Combination of an RXFP1 modulator and an SGLT2 inhibitor

Abstract

This specification generally relates to a combination of an RXFP1 modulator and an SGLT2 inhibitor, and in particular to pharmaceutical compositions containing such combinations and methods of treatment using such combinations.

Description

Technical Field

[0001] This specification describes a combination of a compound (including its salts) that is a modulator of RXFP1 and an SGLT2 inhibitor such as dapagliflozin, a pharmaceutical composition containing such a combination, and the use of such a combination.

Background Art

[0002] Relaxin is a multifaceted hormone known to mediate adaptive changes in systemic hemodynamics and the kidney during pregnancy. Relaxin has anti-fibrotic properties and has also been shown to have 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 (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4).

[0003] In heart failure patients, a significant small population also suffers from pulmonary hypertension (HF+PH patients). Approximately 50% of heart failure patients with preserved ejection fraction also suffer from pulmonary hypertension, which is estimated to increase to 60% in heart failure patients with reduced ejection fraction (Non-Patent Document 5, Non-Patent Document 6). Patients with heart failure accompanied by pulmonary hypertension have been shown to have a reduced survival rate compared to heart failure patients without pulmonary hypertension (Non-Patent Document 7). 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 (Non-Patent Document 8). A 4 mmHg reduction in mPAP is also associated with improvement of dyspnea in patients suffering from heart failure and pulmonary hypertension (Non-Patent Document 9).

[0004] Resistant hypertension (rHT) is defined as the blood pressure of hypertensive patients that remains elevated above the target achieved despite the simultaneous use of three antihypertensive agents at optimized doses 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. For patients with rHT, there are multiple options for what to add next (such as 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 also includes patients whose blood pressure is appropriately controlled when receiving four or more antihypertensive drugs simultaneously (Non-Patent Document 10). rHT patients typically have a long history of severe blood pressure elevation and tend to have a higher cardiovascular risk than treated hypertensive patients with regulated blood pressure (Non-Patent Document 11). It has been suggested that relaxin may have the potential for the treatment of hypertensive disorders (Non-Patent Document 12).

[0005] Clinical trials were conducted using serelaxin, which is unmodified recombinant human relaxin 2. Continuous intravenous administration of serelaxin to inpatients improved cardiac, renal, and hepatic impairments and congestion (Non-Patent Documents 13, 14, 15). However, since serelaxin is rapidly removed from the patient's blood circulation, its therapeutic effect is limited, and the positive effect rapidly disappeared when the intravenous injection was discontinued. Furthermore, since approximately one-third of the patients experienced a significant decrease in blood pressure (>40 mmHg) after intravenous administration of serelaxin, it was concluded that the dose had to be halved or further reduced.

[0006] The cognate receptor for human relaxin is RXFP1, a well-validated pharmacologically important member of the GPCR family 1c, the activation of which by the hormone relaxin is associated with hemodynamic, antifibrotic, and anti-inflammatory properties (Non-Patent Document 16).

[0007] Small molecule modulators of RXFP1 are sought as relaxin mimetics. For example, Patent Document 1, Non-Patent Documents 17 and 18 by Marugan, J. J., et al. discuss small molecule modulators of RXFP1.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Summary of the Invention

Problems to be Solved by the Invention

[0010] Notwithstanding the above, there remains a continuing need for further compounds that are modulators of RXFP1, which can be particularly promising for development as therapeutic agents. Such compounds may also show improved modulation of RXFP1 compared to other known RXFP1 modulators. Such compounds may also show an advantageous pharmacokinetic profile (e.g., lower intrinsic clearance) and / or advantageous physical properties (e.g., higher aqueous solubility) compared to other known RXFP1 modulators. Accordingly, such compounds may be particularly useful for the treatment of medical conditions where modulation of RXFP1 is beneficial. Further, combinations of such compounds with other therapeutic agents may be useful for the treatment of certain medical conditions such as hypertension.

Means for Solving the Problems

[0011] This specification relates to combinations of the RXFP1 modulators described herein with SGLT2 inhibitors. SGLT2 inhibitors such as dapagliflozin have been shown to exhibit a blood pressure-lowering effect (Weber et al., Lancet Diabetes Endocrinol., 2016, 4(3), 211-220). Accordingly, combinations of RXFP1 modulators with SGLT2 inhibitors may provide particular benefits in the treatment of hypertension, particularly resistant hypertension and cardiovascular diseases where an important unmet need still remains.

[0012] Accordingly, this specification describes, in part, RXFP1 modulators for use in the treatment of human patients, wherein the RXFP1 modulators are administered in combination with an SGLT2 inhibitor, and the RXFP1 modulators are

Chemical formula

Chemical formula

Chemical formula

[0013] Similarly, this specification also describes, in part, an RXFP1 modulator for use in the treatment of conditions selected from heart failure, heart failure with preserved ejection fraction, heart failure with mid-range 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, wherein the RXFP1 modulator is administered in combination with an SGLT2 inhibitor, and the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof.

[0014] Similarly, this specification also describes, in part, a method for treating a condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with mid-range 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 a therapeutically effective amount of an RXFP1 modulator to the human patient, wherein the RXFP1 modulator is administered in combination with a therapeutically effective amount of an SGLT2 inhibitor, and the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof.

[0015] Similarly, this specification also describes, in part, a method for treating a condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with mid-range 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 first amount of an RXFP1 modulator and a second amount of an SGLT2 inhibitor, wherein the first amount and the second amount together constitute a therapeutically effective amount, and the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof.

[0016] Similarly, this specification also describes, in part, a pharmaceutical composition comprising an RXFP1 modulator, an SGLT2 inhibitor, and a pharmaceutically acceptable excipient, wherein the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof.

[0017] Similarly, this specification also describes, in part, a first pharmaceutical composition comprising an RXFP1 modulator selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, a second pharmaceutical composition comprising an SGLT2 inhibitor and a pharmaceutically acceptable excipient, and a kit comprising the same.

[0018] Further aspects of the disclosure will be apparent to those skilled in the art upon reading this specification.

DETAILED DESCRIPTION OF THE INVENTION

[0019] 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.

[0020] Terms not specifically defined herein are to be understood as having the meaning that would be ascribed to them by those skilled in the art in light of the disclosure and the context.

[0021] "About" generally may mean an acceptable degree of error for the quantity being measured, taking into account the nature and 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%.

[0022] Embodiments described herein as including one or more features may also be considered as disclosing corresponding embodiments consisting of such features.

[0023] Concentrations, amounts, volumes, percentages, and other numerical values may be presented in range format in this specification. Such range format is used merely for convenience and brevity and should be interpreted flexibly as encompassing 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 numerical value and sub-range were explicitly recited.

[0024] The chemical names of the compounds described in this specification were generated using ChemDraw® Professional version 19.0.0.22 manufactured by 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 in this specification are presented in the form of chemical names and as formulas, in case of any inconsistency, the formula shall prevail.

[0025] RXFP1 modulator In one embodiment, the RXFP1 modulator is

Chem.

Chem.

[0026] In one embodiment, the RXFP1 modulator is Compound 1 or its pharmaceutically acceptable salt.

[0027] In one embodiment, the RXFP1 modulator is Compound 2 or its pharmaceutically acceptable salt.

[0028] In one embodiment, the RXFP1 modulator is Compound 3 or its pharmaceutically acceptable salt.

[0029] In one embodiment, the RXFP1 modulator is Compound 4 or its pharmaceutically acceptable salt.

[0030] In one embodiment, the RXFP1 modulator is compound 5 or a pharmaceutically acceptable salt thereof.

[0031] In one embodiment, the RXFP1 modulator is compound 6 or a pharmaceutically acceptable salt thereof.

[0032] In one embodiment, the RXFP1 modulator is compound 1.

[0033] In one embodiment, the RXFP1 modulator is compound 2.

[0034] In one embodiment, the RXFP1 modulator is compound 3.

[0035] In one embodiment, the RXFP1 modulator is compound 4.

[0036] In one embodiment, the RXFP1 modulator is compound 5.

[0037] In one embodiment, the RXFP1 modulator is compound 6.

[0038] In one embodiment, the RXFP1 modulator is (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-Cyano-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-Cyano-5-(((1S,2R,3S,4R)-3-((cyclopropylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid; (1S,4S)-4-(2-Cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-((neopentylcarbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid; (1S,4S)-4-(2-Cyano-5-(((1S,2R,3S,4R)-3-((3-fluorobicyclo[1.1.1]pentan-1-yl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid; and (1S,4S)-4-(5-(((1S,2R,3S,4R)-3-((cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid; or a pharmaceutically acceptable salt thereof selected from.

[0039] In one embodiment, the RXFP1 modulator is a compound as claimed or exemplified in the specification of International Patent Application No. PCT / EP2021 / 084673 or U.S. Patent Application No. 17 / 457,953, both of which are incorporated by reference in their entirety.

[0040] The term "pharmaceutically acceptable" is used to specify that an object (e.g., a salt, dosage form, or excipient) is suitable for use in a patient. A list of examples of pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts: Properties, Selection and Use, P.H. Stahl and C.G. Wermuth, editors, Weinheim / Zuerich: Wiley-VCH / VHCA, 2002. Suitable pharmaceutically acceptable salts of the compounds described herein are, for example, acid addition salts or base salts. Acid addition salts of the compounds described herein can be formed by contacting the compound with a suitable inorganic or organic acid under conditions known to those of skill in the art. Acid addition salts can be formed using, for example, inorganic acids selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Acid addition salts can also be formed using organic acids selected from the group consisting of trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid, and para-toluenesulfonic acid.

[0041] Thus, in one embodiment, the RXFP1 modulator is a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, sulfate, phosphate, trifluoroacetate, citrate, maleate, oxalate, acetate, formate, benzoate, fumarate, succinate, tartrate, lactate, pyruvate, methanesulfonate, benzenesulfonate, or para-toluenesulfonate.

[0042] The compounds described herein can form base addition salts. The base addition salts of the compounds described herein can be formed by contacting the compound with a suitable inorganic or organic base under conditions known to those skilled in the art. For example, the compound can be treated in an aqueous medium with a hydroxide or alkoxide of an alkali metal or alkaline earth metal (e.g., ethoxide or methoxide) or an organic amine that is appropriately basic (e.g., choline or meglumine) to produce an alkali metal (such as sodium, potassium or lithium) or alkaline earth metal (such as calcium) salt. Thus, in one embodiment, the RXFP1 modulator is a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is a sodium, potassium, lithium, calcium, choline or meglumine salt.

[0043] The compounds and salts described herein can exist in solvated and non-solvated forms. For example, the solvated form can be a hydrate form such as a hemihydrate, monohydrate, dihydrate, trihydrate or an alternative amount thereof. All such solvated and non-solvated forms of the compounds described herein are included herein.

[0044] The atoms of the compounds and salts described herein can exist as their isotopes. The atom is substituted 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), and all compounds described herein are included herein.

[0045] The compounds described herein can 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.

[0046] The compounds described herein contain one or more chiral centers. Unless the structure or chemical name herein indicates chirality, the structure or name is intended to encompass any single stereoisomer and any mixture of stereoisomers (e.g., racemate) corresponding to that structure or name. Here, the structures herein include bonds depicted as solid and dashed wedges (i.e.,

Chemical formula

[0047] 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., racemate) using, for example, chiral chromatography separation. In other embodiments, a single stereoisomer can be obtained, for example, by direct synthesis from chiral starting materials.

[0048] According to one embodiment, the RXFP1 modulator is provided as a single enantiomer having an enantiomeric excess (%ee) of ≧95%, ≧98% or ≧99%. For simplicity, a single enantiomer is present with an enantiomeric excess of 99% or more.

[0049] According to one embodiment, the RXFP1 modulator is provided as a single enantiomer having an enantiomeric excess (%ee) in the range of 95-100%.

[0050] The compounds described herein may exist in one or more tautomeric forms including, but not limited to, the keto and enol forms. Reference to a particular compound includes all tautomeric forms including mixtures thereof. Thus, the structures depicted herein as one tautomer are intended to also include other tautomers.

[0051] The RXFP1 modulators described herein may be administered in the form of prodrugs, which are compounds that are decomposed in the human or animal body to release such RXFP1 modulators. Such pharmaceutically acceptable prodrugs of the RXFP1 modulators also form one embodiment. Various forms of prodrugs are known in the art. See, for example: a) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and Application of Pro-drugs”, by H. Bundgaard p. 113-191 (1991); c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and e) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984).

[0052] Sodium-glucose transporter protein, subtype 2 (SGLT2) inhibitor In some examples, glucose reabsorption in the kidney is controlled by members of the sodium-glucose cotransporter family, which are sodium-dependent glucose transporter proteins. In some examples, glucose reabsorption in the kidney is controlled by the sodium-glucose transporter protein, subtype 2 (SGLT2).

[0053] In one embodiment, the SGLT2 inhibitor is empagliflozin. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, and empagliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin and has the following chemical structure.

Chemical formula

[0054] Dapagliflozin is a potent, highly selective, and orally active inhibitor of human renal sodium-dependent glucose transporter 2 (SGLT2) (SGLT2i) that is approved to improve glycemic control in adults with type 2 diabetes (as an adjunct to diet and exercise) and to reduce the risk of hospitalization for heart failure in adults with type 2 diabetes and established cardiovascular disease or multiple cardiovascular risk factors. Dapagliflozin is disclosed in WO 2003 / 099836 pamphlet together with details regarding its chemical synthesis.

[0055] Treatment with an RXFP1 modulator and an SGLT2 inhibitor The combination comprising an RXFP1 modulator and an SGLT2 inhibitor described herein is expected to be useful in treatment.

[0056] The term "treatment" is intended to have its standard meaning of addressing a disease or condition by reducing, in whole or in part, one, some, or all of its symptoms, or by correcting or compensating for the underlying pathology. The term "treatment" includes "prevention" unless a specific indication to the contrary is provided. The terms "therapeutic" and "therapeutically" should be construed accordingly.

[0057] 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 temporarily or continuously protect a patient from the worsening or exacerbation of a disease or condition that has already occurred or from the occurrence of new symptoms associated with the disease or condition.

[0058] The term "treatment" is used synonymously with "therapy". Similarly, the term "to treat" can be considered to mean "to apply therapy" (where "therapy" is as defined herein).

[0059] The term "therapeutically effective amount" refers to the amount of an RXFP1 modulator as described herein that is effective to provide "treatment" in a subject or to "treat" a disease or disorder in a subject. A therapeutically effective amount can cause any observable or measurable change in a subject as described in the definitions of "treatment", "therapy", and "prevention" above. As will be recognized by those of skill in the art, the effective amount can vary depending on the route of administration, the use of excipients, and combinations with other agents. For example, when combination therapy is used, the amount of the RXFP1 modulator and the amount of the other pharmaceutically active agent are effective together to treat the targeted disorder or condition in the subject when combined. In this regard, the combined amounts are a "therapeutically effective amount" if they are sufficient, when combined, to reduce 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 skill in the art.

[0060] 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.

[0061] Accordingly, in one embodiment, there is provided an RXFP1 modulator for use in the treatment of a human patient, the RXFP1 modulator being administered in combination with an SGLT2 inhibitor, the RXFP1 modulator being selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof. In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

[0062] In one embodiment, there is provided an RXFP1 modulator for use in the treatment of a condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with mid-range 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. The RXFP1 modulator is administered in combination with an SGLT2 inhibitor and is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 or a pharmaceutically acceptable salt thereof. In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin etabonate, sergliflozin etabonate, sotagliflozin and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the condition is hypertension. In one embodiment, the condition is resistant hypertension. In one embodiment, the administration of the RXFP1 modulator and the SGLT2 inhibitor is separate, sequential or simultaneous.

[0063] In one embodiment, there is provided a method for treating a 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 an RXFP1 modulator, wherein the RXFP1 modulator is administered in combination with a therapeutically effective amount of an SGLT2 inhibitor, and the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof. In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the condition is hypertension. In one embodiment, the condition is resistant hypertension. In one embodiment, the administration of the RXFP1 modulator and the SGLT2 inhibitor is separate, sequential, or simultaneous.

[0064] In one embodiment, there is provided a method of treating a condition selected from heart failure, heart failure with preserved ejection fraction, heart failure with mid-range 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 first amount of an RXFP1 modulator and a second amount of an SGLT2 agent, the first amount and the second amount together constituting a therapeutically effective amount, wherein the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 or a pharmaceutically acceptable salt thereof. In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin etabonate, sergliflozin etabonate, sotagliflozin and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the condition is hypertension. In one embodiment, the condition is resistant hypertension. In one embodiment, the administration of the RXFP1 modulator and the SGLT2 inhibitor is separate, sequential or simultaneous.

[0065] In one embodiment, there is provided an RXFP1 modulator for use in the manufacture of a medicament for the treatment of a 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, the treatment comprising administering the RXFP1 modulator in combination with an SGLT2 inhibitor, the RXFP1 modulator being selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 or a pharmaceutically acceptable salt thereof. In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin etabonate, sergliflozin etabonate, sotagliflozin and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the condition is hypertension. In one embodiment, the condition is resistant hypertension. In one embodiment, the administration of the RXFP1 modulator and the SGLT2 inhibitor is separate, sequential or simultaneous.

[0066] In one embodiment, there is provided a method of reducing blood pressure in a human patient in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of an RXFP1 modulator, wherein the RXFP1 modulator is administered in combination with a therapeutically effective amount of an SGLT2 inhibitor, and the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof. In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the administration of the RXFP1 modulator and the SGLT2 inhibitor is separate, sequential, or simultaneous. In one embodiment, the systolic blood pressure of the human patient is reduced. In one embodiment, the diastolic blood pressure of the human patient is reduced. In one embodiment, the systolic blood pressure and the diastolic blood pressure of the human patient are reduced.

[0067] In one embodiment, there is provided a method of effecting a reduction in blood pressure in a human patient in need of such treatment, the method comprising administering to the human patient a first amount of an RXFP1 modulator and a second amount of an SGLT2 inhibitor, the first and second amounts together constituting a therapeutically effective amount, wherein the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof. In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, remogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the administration of the RXFP1 modulator and the SGLT2 inhibitor is separate, sequential, or simultaneous. In one embodiment, the systolic blood pressure of the human patient is reduced. In one embodiment, the diastolic blood pressure of the human patient is reduced. In one embodiment, the systolic and diastolic blood pressures of the human patient are reduced.

[0068] In one embodiment, there is provided a method as disclosed in any of the embodiments herein, wherein the RXFP1 modulator is a compound claimed or exemplified in the specification of International Patent Application No. PCT / EP2021 / 084673 or U.S. Patent Application No. 17 / 457,953, both of which are incorporated by reference in their entirety.

[0069] 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 detailed diagnoses such as heart failure with preserved ejection fraction (HFpEF), heart failure with mid-range ejection fraction (HFmrEF; also referred to as heart failure with mildly reduced ejection fraction), or heart failure with reduced ejection fraction (HFrEF). This may also include heart failure caused by hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0070] As used herein, the term "pulmonary hypertension" can typically be defined as a subject having a mean pulmonary artery pressure of about 20 mmHg or more, optionally 25 mmHg or more, at rest. This can also typically be defined as a mean pulmonary artery pressure of about 30 mmHg or more when the subject is exercising or has recently exercised. Thus, a subject can have a mean pulmonary artery pressure in the range of about 20 mmHg to about 30 mmHg, optionally about 25 mmHg to about 30 mmHg or more. Alternatively or additionally, a subject can have a. a right ventricular systolic pressure of about 40 mmHg or more, and / or b. the following pulmonary vascular resistance: i. less than 3.0 Wood units, or ii. 3.0 Wood units or more .

[0071] Thus, in some cases, pulmonary hypertension can be classified as group 2 pulmonary hypertension by the definition of the World Health Organization. In other cases, pulmonary hypertension can be classified as group 1 pulmonary arterial hypertension by the definition of the World Health Organization (see Ryan et al., 2012, Pulm. Circ. 2(1):107-121).

[0072] The parameters of pulmonary hypertension and heart failure can be measured or estimated using techniques known in the art. For example, these include echocardiography, pulmonary artery catheterization, and implantable monitoring devices. In certain embodiments, a subject can be equipped with a blood pressure monitoring device, optionally a pulmonary artery pressure monitoring device, as known in the art. In certain embodiments, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, this device is worn prior to treatment with the RXFP1 modulators described herein. Alternatively, a subject can wear this device during or after the treatment period.

[0073] As used herein, the term "heart failure with pulmonary hypertension" refers to a small population of heart failure subjects who also have pulmonary hypertension (HF+PH subjects).

[0074] As used herein, the term "resistant hypertension" is defined as the blood pressure of a hypertensive patient that remains elevated above the target achieved despite the concurrent use of three antihypertensive agents at optimized doses of different classes, one of which is a diuretic, or the blood pressure of a patient whose blood pressure is appropriately controlled when receiving four or more antihypertensive agents concurrently (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 patients with RHT, further treatment 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 ≥140 mmHg and / or a diastolic blood pressure ≥90 mmHg when the subject is at rest. Alternatively, subjects with resistant hypertension can typically have a systolic blood pressure ≥130 mmHg and / or a diastolic blood pressure ≥80 mmHg when the subject is at rest. Alternatively, subjects with resistant hypertension can typically have a systolic blood pressure ≥150 mmHg and / or a diastolic blood pressure ≥90 mmHg when the subject is at rest. Optionally, resistant hypertension can be resistant essential hypertension. Essential hypertension, also known as primary hypertension, is a form of hypertension for which no known secondary cause has been identified.

[0075] Pharmaceutical composition The combination of an RXFP1 modulator and an SGLT2 inhibitor described herein can be administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0076] Accordingly, in one embodiment, there is provided a pharmaceutical composition comprising an RXFP1 modulator, an SGLT2 inhibitor, and a pharmaceutically acceptable excipient, wherein the RXFP1 modulator is selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 or a pharmaceutically acceptable salt thereof.

[0077] In one embodiment, the RXFP1 modulator is compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, remogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

[0078] In one embodiment, there is provided a method as disclosed in any of the embodiments herein, wherein the RXFP1 modulator is administered as a pharmaceutical composition comprising the RXFP1 modulator and a pharmaceutically acceptable excipient.

[0079] In one embodiment, there is provided a pharmaceutical composition comprising an RXFP1 modulator, an SGLT2 inhibitor, and a pharmaceutically acceptable excipient for use in a method as described herein, wherein the RXFP1 modulator is selected from compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6 or a pharmaceutically acceptable salt thereof.

[0080] In one embodiment, there is provided a pharmaceutical composition comprising an RXFP1 modulator, an SGLT2 inhibitor, and a pharmaceutically acceptable excipient, wherein the RXFP1 modulator is a compound as claimed or exemplified in International Patent Application No. PCT / EP2021 / 084673 or U.S. Patent Application No. 17 / 457,953 (both applications are incorporated herein by reference in their entirety). In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, remogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

[0081] 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 appreciated by those skilled in the art, certain pharmaceutically acceptable excipients can perform two or more functions and alternative functions depending on the amount of excipient present in the composition and which other excipients are present in the composition.

[0082] The pharmaceutical composition can be in a form suitable for oral use (for example, as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (for example, as creams, ointments, gels or aqueous or oily solutions or suspensions), administration by inhalation (for example, as micronized powders or liquid aerosols), administration by inhalation (for example, as micronized powders) or parenteral administration (for example, as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular or intramuscular dosing) 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.

[0083] Kit In one embodiment, a first pharmaceutical composition comprising an RXFP1 modulator selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, a second pharmaceutical composition comprising an SGLT2 inhibitor and a pharmaceutically acceptable excipient A kit containing the same is provided.

[0084] In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, remogliflozin etabonate, sergliflozin etabonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

Examples

[0085] The compounds described herein are further illustrated in the following examples. These examples are shown for illustrative purposes only and are non-limiting.

[0086] In this example, high-resolution mass spectra were recorded on a Micromass LCT mass spectrometer equipped with an electrospray interface (LC-HRMS).

[0087] 1 1H NMR measurements were performed on Bruker Avance III 300, 400, 500, and 600 spectrometers operating at 300, 400, 500, and 600 MHz 1 for 1H frequency, respectively. The experiments were usually recorded at 25°C. Chemical shifts are given in ppm units by the solvent as an internal standard. Protons on heteroatoms such as NH and OH protons are only reported when detected by NMR, so there may be no description. The following abbreviations are used (and their derivatives, e.g., dd, doublet of doublets, etc.): s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; qn, quintet; p, pentet.

[0088] Flash chromatography was carried out using, unless otherwise stated, normal-phase silica Flash+(registered trademark) (40M, 25M or 12M), Biotage(registered trademark) SNAP Cartridges KP-Sil (340, 100, 50 or 10), or Agela(registered trademark) Flash column silica-CS cartridge (330, 180, 120, 80). Reverse-phase flash chromatography was carried out using, unless otherwise stated, Agela(registered trademark) C-18 spherical 20-35 μm 100A cartridge.

[0089] Generally, all solvents used were commercially available for analysis. Anhydrous solvents were those commonly used for reactions.

[0090] The phase separator used in the examples was an ISOLUTE(registered trademark) Phase Separator column.

[0091] The intermediates and examples named below were named using ChemDraw Professional version 19.0.0.22 manufactured by PerkinElmer.

[0092] The following abbreviations were used. Aq Aq 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 DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DPPA Diphenylphosphoryl azide DTBBPY 4,4'-Di-tert-butyl-2,2'-dipyridyl EDC 3-(Ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine; hydrochloride ESI Electrospray ionization Et Ethyl Et2O Diethyl ether EtOAc Ethyl acetate EtOH Ethanol h / hr Hour HATU (Dimethylamino)-N,N-dimethyl(3-oxide-1H-[1,2,3]triazolo[4,5-b]pyridinyl)methaniminium hexafluorophosphate HOBt 1-Hydroxybenzotriazole; hydrate HPLC High performance liquid chromatography 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 OAc Acetate PE Petroleum ether Pd / C Palladium on carbon Rt Room temperature Sat Saturated SFC Supercritical fluid chromatography T3P 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinan 2,4,6-trioxide TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography

[0093] Intermediate Intermediate 1: Ethyl 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylate

Chemical formula

[0094] Intermediate 2: 8-Methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid

Chem.

[0095] Intermediate 3: 1-Methyl-4-oxocyclohexane-1-carboxylic acid Method A

Chem.

[0096] 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 - 35 °C for 20 h, it was cooled to 0 - 10 °C. Aqueous NaOH solution (918 g, dissolved 22.96 mol in 7.66 L of H2O) 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. When aqueous HCl solution (1.52 L, 6.08 mol, 4 M in H2O) was added following DCM (3.83 L), the pH was adjusted to 3 - 4. 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 under reduced pressure from 2.3 L to 3.1 L. By exchanging the organic reaction solvent from DCM to MeCN under reduced pressure while maintaining the temperature below 45 °C, the title compound was obtained as an 18% solution in MeCN (2.85 kg, 3.32 mol, 87%).1 1H 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。

[0097] Intermediate 4: Naphthalen-1-ylmethyl 1-methyl-4-oxocyclohexane-1-carboxylate Method A

Chemical formula

[0098] 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 - 60 °C for 17 h and then cooled to 25 - 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. MeCN was exchanged with isopropanol under reduced pressure while maintaining the temperature below 50 °C. When the temperature of the mixture was lowered to 20 - 25 °C, a solid precipitate formed. The mixture was further cooled to -10 - 0 °C and 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 H 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。

[0099] Intermediate 5: Methyl 5-(1,3,6,2-dioxazaborinan-2-yl)-4-fluoro-2-methoxybenzoate Method A

Chemical Structure

[0100] 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 refilled with N2 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, whereupon H2 gas was evolved simultaneously. A seed (100 mg of intermediate 5) was added, followed by 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. This 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 H 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。

[0101] Intermediate 6: Methyl 4-fluoro-5-hydroxy-2-methoxybenzoate Method A

Chemical Structure

[0102] Method B Intermediate 5 (32.41 g, 67.3 mmol) was dissolved in 2-Me-THF (100 mL) together with acetic acid (12.13 g, 202 mmol), and the solution was cooled to 0 - 10 °C. 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. The mixture was quenched with an aqueous solution of Na2S2O3·5H2O (20% w / w, 50 mL) to obtain a 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 then further vacuum distilled twice with 2-Me-THF (100 mL) to obtain a dissolved solution at 35 - 45 °C. Nucleation was controlled by the addition of a seed (100 mg of Intermediate 6), followed by the slow addition of 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 H 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。

[0103] Intermediate 7: (1R,2R,3S,4S)-3-(Methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

Chemical Structure

[0104] Intermediate 8: Methyl (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylate hydrochloride

Chemical formula

[0105] Intermediate 11: Naphthalen-1-ylmethyl (1r,4r)-4-hydroxy-1-methylcyclohexane-1-carboxylate

Chemical Structure

[0106] Route B A solution of lithium tri-sec-butylborohydride (1.06 g, 5.6 mmol) in THF (5 mL) was added dropwise over 1 minute under nitrogen 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 gum. 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.

[0107] Intermediate 12: Methyl 4-fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate

Chemical Structure

[0108] Intermediate 13: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid

Chemical formula

[0109] Intermediate 14: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate cyclohexanaminium salt

Chemical formula

[0110] 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

[0111] 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 [Chem.] 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. The pH of the solution was adjusted to 2 by slowly adding HCl (0.5 M, 1.45 L, 2.90 mol) in portions and held at 0 - 5 °C for 1 hour. The heterogeneous slurry was filtered, the solid was washed at 0 °C with 1:3 MeOH:H₂O (600 mL), and the solid was dried under N₂ at 45 °C for 16 hours to obtain the title crude 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, then stirred for 1 hour and filtered. The recovered solid was treated with IPAC (2 × 300 mL) at 0 - 5 °C 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 H NMR (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.

[0112] 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 [Chemical formula] To a solution of DIPEA (6.45 g, 49.9 mmol) in DCM (300 mL) at 0 - 5 °C, intermediate 16 (30.6 g, 49.9 mmol) was added, followed by (1-methylcyclobutyl)methanamine hydrochloride (8.63 g, 62.4 mmol). DIPEA (25.8 g, 200 mmol) was added dropwise while maintaining the temperature at 0 - 5 °C, and then T3P (50.8 g, 79.8 mmol, 50% w / w in EtAOc) was added over 0.5 h. The solution was warmed to 15 - 25 °C, stirred for 1 h, and then H2O (150 mL) was added dropwise while maintaining the temperature below 30 °C. The biphasic solution was separated, the organic phase was washed with H2O (2 × 150 mL), and then the solvent was exchanged to EtOH under reduced pressure to obtain the title compound as a crude solution in EtOH (128 g, 26% w / w, 96% yield), which was used directly in the next step. For the purified compound 1 H NMR (500 MHz, CDCl3) δ 0.98 (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 (2H, 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。

[0113] Intermediate 19: (1R,2S,3R,4S)-3-(5-(((1s,4S)-4-carboxy-4-methylcyclohexyl)oxy)-4-cyano-2-methoxybenzamide)bicyclo[2.2.1]heptane-2-carboxylic acid [Chemical formula] Process A. Intermediate 20: Methyl 4-cyano-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate

Chem.

[0114] Process B. Intermediate 21: 4-Cyano-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid

Chem.

[0115] Step C. Intermediate 22: Methyl (1S,2S,3R,4R)-3-(4-cyano-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

[0116] Process D. Intermediate 23: (1S,4S)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(methoxycarbonyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

Chem.

[0117] Process E. (1R,2S,3R,4S)-3-(5-(((1S,4S)-4-carboxy-4-methylcyclohexyl)oxy)-4-cyano-2-methoxybenzamide)bicyclo[2.2.1]heptane-2-carboxylic acid A solution of LiOH (50 mL, 52.6 mmol, 1.05 M in H2O) was added to a stirred solution of Intermediate 23 (5.1 g, 10.5 mmol) in THF (100 mL) at 10 °C. The reaction mixture was warmed to room temperature, stirred for 14 h, and then acidified to pH 2 using HCl (1 M aqueous solution). The reaction mixture was diluted with EtOAc (350 mL) and washed successively with brine (300 mL, saturated), H2O (300 mL), and brine (300 mL, saturated). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by precipitation from EtOAc / Et2O followed by reverse-phase flash chromatography on a C18 column using a concentration gradient of 0 - 50% MeCN in HCl (0.4% aqueous solution) as the mobile phase to give the title compound as a white solid (4.00 g, 82%); 11H NMR (400 MHz, DMSO-d6) δ 1.13 (s, 3H), 1.20 (s, 1H), 1.23 (s, 2H), 1.33 (t, 2H), 1.46 (q, 4H), 1.84 (d, 1H), 1.92 (d, 2H), 2.03 - 2.14 (m, 4H), 2.38 (d, 1H), 2.67 (d, 1H), 3.89 (s, 3H), 4.23 (t, 1H), 4.43 (dt, 1H), 7.54 (s, 1H), 7.59 (s, 1H), 8.67 (d, 1H), 12.30 (s, 2H). MS (ESI): m / z [M+H] + 471.3。

[0118] Example 1: (1S,4S)-4-(2-Fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methoxycyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (Form A)

Chem.

[0119] Example 2: (1S,4S)-4-(5-(((1S,2R,3S,4R)-3-((Cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid

Chemical formula

[0120] Step B: (1S,4S)-4-(5-(((1S,2R,3S,4R)-3-((Cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid To the EtOH solution of Intermediate 18, 10 wt% Pd / C (290 mg, 5 wt / wt) was added. The vessel was purged 6 times with N2 and then an additional 6 times with H2. The vessel was pressurized with H2 to 0.4 MPa, and the reaction solution was stirred at 20 - 30 °C for 20 h. After completely replacing the H2 atmosphere with N2, the reaction mixture was filtered and washed with EtOH (2 × 12.2 mL). While maintaining the temperature below 45 °C, the solvent was exchanged to EtAOc under reduced pressure. The EtAOc solution (41.0 mL) was heated to 70 - 75 °C for 0.5 h, then cooled to 40 - 45 °C, and n-heptane (34.8 mL) was added dropwise over 0.5 h. The mixture was stirred for 0.5 h, then cooled to 20 - 25 °C over 2 h and then held for an additional 2 h. The heterogeneous slurry was filtered, and the solid was then washed twice with 1:2 EtOAc / n-heptane (11.6 mL) and dried at below 45 °C for 20 h to obtain the title compound as a white solid (3.28 g, 74%). 1 H NMR (500 MHz, DMSO-d6) δ 1.04 - 1.30 (6H, m), overlapping 1.10 (3H, s), 1.35 - 1.58 (5H, m), 1.59 - 1.69 (2H, m), 1.71 - 1.82 (2H, m), 1.83 - 1.92 (2H, m), 1.95 - 2.02 (1H, m), 2.01 - 2.10 (3H, d), 2.18 - 2.31 (2H, m), 2.50 - 2.55 (1H, d), 2.92 - 3.00 (1H, m), 3.06 - 3.14 (1H, m), 3.89 (3H, s), 4.07 - 4.17 (2H, m), 7.11 (1H, d), 7.67 (1H, d), 7.94 (1H, t), 8.83 (1H, d). MS (ESI): m / z [M + H] + 531.3。

[0121] Example 3. (1S,4S)-4-(2-Cyano-5-(((1S,2R,3S,4R)-3-((Cyclopropylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid

Chem.

[0122] Example 4: (1S,4S)-4-(2-Cyano-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

Chemical Structure

[0123] Step B: Intermediate 25: Naphthalen-1-ylmethyl (1S,4s)-4-(5-(((1R,2R,3S,4S)-3-(((1-Methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-5-en-2-yl)carbamoyl)-2-cyano-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylate

Chemical formula

[0124] Step C: (1S,4s)-4-(2-Cyano-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 formula

[0125] Example 5: (1S,4S)-4-(2-Cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(neopentylcarbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

Chem.

[0126] Example 6: (1S,4S)-4-(2-Cyano-5-(((1S,2R,3S,4R)-3-((3-Fluorobicyclo[1.1.1]pentan-1-yl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid

Chemical formula

[0127] Biological and physicochemical data RXFP1 Hu cAMP (Test A) To screen for modulators of hRXFP1, an assay was used to identify compounds that 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.

[0128] 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: Stock standard cAMP provided in the CisBio kit was diluted in 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 in lysis buffer with the HTRF kit.

[0129] Stepwise procedure for performing the assay: 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 the assay. 2. 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 using an Echo acoustic dispenser on the assay day. 3. 4 μL of assay buffer containing 1 mM IBMX (final concentration 0.5 mM) to block phosphodiesterase was added with a Multidrop Combi (ThermoFisher). 4. 1.875 * 4 μL of cell solution at 10^5 cells / ml was added with a Multidrop Combi to obtain 750 cells / well. 5. Incubated at room temperature for 45 minutes. 6. 4 μL of cAMP-d2 in lysis buffer was added with a Multidrop Combi. 7. 4 μL of anti-cAMP cryptate in the lysis buffer was added using a Multidrop Combi. 8. Incubated at room temperature for 2 hours. 9. The homogeneous time-resolved fluorescence (HTRF) signal was detected using an Envision (PerkinElmer) or Pherastar (BMG Labtech) reader (λex = 340 nm, λem = 665 and 615 nm).

[0130] The HTRF data was converted to the amount of cAMP produced in the sample using a cAMP calibration curve and subsequently used for the calculation of the concentration response. The concentration response data was fitted using a four-parameter logistic fit, Hill equation. The results from the assay were reported in Table 1 as EC 50 (μM) and S inf (%). EC 50 is defined as the concentration at which the stimulatory activity reaches 50% of its maximum level. When the assay was performed multiple times for the same compound, the geometric mean was reported. S inf is the fitted activity level, efficacy, at infinite concentration of the test compound. To facilitate comparison of the efficacy data, the efficacy was normalized to the % effect of the response stimulated by the saturating concentration (1 nM) of relaxin. When the assay was performed multiple times for the same compound, the arithmetic mean was reported.

[0131] 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 was performed multiple times for the same compound, the arithmetic mean was reported.

[0132] 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 are reported as CL int (μl / min / mg protein) in Table 1. When the assay was performed multiple times for the same compound, the arithmetic mean value is reported.

[0133] 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 (“reaction stop plate”). 3. Dilute the 10 mM test compound and the control compound to 100 μM by mixing 198 μL of acetonitrile and 2 μL of the 10 mM stock solution in a new 96 - well plate. 4. Remove a vial of cryopreserved (-below -150 °C) human hepatocytes (LiverPool™ 10-donor human hepatocytes obtained from Bioreclamation IVT (product number S01205)) from the storage and ensure that the vial remains at cryogenic temperature until the thawing process is carried out. Thaw the cells as rapidly as possible by placing the vial in a 37 °C water bath and gently agitating. The vial should be left 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 determine the viable cell density. Cells with inadequate viability (<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. 7. Transfer 247.5 μL of the 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. 8. Add 2.5 μL of a 100 μM test compound or control compound to the incubation wells containing the cells to initiate the reaction. 9. 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 "reaction stop plate" and then vortex the sample for 2 minutes. 10. Centrifuge the reaction stop plate at 4,000 rpm for 20 minutes. Transfer 30 μL of the supernatant of each compound to a 96-well analysis plate. Pool four compounds together in one cassette. Then dilute the pooled sample by adding 180 μl of pure water. All incubations are carried out separately.

[0134] All calculations were performed using Microsoft Excel. Peak areas were determined from the extracted ion chromatograms. The in vitro intrinsic clearance (in vitro Cl int , μL / min / 10 6 cells per unit) of the parent compound was determined by regression analysis of the disappearance rate (%) Ln of the parent against the time curve. The in vitro intrinsic clearance (in vitro Cl int , μL / min / 10 6 cells per unit) 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 was performed multiple times for the same compound, the geometric mean value was reported.

[0135] 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 was reported in Table 1 as the mean Cl int (μL / min / 10 6 cells). When the assay was performed multiple times for the same compound, the geometric mean value was reported.

[0136] Solubility (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. Data are reported as solubility (μM) in Table 1. When the assay is performed multiple times for the same compound, the arithmetic mean value is reported.

[0137] [Table 1]

[0138] Human RXFP1 cGMP Production Assay (Test G) The Green GENIe cGMP assay (Montana Molecular; catalog number D800G) was used to profile compounds for RXFP1 agonist activity related to cGMP production. This assay is based on an mNeonGreen fusion protein fluorescent biosensor delivered to mammalian cells with a BacMaM vector. When cGMP binds to the biosensor, fluorescence decreases.

[0139] 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) supplemented with 10% FBS at 0.8 mg / mL to maintain RXFP1 expression.

[0140] Step - by - step protocol for performing the assay: Day 1 1. The cells were split one day before transduction and seeded at 63,000 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 the medium, and collected in a 50 mL tube. 3. The cells were counted using CEDEX (Innovatis) and diluted to 267,000 cells / mL with the medium. 4. The viral transduction master mix was prepared by mixing the reagents at the following ratios for a single well: 6 μL of GENIe BacMAM vector 0.2 μL of 500 mM sodium butyrate 13.8 μL of 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. 50 μL of the cell transduction mix from the above was dispensed per well into a μclear 384-well plate (Greiner; 781946) coated with black poly-D-lysine. 7. The plate was incubated at 37 °C, 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 together with 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.

[0141] The data was processed using Screener software (Genedata AG). After subtraction of the background fluorescence (before the compound point), the area under the curve values from 0 to 90 minutes after compound addition were used for the calculation of the reaction. The concentration - response data was fitted with a four - parameter logistic fit, and the EC 50 value (nM) was reported in Table 2.

[0142] Human RXFP1 phospho - ERK assay (Test H) To profile compounds for RXFP1 agonist activity regarding ERK phosphorylation, an 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 enables the formation of an immune complex with 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.

[0143] 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) supplemented with 10% FBS at 0.8 mg / mL to maintain RXFP1 expression. The assay was performed on cells maintained in continuous culture. 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%. Antibody mixture: EU - and d2 - labeled anti - ERK1 / 2 antibodies were separately diluted 20 - fold with the detection buffer provided in the kit. Shortly before the experiment, equal volumes of each diluted antibody solution were combined to form the antibody mix.

[0144] Step-by-step protocol for performing the assay: Day 1 1. Detach cells from the culture flask using Accutase (Gibco; 1737341), resuspend in phenol red-free DMEM medium containing 10% FBS, and collect in a 50 mL tube. 2. Count the cells using CEDEX (Innovatis) and dilute to 320,000 cells / mL in the above medium. 3. Dispense 100 μL of the cell suspension per well into a μclear 96-well plate (Greiner; 655946) coated with Black μclear poly-D-lysine. 4. Incubate the plate at 37 °C, 5% CO2 for 24 hours. Day 2 5. Serum starvation: 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 hours. 6. Add 50 μL of the test compound solution per well. 7. Incubate the plate at room temperature for 5 minutes. 8. Quickly remove the medium and stop the stimulation by adding 50 μL of lysis buffer (diluted to 1× final concentration before addition) per well. 9. Transfer the plate to -80 °C and freeze the lysates. Day 3 10. Thaw the plate and shake at room temperature for 30 minutes. 11. Homogenize the cell lysates by pipetting. 12. Transfer 16 μL of the homogenate per well to a white low-volume 384-well plate (Greiner; 784075). 13. Add 4 μL of the antibody mix per well. 14. Incubate the plate in the dark at room temperature for 4 hours. 15. The homogeneous time-resolved fluorescence (HTRF) signal was detected using a Pherastar (BMG Labtech) reader (λex = 340 nm, λem = 665 and 615 nm).

[0145] HTRF ratio data were processed using Screener software (Genedata AG). Concentration-response data were fitted with a four-parameter logistic fit and reported as EC 50 values (nM) in Table 2.

[0146] [Table 2]

[0147] One of ordinary skill in the art will understand that the biological assays described above can be performed using minor variations of alternative devices and protocols without significantly affecting the results.

[0148] This description and its specific examples are illustrative only and are intended to show particular embodiments. 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 connection with separate embodiments for clarity can be combined to form a single embodiment. Conversely, various embodiments described in connection with a single embodiment for brevity can be combined to form these partial combinations.

[0149] All publications disclosed herein are hereby incorporated by reference into this specification.

Claims

1. A pharmaceutical composition for use in the treatment of human patients, comprising an RXFP1 modulator and a pharmaceutically acceptable excipient, characterized in that it is administered in combination with an SGLT2 inhibitor, wherein the RXFP1 modulator is 【Chemistry 1】 【Chemistry 2】 A pharmaceutical composition which is selected from or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising an RXFP1 modulator and a pharmaceutically acceptable excipient for use in the treatment of conditions 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 human patients, characterized in that it is administered in combination with an SGLT2 inhibitor, wherein the RXFP1 modulator is 【Transformation 3】 【Chemistry 4】 A pharmaceutical composition which is selected from or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 2, wherein the aforementioned pathological condition is hypertension.

4. The pharmaceutical composition according to claim 2, wherein the aforementioned pathological condition is resistant hypertension.

5. The pharmaceutical composition according to claim 1 or 2, wherein the administration of the RXFP1 modulator and the SGLT2 inhibitor is separate, sequential, or simultaneous.

6. The pharmaceutical composition according to claim 1 or 2, wherein the RXFP1 regulator is compound 1 or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 1 or 2, wherein the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, erzgliflozin, ipragliflozin, remogliflozin etavonate, cergliflozin etavonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 1 or 2, wherein the SGLT2 inhibitor is dapagliflozin.

9. The pharmaceutical composition according to claim 1 or 2, wherein the RXFP1 modulator is compound 1 or a pharmaceutically acceptable salt thereof, and the SGLT2 inhibitor is dapagliflozin.

10. A pharmaceutical composition comprising an RXFP1 modulator, an SGLT2 inhibitor, and a pharmaceutically acceptable excipient, wherein the RXFP1 modulator is 【Transformation 5】 【Transformation 6】 A pharmaceutical composition which is selected from or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition according to claim 10, wherein the RXFP1 regulator is compound 1 or a pharmaceutically acceptable salt thereof.

12. The pharmaceutical composition according to claim 10 or 11, wherein the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, remogliflozin etavonate, cergliflozin etavonate, sotagliflozin, and tofogliflozin or pharmaceutically acceptable salts thereof.

13. The pharmaceutical composition according to claim 10 or 11, wherein the SGLT2 inhibitor is dapagliflozin.

14. The pharmaceutical composition according to claim 10 or 11, wherein the RXFP1 modulator is compound 1 or a pharmaceutically acceptable salt thereof, and the SGLT2 inhibitor is dapagliflozin. 【Request Item 15】 【Chemistry 7】 【Transformation 8】 A first pharmaceutical composition comprising an RXFP1 modifier selected from or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, A second pharmaceutical composition comprising an SGLT2 inhibitor and a pharmaceutically acceptable excipient, A kit that includes this.

16. The kit according to claim 15, wherein the RXFP1 modulator is compound 1 or a pharmaceutically acceptable salt thereof.

17. The kit according to claim 15 or 16, wherein the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, ipragliflozin, remogliflozin etavonate, cergliflozin etavonate, sotagliflozin, and tofogliflozin or a pharmaceutically acceptable salt thereof.

18. The kit according to claim 15 or 16, wherein the SGLT2 inhibitor is dapagliflozin.

19. The kit according to claim 15 or 16, wherein the RXFP1 modulator is compound 1 or a pharmaceutically acceptable salt thereof, and the SGLT2 inhibitor is dapagliflozin.

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