added. After maintaining the reaction temperature at 25 °C - 35 °C for 20 h, it was cooled to 0 °C - 10 °C. Aqueous NaOH solution (dissolved in 7.66 L H2O, 918 g, 22.96 mol) was added to the reaction solution, and the pH of the aqueous phase was adjusted to 9 - 11. The layers were separated, the aqueous layer was retained and cooled to 0 - 10 °C. After adding DCM (3.83 L), the pH was adjusted to 3 - 4 by adding aqueous HCl solution (1.52 L, 6.08 mol, 4 M in H2O). The organic layer was retained, the aqueous layer was extracted with DCM (2 × 3.83 L), and the combined organic phase was washed with brine (2.3 L, 15% w / w NaCl). The organic phase was concentrated from 2.3 to 3.1 L under reduced pressure. The organic reaction solvent was exchanged from DCM to MeCN under reduced pressure while maintaining the temperature below 45 °C, and the title compound was obtained as an 18% solution in MeCN (2.85 kg, 3.32 mol, 87%). 11H NMR (400 MHz, CDCl3) δ 1.39 (3H, s), 1.73 (2H, td), 2.43 (6H, m). MS (ESI): m / z [M+H] + 157.1.

[0095] Intermediate 4: Naphthalen-1-ylmethyl 1-methyl-4-oxocyclohexane-1-carboxylate Method A [Chemical Structure] To a solution of Intermediate 3 (119 g, 192 mmol, 25% w / w in MeCN), 1-chloromethylnaphthalene (32.2 g, 183 mmol) was added, followed by DIPEA (70.0 mL, 49.7 g, 384 mmol) and NaI (2.88 g, 19.2 mmol). The solution was heated at 50 - 60 °C for 8 h and then cooled to 0 - 10 °C. H2O (240 mL) was added and the pH of the reaction mixture was adjusted to 3 - 4 by addition of aqueous HCl solution (55.0 mL, 220 mmol, 4 M in H2O). The reaction mixture was extracted with MTBE (2 × 150 mL) and the combined organic phases were washed with aqueous NaHCO3 solution (150 mL, 144 mmol, 8% w / w in H2O). While maintaining the temperature below 40 °C, the organic reaction solvent was exchanged from MTBE to IPA under reduced pressure. The temperature of the reaction solution was lowered to -10 - 3 °C and the solution was stirred for 2 h, during which a solid precipitate formed. The solid was filtered and dried under N2 for 15 h to give the title compound as a white solid (42.8 g, 144 mmol, 74%); 1 1H NMR (500 MHz, CDCl3) 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.

[0096] Method B To a solution of Intermediate 3 (2.66 kg, 3.09 mol, 18.2% in MeCN), 1-chloromethylnaphthalene (535 g, 2.94 mol) was added, followed by potassium carbonate (513 g, 3.71 mol) and a further portion of fresh MeCN (714 mL). The suspension was heated at 50 °C to 60 °C for 17 h and then cooled to 25 °C to 30 °C. The solid was removed by filtration through a Celite pad and washed with MeCN (2 × 967 mL). The filtrate was concentrated to 1.45 - 1.93 L under reduced pressure. While maintaining the temperature below 50 °C, MeCN was exchanged with isopropanol under reduced pressure. The temperature of the mixture was lowered to 20 - 25 °C and a solid precipitate formed thereon. The mixture was further cooled to -10 °C to 0 °C, then the solid was filtered, washed with isopropanol and dried under N2 to give the title compound as a white solid (752.6 g, 2.49 mol, 80.5%); 1 Hnm R(500 MHz,CDCl3)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.

[0097] Intermediate 5: Methyl 5-(1,3,6,2-dioxazaborocan-2-yl)-4-fluoro-2-methoxybenzoate Method A

Chemical Structure

[0098] Method B B2Pin2 (29.0 g, 114 mmol) and methyl 4-fluoro-2-methoxybenzoate (20.6 g, 109 mmol) were added to 2-Me-THF (140 mL) degassed with N2 to less than 1% oxygen. The solution was maintained at 20 - 30 °C, then DTBBPY (88 mg, 0.33 mmol) and [Ir(COD)OMe]2 (108 mg, 0.16 mmol) were added, the reaction vessel was evacuated, and N2 was refilled until the oxygen level was less than 0.5%. The reaction mixture was heated to 80 - 85 °C and held at that temperature for an additional 3 hours. The reaction mixture was cooled to 0 - 10 °C, and then isopropanol (12.4 mL, 218 mmol) was slowly added while generating H2 gas. A seed crystal (100 mg of Intermediate 5) was added, followed by the addition of diethanolamine (22.84 g, 218 mmol) dissolved in IPA (20 mL) to obtain a mobile slurry. The slurry was warmed to 20 - 30 °C, and the solid was recovered by filtration. It was then washed with 2-Me-THF (160 mL), and the solid was dried under N2 for 10 hours to obtain the title compound as a white solid (29.1 g, 96 mol, 88%); 1 1H NMR (500 MHz, DMSO-d6) δ 2.81 - 2.89 (2H, m), 3.14 (2H, dq), 3.71 (2H, ddd), 3.74 (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.

[0099] Intermediate 6: Methyl 4-fluoro-5-hydroxy-2-methoxybenzoate Method A

Chemical Structure

[0100] Method B Intermediate 5 (32.41 g, 67.3 mmol) was dissolved in 2-Me-THF (100 mL) containing acetic acid (12.13 g, 202 mmol) and cooled to 0 - 10 °C. A hydrogen peroxide solution (30% w / w, 9.16 g, 80.8 mmol) was added over 2 hours, then the reaction temperature was adjusted to 20 - 30 °C and maintained for 18 hours. An aqueous solution of Na2S2O3·5H2O (20% w / w, 50 mL) quenched the mixture, resulting in phase separation. The aqueous phase was discarded and the organic phase was washed twice with an aqueous solution of Na2S2O3·5H2O (5% w / w, 100 mL). The organic phase was concentrated to 60 mL under reduced pressure and subsequently vacuum distilled twice more with 2-Me-THF (100 mL) to obtain a solution that was dissolved at 35 - 45 °C. Seeding (100 mg of Intermediate 6) was added and nucleation was controlled by slowly adding 300 mL of n-heptane over 5 hours. The resulting slurry was adjusted to 20 - 30 °C, stirred overnight, and then filtered. The recovered solid was washed with n-heptane (2 × 60 mL) and dried to obtain the title compound as a white solid (12.5 g, 62.5 mmol, 93% yield); 1 1H NMR (500 MHz, CDCl3) δ 3.82 (3H, s), 3.86 (3H, s), 6.72 (1H, d), 7.54 (1H, d). MS (ESI): m / z [M + H] + 201.0.

[0101] Intermediate 7: (1R,2R,3S,4S)-3-(Methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

Chemical Structure

[0102] Intermediate 8: Methyl (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylate hydrochloride

Chemical Structure

[0103] Intermediate 11: Naphthalen-1-ylmethyl (1r,4r)-4-hydroxy-1-methylcyclohexane-1-carboxylate

Chemical Structure

[0104] Route B Under nitrogen, a solution of lithium tri-sec-butylborohydride (1.06 g, 5.6 mmol) in THF (5 mL) was added dropwise over 1 minute 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 hours. The reaction mixture was quenched with 0.1 M HCl (10 mL) at -78 °C and then extracted with EtOAc (3 × 50 mL). The organic layers were pooled, dried over Na2SO4, 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 pale yellow rubber. The isolated material had a cis / trans ratio of 3:100. 1 1H NMR (400 MHz, CDCl3) δ 1.21 - 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.

[0105] Intermediate 12: Methyl 4-fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate

Chemical Structure

[0106] Intermediate 13: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid

Chemical formula

[0107] Intermediate 14: 4 - Fluoro - 2 - methoxy - 5 - (((1s,4s) - 4 - methyl - 4 - ((naphthalen - 1 - ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate cyclohexanaminium salt [Chemical formula] To the crude solution of Intermediate 13 directly used from the previous step in IPAC, a solution of cyclohexylamine (280 mL, 699 mmol, 2.5 M in IPAC) was added dropwise at 50 - 55 °C over 3 hours. The heterogeneous slurry was stirred at 50 °C - 55 °C for 0.5 hour and then at 40 °C - 45 °C for an additional 1 hour. The reaction mixture was filtered, and the solid was washed with preheated IPAC (3 × 0.98 L) at 40 - 45 °C and dried at 45 °C under N₂ for 16 hours. To the dried and recovered solid, MeOH (3.64 L) was added, and the mixture was heated to 55 °C - 56 °C. H₂O (1.58 L) was added dropwise over 1 hour, then the mixture was stirred for 1 hour and then cooled to 0 °C - 5 °C over 3 hours. The heterogeneous slurry was held for an additional 1 hour and then filtered and washed with 5:3 MeOH:H₂O at 0 °C (2 × 750 mL), and the solid was dried at 45 °C under N₂ for 16 hours to obtain the title compound as a white solid (332 g, 85% from methyl 4-fluoro-5-hydroxy-2-methoxybenzoate). 1 HnmR(500 MHz,CDCl3)δ 0.96(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.

[0108] Intermediate 15: Methyl (1S,2S,3R,4R)-3-(4-fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamide)bicyclo[2.2.1]hepta-5-ene-2-carboxylate

Chemical Structure

[0109] Intermediate 16: (1S,2S,3R,4R)-3-(4-Fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamide)bicyclo[2.2.1]hepta-5-ene-2-carboxylic acid [Chemical formula] The crude solution of intermediate 15 in THF (750 mL) from the previous step was cooled to 0 - 5 °C. An aqueous solution of LiOH·2H₂O (27.7 g, 661 mmol, in 150 mL of H₂O) was added, and the solution was held for 36 hours. HCl (0.5 M, 1.45 L, 2.90 mol) was added slowly in portions to adjust the pH of the solution to 2, and it was held at 0 - 5 °C for 1 hour. The heterogeneous slurry was filtered, and the solid was washed with 1:3 MeOH. H₂O was added at 0 °C (600 mL), and the solid was dried under N₂ at 45 °C for 16 hours to obtain the crude title compound as a white solid (158 g, 99%). The crude product (150 g) was slurried in IPAC (1.13 L) at 60 - 65 °C for 0.5 hour. The heterogeneous mixture was cooled to 0 - 5 °C over 3 hours and then stirred for an additional 1 hour, followed by filtration. The recovered solid was dried with IPAC at 0 - 5 °C (2 × 300 mL) and then dried under N₂ at 45 °C for 12 hours to obtain the title compound as a white solid (127 g, 82% from intermediate 14); 1 HnmR (500 MHz, CDCl₃) δ 1.16 (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 (1H, 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.

[0110] Intermediate 17: Naphthalen-1-ylmethyl (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-carboxylate

Chem.

[0111] Compound (I): (1S,4S)-4-(2-Fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-Methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

Chemical Structure

[0112] Part 1: The crude title compound (2.50 g, 4.59 mmol) was dissolved in EtOH (15.0 mL). While adding water (7.50 mL) dropwise, the temperature of the solution was maintained at 25.0 ± 2.0 °C, during which a precipitate formed. The heterogeneous slurry was stirred for an additional 1.0 h and then recovered by filtration. The solid was washed with a (2:3) mixture of EtOH / water (2 × 5.00 mL), recovered, and dried under N2 to give the title compound as a white solid (1.80 g, 72%). This material was characterized as Form A and used as a seed according to the method described in Part 2.

[0113] Part 2: The crude title compound (50.0 g, 91.8 mmol) was dissolved in EtOH (350 mL) and then filtered. EtOH (100 mL) was added to the vessel and then filtered to obtain a combined EtOH solution. While adding H2O (150 mL) slowly over 0.5 h, the temperature of the solution was maintained at 25.0 ± 2.0 °C. The solution was stirred for an additional 0.5 h and then the seed material from Part 1 (0.005 g, 0.1% w / w) was added. The solution was held for 6 h, then cooled to 20.0 ± 0.5 °C over 2 h and then held for an additional 6 h. H2O (150 mL) was added slowly over 6 h and then the mixture was held for an additional 2 h before filtration. EtOH (45 mL) and H2O (30 mL) were added to the vessel and then used to wash the filter cake. The solid was recovered and dried under N2 at < 45 °C for 12 h to give the title compound Form A as a white solid (42.2 g, 85%); 1 1H NMR (500 MHz, CDCl3) δ 0.97 (3H, s), 1.12 - 1.42 (5H, m), overlap 1.25 (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). Calculated HRMS (ESI) m / z for C30H42FN2O6 [M + H] +: 545.3022, Measured value: 545.3019. The XRPD pattern (XRPD method A) is shown in Figure 1.

[0114] Example 1 Amorphous (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-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 dried overnight in a freeze dryer (Christ Alpha 2-4LD) to obtain amorphous (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-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.

[0115] Example 2 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]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid Polymers compatible with compound (I) were screened to select a polymer suitable for the solid dispersion. The following polymers were found to be stable at 50 °C and 40 °C / 75% relative humidity (RH) for 4 weeks without signs of crystallization and were found to be suitable at drug loadings of 10%, 20% and 40%; · Kollidon® 30 (poly(1-vinyl-2-pyrrolidone), available from BASF Pharma) · Kollidon® VA 64 (copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate in a mass ratio of 6:4, available from BASF Pharma) · Soluplus® (polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, available from BASF Pharma) · Eudragit® L100-55S (methacrylic acid-ethyl acrylate copolymer (1:1), available from Evonik Industries AG) · polyacrylic acid · AQOAT® HPMC AS-LF (hypromellose acetate succinate, available from Shin-Etsu Chemical Co., Ltd.) · AQOAT® HPMC AS-HF (hypromellose acetate succinate, available from Shin-Etsu Chemical Co., Ltd.) · cellulose acetate phthalate · polyvinyl acetate phthalate

[0116] Three amorphous solid dispersions of compound (I) were prepared at drug loadings of 20%, 30% and 40% w / w according to the following process: 1. Compound (I) was weighed into a glass vessel and then Kollidon® VA 64 (copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate in a mass ratio of 6:4, available from BASF Pharma) was added. The powder was then mixed for 5 minutes at 32 rpm using a Turbula® blender. 2. Before extruding the mixture, the hot melt extruder was first washed with molten beads. The 20% drug loading solid dispersion was extruded at 200 °C and the filaments were collected in a clean glass vessel. The 30% drug loading solid dispersion was also extruded at 200 °C and the 40% drug loading solid dispersion was extruded at 210 °C. 3. Next, the collected filaments were manually crushed using a laboratory mill.

[0117] The samples were stabilized at 40 °C / 75% relative humidity (RH) (open container) and 50 °C (sealed container, ambient RH) for 4 weeks, and the XRPD of the samples was recorded weekly. Figure 3 shows the XRPD diffraction patterns (XRPD method B) of each sample after formation and after storage at 40 °C / 75% relative humidity (RH) (open container) for 4 weeks and at 50 °C (sealed container, ambient RH) for 4 weeks. The XRPD patterns indicated that crystallization of compound (I) did not occur when the amorphous solid dispersion was stored at 40 °C / 75% relative humidity (RH) for 4 weeks (open container) and at 50 °C for 4 weeks (sealed container, ambient RH). Dissolution experiments using 30% drug-loaded solid dispersions showed improved dissolution in simulated gastric fluid pH 1.8 and fasted-state simulated intestinal fluid (FaSSIF) pH 6.5 compared to granules based on crystalline form A of compound (I).

[0118] Example 3 (1S,4S)-4-(2-Fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid, Form G When 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]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid, DSC shows an endothermic transition starting at about 43.9 °C. This new endothermic peak is assigned to a solid-solid phase transition, and the phase obtained at a temperature above the completion of the endothermic peak observed at about 43.9 °C is designated as form G. This new anhydrous phase form G exists up to the last endothermic peak in the DSC corresponding to the onset of melting at 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.

[0119] Biological and Physicochemical Data of Compound (I) RXFP1 Hu cAMP (Test A) To screen for modulators of hRXFP1, a compound identification assay was used to stimulate cAMP production via the GS-coupled hRXFP1 receptor. The cAMP HiRange HTRF kit (available from CisBio Bioassays, France; catalog number 62AM6PEJ) was used extensively according to the manufacturer's recommendations for the detection of cAMP. The HTRF method is a competitive immunoassay between natural cAMP produced by cells and cAMP labeled with the dye d2. The tracer binding is visualized with a cryptate-labeled antibody against cAMP, and thus the signal is inversely proportional to the amount of cAMP produced.

[0120] Preparation of assay reagents: Assay buffer: HBSS (ThermoFisher, 14065) with 5 mM Hepes (ThermoFisher, 15630) pH 7.4 containing 0.1% BSA (Sigma, A8806) Cells: Jump-In™ T-REx™ CHO-K1 cells (ThermoFisher) stably transfected with human RXFP1 were used. The cells were induced to express human RXFP1 by treatment with 10 ng / ml doxycycline for 24 hours. The cells were then cryopreserved for long-term storage. At the start of each experiment, the cells were thawed, washed with PBS, and resuspended in assay buffer to 1.875×10^5 cells / ml. cAMP standard: The stock standard cAMP provided in the CisBio kit was diluted with assay buffer to a maximum final concentration of 2.8 μM in the assay. HTRF detection reagent: cAMP-d2 and anti-cAMP cryptate reconstituted according to the CisBio instructions were diluted 1:40 with the lysis buffer provided with the HTRF-kit.

[0121] Step-by-step procedure for performing the assay: 40 nL of the test compound dissolved in DMSO was aqueously dispensed (Labcyte Echo) into a white 384-well plate (Greiner; 784075), sealed, and stored at room temperature until assayed. On the day of the assay, using an Echo acoustic dispenser, 40 nL of 200 nM relaxin-2 in DMSO (final concentration 1 nM) was added to 100% control wells, and 40 nL of DMSO was added to 0% wells. 4 μL of assay buffer containing 1 mM IBMX (final concentration 0.5 mM) to block phosphodiesterase was added using a Multidrop Combi (ThermoFisher). 4 μL of cell solution (1.875×10^5 cells / ml) was added using a Multidrop Combi to obtain 750 cells / well. Incubation at room temperature for 45 minutes. 4 μL of cAMP-d2 in lysis buffer was added using a Multidrop Combi. 4 μL of anti-cAMP cryptate in lysis buffer was added using a Multidrop Combi. Incubation at room temperature for 2 hours. The homogeneous time-resolved fluorescence (HTRF) signal was detected with an Envision (PerkinElmer) or Pherastar (BMG Labtech) reader (λex = 340 nm, λem = 665 and 615 nm).

[0122] Using a cAMP standard curve, the HTRF data was converted to the amount of cAMP produced in the samples, which was subsequently used for the calculation of concentration-response. The concentration-response data was fitted to Hill's equation using a four-parameter logistic fit. The results of the assay were reported in Table 1 as EC 50 (μM) and S inf (%).

[0123] EC 50It is defined as the concentration at which the stimulating activity reaches 50% of its maximum level. When the assay is performed multiple times for the same compound, the geometric mean is reported.

[0124] S inf is the applied activity level at infinite concentration of the test compound and is the efficacy. To facilitate comparison of efficacy data, the efficacy was normalized against the % effect of the response stimulated by saturating concentration of relaxin (1 nM). When the assay is performed multiple times for the same compound, the arithmetic mean is reported.

[0125] Human plasma protein binding (Test B) The assay was performed according to the human plasma protein binding assay described on pages 167 - 170 of 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. The data was reported in Table 1 as the unbound fraction (f u )(free %). When the assay is performed multiple times for the same compound, the arithmetic mean is reported.

[0126] Human liver microsome stability (Test C) The assay was performed according to the human liver microsome stability assay described on pages 170 - 174 of 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. The data was reported in Table 1 as CL int (μl / min / mg protein). When the assay is performed multiple times for the same compound, the arithmetic mean is reported.

[0127] Human Hepatocyte Stability (Test D) The metabolic stability of the compound in human hepatocytes was evaluated using the following protocol: 1. Prepare 10 mM stock solutions of the compound and the control compound in a suitable solvent (DMSO). Place the incubation medium (L-15 medium) in a 37 °C water bath and warm it for at least 15 minutes before use. 2. Add 80 μL of acetonitrile to each well of a 96-well deep well plate (“quench plate”). 3. In a new 96-well plate, dilute the 10 mM test compound and the control compound to 100 μM by combining 198 μL of acetonitrile and 2 μL of the 10 mM stock solution. 4. Remove a vial of cryopreserved (-150 °C or less) human hepatocytes (LiverPool™ 10-donor human hepatocytes obtained from Bioreclamation IVT (product number S01205)) from the storage and keep the vial at cryogenic temperature until the thawing process occurs. Thaw the cells as rapidly as possible by placing the vial in a 37 °C water bath and gently shaking. The vial should be kept in the water bath until all ice crystals have dissolved and are no longer visible. After thawing is complete, spray 70% ethanol on the vial and transfer the vial to a biosafety cabinet. 5. Open the vial and pour the contents into a 50 mL conical tube containing thawing medium. Place the 50 mL conical tube in a centrifuge and spin at 100 g for 10 minutes (room temperature). After spinning is complete, aspirate the thawing medium and resuspend the hepatocytes in sufficient incubation medium to yield approximately 1.5×10 6 cells / mL. 6. Use a Cellometer® Vision to count the cells and measure the viable cell density. Cells with insufficient viability (less than 80% viability) are not acceptable for use. Dilute the cells in incubation medium to a research cell density of 1.0×10 6 viable cells / mL. Transfer 7.247.5 μL of hepatocytes to each well of a 96-well cell incubation plate. Place this plate on an Eppendorf Thermomixer Comfort plate shaker and warm the hepatocytes for 10 minutes. Add 2.5 μL of 100 μM test compound or control compound to the incubation well containing the cells to initiate the reaction. Incubate this plate on an Eppendorf Thermomixer Comfort plate shaker at 37 °C and 900 rpm. At 0.5, 5, 15, 30, 45, 60, 80, 100, and 120 minutes, transfer 20 μL of the incubated mixture to a separate "quench plate" and then mix the sample by vortexing for 2 minutes. Centrifuge the quench plate at 4,000 rpm for 20 minutes. Transfer 30 μL of the supernatant of each compound to a 96-well analysis plate. Pool 4 compounds into one cassette. Then, dilute the pooled sample by adding 180 μl of pure water. All incubations are performed in a single run.

[0128] All calculations were performed using Microsoft Excel. The peak area was determined from the extracted ion chromatogram. The in vitro intrinsic clearance (in vitro Cl int (L / min / 10 6 cells)) was determined by regression analysis of the disappearance rate (%) Ln of the parent with respect to the time curve. The in vitro intrinsic clearance (in vitro Cl int , μL / min / 10 6 cells) was reported in Table 1 and determined from the gradient value using the following equation: In vitro Cl int = kV / N V = incubation volume (0.25 mL); N = number of hepatocytes per well (0.25 × 10 6 cells). When the assay is performed multiple times for the same compound, report the geometric mean.

[0129] Rat hepatocyte stability (Test E) The assay was performed according to the rat hepatocyte stability assay described on pages 170 - 174 of 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. The data are reported in Table 1 as mean Cl int (μl / min / 10 6 cells). When the assay was performed multiple times for the same compound, the geometric mean is reported.

[0130] Solubility (pH 7.4) (Test F) The assay was performed according to the solubility assay described on pages 164 - 167 of 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. The data are reported in Table 1 as solubility (μM). When the assay was performed multiple times for the same compound, the arithmetic mean is reported.

[0131]

Table 1

[0132] Human RXFP1 cGMP production assay (Test G) To profile compounds for RXFP1 agonist activity related to cGMP production, the GreengENIe cGMP assay (Montanamolecular; catalog number D800G) was used. This assay is based on an mNeonGreen fusion protein fluorescent biosensor delivered to mammalian cells in a BacMam vector. When cGMP binds to the biosensor, fluorescence decreases.

[0133] Preparation of assay reagents: Assay buffer: DPBS (Gibco; 14040133) containing 0.1% BSA (Sigma; A8806) Cells: HEK293s cells stably transfected with human RXFP1 in pIRESneo3 were used. The cells were cultured in DMEM medium (Gibco; 31966) containing 10% FBS supplemented at 0.8 mg / mL to maintain RXFP1 expression.

[0134] Step - by - step protocol for running the assay: Day 1 1. One day before transduction, the cells were split and seeded at 63000 cells / cm2 in DMEM medium containing 10% FBS without antibiotics in a tissue culture flask. Day 2 2. After washing with PBS, the cells were detached using Accutase (Gibco; 1737341), resuspended in medium, and collected in a 50 mL tube. 3. The cells were counted with a CEDEX (Innovatis) and diluted to 267000 cells / mL with medium. 4. The viral transduction master mix was prepared by mixing the reagents at the following ratios for a single well: 6 μL gENIe BacMAM vector 0.2 μL 500 mM sodium butyrate 13.8 μL DMEM medium containing 10% FBS Total volume 20 μL 5. The cells and the transduction master mix were mixed at a ratio of 30 μL of cells and 20 μL of master mix for a single well. 6. The above-derived cell transduction mixture was dispensed at 50 μL per well into a black poly-D-lysine-coated μclear 384-well plate (Greiner; 781946). 7. The plate was incubated at 37 °C and 5% CO2 in the dark for 24 hours. Day 3 8. The medium was removed from the plate using a Bluewasher (BluCatBio). 9. 20 μL of assay buffer was added using a Multidrop Combi (ThermoFisher). 10. The plate was incubated at room temperature in the dark for 30 minutes before the assay. 11. The plate was assayed using a FLIPR Tetra (Molecular Devices): 10 μL of the compound diluted in assay buffer was added to each well by the FLIPR Tetra, and the green fluorescence was measured over time for up to 3 hours.

[0135] The data were processed using Screener software (Genedata AG). After subtracting the background fluorescence (before the addition of the compound), the area under the curve values from 0 to 90 minutes after the addition of the compound were used for the calculation of the response. The concentration-response data were fitted with a four-parameter logistic fit, and the EC 50 value (nM) was reported in Table 2.

[0136] Human RXFP1 phosphorylation-ERK assay (Test H) To profile compounds for RXFP1 agonist activity related to ERK phosphorylation, a Go-ERK (Thr202 / Tyr204) cell kit (CisBio; 64AERPEH) was used. This 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 specifically binds to phosphorylated ERK, and the second antibody binds to another motif of ERK and is independent of its phosphorylation state. ERK phosphorylation enables 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.

[0137] Preparation of assay reagents: Cells: HEK293s cells stably transfected with human RXFP1 in pIRESneo3 were used. The cells were cultured in DMEM medium (Gibco; 31966) containing 10% FBS supplemented at 0.8 mg / mL to maintain RXFP1 expression. Assays were performed on continuously cultured cells.

[0138] Dilution of test compounds: Compounds were diluted to the desired concentration with serum-free DMEM without phenol red (Gibco; 31053-038). The DMSO concentration was adjusted to 0.4%.

[0139] Antibody mixture: Eu- and d2-labeled anti-ERK1 / 2 antibodies were separately diluted 20-fold with the detection buffer provided in the kit. Immediately before the experiment, equal volumes of each diluted antibody solution were combined to form the antibody mixture.

[0140] Step-by-step protocol for performing the assay: Day 1 1. Cells were detached from the culture flask using Accutase (GIbco; 1737341), resuspended in phenol red-free DMEM medium containing 10% FBS, and collected in 50 mL tubes. 2. Cells were counted using a CEDEX (Innovatis) and diluted to 320,000 cells / mL in the above medium. 3. 100 μL of the cell suspension per well was dispensed into a black μclear poly-D-lysine-coated μclear 96-well plate (Greiner; 655946). 4. The plate was incubated at 37 °C and 5% CO2 for 24 hours. Day 2 5. Serum starvation: The medium was removed and replaced with 50 μL of serum-free DMEM without phenol red. The plate was incubated at 37 °C and 5% CO2 for 5 hours. 6. 50 μL of the test compound solution was added per well. 7. The plate was incubated at room temperature for 5 minutes. 8. The stimulation was stopped by quickly removing the medium and adding 50 μL of lysis buffer (diluted to 1-fold the final concentration before addition) per well. 9. The plate was transferred to -80 °C to freeze the lysates. Day 3 10. The plate was thawed and shaken at room temperature for 30 minutes. 11. The cell lysates were homogenized by pipetting. 12. 16 μL of the homogenate per well was transferred to a white low-volume 384-well plate (Greiner; 784075). 13. 4 μL of the antibody mixture was added per well. 14. The plate was incubated at room temperature in the dark for 4 hours. 15. The homogeneous time-resolved fluorescence (HTRF) signal was detected using a Pherastar (BMGLabtech) reader (λex = 340 nm, λem = 665 and 615 nm).

[0141] HTRF ratio data were processed using Screener software (Genedata AG). The concentration-response data were fitted to a four-parameter logistic fit and EC 50 values (nM) as reported in Table 2.

[0142]

Table 2

[0143] Solubility of Amorphous Compound (I) in SGF at pH 1.2 (Test I) Preparation of Amorphous Nanosuspension of Compound (I) A 100 mM stock solution of Compound (I) was prepared in DMA and Miglyol 812 (9.44 mg in 140.2 μL of DMA and 23.6 μL in Miglyol 812) by sonication and stirring. 10 μL of the stock solution was rapidly injected under sonication for several seconds into 990 μL of stabilizer solution containing 0.2% w / w PVPK30 and 0.2% w / w Pluronic F127 and 0.25 mM SDS aqueous solution to prepare a 1 mM amorphous nanosuspension. This formulation was further diluted to 0.25 mM with purified water.

[0144] Solubility Experiment The bulk concentration in equilibrium with the amorphous particles was determined at 37 °C by turbidimetry (described in Lindfors et.al., Langmuir, 2006, 22(3), 911 - 916). A low volume of drug suspension was continuously added directly to a fluorescence cuvette containing 2000 μL of simulated gastric fluid pH 1.2 (SGF) and then mixed to obtain 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, and both the emission wavelength and the excitation wavelength were set to 700 nm. The slit excitation was set to 2.5 nm and the slit emission was set to 5 nm. The particles were dissolved for at least 30 seconds before starting the measurement. Then, data was collected for 1 minute and the average light scattering intensity was calculated. The solubility test was performed within 45 minutes from the formation of the amorphous nanosuspension.

[0145]

Table 3

[0146] Light scattering was plotted as a function of the total concentration of compound (I). At low concentrations, the particles dissolved, but at concentrations above the solubility, the particles remained suspended and the light scattering intensity increased almost linearly as a function of the total concentration. The line fitted to the high-concentration data was extrapolated to zero intensity to estimate the amorphous solubility of compound (I) in SGF at pH 1.2 at 37 °C, which was determined to be 8 μM. The light scattering intensity as a function of the total concentration is shown in Figure 6.

[0147] Solubility of crystalline form A of compound (I) in SGF at pH 1.2 (Test J) The crystalline solubility in simulated gastric fluid pH 1.2 (SGF) was measured using the small shaking flask method (stirred at 37 °C for 24 h). An excess of form A of compound (I) was weighed into a vial, 1.2 mL of SGF at pH 1.2 was added, and the sample was sonicated for 45 min.

[0148] The sample was stirred at 37 °C for 24 h on an Eppendorf shaker at a stirring speed of 1000 rpm. The sample was then centrifuged at 10000 RPM for 15 min at 37 °C. Approximately 500 μL of the supernatant was removed with an automatic pipette into a new vial and centrifuged again at 10000 rpm at 37 °C for 15 min. The sample was then taken by automatic pipette from the second centrifugation and finally diluted. The pH of the equilibrated remaining sample solution after the second centrifugation was measured. XRPD was used to confirm that no morphological changes had occurred. UHPLC was used to quantify the concentration in the sample, and the solubility of form A of compound (I) in SGF at pH 1.2 at 37 °C was found to be 0.4 μM.

[0149] Those skilled in the art will understand that the above assays can be carried out using alternative equipment and minor modifications to the protocol without significantly affecting the results.

[0150] This description and its specific examples illustrate particular embodiments, but are for illustrative purposes only. Accordingly, the present disclosure is not limited to the exemplary embodiments described herein and can be variously modified. Additionally, it should be recognized that various embodiments described in relation to separate embodiments for clarity reasons can be combined to form a single embodiment. Conversely, various embodiments described in relation to a single embodiment for brevity reasons can be combined to form these partial combinations as well.

[0151] Any publications disclosed within this specification are hereby incorporated by reference into this specification.

Claims

1. Amorphous compounds (I). 【Chemistry 1】

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

3. The pharmaceutical composition according to claim 2, wherein at least 80 wt% of compound (I) in the pharmaceutical composition is amorphous.

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

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

6. The solid dispersion according to claim 4 or 5, wherein at least 80 wt% of compound (I) in the solid dispersion is amorphous.

7. The solid dispersion according to claim 4 or 5, wherein less than 20 wt% of compound (I) in the solid dispersion is crystalline.

8. The solid dispersion according to claim 4 or 5, wherein compound (I) is present in an amount of 5 wt% to 95 wt%.

9. The solid dispersion according to claim 4 or 5, wherein one or more of the polymers are present in an amount of 5 wt% to 95 wt%.

10. The solid dispersion according to claim 4 or 5, wherein one or more of the polymers are water-soluble.

11. The solid dispersion according to claim 4 or 5, wherein the one or more polymers are selected from poly(1-vinyl-2-pyrrolidone), a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, methacrylate-ethyl acrylate copolymer, polyacrylic acid, hypromellose succinate acetate, cellulose phthalate acetate, and polyvinyl phthalate acetate.

12. The solid dispersion according to claim 4 or 5, wherein the one or more polymers is a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate, and optionally a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate in a weight ratio of 6:

4.

13. A pharmaceutical composition comprising the solid dispersion described in claim 4 or 5.

14. The pharmaceutical composition according to claim 2 or 3, in the form of a tablet or capsule.

15. The pharmaceutical composition according to claim 13, in the form of a tablet or capsule.

16. A pharmaceutical composition according to claim 2 or 3, suitable for oral administration.

17. The pharmaceutical composition according to claim 13, which is suitable for oral administration.

18. The pharmaceutical composition according to claim 2 or 3 for use in the treatment of a subject having a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension.

19. The pharmaceutical composition according to claim 13, for use in the treatment of subjects having a disease or condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with moderate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension.

20. The crystalline form of compound (I), which has a melting onset temperature of approximately 217.5°C.

21. A crystalline form of compound (I) that can be obtained by heating crystalline form A of compound (I) to a temperature of approximately 43.9°C, sometimes approximately 45°C, sometimes approximately 50°C, sometimes approximately 55°C, sometimes approximately 60°C, and sometimes approximately 65°C.

22. A method for preparing the crystalline form (A) of compound (I) according to claim 20 or 21, comprising heating the crystalline form (A) of compound (I) to a temperature of approximately 43.9°C, optionally approximately 45°C, optionally approximately 50°C, optionally approximately 55°C, optionally approximately 60°C, and optionally approximately 65°C.

23. The morphology (A) of compound (I) has an X-ray powder diffraction pattern (CuK) having at least five specific peaks at approximately 2θ = 7.5, 10.7, 12.8, 14.5 and 15.8°. α The crystal morphology according to claim 21, having a line.

24. The method according to claim 22, wherein the form (A) of compound (I) has an X-ray powder diffraction pattern (CuK α-ray) having at least five specific peaks at about 2θ = 7.5, 10.7, 12.8, 14.5 and 15.8°.

25. Form A of compound (I) exhibits an X-ray powder diffraction pattern substantially the same as the one shown in Figure 1 (CuK α The crystal morphology according to claim 21, having a line.

26. The method according to claim 22, wherein form A of compound (I) has substantially the same X-ray powder diffraction pattern (CuK α-ray) as the X-ray powder diffraction pattern shown in Figure 1.

27. A pharmaceutical composition comprising the crystalline form described in claim 21 and a pharmaceutically acceptable excipient.

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