Combination of RXFP1 modulators and SGLT2 inhibitors

Abstract

The present specification relates generally to combinations of RXFP1 modulators and SGLT2 inhibitors, in particular pharmaceutical compositions comprising such combinations, and methods of treatment using such combinations.

Description

Technical Field

[0001] The present specification describes a combination of a compound (including a salt thereof) that is a RXFP1 modulator and an SGLT2 inhibitor such as dapagliflozin; a pharmaceutical composition comprising such a combination and the use of such a combination. Background Art

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

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

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

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

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

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

[0008] Despite the foregoing, there is a continuing need for additional compounds that are modulators of RXFP1, which may make these compounds particularly promising for development as therapeutic agents. Such one or more compounds may also exhibit improved RXFP1 regulation compared to other known RXFP1 modulators. Such one or more compounds may also exhibit favorable pharmacokinetic profiles (e.g., lower intrinsic clearance) and / or favorable physical properties (e.g., higher water solubility) compared to other known RXFP1 modulators. Therefore, such one or more compounds may be particularly useful for treating disease states in which RXFP1 regulation is beneficial. In addition, such one or more compounds in combination with other therapeutic agents may be used to treat certain conditions, such as hypertension. Summary of the invention

[0009] This specification relates to the combination of RXFP1 regulators and SGLT2 inhibitors as described herein. SGLT2 inhibitors (such as dapagliflozin) have been shown to have a blood pressure lowering effect (Weber et al., Lancet Diabetes Endocrinol. [Lancet Diabetes and Endocrinology], 2016, 4 (3), 211-220). Therefore, the combination of RXFP1 regulators and SGLT2 inhibitors can provide special benefits in the treatment of hypertension (especially refractory hypertension) and cardiovascular diseases, and there is still a significant unmet need for this.

[0010] Thus, the present specification describes, in part, a RXFP1 modulator for use in therapy in a human patient, wherein the RXFP1 modulator is administered in combination with a SGLT2 inhibitor, and wherein the RXFP1 modulator is selected from:

[0011]

[0012]

[0013] or a pharmaceutically acceptable salt thereof.

[0014] Similarly, the present specification describes, in part, an RXFP1 modulator for use in treating a condition in a human patient selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, wherein the RXFP1 modulator is administered in combination with an SGLT2 inhibitor, and wherein the RXFP1 modulator is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6, or a pharmaceutically acceptable salt thereof.

[0015] Similarly, the present specification describes, in part, a method for treating a condition in a human patient selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, wherein the human patient is in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of a RXFP1 modulator, wherein the RXFP1 modulator is administered in combination with a therapeutically effective amount of a SGLT2 inhibitor, and wherein the RXFP1 modulator is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6, or a pharmaceutically acceptable salt thereof.

[0016] Similarly, the present specification describes, in part, a method for treating a condition in a human patient selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, wherein the human patient is 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 wherein the RXFP1 modulator is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6, or a pharmaceutically acceptable salt thereof.

[0017] Similarly, the present specification describes, in part, a pharmaceutical composition comprising a RXFP1 modulator, a SGLT2 inhibitor, and a pharmaceutically acceptable excipient, and 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.

[0018] Similarly, the specification describes, in part, a kit comprising:

[0019] a first pharmaceutical composition comprising an RXFP1 modulator selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; and

[0020] The second pharmaceutical composition comprises an SGLT2 inhibitor and a pharmaceutically acceptable excipient.

[0021] Additional aspects of the present disclosure will be apparent to those skilled in the art from reading this specification. DETAILED DESCRIPTION

[0022] Many embodiments are described in detail throughout the specification and will be apparent to the skilled reader.This specification is not to be construed as limited to any particular embodiment or embodiments described herein.

[0023] Terms not expressly defined herein should be understood to have the meaning that one skilled in the art would give them based on the disclosure and context.

[0024] "About" can generally mean an acceptable degree of error for a measured amount given the nature or precision of the measurements. Exemplary degrees of error are within a percentage (%) of a given value or range of values, typically within 10%, and more typically within 5%.

[0025] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of corresponding embodiments “consisting of such features”.

[0026] Concentrations, amounts, volumes, percentages and other numerical values ​​can be presented in a range format. It should also be understood that such a range format is used only for convenience and brevity, and should be flexibly interpreted to include not only the numerical values ​​explicitly listed as the limits of the range, but also all individual numerical values ​​or sub-ranges contained within the range, just as each numerical value and sub-range is explicitly listed.

[0027] The chemical names of the compounds described in this specification are derived from of Produced by Professional Edition 19.0.0.22. The skilled person will appreciate that different chemical naming software may generate different chemical names for a particular compound. Where the compounds described herein are described in the form of a chemical name and a chemical formula, in the event of any inconsistency, the chemical formula shall prevail.

[0028] RXFP1 modulators

[0029] In one embodiment, the RXFP1 modulator is selected from:

[0030]

[0031]

[0032] or a pharmaceutically acceptable salt thereof.

[0033] In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof.

[0034] In one embodiment, the RXFP1 modulator is Compound 2 or a pharmaceutically acceptable salt thereof.

[0035] In one embodiment, the RXFP1 modulator is Compound 3 or a pharmaceutically acceptable salt thereof.

[0036] In one embodiment, the RXFP1 modulator is Compound 4 or a pharmaceutically acceptable salt thereof.

[0037] In one embodiment, the RXFP1 modulator is Compound 5 or a pharmaceutically acceptable salt thereof.

[0038] In one embodiment, the RXFP1 modulator is Compound 6 or a pharmaceutically acceptable salt thereof.

[0039] In one embodiment, the RXFP1 modulator is Compound 1.

[0040] In one embodiment, the RXFP1 modulator is Compound 2.

[0041] In one embodiment, the RXFP1 modulator is Compound 3.

[0042] In one embodiment, the RXFP1 modulator is Compound 4.

[0043] In one embodiment, the RXFP1 modulator is Compound 5.

[0044] In one embodiment, the RXFP1 modulator is Compound 6.

[0045] In one embodiment, the RXFP1 modulator is selected from:

[0046] (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid;

[0047] (1S,4s)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid;

[0048] (1S,4s)-4-(2-cyano-5-(((1S,2R,3S,4R)-3-((cyclopropylmethyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid;

[0049] (1S,4s)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(neopentylcarbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid;

[0050] (1S,4s)-4-(2-cyano-5-(((1S,2R,3S,4R)-3-((3-fluorobicyclo[1.1.1]pentan-1-yl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid; and

[0051] (1S,4s)-4-(5-(((1S,2R,3S,4R)-3-((cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid;

[0052] or a pharmaceutically acceptable salt thereof.

[0053] In one embodiment, 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 by reference in their entirety).

[0054] The term "pharmaceutically acceptable" is used to specify that an object (e.g., a salt, a dosage form, or an 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, edited by PHStahl and CGWermuth, Weinheim / Zürich: Wiley-VCH / VHCA, 2002. Suitable pharmaceutically acceptable salts of the compounds described herein are, for example, acid addition salts or base addition salts. Acid addition salts of the compounds described herein can be formed by contacting the compound with a suitable inorganic acid or organic acid under conditions known to the skilled person. Acid addition salts can be formed, for example, using an inorganic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Acid addition salts can also be formed using an organic acid 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 p-toluenesulfonic acid.

[0055] Thus, in one embodiment, the RXFP1 modulator is a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, sulfate, phosphate, trifluoroacetate, citrate, maleate, oxalate, acetate, formate, benzoate, fumarate, succinate, tartrate, lactate, pyruvate, methanesulfonate, benzenesulfonate or p-toluenesulfonate.

[0056] The compounds described in this specification can form base addition salts. Base addition salts of compounds described herein can be formed by contacting the compound with a suitable inorganic base or organic base under conditions known to the technician. For example, alkali metal (such as sodium, potassium, or lithium) or alkaline earth metal (such as calcium) salts can be prepared by treating the compound with an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g., ethanolate or methanolate) or a suitable alkaline organic amine (e.g., choline or meglumine) in an aqueous medium. Therefore, in one embodiment, the RXFP1 modulator is a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a lithium salt, a calcium salt, a choline salt or a meglumine salt.

[0057] The compounds and salts described in this specification may exist in solvated and non-solvated forms. For example, the solvated form may be a hydrated form, such as a hemihydrate, a monohydrate, a dihydrate, a trihydrate or an alternative amount thereof. All such solvated and non-solvated forms of the compounds described herein are encompassed herein.

[0058] The atoms of the compounds and salts described in this specification may exist as their isotopes. This document covers all compounds described herein in which atoms are replaced by one or more of their isotopes (e.g., compounds described herein in which one or more carbon atoms are 11 C or 13 C carbon isotope, or one or more hydrogen atoms are 2 H or 3 H isotope).

[0059] The compounds described herein may exist in one or more geometric forms, optical forms, enantiomeric forms, and diastereomeric forms (including but not limited to cis and trans, E- and Z-forms, and R-, S- and meso forms). Unless otherwise indicated, reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Where appropriate, such isomers may be separated from their mixtures by application or modification of known methods (e.g., chromatography techniques and recrystallization techniques). Where appropriate, such isomers may be prepared by application or modification of known methods.

[0060] The compounds described herein may include one or more chiral centers. Where a structure or chemical name in this specification does not indicate chirality, the structure or name is intended to encompass any single stereoisomer corresponding to the structure or name, as well as any mixture of stereoisomers (e.g., racemates). Structures in this specification include bonds drawn as solid and dashed wedges (i.e., and ), the real and imaginary wedges are intended to represent the absolute configuration of the chiral center.

[0061] It is well known in the art how such optically active forms are separated. For example, a single stereoisomer can be obtained by separating it from a mixture of isomers (e.g., a racemate) using, for example, chiral chromatographic separation. In other embodiments, a single stereoisomer is obtained by, for example, direct synthesis of chiral starting materials.

[0062] According to one embodiment, the RXFP1 modulator is provided as a single enantiomer in an enantiomeric excess (%ee) ≥ 95%, ≥ 98% or ≥ 99%. Conveniently, the single enantiomer is present in an enantiomeric excess of ≥ 99%.

[0063] According to one embodiment, the RXFP1 modulator is provided as a single enantiomer in an enantiomeric excess (%ee) ranging from 95% to 100%.

[0064] The compounds described herein can exist in one or more tautomeric forms, including but not limited to keto- and enol-forms. Reference to a particular compound includes all tautomeric forms, including mixtures thereof. Therefore, structures depicted herein as one tautomer are also intended to include other tautomers.

[0065] The RXFP1 modulators described herein can be administered in the form of prodrugs, which are compounds that break down in the human or animal body to release such RXFP1 modulators. Thus, pharmaceutically acceptable prodrugs of RXFP1 modulators also form embodiments. Various forms of prodrugs are known in the art. For example, see

[0066] a) Design of Pro-drugs, edited by H. Bundgaard (Elsevier, 1985);

[0067] b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H.Bundgaard, Chapter 5 “Design and Application of Prodrugs”, H.Bundgaard, pp. 113-191 (1991);

[0068] c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);

[0069] d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and

[0070] e) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984).

[0071] Sodium-glucose transporter subtype 2 (SGLT2) inhibitors

[0072] In some instances, the reabsorption of glucose in the kidney is regulated by members of the sodium-glucose co-transporter family, which are sodium-dependent glucose transporters. In some instances, the reabsorption of glucose in the kidney is regulated by sodium-glucose transporter subtype 2 (SGLT2).

[0073] In one embodiment, the SGLT2 inhibitor is gliflozin. In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, erpagliflozin, empagliflozin, reglipgliflozin etabonate, sergliflozin etabonate, sogliflozin and togliflozin, 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, which has the following chemical structure:

[0074]

[0075] Dapagliflozin is a potent, highly selective and orally active human renal sodium-dependent glucose transporter 2 (SGLT2) inhibitor (SGLT2i) 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 has been disclosed in WO 2003 / 099836 together with details of its chemical synthesis.

[0076] Treatment with RXFP1 modulators and SGLT2 inhibitors

[0077] The combinations described herein comprising a RXFP1 modulator and a SGLT2 inhibitor are expected to be useful in therapy.

[0078] The term "therapy" is intended to have its normal meaning: treating a disease or condition so as to fully or partially relieve one, some or all of its symptoms, or so as to correct or compensate for an underlying pathology. The term "therapy" also includes "prophylaxis," unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted accordingly.

[0079] The term "prophylaxis" is intended to have its normal meaning and includes primary prevention, which prevents the development of a disease or condition, and secondary prevention, wherein the disease or condition has already developed and the patient is temporarily or permanently protected against an exacerbation or worsening of the disease or condition or against the development of new symptoms associated with the disease or condition.

[0080] The term "treatment" is used synonymously with "therapy". Similarly, the term "treat" may be viewed as "applying therapy", where "therapy" is as defined herein.

[0081] The term "therapeutically effective amount" refers to an amount of an RXFP1 modulator as described herein that effectively provides "therapy" in a subject, or effectively "treats" a disease or condition in a subject. As described in the definitions of "therapy", "treatment" and "prophylaxis" above, a therapeutically effective amount can cause any observable or measurable change in a subject. As recognized by those skilled in the art, the effective amount can vary depending on the route of administration, the use of excipients, and the use in conjunction with other agents. For example, in the case of a combination therapy, the amount of the RXFP1 modulator and the amount of the other one or more pharmaceutically active agents are combined to effectively treat the subject's targeted disorder or condition. In this context, if they are combined to reduce the symptoms of a disease or condition that is responsive to the regulation and / or excitement of RXFP1 as described above, the combined amount is a "therapeutically effective amount". Typically, such an amount can be determined by those skilled in the art.

[0082] As used herein, the terms "subject" and "patient" are used interchangeably. "Subject" includes, for example, a mammal, such as a human. In some embodiments, the subject is a human.

[0083] Therefore, in one embodiment, a RXFP1 modulator for use in a therapy of a human patient is provided, wherein the RXFP1 modulator is administered in combination with an SGLT2 inhibitor, and 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, erpagliflozin, ipagliptin, reglipgliflozin efate, sergliflozin efate, sogliflozin, and togliflozin, or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

[0084] In one embodiment, a RXFP1 modulator is provided for use in treating a condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, wherein the RXFP1 modulator is administered in combination with an SGLT2 inhibitor, and 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, ergliflozin, ipagliptin, reglipflozin efacic acid, sergliflozin efacic acid, sogliflozin, and togliflozin, 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 RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially or simultaneously.

[0085] In one embodiment, a method for treating a human patient selected from the following conditions is provided: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension and resistant hypertension, the human patient needs 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 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, ergliflozin, ipagliptin, reglipflozin efacic acid, sergliflozin efacic acid, sogliflozin and togliflozin, 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 RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially or simultaneously.

[0086] In one embodiment, a method for treating a human patient selected from the following conditions is provided: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, the human patient needs 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 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, ergliflozin, ipagliptin, reglipflozin efacic acid, sergliflozin efacic acid, sogliflozin and togliflozin, 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 RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially or simultaneously.

[0087] In one embodiment, a RXFP1 modulator is provided for use in the manufacture of a medicament for treating a condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, wherein the treatment comprises administering a combination of the RXFP1 modulator and an SGLT2 inhibitor, and 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, erpa ... In one embodiment, the RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially or simultaneously.

[0088] In one embodiment, a method for reducing blood pressure in a human patient is provided, the human patient being in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of a RXFP1 modulator, wherein the RXFP1 modulator is administered in combination with a therapeutically effective amount of a SGLT2 inhibitor, and 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, ergliflozin, ipagliptin, efagagliflozin, efagagliflozin, soglipflozin, and togliflozin, or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially, or simultaneously. 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, both the systolic blood pressure and the diastolic blood pressure of the human patient are reduced.

[0089] In one embodiment, a method for reducing blood pressure in a human patient is provided, the human patient being 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 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, ergliflozin, ipagliptin, reglipflozin efate, sergliflozin efate, sogliflozin, and togliflozin, or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin. In one embodiment, the RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially, or simultaneously. 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, both the systolic blood pressure and the diastolic blood pressure of the human patient are reduced.

[0090] In one embodiment, a method as disclosed in any of the embodiments herein is provided, 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 by reference in their entirety).

[0091] As used herein, the term "heart failure" includes acute heart failure, chronic heart failure (CHF), and acute decompensated heart failure (ADHF). The term "heart failure" may also include more specific diagnoses such as heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate ejection fraction (HFmrEF; also known as heart failure with mildly reduced ejection fraction), or heart failure with reduced ejection fraction (HFrEF). This may also include heart failure caused by hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0092] As used herein, the term "pulmonary hypertension" may be defined as a subject having a mean pulmonary artery pressure of about 20 mmHg or more, optionally 25 mmHg or more, typically when the subject is at rest. It may also be defined as a mean pulmonary artery pressure of about 30 mmHg or more, typically when the subject is or has recently been exercising. Thus, the subject's mean pulmonary artery pressure may be in the range of about 20 mmHg to about 30 mmHg, optionally about 25 mmHg to about 30 mmHg or more. Alternatively or in addition, the subject may have:

[0093] a. right ventricular systolic pressure of approximately 40 mmHg or greater; and / or

[0094] b. Pulmonary vascular resistance of:

[0095] i. less than 3.0 wood units; or

[0096] ii.3.0 or more Wood units.

[0097] Thus, in some cases, pulmonary hypertension can be classified as Group 2 pulmonary hypertension as defined by the World Health Organization. In other cases, pulmonary hypertension can be classified as Group 1 pulmonary hypertension as defined by the World Health Organization (see Ryan et al., 2012, Pulm. Circ. 2(1):107-121).

[0098] 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 catheters, and implantable monitoring devices. In certain embodiments, the subject can be equipped with a blood pressure monitoring device as known in the art, optionally a pulmonary artery pressure monitoring device. In a specific embodiment, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, the device is equipped before treatment with an RXFP1 modulator as described herein. Alternatively, the subject is equipped with the device during or after treatment.

[0099] As used herein, the term "heart failure with pulmonary hypertension" refers to a subgroup of heart failure subjects who also suffer from pulmonary hypertension (HF+PH subjects).

[0100] As used herein, the term "resistant hypertension" is defined as the blood pressure of a hypertensive patient that is above target despite the simultaneous use of optimized doses of 3 different classes of antihypertensive agents (one of which is a diuretic), or the blood pressure of a patient whose blood pressure is adequately controlled when receiving 4 or more antihypertensive drugs simultaneously (Carey et al., Hypertension, 2018, 72, e53-e90). The initial treatment of hypertension can be a calcium channel blocker (CCB), a renin-angiotensin system blocker (angiotensin converting enzyme [ACE] inhibitor or angiotensin receptor blocker [ARB]), and a diuretic. For rHT patients, additional treatment may include mineralocorticoid receptor antagonists (MRAs), beta-blockers, and / or alpha-blockers. Typically, subjects with resistant hypertension have a systolic blood pressure ≥140 mmHg, and / or a diastolic blood pressure ≥90 mmHg when the subject is at rest. Alternatively, a subject with resistant hypertension typically has a systolic blood pressure of ≥130 mmHg, and / or a diastolic blood pressure of ≥80 mmHg, when the subject is at rest. Alternatively, a subject with resistant hypertension typically has a systolic blood pressure of ≥150 mmHg, and / or a diastolic blood pressure of ≥90 mmHg, when the subject is at rest. In some cases, resistant hypertension may be resistant essential hypertension. Essential hypertension (essential hypertension / primaryhypertension) is hypertension without a known definite secondary cause.

[0101] Pharmaceutical composition

[0102] The combinations of RXFP1 modulators and SGLT2 inhibitors described herein may be administered as pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients.

[0103] Therefore, in one embodiment, a pharmaceutical composition is provided, which comprises a RXFP1 modulator, a 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.

[0104] 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, erpagliflozin, ipagliptin, reglipgliflozin etabonate, sergliflozin etabonate, sogliflozin and togliflozin, or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

[0105] 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.

[0106] In one embodiment, a pharmaceutical composition for use in the methods described herein is provided, the pharmaceutical composition comprising a RXFP1 modulator, a 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.

[0107] In one embodiment, a pharmaceutical composition is provided, the pharmaceutical composition comprising an RXFP1 regulator, an SGLT2 inhibitor and a pharmaceutically acceptable excipient, wherein the RXFP1 regulator is a compound as claimed or illustrated in International Patent Application No. PCT / EP2021 / 084673 or U.S. Patent Application No. 17 / 457,953 (both applications are incorporated by reference in their entirety). In one embodiment, the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, ergliflozin, ipagliptin, reglipgliflozin efate, sergliflozin efate, sogliflozin and togliflozin or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

[0108] The one or more excipients selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art and are described, for example, in Handbook of Pharmaceutical Excipients, Sixth Edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmaceutically acceptable excipients can be used, for example, as adjuvants, diluents, carriers, stabilizers, flavoring agents, coloring agents, fillers, binders, disintegrants, lubricants, glidants, thickeners, and coating agents. As will be appreciated by those skilled in the art, certain pharmaceutically acceptable excipients can be used for more than one function and can be used for alternative effects, depending on how many excipients are present in the composition and which other excipients are present in the composition.

[0109] The pharmaceutical composition may be in a form suitable for oral use (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration by inhalation (e.g., as finely divided powders or liquid aerosols), administration by insufflation (e.g., as finely divided powders), or parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular or intramuscular administration), or as suppositories for rectal administration. The composition may be obtained by conventional procedures well known in the art. Compositions intended for oral use may contain additional components, for example, one or more coloring agents, sweeteners, flavoring agents and / or preservatives.

[0110] Reagent test kit

[0111] In one embodiment, a kit is provided, the kit comprising:

[0112] a first pharmaceutical composition comprising an RXFP1 modulator selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; and

[0113] The second pharmaceutical composition comprises an SGLT2 inhibitor and a pharmaceutically acceptable excipient.

[0114] 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, erpagliflozin, ipagliptin, reglipgliflozin etabonate, sergliflozin etabonate, sogliflozin and togliflozin, or a pharmaceutically acceptable salt thereof. In one embodiment, the SGLT2 inhibitor is dapagliflozin.

[0115] Examples

[0116] The compounds described in this specification are further illustrated in the following examples. These examples are given by way of illustration only and are non-limiting.

[0117] In these examples, high resolution mass spectra were recorded on a Micromass LCT mass spectrometer equipped with an electrospray interface (LC-HRMS).

[0118] 1 H NMR measurements were performed at 300, 400, 500, and 600 MHz. 1 The measurements were performed on Bruker Avance III 300, 400, 500 and 600 spectrometers operating at 100 MHz and 500 MHz. Typically, the experiments were recorded at 25°C. Chemical shifts are given in ppm with the solvent as internal standard. Protons on heteroatoms such as NH and OH protons are reported only when detected in the NMR and may therefore be missing. The following abbreviations (and their derivatives, such as dd, doublet, etc.) are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; qn, quintet; p, quintet.

[0119] Unless otherwise stated, normal phase silica was used. (40M, 25M or 12M), SNAP Cartridges KP-Sil (340, 100, 50 or 10), or Flash chromatography was performed using Flash Column Silica-CS Cartridges (330, 180, 120, 80).

[0120] Unless otherwise stated, use Reversed phase flash chromatography was performed using a C-18 spherical 20-35 μm 100A cartridge.

[0121] Generally, all solvents used were commercially available and of analytical grade. Anhydrous solvents were routinely used for the reactions.

[0122] The phase separator used in these examples is Phase separation column.

[0123] The intermediates and examples named below were named using ChemDraw Professional version 19.0.0.22 from PerkinElmer.

[0124] The following abbreviations are used

[0125] Aq water based

[0126] B2Pin2 4,4,5,5-Tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1,3,2-dioxaborolane

[0127] Calcd

[0128] DCM Dichloromethane

[0129] DIA Diisopropylamine

[0130] DIAD (E)-Diisopropyl diazene-1,2-dicarboxylate

[0131] DIPEA N-ethyl-N-isopropyl-propan-2-amine

[0132] DMF N,N-Dimethylformamide

[0133] DMSO Dimethyl sulfoxide

[0134] DPPA Diphenylphosphorylazide

[0135] DTBBPY 4,4'-di-tert-butyl-2,2'-bipyridine

[0136] EDC 3-(Ethyliminomethylamino)-N,N-dimethyl-propan-1-amine; hydrochloride

[0137] ESI Electrospray ionization

[0138] Et Ethyl

[0139] Et2O Ether

[0140] EtOAc Ethyl acetate

[0141] EtOH

[0142] h / hr hour

[0143] HATU (dimethylamino)-N,N-dimethyl(3-oxido-1H-[1,2,3]triazolo[4,5-b]pyridinyl)methanilinium hexafluorophosphate

[0144] HOBt 1-Hydroxybenzotriazole; hydrate

[0145] HPLC High Performance Liquid Chromatography

[0146] HRMS High Resolution Mass Spectrometry

[0147] IPA Isopropyl Alcohol

[0148] IPAC Isopropyl Acetate

[0149] [Ir(COD)OMe]2 Bis(1,5-cyclooctadiene)di-μ-methoxyiridium(I)

[0150] L Liter

[0151] Me Methyl

[0152] MeCN Acetonitrile

[0153] mL milliliters

[0154] MeOH Methanol

[0155] 2-Me-THF 2-Methyltetrahydrofuran

[0156] Min Minutes

[0157] MS mass spectrometry

[0158] MTBE Methyl tert-butyl ether

[0159] NMR Nuclear Magnetic Resonance

[0160] OAc acetate

[0161] PE Petroleum Ether

[0162] Pd / C Palladium Carbon

[0163] Rt Room temperature

[0164] Sat

[0165] SFC Supercritical Fluid Chromatography

[0166] T3P 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide

[0167] TEA Triethylamine

[0168] TFA Trifluoroacetic acid

[0169] THF Tetrahydrofuran

[0170] TLC Thin layer chromatography

[0171] Intermediates

[0172] Intermediate 1: 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid ethyl ester

[0173]

[0174] A solution of DIA (576 mL, 413 g, 4.08 mol) in THF (3.50 L) was cooled to -50 to -40 °C and a solution of n-BuLi (2.5 M in hexanes, 1.09 kg, 3.92 mol) was added over 3 h, with the temperature maintained between -50 to -40 °C. The solution was stirred at -50 to -40 °C for 3 h, followed by the addition of a solution of ethyl 1,4-dioxaspiro[4.5]decane-8-carboxylate (700 g, 3.27 mol, 2.34 M in THF) over 2 h, with the temperature maintained between -50 to -40 °C. The reaction mixture was stirred at -50 to -40 °C for 4 h, and then iodomethane (603 g, 4.25 mol, 3.04 M in THF) was added over 3 h, with the temperature maintained between -50 to -30 °C. The reaction mixture was further stirred at -50°C to -30°C for 2h, followed by the addition of aqueous NH4Cl (3.50 L, 20% w / w in H2O) over 1h, with the temperature maintained at <0°C. The solution was warmed to between 15°C and 25°C for 0.5h, then the layers were separated and the organic layer was washed with aqueous NH4Cl (2x3.50 L, 20% w / w in H2O). The organic reaction solvent was exchanged from THF to EtOH under reduced pressure (with the temperature maintained at <45°C), giving the title compound (2.51 kg, 2.91 mol, 89%) as a 27% w / w solution in EtOH. The purified compound 1 H NMR(400MHz CDCl3)δ1.18(3H,s),1.27-1.22(3H,m),1.47-1.35(2H,m),1.70-1.56(4H,m),2.13(2H,d),3.92(4H,s),4.14(2H,q),MS(ESI):m / z[M+H] + 229.2.

[0175] Intermediate 2: 8-Methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid

[0176]

[0177] To a solution of intermediate 1 (1.13 kg, 1.31 mol, 27% w / w in EtOH) was added EtOH (900 mL) followed by aqueous NaOH (2.63 L, 5.26 mol, 2 M in H2O) with the temperature maintained between 15°C and 30°C. The solution was heated to between 50°C and 60°C for 6 h, then cooled to 15°C to 30°C, and the solution was concentrated to 1.8 to 2.4 L under reduced pressure. Hexane (1.50 L) was added, and the layers were separated. The aqueous layer was collected and the pH was adjusted to between 3 and 4 by adding aqueous HCl (1.30 L, 5.2 mol, 4 M in H2O) with the temperature maintained at <20°C. This aqueous solution was extracted with DCM (2 x 1.50 L) and the combined organic phases were concentrated under reduced pressure (with the temperature maintained at < 30 °C) to give the title compound (1.43 kg, 1.24 mol, 94%) as a 17% w / w solution in DCM. 1 H NMR (500MHz CDCl3) δ1.25(3H,s),1.53(2H,dt),1.62-1.72(4H,m),2.05-2.19(2H,m),3.93(4H,s). MS(ESI):m / z[M+Na] + 223.1.

[0178] Intermediate 3: 1-Methyl-4-oxocyclohexane-1-carboxylic acid

[0179] Method A

[0180]

[0181] To a solution of intermediate 2 (367 g, 250 mmol, 14% w / w in DCM) was added TFA (95.3 mL, 142 g, 1.25 mol). The reaction temperature was maintained between 25°C and 35°C for 20 h, then cooled to between 0°C and 10°C. H2O (250 mL) was added to the reaction solution, and the pH of the aqueous phase was adjusted to between 9 and 10 by adding aqueous NaOH (440 mL, 1.76 mol, 4 M in H2O). The layers were separated, and the aqueous layer was retained and cooled to between 0°C and 10°C. The pH was adjusted to between 2 and 3 by adding aqueous HCl (73.5 mL, 294 mmol, 4 M in H2O), then extracted with DCM (3x250 mL), and the combined DCM solution was concentrated to between 150 and 200 mL under reduced pressure. The organic reaction solvent was exchanged from DCM to MeCN under reduced pressure (with the temperature maintained at <40°C) to give the title compound (119 g, 227 mmol, 91%) as a 30% w / w solution in MeCN.1 H NMR (400MHz CDCl3) δ1.39(3H,s),1.73(2H,td),2.43(6H,m). MS(ESI):m / z[M+H] + 157.1.

[0182] Method B

[0183] To a solution of intermediate 2 (6.17 kg, 3.83 mol, 12.4% in DCM) was added TFA (1.42 L, 2.18 kg, 19.13 mol). The reaction temperature was maintained between 25°C and 35°C for 20 h and then cooled to between 0°C and 10°C. An aqueous solution of NaOH (918 g, 22.96 mol, dissolved in 7.66 L H2O) was added to the reaction solution and the pH of the aqueous phase was adjusted to between 9 and 11. The layers were separated and the aqueous layer was retained and cooled to between 0°C and 10°C. DCM (3.83 L) was added followed by aqueous HCl (1.52 L, 6.08 mol, 4 M in H2O) to adjust the pH to between 3 and 4. The organic layer was retained and the aqueous layer was extracted with DCM (2 x 3.83 L) and the combined organic phases were washed with brine (2.3 L, 15% w / w NaCl). The organic phase was concentrated under reduced pressure to 2.3 to 3.1 L. The organic reaction solvent was exchanged from DCM to MeCN under reduced pressure (with the temperature maintained at <45° C.) to give the title compound (2.85 kg, 3.32 mol, 87%) as an 18% solution in MeCN. 1 H NMR (400MHz CDCl3) δ1.39(3H,s),1.73(2H,td),2.43(6H,m). MS(ESI):m / z[M+H] + 157.1.

[0184] Intermediate 4: 1-Methyl-4-oxocyclohexane-1-carboxylic acid naphth-1-ylmethyl ester

[0185] Method A

[0186]

[0187] To a solution of intermediate 3 (119 g, 192 mmol, 25% w / w in MeCN) was added 1-chloromethylnaphthalene (32.2 g, 183 mmol), followed by DIPEA (70.0 mL, 49.7 g, 384 mmol) and NaI (2.88 g, 19.2 mmol). The solution was heated to between 50°C and 60°C for 8 h, then cooled to between 0°C and 10°C. H2O (240 mL) was added, and the pH of the reaction mixture was adjusted to between 3 and 4 by adding aqueous HCl (55.0 mL, 220 mmol, 4 M in H2O). The reaction mixture was extracted with MTBE (2x150 mL), and the combined organic phases were washed with aqueous NaHCO3 (150 mL, 144 mmol, 8% w / w in H2O). The organic reaction solvent was exchanged from MTBE to IPA under reduced pressure, with the temperature maintained at <40°C. The temperature of the reaction solution was lowered to between -10°C and 3°C, and the solution was stirred for 2 h, at which time a solid precipitate formed. The solid was filtered and dried under N2 for 15 h to give the title compound (42.8 g, 144 mmol, 74%) as a white solid; 1 H NMR(500MHz, 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.

[0188] Method B

[0189] To the solution of intermediate 3 (2.66kg, 3.09mol, 18.2%, in MeCN) add 1-chloromethylnaphthalene (535g, 2.94mol), then add potassium carbonate (513g, 3.71mol) and another part of fresh MeCN (714mL). The suspension is heated to between 50 ℃ and 60 ℃ for 17h, then cooled to between 25 ℃ and 30 ℃. The solid is removed by filtering through a Celite pad and washed with MeCN (2x967mL). The filtrate is concentrated to 1.45 to 1.93L under reduced pressure. Under reduced pressure, MeCN is exchanged into isopropanol, wherein the temperature is maintained at <50 ℃. The temperature of the mixture is reduced to between 20 ℃ and 25 ℃, and a solid precipitate is formed. 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%); 1H NMR(500MHz, 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.

[0190] Intermediate 5: 5-(1,3,6,2-dioxaboroctan-2-yl)-4-fluoro-2-methoxybenzoic acid methyl ester

[0191] Method A

[0192]

[0193] B2Pin2 (362g, 1.43mol) is added to 2-Me-THF (1.75L) degassed to <1% oxygen with N2. The solution is maintained between 20°C and 30°C, and 4-fluoro-2-methoxybenzoic acid methyl ester (250g, 1.36mol) is added. DTBBPY (1.09g, 4.10mmol) is added, and the reaction vessel is evacuated and refilled with N2 until the oxygen level is <0.5%. [Ir(COD)OMe]2 (1.35g, 2.04mmol) is added, and the reaction vessel is evacuated and refilled with N2 until the oxygen level is <0.5%. The reaction mixture is heated to between 80°C and 85°C and kept for another 2h at this temperature. The reaction mixture is cooled to between 0°C and 5°C, and then diethanolamine (428g, 4.07mol, 10.9M, in IPA) is slowly added over a period of 2.5h, while producing H2 gas. The reaction mixture was stirred between 0°C and 5°C for 2.5h, then filtered and the solid was washed with 2-Me-THF (3x750mL). The solid was dried under N2 for 10h to give the title compound (356g, 1.20mol, 88%) as a white solid; 1 H NMR(500MHz,DMSO-d6)δ2.81-2.89(2H,m),3.14(2H,dq),3.71(2H,ddd),3.7 4(3H,s),3.78(3H,s),3.84(2H,td),6.77(1H,d),7.10(1H,s),7.83(1H,d). MS(ESI):m / z[M+H] + 297.1.

[0194] Method B

[0195] B2Pin2 (29.0g, 114mmol) and 4-fluoro-2-methoxybenzoic acid methyl ester (20.6g, 109mmol) are added to 2-Me-THF (140mL) degassed to <1% oxygen with N2. The solution is maintained between 20 ℃ and 30 ℃, then DTBBPY (88mg, 0.33mmol) and [Ir (COD) OMe]2 (108mg, 0.16mmol) are added, and the reaction vessel is evacuated and refilled with N2 until the oxygen level is <0.5%. The reaction mixture is heated to between 80 ℃ and 85 ℃ and kept for 3h at this temperature. The reaction mixture is cooled to between 0 ℃ and 10 ℃, and isopropanol (12.4mL, 218mmol) is slowly added subsequently, while producing H2 gas. Seed crystals (100 mg of intermediate 5) were added, followed by diethanolamine (22.84 g, 218 mmol) dissolved in IPA (20 mL) to give a mobile slurry. The slurry was warmed to 20° C. to 30° C., and the solid was collected by filtration. It was then washed with 2-Me-THF (160 ml), and the solid was dried under N2 for 10 h to give the title compound (29.1 g, 96 mol, 88%) as a white solid; 1 H NMR(500MHz,DMSO-d6)δ2.81-2.89(2H,m),3.14(2H,dq),3.71(2H,ddd),3.7 4(3H,s),3.78(3H,s),3.84(2H,td),6.77(1H,d),7.10(1H,s),7.83(1H,d). MS(ESI):m / z[M+H] + 297.1.

[0196] Intermediate 6: 4-Fluoro-5-hydroxy-2-methoxybenzoic acid methyl ester

[0197] Method A

[0198]

[0199] To a suspension of intermediate 5 (350 g, 1.18 mol) in H2O (1.05 L) was added THF (1.75 L) and the reaction mixture was stirred until a clear solution was obtained. (NH4)2CO3 (136 g, 1.41 mol) was added and the heterogeneous mixture was cooled to between 0°C and 10°C. NaBO3.4H2O (217 g, 1.41 mol) was added in 10 equal portions over a period of 2 h, with the reaction temperature being maintained between 0°C and 30°C. The reaction temperature was adjusted to between 20°C and 30°C and maintained for 1 h. An aqueous solution of NaHSO3 (1.96 L, 942 mmol, 0.48 M in H2O) was added over 3 h and the reaction mixture was stirred for an additional 0.5 h. The reaction mixture was filtered, the solid was washed with ethyl acetate (700 mL), and the filtrate and washings were combined to give a two-phase solution. The solution was separated and the remaining organic phase solvent was exchanged from THF / ethyl acetate to MeOH under reduced pressure, wherein the temperature was maintained at <40°C. H2O (3.50 L) was added dropwise over a period of 4 h, and the reaction mixture was cooled to between 0°C and 5°C and maintained for 2 h. The reaction mixture was filtered, and the collected solids were washed with H2O (3x350 mL) and dried under hot air at <40°C to give the title compound (195 g, 974 mmol, 83% yield) as a white solid; 1 H NMR (500MHz, CDCl3) δ3.82(3H,s),3.86(3H,s),6.72(1H,d),7.54(1H,d). MS(ESI):m / z[M+H] + 201.0.

[0200] Method B

[0201] Intermediate 5 (32.41 g, 67.3 mmol) and acetic acid (12.13 g, 202 mmol) were dissolved in 2-Me-THF (100 mL) and cooled to between 0°C and 10°C. Hydrogen peroxide solution (30% w / w, 9.16 g, 80.8 mmol) was added over 2 hours, and then the reaction temperature was adjusted to between 20°C and 30°C and maintained for 18 hours. An aqueous solution of Na2S2O3.5H2O (20% w / w, 50 mL) quenched the mixture and caused 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, followed by another 2 vacuum distillations with 2-Me-THF (100 mL) to give a dissolved solution (at 35°C to 45°C). Nucleation was controlled by adding seed crystals (100 mg of intermediate 6) followed by slow addition of 300 mL of n-heptane over 5 hours. The resulting slurry was adjusted to between 20°C and 30°C and stirred overnight before filtering. The collected solid was washed with n-heptane (2x60 mL) and dried to give the title compound (12.5 g, 62.5 mmol, 93% yield) as a white solid; 1 H NMR (500MHz, CDCl3) δ3.82(3H,s),3.86(3H,s),6.72(1H,d),7.54(1H,d). MS(ESI):m / z[M+H] + 201.0.

[0202] Intermediate 7: (1R,2R,3S,4S)-3-(Methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

[0203]

[0204] To a solution of (3aR,4R,7S,7aS)-3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran-1,3-dione (387 g, 2.36 mol) in toluene (4.64 L) was added quinidine (843 g, 2.60 mol) followed by toluene (774 mL). The reaction mixture was cooled to between -10°C and -5°C and MeOH (227 g, 286 mL, 7.08 mol) was added dropwise over 1.5 h and then maintained at -10°C to -5°C for 14 h. The reaction mixture was warmed to between -5°C and 5°C, maintained for 2 h, and then filtered. The solid was washed with toluene (3x387 mL), and the filtrate and washings were combined and cooled to between 0°C and 10°C. In a separate container, an aqueous solution of HCl (590 mL, 7.08 mol, 12 M in H2O) and NaCl (1.24 kg, 21.2 mol) were added to H2O (6.39 L), and the resulting solution was added dropwise to the main reaction vessel, wherein the reaction solution was maintained at <10°C. The reaction mixture was warmed to between 10°C and 20°C for 0.5 h, then filtered. The solid was washed with toluene (1.94 L), the filtrate and washings were combined, and the two phases of the solution were separated. The organic phase was washed with aqueous NaCl (3.87 L, 20% w / w in H2O) and stored at <5°C to give the title compound (6.19 kg, 1.83 mol, 78%) as a 5.9% w / w solution in toluene; the purified compound 1 H NMR(400MHzDMSO-d6)δ1.25-1.32(1H,m),1.95(1H,d),2.48-2.50(2H,m),2.93(2H,s),3.51(3H,s),6.15-6.22(2H,m),12.21(1H,s). MS(ESI):m / z[M+Na]+219.1.

[0205] Intermediate 8: (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester hydrochloride

[0206]

[0207] To a solution of intermediate 7 in toluene (6.19 kg, 5.9% w / w, 1.85 mol) between -5°C and 5°C was added TEA (307 mL, 223 g, 2.22 mol) followed by DPPA (538 g, 1.94 mol), wherein the reaction solution was maintained at <5°C. The reaction mixture was stirred between -5°C and 5°C for 4 h, then TEA (767 mL, 557 g, 5.55 mol) was added followed by citric acid (352 g, 1.85 mol). The reaction mixture was stirred between -5°C and 5°C for 6 h, then H2O (3.6 L) was added, wherein the reaction solution was maintained at <10°C. The biphasic reaction solution was stirred for 0.5 h, the phases were separated and the organic phase was washed with H2O (3.6 L) and aqueous NaCl (3.6 L, 15% w / w in H2O) and then stored between 2°C and 8°C to give (1S,2S,3R,4R)-3-(azidocarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester (Intermediate 9) as a solution in toluene, which was used directly in the next step. Intermediate 9 as a solution in toluene between 2°C and 8°C was added to a reactor containing toluene (1.80 L) between 70°C and 80°C over 2h, maintaining the reaction temperature at <80°C. The resulting solution was stirred for 1 h and then cooled to between 20°C and 30°C. The organic reaction solvent was exchanged from toluene to 1,4-dioxane under reduced pressure, and the temperature was maintained at <50°C to give (1S, 2S, 3R, 4R)-3-isocyanatobicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester (intermediate 10) as a solution in 1,4-dioxane, which was used directly in the next step. To a solution of intermediate 10 in 1,4-dioxane between 10°C and 20°C, HCl (420 mL, 1.68 mol, 4M in 1,4-dioxane) was added, followed by H2O (360 mL, 1.68 mol, 4.67M in 1,4-dioxane). The reaction mixture was warmed to between 25°C and 35°C and maintained for 16h. MTBE (1.65 L) was added dropwise, and the reaction mixture was filtered, the solid was washed with MTBE / 1,4-dioxane (1:1, 660 mL) and MTBE (660 mL), then dried under vacuum between 30°C and 40°C to give the title compound as a white solid (258 g, 1.27 mol, >99% ee, 75%); 1 H NMR(400MHz,DMSO-d6)δ1.45(1H,d),2.04(1H,d),2.52-2.67(1H,m),2.94-3 .10(2H,m),3.19(1H,d),3.65(3H,s),6.21(1H,m),6.30(1H,m),8.34(3H,s). MS(ESI):m / z[M+H] +168.1.

[0208] Intermediate 11: (1r,4r)-4-hydroxy-1-methylcyclohexane-1-carboxylic acid naphth-1-ylmethyl ester

[0209]

[0210] To a solution of Na2HPO4.12H2O (8.25 g, 23.0 mmol), NaH2PO4 (0.55 g, 4.48 mmol) and MgCl2 (0.11 g, 1.10 mmol) in H2O (550 mL) at 20°C to 30°C was added intermediate 4 (50.0 g, 169 mmol) in IPA (450 mL). The pH of the reaction solution was adjusted to between 7.3 and 7.8 using 6M HCl, and NAD+ (0.66 g, 1.00 mmol) was added, followed by ADH-230 (7.50 g, 0.15 wt%). ADH-230 is an alcohol dehydrogenase (Catalog No. ADH-230) obtained from Johnson Matthey PLC, UK. The reaction mixture was then maintained at 33°C to 37°C for 18 h and then concentrated under reduced pressure to between 300 and 400 mL, with the temperature maintained at <45°C. NaCl (150 g), (20.0g, 0.4wt%) and MTBE (500mL), and the reaction was kept for 0.5h. The mixture was filtered, and the filter cake was washed with MTBE (250mL). The combined filtrate was separated, and the aqueous phase was extracted with MTBE (500mL). The organic phases were combined and washed with H2O (250mL), and then the solvent was exchanged to THF (wherein the temperature was maintained at <45°C) under reduced pressure to give the title compound (138g, 33% w / w%, >99:1 trans:cis, <0.1% IPA, 92% yield) as a solution in THF, which was used directly in the next step. The purified compound 1 H NMR(500MHz, CDCl3)δ1.21(3H,s),1.48-1.58(2H,m),1.62-1.77(4H,m),1.82-1.93(2H,m),3.74-3.77( 1H,m),5.57(2H,s),7.41-7.48(1H,m),7.48-7.57(3H,m),7.85(1H,d),7.87-7.91(1H,m),7.98(1H,d). MS(ESI):m / z[M+Na]+321.1.

[0211] Route B

[0212] Under nitrogen, a solution of tri-sec-butyl lithium borohydride (1.06g, 5.6mmol) in THF (5mL) was added dropwise to a stirred solution of intermediate 4 (1.00g, 3.37mmol) in THF (10mL) cooled to -78°C over a period of 1min. The resulting solution was stirred at -78°C for 2h. The reaction mixture was quenched with 0.1M HCl (10mL) (at -78°C) and then extracted with EtOAc (3x50mL). The organic layers were merged and dried over Na2SO4, filtered and evaporated. The residue was purified by preparative TLC (EtOAc / PE, 1:3) to obtain the title compound (0.488g, 48.5%) in a light yellow jelly. The separated material has a cis / trans ratio of 3:100. 1 H NMR (400MHz, CDCl3) δ1.21-1.25(s,3H),1.37-1.49(m,1H),1.49-1.61(m,2H),1.61-1.74(m,4H),1.83-1.95(m,2H),3 .74-3.83(dq,1H),5.57-5.61(s,2H),7.43-7.54(dd,1H),7.50-7.61(m,3H),7.84-7.94(m,2H),7.97-8.04(m,1H).). MS(ESI):m / z[M+Na] + 321.

[0213] Intermediate 12: methyl 4-fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate

[0214]

[0215] To a solution of intermediate 11 in THF (736 g, 34% w / w, 839 mmol) was added THF (156 mL), PPh3 (248 g, 944 mmol) and intermediate 6 (140 g, 699 mmol). The solution was heated to 30 °C and DIAD (184 g, 909 mmol) was added dropwise over 1 h, with the reaction temperature maintained at <40 °C. The solution was kept between 30 °C and 40 °C for 1 h, then cooled to between 20 °C and 30 °C, followed by the addition of an aqueous solution of NaCl (700 mL, 20% w / w in H2O). The layers were separated and the crude solution of the title compound in THF was used directly in the next step. Purification of the compound 1H NMR(500MHz, CDCl3)δ1.17(3H,s),1.20-1.30(2H,m),1.58(2H,qd),1.88-1.98(2H,m),2.29(2H,d),3.84(3H ,s),3.88(3H,s),4.05(1H,tq),5.61(2H,s),6.72(1H,d),7.43-7.58(5H,m),7.82-7.94(2H,m),8.00(1H,d). MS(ESI):m / z[M+Na]+503.2.

[0216] Intermediate 13: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid

[0217]

[0218] To a crude solution of intermediate 12 from the previous step, used directly, was added an aqueous solution of LiOH.2H2O (88.0 g, 2.10 mol in 525 mL of H2O) between 0°C and 5°C over 1 h, with the reaction temperature maintained at <10°C. The solution was warmed to between 15°C and 30°C and stirred vigorously for 16 h. IPAC (1.68 L) was added, and the solution was cooled to between 0°C and 10°C, followed by dropwise addition of H3PO4 (1.26 L, 2.52 M, 2 M in H2O) (with the reaction temperature maintained at <10°C) to give a solution with a pH between 4.0 and 5.0. The organic layer was separated and washed with an aqueous solution of NaCl (700 mL, 20% w / w in H2O). THF was removed under reduced pressure, with the temperature maintained at <50°C, and IPAC (4.20 L) was added to give the title compound in IPAC, which was used directly in the next step. Purification of the compound 1 H NMR(500MHz, CDCl3)δ1.18(3H,s),1.22-1.36(2H,m),1.58(2H,qd),1.95(2H,dt),2.29(2H,d),4.02(3H,s),4.19 (1H,td),5.60(2H,s),6.82(1H,d),7.46(1H,dd),7.49-7.62(3H,m),7.78(1H,d),7.82-7.94(2H,m),7.99(1H,d).

[0219] Intermediate 14: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid cyclohexylamine salt

[0220]

[0221] To a crude solution of the intermediate 13 used directly from the previous step in IPAC between 50°C and 55°C was added a solution of cyclohexylamine (280 mL, 699 mmol, 2.5 M in IPAC) over 3 h. The heterogeneous slurry was stirred between 50°C and 55°C for 0.5 h, then stirred between 40°C and 45°C for another 1 h. The reaction mixture was filtered and the solid was washed with IPAC (3x0.98 L) preheated to between 40°C and 45°C, and dried at 45°C under N2 flow for 16 h. MeOH (3.64 L) was added to the dried collected solid and the mixture was heated to between 55°C and 56°C. H2O (1.58 L) was added dropwise over 1 h, and the mixture was then stirred for 1 h, then cooled to between 0°C and 5°C over 3 h. The heterogeneous slurry was maintained for another 1 h, then filtered, washed with 5:3 MeOH:H2O (2x750 mL) at 0°C, and the solid was dried under N2 at 45°C for 16 h to give the title compound as a white solid (332 g, 85% from methyl 4-fluoro-5-hydroxy-2-methoxybenzoate); 1 H NMR (500MHz, CDCl3) δ0.96 (1H, ddt), 1.03-1.36 (6H, m), overlap 1.14 (3H, S), 1.46-1.7 (5H, m), 1.91 (4H, dt), 2.26 (2H, d), 2.81 (1H, t),3.76(3H,s),4.03(1H,tt),5.59(2H,s),6.65(1H,d),7.37-7.49(2H,m),7.49-7.6(3H,m),7.81-7.93(2H,m),7.98(1H,d). MS(ESI):m / z[M+Na]+489.2.

[0222] Intermediate 15: (1S,2S,3R,4R)-3-(4-fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamido)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester

[0223]

[0224] To a solution of intermediate 14 (149 g, 264 mmol) in DCM (750 mL) between 15°C and 30°C was added HO (450 mL) followed by the slow addition of HCl (300 mL, 1 M in HO). The two-phase solution was stirred for 0.5 h and then separated and the organic phase was washed with HCl (750 mL, 0.2 M in HO) and then with HO (3x750 mL). The organic solution was concentrated under reduced pressure (wherein the temperature was maintained below 30°C) to dryness to <0.1% HO. The solution was diluted with DCM (450 mL) to bring the total volume to 750 mL and then intermediate 8 (59.3 g, 291 mmol) was added to give a heterogeneous slurry. To this mixture was added DIPEA (137 g, 1.06 mol) followed by T3P (252 g, 397 mmol, 50% w / w in EtAOc) and the solution was stirred for 1 h. The solution was cooled to between 0°C and 10°C before H2O (750 mL) was added and then stirred for an additional 0.5 h. The biphasic solution was separated and the organic phase was washed with H2O (2 x 750 mL) and then solvent exchanged to THF under reduced pressure to give the title compound in THF which was used directly in the next step. Purification of the compound 1 H NMR(500MHz, CDCl3)δ1.16(3H,s),1.25(2H,td),1.49-1.69(3H,m),1.92-2.01(2H,m ),2.04-2.1(1H,m),2.28(2H,d),2.71(1H,dd),2.83(1H,s),2.92-3.05(1H,m),3.61 (3H,s),3.93(3H,s),4.17(1H,td),4.46(1H,td),5.60(2H,s),6.25(2H,ddd),6.72( 1H,d),7.46(1H,dd),7.48-7.6(3H,m),7.81-7.95(3H,m),7.99(1H,d),8.60(1H,d). MS(ESI):m / z[M+H] + 616.3.

[0225] Intermediate 16: (1S,2S,3R,4R)-3-(4-fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamido)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

[0226]

[0227] The crude solution (750 mL) of intermediate 15 in THF from the previous step was cooled to between 0°C and 5°C. An aqueous solution of LiOH.2H2O (27.7 g, 661 mmol in 150 mL of H2O) was added and the solution was kept for 36 h. 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 kept for 1 h between 0°C and 5°C. The heterogeneous slurry was filtered, and the solid was washed with 1:3 MeOH:H2O (600 mL) at 0°C, and the solid was dried at 45°C under N2 for 16 h to give the crude title compound (158 g, 99%) as a white solid. The crude product (150 g) was slurried in IPAC (1.13 L) between 60°C and 65°C for 0.5 h. The heterogeneous mixture was cooled to between 0°C and 5°C over 3 h, then stirred for 1 h, and then filtered. The collected solid was treated with IPAC between 0 and 5 °C (2 x 300 mL) and then dried at 45 °C under N2 for 12 h to give the title compound as a white solid (127 g, 82% from intermediate 14); 1 H NMR(500MHz, CDCl3)δ1.16(3H,s),1.2-1.35(2H,m),1.50-1.69(3H,m),1.89-2 .08(3H,m),2.27(2H,ddd),2.72(1H,dd),2.80(1H,s),3.06(1H,s),3.75(3H,s) ,4.15(1H,tt),4.43-4.54(1H,m),5.59(2H,s),6.24(2H,ddd),6.53(1H,d),7. 45(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.

[0228] Intermediate 17: (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1R,2R,3S,4S)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-5-en-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid naphth-1-ylmethyl ester

[0229]

[0230] To a solution of DIPEA (6.45 g, 49.9 mmol) in DCM (300 mL) between 0°C and 5°C was added intermediate 16 (30.6 g, 49.9 mmol) followed by (1-methylcyclobutyl)methanamine hydrochloride (8.63 g, 62.4 mmol). DIPEA (25.8 g, 200 mmol) was added dropwise with the temperature maintained between 0°C and 5°C followed by T3P (50.8 g, 79.8 mmol, 50% w / w in EtAOc) over 0.5 h. The solution was warmed to between 15°C and 25°C and stirred for 1 h followed by the addition of H2O (150 mL) dropwise with the temperature maintained below 30°C. The biphasic solution was separated and the organic phase was washed with H2O (2 x 150 mL) and then the solvent was exchanged to EtOH under reduced pressure to give the title compound as a crude solution in EtOH (128 g, 26% w / w, 96% yield) which was used directly in the next step. Purification of the compound 1 H NMR(500MHz, CDCl3)δ0.98(3H,s),1.16(3H,s),1.21-1.29(2H,m),1.51-1.66(5H,m),1.66-1.76(3H, m),1.76-1.82(1H,m),1.88-2.02(2H,m),2.26(3H,dd),2.40(1H,dd),2.80(1H,s),3.00(1H,s),3.05( 1H,dd),3.21(1H,dd),3.93(3H,s),4.06-4.2(1H,m),4.39(1H,td),5.60(2H,s),5.64(1H,t),6.19-6. 38(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.

[0231] Intermediate 19: (1R,2S,3R,4S)-3-(5-(((1s,4S)-4-carboxy-4-methylcyclohexyl)oxy)-4-cyano-2-methoxybenzamido)bicyclo[2.2.1]heptane-2-carboxylic acid

[0232]

[0233] Step A. Intermediate 20: Methyl 4-cyano-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate

[0234]

[0235] A solution of 4-cyano-5-hydroxy-2-methoxybenzoic acid methyl ester (1.4 g, 6.7 mmol), intermediate 11 and PPh3 (2.6 g, 10.1 mmol) in THF (30 mL) was stirred at 60 ° C for 10 min. After slowly adding DIAD (1.97 mL, 10.1 mmol), the reaction mixture was stirred at 60 ° C for 14 h. The solvent was then removed under reduced pressure and the residue was redissolved in EtOAc (150 mL), washed successively with NaHCO3 (saturated, 200 mL), NH4Cl (saturated, 250 mL) and brine (saturated, 250 mL). The organic layer was separated and dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (using a gradient of 0%-18% EtOAc in PE as a mobile phase) to give the title compound (3.25 g, 99%) as a white solid. MS (ESI): m / z[M+Na] + 510.3.

[0236] Step B. Intermediate 21: 4-Cyano-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid

[0237]

[0238] At 10 ° C, LiOH (1.6 g, 66.7 mmol) was added in batches to a stirred solution of intermediate 20 (3.25 g, 6.7 mmol) in H2O:THF 1:3 (80 mL), and the resulting suspension was stirred at 20 ° C. After 3 h, the pH of the reaction mixture was adjusted to pH 3 by adding HCl (2M). The reaction mixture was diluted with EtOAc (350 mL) and washed with brine (saturated, 350 mL), H2O (350 mL) and brine (350 mL) in sequence. The organic layer was separated, dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude product was purified by crystallization from IPA / EtOAc to obtain the title compound (3.16 g, 100%) as a white solid. MS (ESI): m / z[M+Na] + 496.3.

[0239] Step C. Intermediate 22: (1S,2S,3R,4R)-3-(4-cyano-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamido)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester

[0240]

[0241] DIPEA (3.5 mL, 20 mmol) was added to a solution of intermediate 21 (3.16 g, 6.67 mmol), intermediate 8 (1.291 g, 6.34 mmol), EDC (1.9 g, 10 mmol) and HOBt (1.533 g, 10.01 mmol) in DMF (60 mL) at 10 °C, and the resulting suspension was stirred at room temperature for 13 hours. The reaction mixture was diluted with EtOAc (500 mL) and washed successively with NH4Cl (saturated, 200 mL), H2O (300 mL) and brine (saturated, 250 mL). The organic layer was dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (using a gradient of 0%-20% EtOAc in PE as mobile phase) to give the title compound (2.6 g, 62%) as a white solid. MS (ESI): m / z[M+H] + 623.4.

[0242] Step D. Intermediate 23: (1S,4s)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(methoxycarbonyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0243]

[0244] Intermediate 22 (5.7 g, 9.15 mmol) and Pd / C (0.584 g, 0.55 mmol) in MeOH (100 mL) were stirred at 20 °C under hydrogen atmosphere (1.5 atm) for 14 h. The mixture was stirred by The mixture was filtered through a pad of Celite® and the solvent was removed under reduced pressure. The crude product was purified by crystallization from EtOAc / EtOH to give the title compound (5.1 g) as a pale yellow solid. MS (ESI): m / z [M+H] + 485.4.

[0245] Step E. (1R,2S,3R,4S)-3-(5-(((1s,4S)-4-carboxy-4-methylcyclohexyl)oxy)-4-cyano-2-methoxybenzamido)bicyclo[2.2.1]heptane-2-carboxylic acid

[0246] At 10 ° C, a solution of LiOH (50mL, 52.6mmol, 1.05M in H2O) was added to a stirred solution of intermediate 23 (5.1g, 10.5mmol) in THF (100mL). The reaction mixture was allowed to warm to room temperature and stirred for 14h, then acidified to pH 2 using HCl (1M, aqueous). The reaction mixture was diluted with EtOAc (350mL) and washed successively with brine (300mL, saturated), H2O (300mL) and brine (300mL, saturated). The organic phase was separated and dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude product was precipitated from EtOAc / Et2O, then purified by reversed-phase flash chromatography (on a C18 column, using a gradient of 0%-50% MeCN in HCl (0.4%, aqueous) as a mobile phase) to obtain the title compound (4.00g, 82%) as a white solid; 1 H NMR (400MHz, DMSO-d6) δ1.13(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.

[0247] Examples

[0248] Example 1: (1S,4s)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (Form A)

[0249]

[0250] To a solution of intermediate 17 in EtOH (206 g, 19% w / w, 57.9 mmol) was added EtOH (385 mL), followed by 10 wt% Pd / C (3.96 g, 5% w / w). The vessel was purged six times with N2, followed by six more times with H2. The vessel was pressurized to 0.4 MPa with H2, and the reaction solution was stirred between 20°C and 30°C for 20 h. The H2 atmosphere was completely replaced with N2, and then the reaction mixture was filtered and the solid was washed with EtOH (3x80 mL). A second batch of the same operation was performed, and the collected EtOH solutions were combined to give a single solution. Solvent was exchanged into EtOAc under reduced pressure, wherein the temperature was maintained below 45°C. EtOAc solution (280 mL) was heated to between 70°C and 75°C for 0.5 h, then cooled to between 40°C and 45°C, and n-heptane (475 mL) was added dropwise over 0.5 h. The mixture was stirred for 0.5 h then cooled to between 20°C and 25°C over 2 h and then held for another 2 h. The heterogeneous slurry was filtered and the solid was washed twice with 1:2 EtOAc / n-heptane (160 mL) and then dried below 45°C for 20 h to give the crude title compound (55.7 g, 87%) as a white solid.

[0251] Part 1: The crude title compound (2.50g, 4.59mmol) was dissolved in EtOH (15.0mL). The temperature of the solution was maintained at 25.0°C ± 2.0°C during the addition of water (7.50mL), during which a precipitate was formed. The heterogeneous slurry was stirred for another 1.0h and then collected via filtration. The solid was washed with a (2:3) mixture of EtOH / water (2x5.00mL), collected and dried under N2 to give the title compound (1.80g, 72%) as a white solid. The material is characterized as Form A and is used as a crystal seed according to the method described in Part 2.

[0252] Part 2: The crude title compound (50.0 g, 91.8 mmol) was dissolved in EtOH (350 mL) and then passed through a filter. EtOH (100 mL) was added to a container and then passed through a filter to give a combined EtOH solution. During the slow addition of H2O (150 mL) over 0.5 h, the temperature of the solution was maintained at 25.0 ° C ± 2.0 ° C. The solution was stirred for another 0.5 h and then the seed material (0.005 g, 0.1% w / w) from Part 1 was added. The solution was kept for 6 h and then cooled to 20.0 ° C ± 0.5 ° C over 2 h and then kept for another 6 h. H2O (150 mL) was slowly added over 6 h and then the mixture was kept for another 2 h and then filtered. EtOH (45 mL) and H2O (30 mL) were added to a container and then used to wash the filter cake. The solid was collected and dried below 45 °C under N2 for 12 h to give the title compound Form A (42.2 g, 85%) as a white solid; 1 H 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). For C30H42FN2O6, HRMS (ESI) m / z [M+H] + Calculated value: 545.3022, measured value: 545.3019.

[0253] Example 2: (1S,4s)-4-(5-(((1S,2R,3S,4R)-3-((cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid

[0254]

[0255] Step A Intermediate 18: (1S,4s)-4-(5-(((1R,2R,3S,4S)-3-((cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]hept-5-en-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid naphth-1-ylmethyl ester

[0256] To a solution of DIPEA (1.07 g, 8.31 mmol) in DCM (50.5 mL) between 0°C and 5°C was added intermediate 16 (5.00 g, 8.31 mmol) followed by cyclobutylmethylamine hydrochloride (1.26 g, 10.4 mmol). DIPEA (4.21 g, 32.6 mmol) was added dropwise with the temperature maintained between 0°C and 5°C followed by the addition of T3P (8.46 g, 13.3 mmol, 50% w / w in EtOAc) over 0.5 h. The solution was warmed to between 15°C and 25°C and stirred for 2 h followed by the addition of water (25.0 mL) dropwise with the temperature maintained below 30°C. The two phase solution was separated and the organic phase was washed with water (2x25.0 mL) and then the solvent was exchanged to EtOH (75.0 mL) under reduced pressure to give the title compound as a solution in EtOH which was used directly in the next step.

[0257] Step B: (1S,4s)-4-(5-(((1S,2R,3S,4R)-3-((cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid

[0258] 10wt% Pd / C (290mg, 5%w / w) is added to the EtOH solution of intermediate 18. The container is purged six times with N2 and then purged six times with H2. The container is pressurized to 0.4MPa with H2, and the reaction solution is stirred for 20h between 20°C and 30°C. The H2 atmosphere is completely replaced with N2, and then the reaction mixture is filtered and the solid is washed with EtOH (2x12.2ml). Under reduced pressure, the solvent is exchanged into EtAOc, wherein the temperature is maintained at less than 45°C. EtAOc solution (41.0mL) is heated to between 70°C and 75°C for 0.5h, then cooled to between 40°C and 45°C, and n-heptane (34.8mL) is added dropwise over 0.5h. The mixture is stirred for 0.5h and then cooled to between 20°C and 25°C over 2h, and then kept for 2h. The heterogeneous slurry was filtered and the solid was washed twice with 1:2 EtOAc / n-heptane (11.6 mL) and then dried below 45 °C for 20 h to give the title compound (3.28 g, 74%) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 1.04-1.30 (6H, m), overlap 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 (3 H,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.

[0259] Example 3. (1S,4s)-4-(2-cyano-5-(((1S,2R,3S,4R)-3-((cyclopropylmethyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid

[0260]

[0261] At room temperature, a solution of intermediate 19 (500 μL, 0.024 g, 0.05 mmol, 0.1 M in DMF), a solution of cyclopropylmethylamine (500 μL, 0.05 mmol, 0.1 M in DMF) and a solution of DIPEA (500 μL, 0.15 mmol, 0.3 M in DMF) were added to a vial. A solution of HATU (500 μL, 0.15 mmol, 0.3 M in DMF) was added and the reaction mixture was stirred overnight at 40 °C. The crude mixture was washed with DMSO (3x500 μL), filtered and the filtrate was evaporated under reduced pressure. The crude product was purified by preparative SFC (on a Phenomenex Luna HILIC column (5 μm 250x30 ID mm) using MeOH / 20 mM NH in CO2 as mobile phase) to obtain the title compound (10.3 mg, 39%). For C 29 H 38 N3O6, HRMS (ESI) m / z [M+H] + Calculated value: 524.2756, measured value: 524.2752.

[0262] Example 4: (1S,4s)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0263]

[0264] Step A: Intermediate 24: (1S,2S,3R,4R)-3-(4-cyano-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamido)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

[0265]

[0266] 2M aqueous LiOH (10.6 mL, 21.12 mmol) was added to a solution of intermediate 22 (2.60 g, 4.18 mmol) in DME (50 mL), and the reaction mixture was stirred at room temperature for 12 hours. 10% aqueous citric acid was added to the reaction mixture until pH <3, and then the reaction mixture was extracted twice with EtOAc and the combined organic layers were concentrated in vacuo to give the title compound (2.5 g, 97%). MS (ESI) m / z 609.3 [M+H] + .

[0267] Step B: Intermediate 25: (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-carboxylic acid naphth-1-ylmethyl ester

[0268]

[0269] HATU (344 mg, 0.904 mmol) and DIPEA (0.43 mL, 2.46 mmol) were added to intermediate 24 (500 mg, 0.821 mmol) and (1-methylcyclobutyl)methylamine hydrochloride (134 mg, 0.986 mmol) in DMF (4 mL), and the mixture was stirred at room temperature for 30 min. H2O (50 mL) was added to the reaction mixture and the residual precipitate was collected by filtration, and the residue was then dried under a vacuum pump to give the title compound (611 mg, 100%). MS (ESI) m / z 690.4 [M+H] + .

[0270] Step C: (1S,4s)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0271]

[0272] Palladium (10% Pd / C, 50% H2O, 200 mg) was added to a solution of intermediate 25 (609 mg, 0.883 mmol) in MeOH (4.4 mL). The reaction mixture was stirred at room temperature for 3 hours under 1 atm of hydrogen atmosphere. The hydrogen in the reaction vessel was replaced with argon and the reaction mixture was washed with water. After the filtrate was concentrated in vacuo, the crude product was purified by flash chromatography (using a gradient of 0%-5% MeOH in CHCl 3 as mobile phase) to give the title compound (389 mg, 80%). 1 H NMR(400MHz,DMSO-d6)δ0.99(s,3H),1.21(s,3H),1.23-1.41(m,5H),1.45-1.85(m,10 H),1.96-2.13(m,3H),2.18-2.29(m,3H),2.35-2.40(m,1H),2.65-2.71(m,1H),3.06( dd, J = 13.2, 5.8 Hz, 1H), 3.17 (dd, J = 13.2, 6.3 Hz, 1H), 3.98 (s, 3H), 4.26-4.33 (m, 1H), 4.34-4.43 (m, 1H), 7.37 (s, 1H), 7.72 (s, 3H), 7.84-7.91 (m, 1H), 9.09 (d, J = 8.5 Hz, 1H). For C31H42N3O6, HRMS (ESI) m / z [M+H] + Calculated value: 552.3068, measured value: 552.3064.

[0273] Example 5: (1S,4s)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(neopentylcarbamoyl)bicyclo[2.2.1]hept-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid

[0274]

[0275] The title compound was prepared similarly to Example 3 using 2,2-dimethylpropan-1-amine instead of cyclopropylmethylamine. For C30H42N3O6, HRMS (ESI) m / z [M+H] + Calculated value: 540.3068, measured value: 540.3076.

[0276] 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]hept-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid

[0277]

[0278] The title compound was prepared analogously to Example 3 using 3-fluorobicyclo[1.1.1]pentan-1-amine instead of cyclopropylmethylamine. For C30H37FN3O6, HRMS (ESI) m / z [M+H] + Calculated value: 554.2666, measured value: 554.2670.

[0279] Biological and physicochemical data

[0280] RXFP1 Hu cAMP (Test A)

[0281] To screen for modulators of hRXFP1, an assay to identify compounds that stimulate cAMP production via the Gs-coupled hRXFP1 receptor was used. cAMP was detected using a large number of cAMP HiRange HTRF kits (purchased from CisBio Bioassays, France; catalog number 62AM6PEJ) according to the manufacturer's recommendations. The HTRF method is a competitive immunoassay between native cAMP produced by cells and cAMP labeled with the dye d2. Tracer binding is visualized by antibodies labeled with cryptates against cAMP, and the signal is therefore inversely proportional to the amount of cAMP produced.

[0282] Preparation of assay reagents:

[0283] Assay buffer: HBSS (ThermoFisher, 14065) with 5 mM Hepes (ThermoFisher, 15630), pH 7.4, containing 0.1% BSA (Sigma, A8806)

[0284] Cells: Jump-In cells stably transfected with human RXFP1 TM T-REx TMCHO-K1 cells (Thermo Fisher Scientific). Cells were induced to express human RXFP1 by treating with 10 ng / ml doxycycline for 24 h. They were then cryopreserved for long-term storage. At the beginning of each experiment, cells were thawed, washed with PBS and resuspended in assay buffer to 1.875*10^5 cells / ml

[0285] cAMP Standard: Dilute the stock standard cAMP provided in the CisBio kit in assay buffer to a maximum final concentration of 2.8 μM in the assay.

[0286] HTRF detection reagents: cAMP-d2 and anti-cAMP cryptate reconstituted according to the CisBio instructions were diluted 1:40 in the lysis buffer provided in the HTRF-kit.

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

[0288] 1. 40 nL of test compound dissolved in DMSO was acoustically dispensed (Labcyte Echo) into a white 384-well plate (Greiner; 784075), sealed and stored at room temperature until assayed.

[0289] 2. On the day of the assay, add 40 nL of 200 nM Relaxin-2 in DMSO (1 nM final concentration) to the 100% control wells and 40 nL of DMSO to the 0% wells using an Echo acoustic dispenser.

[0290] 3. Using a Multidrop Combi (Thermo Fisher Scientific), add 4 μL of assay buffer containing 1 mM phosphodiesterase-blocking IBMX (0.5 mM final concentration).

[0291] 4. Add 4 μL of cell solution (1.875*10^5 cells / ml) using Multidrop Combi to obtain 750 cells / well.

[0292] 5. Incubate at room temperature for 45 minutes.

[0293] 6. Add 4 μL of cAMP-d2 in lysis buffer using the Multidrop Combi.

[0294] 7. Add 4 μL of anti-cAMP cryptate in lysis buffer using Multidrop Combi

[0295] 8. Incubate at room temperature for 2 hours

[0296] 9. Homogeneous time-resolved fluorescence (HTRF) signals were detected using an Envision (PerkinElmer) or Pherastar (BMG Labtech) reader (λex = 340 nm, λem = 665 and 615 nm).

[0297] Using a cAMP standard curve, the HTRF data were converted to the amount of cAMP produced in the sample, which was then used to calculate the concentration response. The concentration response data were fit using a four-parameter logistic fit, the Hill equation. The results from the assay are reported in Table 1 as EC 50 (μM) and S inf (%).

[0298] EC 50 It is defined as the concentration at which the stimulatory activity reaches 50% of its maximal level. When multiple assays were run for the same compound, the geometric mean is reported.

[0299] S inf is the activity level, potency, fitted at infinite concentration of the test compound. To facilitate comparison of potency data, potency was normalized to the % effect of the response stimulated by a saturating concentration of relaxin (1 nM). When multiple assays were run for the same compound, the arithmetic mean was reported.

[0300] Human plasma protein binding (Test B)

[0301] The assay was performed according to the human plasma protein binding assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), 157-179, pp. 167-170. The data are reported in Table 1 as the unbound fraction (f u ) (% free). When multiple assays were run for the same compound, the arithmetic mean is reported.

[0302] Human liver microsome stability (Test C)

[0303] The assay was performed according to the human liver microsomal stability assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), 157-179, pp. 170-174. Data are reported in Table 1 as CL int (μl / min / mg protein). When multiple assays were run for the same compound, the arithmetic mean is reported.

[0304] Human hepatocyte stability (Test D)

[0305] The metabolic stability of compounds in human hepatocytes was assessed using the following protocol:

[0306] 1. Prepare 10 mM stock solutions of compound and control compounds in appropriate solvent (DMSO). Place incubation medium (L-15 medium) in a 37°C water bath and allow to warm for at least 15 minutes before use.

[0307] 2. Add 80 μL of acetonitrile to each well of a 96-well deep well plate ("Quench Plate").

[0308] 3. In a new 96-well plate, dilute the 10 mM test and control compounds to 100 μM by combining 198 μL of acetonitrile and 2 μL of the 10 mM stock solution.

[0309] 4. A vial of cryopreserved (below -150°C) human hepatocytes (LiverPool from Bioreclamation IVT) was placed in the TM 10-Donor human hepatocytes (Product No. S01205)) were removed from storage, ensuring that the vials remained cold until thawing. As quickly as possible, the cells were thawed by placing the vials in a 37°C water bath and gently shaking the vials. The vials were kept in the water bath until all ice crystals were dissolved and no longer visible. After thawing was complete, the vials were sprayed with 70% ethanol and transferred to a biosafety cabinet.

[0310] 5. Open the vial and pour the contents into a 50 mL conical tube containing lysis medium. Place the 50 mL conical tube in a centrifuge and spin at 100 g for 10 minutes (room temperature). After the spin is complete, aspirate the lysis medium and resuspend hepatocytes in enough incubation medium to produce approximately 1.5 × 106 cells / mL.

[0311] 6. Use Vision, count cells and determine viable cell density. Cells with poor viability (<80% viability) are unacceptable for use. Dilute cells with incubation medium to reach 1.0 × 10 6 The working cell density was 10 viable cells / mL.

[0312] 7. Transfer 247.5 μL of hepatocytes to each well of a 96-well cell incubation plate. Place the plate on an Eppendorf Thermomixer Comfort plate shaker to allow the hepatocytes to warm for 10 minutes.

[0313] 8. Add 2.5 μL of 100 μM test compound or control compound to the incubation wells containing cells to initiate the reaction.

[0314] 9. Incubate the plate at 37°C and 900 rpm on an Eppendorf Thermomixer Comfort plate shaker. At 0.5, 5, 15, 30, 45, 60, 80, 100 and 120 min, transfer 20 μL of the incubated mixture to a separate "quench plate" and mix the samples by vortexing for 2 min.

[0315] 10. Centrifuge the quenched plates at 4,000 rpm for 20 minutes. Transfer 30 μL of supernatant of each compound to a 96-well assay plate. Combine the 4 compounds in one box. Then dilute the combined sample by adding 180 μL of purified water. All incubations were performed in duplicate.

[0316] All calculations were performed using Microsoft Excel. Peak areas were determined from the extracted ion chromatograms. In vitro intrinsic clearance (IVCl) of the parent compound was determined by regression analysis of the Ln parent percent disappearance versus time curve. int , in μL / min / 10 6 In vitro intrinsic clearance (in vitro Cl int , in μL / min / 10 6 The cell counts (per cell) are reported in Table 1 and determined from the slope values ​​using the following equation:

[0317] In vitro Cl int =kV / N

[0318] V = incubation volume (0.25 mL);

[0319] N = number of hepatocytes per well (0.25×10 6 cells)

[0320] When multiple assays were run for the same compound, the geometric mean was reported.

[0321] Rat hepatocyte stability (Test E)

[0322] The assay was performed according to the rat hepatocyte stability assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), 157-179, pp. 170-174. Data are reported in Table 1 as mean Cl int (μl / min / 10 6 When multiple assays were run for the same compound, the geometric mean is reported.

[0323] Solubility (Test F)

[0324] The assay was performed according to the solubility assay described in Wernevik, J. et al., "A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling", Assay and Drug Development Technologies, 2020, 18(4), pp. 164-167, 157-179. The data are reported as solubility (μM) in Table 1. When multiple assays were run for the same compound, the arithmetic mean was reported.

[0325] Table 1 - Measurement data

[0326]

[0327]

[0328] Human RXFP1 cGMP Production Assay (Test G)

[0329] To profile the RXFP1 agonist activity of compounds with respect to cGMP production, the Green GENIecGMP assay (Montana Molecular; Cat. No. D800G) was used. The assay is based on a mNeonGreen fusion protein fluorescent biosensor delivered to mammalian cells in a BacMam vector. When cGMP binds to the biosensor, fluorescence decreases.

[0330] Preparation of assay reagents:

[0331] Assay buffer: DPBS (Gibco; 14040133) containing 0.1% BSA (Sigma; A8806)

[0332] 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.

[0333] Step-by-step protocol for running the assay:

[0334] Day 1

[0335] 1. The cells were split one day before transduction and seeded at 63,000 cells / cm2 in DMEM medium containing 10% FBS but without antibiotics in tissue culture flasks.

[0336] Day 2

[0337] 2. After washing with PBS, the cells were detached using accutase (Gibco; 1737341), resuspended in culture medium and collected in a 50 mL tube.

[0338] 3. The cells were counted using CEDEX (Innovatis) and diluted to 267,000 cells / mL with culture medium.

[0339] 4. Prepare viral transduction mastermix by mixing reagents in the following ratios (for a single well):

[0340] 6 μL GENIe BacMAM vector

[0341] 0.2 μL 500 mM sodium butyrate

[0342] 13.8 μL DMEM medium containing 10% FBS

[0343] 20 μL total volume

[0344] 5. Combine cells and transduction mastermix at a ratio of 30 μL cells to 20 μL mastermix for a single well.

[0345] 6. In a black poly-D-lysine-coated μclear 384-well plate (Greiner; 781946), dispense 50 μL of the cell transduction mixture from above into each well.

[0346] 7. Incubate the plate in the dark at 37°C, 5% CO2 for 24 h.

[0347] Day 3

[0348] 8. Remove the culture medium from the plate using Bluewasher (BluCatBio).

[0349] 9. Add 20 μL of assay buffer using a Multidrop Combi (Thermo Fisher Scientific).

[0350] 10. Incubate the plate at room temperature in the dark for 30 min prior to assay.

[0351] 11. Assay plates using FLIPR Tetra (Molecular Devices): 10 μL of compound diluted in assay buffer was added to each well by FLIPR Tetra and green fluorescence was measured over time for up to 3 h.

[0352] Data were processed using Screener software (Genedata AG). After subtracting background fluorescence (before compound addition), the area under the curve values ​​from 0 to 90 min after compound addition were used to calculate the response. The concentration response data were fitted using a four-parameter logistic fit, and the EC 50 The values ​​(nM) are reported in Table 2.

[0353] Human RXFP1 phosphorylation ERK (phospho-ERK) assay (Test H)

[0354] To analyze the RXFP1 agonist activity of compounds with respect to ERK phosphorylation, the Advanced Phospho-ERK (Thr202 / Tyr204) Cell Kit (CisBio; 64AERPEH) was used. The assay uses two antibodies. One is labeled with a donor fluorophore (Eu cryptate) and the second is labeled with an acceptor (d2). The first antibody binds specifically to phosphorylated ERK and the second antibody binds to another motif of ERK and is independent of its phosphorylation state. ERK phosphorylation allows the formation of an immune complex involving the two antibodies, thereby generating a FRET signal. Its intensity is proportional to the concentration of phosphorylated ERK in the sample. The assay was performed according to the manufacturer's recommendations.

[0355] Preparation of assay reagents:

[0356] Cells: HEK293s cells stably transfected with human RXFP1 in pIRESneo3 were used. Cells were cultured in DMEM medium (Gibco; 31966) supplemented with 10% FBS at 0.8 mg / mL to maintain RXFP1 expression. Cells maintained in continuous culture were assayed.

[0357] Dilution of test compounds: Compounds were diluted to the desired concentration using serum-free DMEM without phenol red (Gibco; 31053-038). DMSO concentration was adjusted to 0.4%.

[0358] Antibody mixture: Eu- and D2-labeled anti-ERK1 / 2 antibodies were diluted 20-fold with the detection buffer provided in the kit. Shortly before the experiment, equal volumes of each diluted antibody solution were combined into the antibody mixture.

[0359] Step-by-step protocol for running the assay:

[0360] Day 1

[0361] 1. The cells were detached from the culture flask using accutase (Gibco; 1737341), resuspended in phenol red-free DMEM medium containing 10% FBS and collected in a 50 mL tube.

[0362] 2. The cells were counted using CEDEX (Innovatis) and diluted to 320,000 cells / mL using the above culture medium.

[0363] 3. Dispense 100 μL of the cell suspension into each well of a black μclear poly-D-lysine-coated μclear 96-well plate (Greiner; 655946).

[0364] 4. Incubate the plate at 37°C, 5% CO2 for 24 h.

[0365] Day 2

[0366] 5. Serum starvation method: Remove the medium and replace with 50 μL of serum-free DMEM without phenol red. Incubate the plate at 37°C, 5% CO2 for 5 h.

[0367] 6. Add 50 μL of test compound solution to each well.

[0368] 7. Incubate the plate at room temperature for 5 min.

[0369] 8. Stop stimulation by quickly removing the medium and adding 50 μL of lysis buffer per well (diluted to 1x final concentration before addition).

[0370] 9. Transfer the plates to -80°C and freeze the lysates.

[0371] Day 3

[0372] 10. Thaw the plate and shake at room temperature for 30 min.

[0373] 11. Homogenize the cell lysate by pipetting.

[0374] 12. Transfer 16 μL of homogenate per well to a white low-volume 384-well plate (Greiner; 784075)

[0375] 13. Add 4 μL of antibody mixture to each well.

[0376] 14. Incubate the plate at room temperature in the dark for 4 h.

[0377] 15. Homogeneous time-resolved fluorescence (HTRF) signals were detected using a Pherastar (BMG Laboratory Technologies) reader (λex = 340 nm, λem = 665 and 615 nm).

[0378] HTRF ratio data were processed using Screener software (Gene Data). Concentration response data were fitted with a four-parameter logistic fit, and EC 50 The values ​​(nM) are reported in Table 2.

[0379] Table 2 - Measurement data

[0380]

[0381] Those skilled in the art will appreciate that the bioassays described above may be performed using alternative equipment and minor changes in the protocol without significantly affecting the results.

[0382] Although this specification and its specific examples show certain embodiments, they are intended for illustrative purposes only. Therefore, this disclosure is not limited to the illustrative embodiments described in this specification and can be modified differently. In addition, it should be understood that, for reasons of clarity, the various embodiments described in the context of the separate embodiments can also be combined to form a single embodiment. On the contrary, for the sake of brevity, the various embodiments described in the context of a single embodiment can also be combined to form a sub-combination thereof.

[0383] Any publications disclosed within this specification are hereby incorporated by reference.

Claims

1. A RXFP1 modulator for use in therapy of a human patient, wherein the RXFP1 modulator is administered in combination with a SGLT2 inhibitor, and wherein the RXFP1 modulator is selected from: or a pharmaceutically acceptable salt thereof.

2. An RXFP1 modulator for use in treating a condition in a human patient selected from the group consisting of heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension, and resistant hypertension, wherein the RXFP1 modulator is administered in combination with an SGLT2 inhibitor, and wherein the RXFP1 modulator is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6, or a pharmaceutically acceptable salt thereof.

3. A RXFP1 modulator for use as claimed in claim 2, wherein the disorder is hypertension.

4. A RXFP1 modulator for use as claimed in claim 2, wherein the disorder is resistant hypertension.

5. The RXFP1 modulator for use according to any of the preceding claims, wherein the RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially or simultaneously.

6. A RXFP1 modulator for use as claimed in any one of the preceding claims, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof.

7. An RXFP1 modulator for use as claimed in any one of the preceding claims, wherein the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, erpagliflozin, ipagliptin, repagliflozin etabonate, sergliflozin etabonate, sogliflozin and togliflozin, or a pharmaceutically acceptable salt thereof.

8. A RXFP1 modulator for use as claimed in any preceding claim, wherein the SGLT2 inhibitor is dapagliflozin.

9. A RXFP1 modulator for use as claimed in any one of the preceding claims, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof, and the SGLT2 inhibitor is dapagliflozin.

10. A method for treating a condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension and resistant hypertension, the human patient being in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of a RXFP1 modulator, wherein the RXFP1 modulator is administered in combination with a therapeutically effective amount of an SGLT2 inhibitor, and 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.

11. A method for treating a condition selected from the following in a human patient: heart failure, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction, heart failure with reduced ejection fraction, heart failure with pulmonary hypertension, chronic kidney disease, acute kidney injury, hypertension and resistant hypertension, the human patient being 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 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.

12. The method of claim 10 or 11, wherein the disorder is hypertension.

13. The method of claim 10 or 11, wherein the condition is resistant hypertension.

14. A method for lowering blood pressure in a human patient in need of such treatment, the method comprising administering to the human patient a therapeutically effective amount of a RXFP1 modulator, wherein the RXFP1 modulator is administered in combination with a therapeutically effective amount of an SGLT2 inhibitor, and 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.

15. A method for lowering blood pressure in a human patient in need of such treatment, the method comprising administering to the human patient a first amount of a RXFP1 modulator and a second amount of a SGLT2 inhibitor, wherein the first amount and the second amount together constitute a therapeutically effective amount, and 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.

16. The method of any one of claims 10 to 15, wherein the RXFP1 modulator and the SGLT2 inhibitor are administered separately, sequentially or simultaneously.

17. The method of any one of claims 10 to 16, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof.

18. The method of any one of claims 10 to 17, wherein the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, erpagliflozin, ipragliflozin, repagliflozin etabonate, sergliflozin etabonate, sogliflozin and togliflozin, or a pharmaceutically acceptable salt thereof.

19. The method of any one of claims 10 to 18, wherein the SGLT2 inhibitor is dapagliflozin.

20. The method of any one of claims 10 to 19, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof, and the SGLT2 inhibitor is dapagliflozin.

21. A pharmaceutical composition comprising a RXFP1 modulator, a 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.

22. The pharmaceutical composition of claim 21, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof.

23. The pharmaceutical composition of claim 21 or 22, wherein the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, erpagliflozin, ipragliflozin, repagliflozin ectoate, sergliflozin ectoate, sogliflozin and togliflozin, or a pharmaceutically acceptable salt thereof.

24. The pharmaceutical composition of any one of claims 21 to 23, wherein the SGLT2 inhibitor is dapagliflozin.

25. The pharmaceutical composition of any one of claims 21 to 24, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof, and the SGLT2 inhibitor is dapagliflozin.

26. A pharmaceutical composition as claimed in any one of claims 21 to 25 for use in a method as claimed in any one of claims 10 to 20.

27. A kit comprising: a first pharmaceutical composition comprising an RXFP1 modulator selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; and The second pharmaceutical composition comprises an SGLT2 inhibitor and a pharmaceutically acceptable excipient.

28. The kit of claim 27, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof.

29. The kit of claim 27 or 28, wherein the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, empagliflozin, erpagliflozin, ipragliflozin, repagliflozin etabonate, sergliflozin etabonate, sogliflozin and togliflozin, or a pharmaceutically acceptable salt thereof.

30. The kit of any one of claims 27 to 29, wherein the SGLT2 inhibitor is dapagliflozin.

31. The kit of any one of claims 27 to 30, wherein the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof, and the SGLT2 inhibitor is dapagliflozin.

32. A kit as claimed in any one of claims 27 to 31 for use in a method as claimed in any one of claims 10 to 20.

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