RXFP1 modulator for treating heart failure associated with resistant hypertension or pulmonary hypertension

Abstract

This specification generally relates to the use of RXFP1 modulators, particularly methods of treating heart failure associated with resistant hypertension and pulmonary hypertension.

Description

【Technical Field】 【0001】 This specification describes a treatment method using a compound (including its salts) that is a modulator of RXFP1. 【Background Art】 【0002】 Relaxin is a multifaceted hormone known to mediate adaptive changes in systemic hemodynamics and the kidney during pregnancy. Relaxin has anti-fibrotic properties and has also been shown to have beneficial effects in heart failure, such as acute decompensated heart failure (ADHF). Heart failure is associated with significant morbidity and mortality. Heart failure is characterized by complex tissue remodeling involving increased cardiomyocyte death and interstitial fibrosis. Relaxin activates many signaling cascades that have been shown to be beneficial in situations such as ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and activation of the nitric oxide signaling pathway (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). 【0003】 In heart failure patients, a significant small population also suffers from pulmonary hypertension (HF+PH patients). Approximately 50% of heart failure patients with preserved ejection fraction further suffer from pulmonary hypertension, which is estimated to increase to 60% in heart failure patients with reduced ejection fraction (Non-Patent Document 5, Non-Patent Document 6). Patients with heart failure accompanied by pulmonary hypertension have been shown to have a reduced survival rate compared to heart failure patients without pulmonary hypertension (Non-Patent Document 7). In heart failure patients, a 3 mmHg increase or decrease in estimated pulmonary artery diastolic pressure (ePAD) (corresponding to an approximately 4 mmHg increase or decrease in mean pulmonary artery pressure (mPAP)) was associated with a 24% increase or 19% decrease in cardiovascular mortality, respectively (Non-Patent Document 8). A 4 mmHg reduction in mPAP is also associated with improvement in dyspnea in patients with heart failure and pulmonary hypertension (Non-Patent Document 9). 【0004】 Resistant hypertension (rHT) is defined as the blood pressure of hypertensive patients that remains elevated above the target achieved despite the concurrent use of three antihypertensive agents at optimized doses from different classes, one of which is a diuretic. The current standard of care (SoC) for the initial treatment of hypertension is calcium channel blockers (CCBs), renin-angiotensin system blockers (angiotensin-converting enzyme [ACE] inhibitors or angiotensin receptor blockers [ARBs]), and diuretics. For patients with rHT, there are multiple options for what to add next (such as mineralocorticoid receptor antagonists [MRAs], β-blockers, or α-blockers), and the guidelines currently recommend MRAs as the preferred option for the treatment of rHT. rHT also includes patients whose blood pressure is appropriately controlled when receiving four or more antihypertensive agents simultaneously (Non-Patent Document 10). rHT patients typically have a long history of severe blood pressure elevation and tend to have a higher cardiovascular risk than treated hypertensive patients with regulated blood pressure (Non-Patent Document 11). It has been suggested that relaxin may have therapeutic potential for hypertensive disorders (Non-Patent Document 12). 【0005】 Clinical trials were conducted using serelaxin, which is unmodified recombinant human relaxin 2. Continuous intravenous administration of serelaxin to inpatients improved cardiac, renal, and hepatic impairment and congestion (Non-Patent Documents 13, 14, 15). However, since serelaxin is rapidly removed from the patient's blood circulation, its therapeutic effect is limited, and the positive effect rapidly disappeared when the intravenous injection was discontinued. Furthermore, since approximately one-third of the patients experienced a significant decrease in blood pressure (>40 mmHg) after intravenous administration of serelaxin, the conclusion was reached that the dose had to be halved or further reduced. 【0006】 The cognate receptor for human relaxin is RXFP1, a well-validated pharmacologically important member of the GPCR family 1c, whose activation by the hormone relaxin is associated with hemodynamic, antifibrotic, and anti-inflammatory properties (Non-Patent Document 16). 【0007】 Small molecule modulators of RXFP1 are sought as relaxin mimetics. For example, Patent Document 1, Non-Patent Documents 17 and 18 by Marugan, J.J., et al. discuss small molecule modulators of RXFP1. 【Prior Art Documents】 【Patent Documents】 【0008】 【Patent Document 1】 Pamphlet of International Publication No. WO2013 / 165606A1 【Non-Patent Documents】 【0009】 【Non-Patent Document 1】 Bathgate RA et al. (2013) Physiol. Rev. 93(1):405 - 480 【Non-Patent Document 2】 Mentz RJ et al. (2013) Am. Heart J. 165(2):193 - 199 【Non-Patent Document 3】 Tietjens J et al. (2016) Heart 102:95 - 99 【Non-Patent Document 4】 Wilson SS et al. (2015) Pharmacology 35:315 - 327 【Non-Patent Document 5】 Guazzi, (2014) Circ Heart Fail., 7:367 - 377 【Non-Patent Document 6】 Miller et al., (2013) JACC Heart Fail., 1(4):290 - 299 【Non-Patent Document 7】 Barnett and De Marco, (2012) Heart Fail. Clin. 8:447 - 459 【Non-Patent Document 8】 Zile MR, et al. (2017) Circ Heart Fail., 10:e003594 【Non-Patent Document 9】 Solomonica A,et al.(2013)Circ Heart Fail.,6:53-60 【Non-Patent Document 10】 Carey et al.,Hypertension,2018,72,e53-e90 【Non-Patent Document 11】 Acelajado et al.,Circulation Research,2019,124,1061-1070 【Non-Patent Document 12】 Lekgabe et al.,Hypertension,2005,46,412-8 【Non-Patent Document 13】 Felker GM et al.(2014)J.Am.Coll.Cardiol.64(15):1591-1598 【Non-Patent Document 14】 Metra M et al.(2013)J.Am.Coll.Cardiol.61(2):196-206 【Non-Patent Document 15】 Teerlink JR et al.(2013)Lancet 381(9860):29-39 【Non-Patent Document 16】 Halls ML et al.,(2015),Pharmacol Rev.67(2):389-440 【Non-Patent Document 17】 Xiao J et al.(2013)Nat.Commun.4:1953 【Non-Patent Document 18】 McBride A et al.(2017)Scientific Reports 7:10806 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0010】 Notwithstanding the above, there remains a continuing need for additional compounds that modulate RXFP1, which can be particularly promising for development as therapeutic agents. Such compounds may also show improved modulation of RXFP1 compared to other known RXFP1 modulators. Such compounds may also show advantageous pharmacokinetic profiles (e.g., lower intrinsic clearance) and / or advantageous physical properties (e.g., higher aqueous solubility) compared to other known RXFP1 modulators. Accordingly, such compounds may be particularly useful for the treatment of medical conditions where modulation of RXFP1 is beneficial. 【Means for Solving the Problems】 【0011】 This specification relates to the use of the RXFP1 modulators described herein in the treatment of subjects suffering from resistant hypertension and subjects suffering from heart failure with pulmonary hypertension (HF+PH). The treatment of HF+PH subjects and subjects with resistant hypertension remains a significant unmet need. 【0012】 This specification describes, in part, a method of treating a subject having a medical condition that is heart failure with resistant hypertension or heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of an RXFP1 modulator described herein. 【0013】 Similarly, this specification describes, in part, an RXFP1 modulator described herein for use in the treatment of a subject having a medical condition that is heart failure with resistant hypertension or heart failure with pulmonary hypertension. 【0014】 Similarly, this specification describes, in part, the use of an RXFP1 modulator described herein in the manufacture of a medicament for treating a subject having a medical condition that is heart failure with resistant hypertension or heart failure with pulmonary hypertension. 【0015】 In one embodiment, the RXFP1 modulator is 【Chemical Formula】 【Chemical Formula】 [Chem.] is selected from or a pharmaceutically acceptable salt thereof. 【0016】 Further aspects of the present disclosure will be apparent to those skilled in the art upon reading this specification. [Mode for Carrying Out the Invention] 【0017】 Numerous embodiments are detailed throughout this specification and will be apparent to those skilled in the art. This specification should not be construed as being limited to any particular embodiment described herein. 【0018】 Terms not specifically defined herein are to be understood as having the meaning that would be ascribed to them by those skilled in the art in light of the present disclosure and the context. 【0019】 "About" generally can mean an acceptable degree of error for the quantity being measured, considering the nature or precision of the measurement method. Exemplary degrees of error are within a percentage (%) of the given value or range of values, typically within 10%, more typically within 5%. 【0020】 Embodiments described herein as including one or more features can also be regarded as disclosing corresponding embodiments "consisting of" such features. 【0021】 Concentrations, amounts, volumes, percentages, and other numerical values can be presented herein in a range format. Such range formats are used merely for convenience and brevity and should be interpreted flexibly as including not only the numerical values explicitly recited as the limits of the range but also all the individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly recited. 【0022】 The chemical names of the compounds described in this specification were generated using ChemDraw® Professional version 19.0.0.22 manufactured by PerkinElmer®. Those skilled in the art will understand that different chemical naming software may generate different chemical names for a particular compound. When the compounds described in this specification are presented in the form of chemical names and as formulas, in case of any discrepancies, the formula shall prevail. 【0023】 RXFP1 modulator Disclosed herein is a method for treating a subject having a condition that is heart failure associated with resistant hypertension or pulmonary hypertension, the method comprising administering to the subject an effective amount of an RXFP1 modulator. 【0024】 In one embodiment, the RXFP1 modulator is 【Chem.】 【Chem.】 【Chem.】 or a pharmaceutically acceptable salt thereof. 【0025】 In one embodiment, the RXFP1 modulator is Compound 1 or a pharmaceutically acceptable salt thereof. 【0026】 In one embodiment, the RXFP1 modulator is Compound 2 or a pharmaceutically acceptable salt thereof. 【0027】 In one embodiment, the RXFP1 modulator is Compound 3 or a pharmaceutically acceptable salt thereof. 【0028】 In one embodiment, the RXFP1 modulator is Compound 4 or a pharmaceutically acceptable salt thereof. 【0029】 In one embodiment, the RXFP1 modulator is Compound 5 or a pharmaceutically acceptable salt thereof. 【0030】 In one embodiment, the RXFP1 modulator is compound 6 or a pharmaceutically acceptable salt thereof. 【0031】 In one embodiment, the RXFP1 modulator is compound 1. 【0032】 In one embodiment, the RXFP1 modulator is compound 2. 【0033】 In one embodiment, the RXFP1 modulator is compound 3. 【0034】 In one embodiment, the RXFP1 modulator is compound 4. 【0035】 In one embodiment, the RXFP1 modulator is compound 5. 【0036】 In one embodiment, the RXFP1 modulator is compound 6. 【0037】 In one embodiment, the RXFP1 modulator is (1S,4S)-4-(2-fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid; (1S,4S)-4-(2-cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid; (1S,4S)-4-(2-cyano-5-(((1S,2R,3S,4R)-3-((cyclopropylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid; (1S,4S)-4-(2-Cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-((neopentylcarbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid; (1S,4S)-4-(2-Cyano-5-(((1S,2R,3S,4R)-3-((3-fluoro[1.1.1]pentan-1-yl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid; and (1S,4S)-4-(5-(((1S,2R,3S,4R)-3-((cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid; or a pharmaceutically acceptable salt thereof selected from. 【0038】 In one embodiment, the RXFP1 modulator is a compound as claimed or exemplified in the specification of International Patent Application No. PCT / EP2021 / 084673 or US Patent Application No. 17 / 457,953, both of which are incorporated by reference in their entirety. 【0039】 The term "pharmaceutically acceptable" is used to specify that an object (e.g., a salt, dosage form, or excipient) is suitable for use in a patient. A list of examples of pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts: Properties, Selection and Use, P.H. Stahl and C.G. Wermuth, editors, Weinheim / Zuerich: Wiley-VCH / VHCA, 2002. Suitable pharmaceutically acceptable salts of the compounds described herein are, for example, acid addition salts or base salts. Acid addition salts of the compounds described herein can be formed by contacting the compound with a suitable inorganic or organic acid under conditions known to those of skill in the art. Acid addition salts can be formed using, for example, 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 para-toluenesulfonic acid. 【0040】 Thus, in one embodiment, the RXFP1 modulator is a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, sulfate, phosphate, trifluoroacetate, citrate, maleate, oxalate, acetate, formate, benzoate, fumarate, succinate, tartrate, lactate, pyruvate, methanesulfonate, benzenesulfonate, or para-toluenesulfonate. 【0041】 The compounds described herein may form base addition salts. The base addition salts of the compounds described herein can be formed by contacting the compound with a suitable inorganic or organic base under conditions known to those skilled in the art. For example, the compound can be treated in an aqueous medium with a hydroxide or alkoxide of an alkali metal or alkaline earth metal (e.g., ethoxide or methoxide) or an organic amine that is suitably basic (e.g., choline or meglumine) to produce an alkali metal (such as sodium, potassium or lithium) or alkaline earth metal (such as calcium) salt. Thus, in one embodiment, the RXFP1 modulator is a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is a sodium, potassium, lithium, calcium, choline or meglumine salt. 【0042】 The compounds and salts described herein may exist in solvated and non-solvated forms. For example, the solvated form may be a hydrate form such as a hemihydrate, monohydrate, dihydrate, trihydrate or alternative amounts thereof. All such solvated and non-solvated forms of the compounds described herein are included herein. 【0043】 The atoms of the compounds and salts described herein may exist as their isotopes. The atom is substituted by one or more of its isotopes (e.g., one or more carbon atoms are 11 C or 13 C carbon isotopes, or one or more hydrogen atoms are 2 H or 3 H isotopes), and all compounds described herein are included herein. 【0044】 The compounds described herein may exist in one or more geometric, optical, enantiomeric and diastereomeric forms including, but not limited to, cis- and trans-forms, E- and Z-forms and R-, S- and meso-forms. Unless otherwise specified, references to a particular compound include all such isomeric forms including racemic mixtures and other mixtures. Where appropriate, such isomers can be separated from their mixtures by the application or adaptation of known methods (e.g., chromatographic techniques and recrystallization techniques). Where appropriate, such isomers can be prepared by the application or adaptation of known methods. 【0045】 The compounds described herein contain one or more chiral centers. Unless the structure or chemical name herein indicates chirality, the structure or name is intended to encompass any single stereoisomer and any mixture of stereoisomers (e.g., a racemate) corresponding to that structure or name. Here, the structures herein include bonds depicted as solid and dashed wedges (i.e., 【Chem.】 ) and it is intended that the solid and dashed wedges indicate the absolute configuration of the chiral center. 【0046】 How such optically active forms can be separated is well known in the art. For example, a single stereoisomer can be obtained by isolating it from a mixture of isomers (e.g., a racemate) using, for example, chiral chromatographic separation. In other embodiments, a single stereoisomer is obtained, for example, by direct synthesis from a chiral starting material. 【0047】 According to one embodiment, the RXFP1 modulator is provided as a single enantiomer having an enantiomeric excess (%ee) of ≧95%, ≧98% or ≧99%. For simplicity, a single enantiomer is present with an enantiomeric excess of greater than 99%. 【0048】 According to one embodiment, the RXFP1 modulator is provided as a single enantiomer having an enantiomeric excess (%ee) in the range of 95 to 100%. 【0049】 The compounds described herein may exist in one or more tautomeric forms including, but not limited to, the keto and enol forms. Reference to a particular compound includes all tautomeric forms including mixtures thereof. Accordingly, the structures depicted herein as one tautomer are intended to also include the other tautomers. 【0050】 The RXFP1 modulators described herein may be administered in the form of prodrugs, which are compounds that are decomposed in the human or animal body to release such RXFP1 modulators. Such pharmaceutically acceptable prodrugs of the RXFP1 modulators also form one embodiment. Various forms of prodrugs are known in the art. See, for example: a) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and Application of Pro-drugs”, by H. Bundgaard p. 113-191 (1991); c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and e) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984). 【0051】 Treatment using RXFP1 modulators As a result of their regulation of RXFP1, the RXFP1 modulators described herein are expected to be useful in treatment. 【0052】 The term "treatment" is intended to have its standard meaning of addressing a disease or condition by alleviating, in whole or in part, one, some, or all of its symptoms, or by correcting or compensating for the underlying pathology. The term "treatment" includes "prevention" unless specifically indicated to the contrary. The terms "therapeutic" and "therapeutically" are to be construed accordingly. 【0053】 The term "prevention" is intended to have its standard meaning and includes primary prevention to prevent the occurrence of a disease or condition and secondary prevention to temporarily or continuously protect a patient against the worsening or exacerbation of a disease or condition that has already occurred or against the development of new symptoms associated with the disease or condition. 【0054】 The term "treatment" is used synonymously with "therapy". Similarly, the term "treating" may be regarded as "applying therapy" (where "therapy" is as defined herein). 【0055】 It will be understood that the RXFP1 modulators described herein can be used in the treatment of conditions such as heart failure associated with resistant hypertension and pulmonary hypertension. 【0056】 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" can also include more detailed diagnoses such as heart failure with preserved ejection fraction (HFpEF), heart failure with mid-range ejection fraction (HFmrEF; also referred to as heart failure with mildly reduced ejection fraction), or heart failure with reduced ejection fraction (HFrEF). This can also include heart failure caused by hypertrophic cardiomyopathy or dilated cardiomyopathy. 【0057】 As used herein, the term "pulmonary hypertension" can typically be defined as a subject having a mean pulmonary artery pressure of about 20 mmHg or more, optionally 25 mmHg or more, at rest. This can also typically be defined as a mean pulmonary artery pressure of about 30 mmHg or more when the subject is exercising or has recently exercised. Thus, a subject can have a mean pulmonary artery pressure in the range of about 20 mmHg to about 30 mmHg, optionally about 25 mmHg to about 30 mmHg or more. Alternatively or additionally, a subject can have a. a right ventricular systolic pressure of about 40 mmHg or more, and / or b. the following pulmonary vascular resistance: i. less than 3.0 Wood units, or ii. 3.0 Wood units or more . 【0058】 Thus, in some cases, pulmonary hypertension can be classified as group 2 pulmonary hypertension according to the definition by the World Health Organization. In other cases, pulmonary hypertension can be classified as group 1 pulmonary arterial hypertension according to the definition by the World Health Organization (see Ryan et al., 2012, Pulm. Circ. 2(1):107-121). 【0059】 The parameters of pulmonary hypertension and heart failure can be measured or estimated using techniques known in the art. For example, these include echocardiography, pulmonary artery catheter, and implantable monitoring devices. In certain embodiments, a subject can be equipped with a blood pressure monitoring device, optionally a pulmonary artery pressure monitoring device, as known in the art. In certain embodiments, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, this device is worn before treatment with the RXFP1 modulator described herein. Alternatively, the subject wears this device during or after the treatment period. 【0060】 As used herein, the term "heart failure with pulmonary hypertension" refers to a small subset of heart failure subjects who simultaneously suffer from pulmonary hypertension (HF+PH subjects). 【0061】 As used herein, the term "resistant hypertension" is defined as the blood pressure of a hypertensive patient that remains elevated above the target achieved despite the simultaneous use of three antihypertensive agents at optimized doses of different classes, one of which is a diuretic, or the blood pressure of a patient whose blood pressure is appropriately controlled when receiving four or more antihypertensive agents simultaneously (Carey et al., Hypertension, 2018, 72, e53 - e90). Initial treatment of hypertension can be calcium channel blockers (CCBs), renin - angiotensin system blockers (angiotensin - converting enzyme [ACE] inhibitors or angiotensin receptor blockers [ARBs]), and diuretics. In the case of patients with rHT, further treatment can include mineralocorticoid receptor antagonists (MRAs), β - blockers, and / or α - blockers. Subjects with resistant hypertension can typically have a systolic blood pressure ≥140 mmHg and / or a diastolic blood pressure ≥90 mmHg when the subject is at rest. Alternatively, subjects with resistant hypertension can typically have a systolic blood pressure ≥130 mmHg and / or a diastolic blood pressure ≥80 mmHg when the subject is at rest. Alternatively, subjects with resistant hypertension can typically have a systolic blood pressure ≥150 mmHg and / or a diastolic blood pressure ≥90 mmHg when the subject is at rest. Optionally, resistant hypertension can be resistant essential hypertension. Essential hypertension, also known as primary hypertension, is a form of hypertension for which no known secondary cause has been identified. 【0062】 "Treatment" refers to the improvement and / or elimination of one or more symptoms or causes of a target disease. In some embodiments, this may involve changing the level of one or more biological markers or functions to be within the normal range (compared to a healthy cohort). For example, the RXFP1 modulators disclosed herein can reduce the mean pulmonary artery pressure (mPAP) in a subject. For example, mPAP can be reduced by at least 1 mmHg to 15 mmHg or more. Thus, the RXFP1 modulators disclosed herein can reduce the mean pulmonary artery pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg or at least 15 mmHg or more. Similarly, the RXFP1 modulators disclosed herein can reduce the estimated pulmonary artery diastolic pressure (ePAD) in a subject. For example, ePAD can be reduced by at least 1 mmHg to 15 mmHg or more. Thus, the RXFP1 modulators disclosed herein can reduce the estimated pulmonary artery diastolic pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg or at least 15 mmHg or more. Further or alternatively, the RXFP1 modulators disclosed herein can increase the percent ejection fraction (EF%) in a subject as an indicator of cardiac output. For example, EF% can increase by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40% or 1% to 50% or more. Thus, the RXFP1 modulators disclosed herein can increase the percent ejection fraction (EF%) in a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more.Additionally or alternatively, the RXFP1 modulators disclosed herein can lower systolic and / or diastolic blood pressure in a subject. 【0063】 In certain embodiments, as described in Solomonica A, et al. (2013) Circ Heart Fail. 6:53 - 60, a decrease in mPAP as described herein can cause an improvement in dyspnea. 【0064】 In one embodiment, a method of treating a subject having a condition that is heart failure with resistant hypertension or pulmonary hypertension is provided, the method comprising administering to the subject an effective amount of an RXFP1 modulator. In one embodiment, a method of treating a subject having heart failure with pulmonary hypertension is provided, the method comprising administering to the subject an effective amount of an RXFP1 modulator. In one embodiment, the heart failure is heart failure with reduced ejection fraction, heart failure with mid - range ejection fraction, or heart failure with preserved ejection fraction. 【0065】 In one embodiment, a method of treating a subject having resistant hypertension is provided, the method comprising administering to the subject an effective amount of an RXFP1 modulator. In one embodiment, the resistant hypertension is resistant essential hypertension. 【0066】 In one embodiment, the subject has a mean pulmonary artery pressure of about 25 mmHg or greater and / or a right ventricular systolic pressure of about 40 mmHg or greater. In one embodiment, the subject has a pulmonary vascular resistance of less than 3.0 Wood units. In one embodiment, the subject has a pulmonary vascular resistance of 3.0 Wood units or greater. In one embodiment, the subject is equipped with a blood pressure monitoring device, optionally a pulmonary artery pressure monitoring device. In one embodiment, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. 【0067】 The term "therapeutically effective amount" refers to the amount of an RXFP1 modulator as described herein that is effective to provide "treatment" in a subject or to "treat" a disease or disorder in a subject. A therapeutically effective amount can cause any observable or measurable change in a subject, as defined in the definitions of "treatment", "treating" and "preventing" above. As will be appreciated by those skilled in the art, the effective amount can vary depending on the route of administration, the use of excipients and combinations with other agents. For example, when combination therapy is used, the amount of the RXFP1 modulator and the amount of other pharmaceutically active agents are effective together to treat the targeted disorder or condition in a subject when combined. In this context, the combined amounts are a "therapeutically effective amount" if they are sufficient to reduce the symptoms of a disease or condition responsive to the modulation and / or agonism of RXFP1 as described above when combined. Typically, such amounts can be determined by those skilled in the art. 【0068】 As used herein, the terms "subject" and "patient" are used interchangeably. "Subject" includes, for example, mammals such as humans. In some embodiments, the subject is a human. 【0069】 In one embodiment, an RXFP1 modulator for use in the treatment of a condition that is heart failure with resistant hypertension or pulmonary hypertension is provided. In one embodiment, an RXFP1 modulator for use in the treatment of heart failure with pulmonary hypertension is provided. In one embodiment, an RXFP1 modulator for use in the treatment of resistant hypertension is provided. In one embodiment, the resistant hypertension is resistant essential hypertension. 【0070】 In one embodiment, the use of an RXFP1 modulator in the manufacture of a medicament for the treatment of a condition that is heart failure with resistant hypertension or pulmonary hypertension is provided. In one embodiment, the use of an RXFP1 modulator in the manufacture of a medicament for the treatment of heart failure with pulmonary hypertension is provided. In one embodiment, the use of an RXFP1 modulator in the manufacture of a medicament for the treatment of resistant hypertension is provided. In one embodiment, the resistant hypertension is resistant essential hypertension. 【0071】 Pharmaceutical composition The RXFP1 modulator described herein can be administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. 【0072】 Accordingly, in one embodiment, provided is a method described herein, wherein the RXFP1 modulator is administered as a pharmaceutical composition comprising the RXFP1 modulator and a pharmaceutically acceptable excipient. 【0073】 In one embodiment, provided is a pharmaceutical composition comprising an RXFP1 modulator and at least one pharmaceutically acceptable excipient for use in the method described herein. 【0074】 The excipients selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition to be provided. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art and are described, for example, in Handbook of Pharmaceutical Excipients, Sixth edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmaceutically acceptable excipients can function, for example, as adjuvants, diluents, carriers, stabilizers, flavoring agents, coloring agents, fillers, binders, disintegrants, lubricants, glidants, thickening agents, and coating agents. As will be appreciated by those skilled in the art, a particular pharmaceutically acceptable excipient can perform two or more functions and alternative functions depending on the amount of excipient present in the composition and which other excipients are present in the composition. 【0075】 The pharmaceutical composition may be in a suitable form 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 a finely divided powder or liquid aerosol), administration by inhalation (e.g., as a finely divided powder) or parenteral administration (e.g., as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular administration) or as a suppository for rectal administration. The composition may be obtained by the usual procedures known in the art. Compositions intended for oral use may contain additional ingredients, such as one or more colorants, sweeteners, flavorings and / or preservatives. EXAMPLES 【0076】 The compounds described herein are further illustrated in the following examples, which are offered by way of illustration only and are not limiting. 【0077】 In this example, high-resolution mass spectra were recorded on a Micromass LCT mass spectrometer equipped with an electrospray interface (LC-HRMS). 【0078】 1 H NMR measurements were performed at 300, 400, 500, and 600 MHz, respectively. 1 The experiments were carried out on Bruker Avance III 300, 400, 500 and 600 spectrometers operating at H frequencies. Experiments were typically 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 may be absent as they are only reported when detected by NMR. The following abbreviations have been used (and their derivatives, e.g. dd, doublet of doublets, etc.): s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; qn, quintet; p, quintet. 【0079】 Flash chromatography was carried out using either normal-phase silica Flash+(registered trademark) (40M, 25M or 12M), Biotage(registered trademark) SNAP Cartridges KP-Sil (340, 100, 50 or 10), or Agela(registered trademark) Flash column silica-CS cartridge (330, 180, 120, 80) unless otherwise stated. 【0080】 Reverse-phase flash chromatography was carried out using Agela(registered trademark) C-18 spherical 20 - 35μm 100A cartridge unless otherwise stated. 【0081】 Generally, all solvents used were commercially available for analysis. Aprotic solvents were those commonly used for reactions. 【0082】 The phase separator used in the examples was an ISOLUTE(registered trademark) Phase Separator column. 【0083】 The intermediates and examples named below were named using ChemDraw Professional version 19.0.0.22 manufactured by PerkinElmer. 【0084】 The following abbreviations were used. Aq Aq B2Pin2 4,4,5,5 - tetramethyl - 2 - (4,4,5,5 - tetramethyl - 1,3,2 - dioxaborolan - 2 - yl) - 1,3,2 - dioxaborolane Calcd Calculated DCM Dichloromethane DIA Diisopropylamine DIAD Diisopropyl (E) - diazene - 1,2 - dicarboxylate DIPEA N - ethyl - N - isopropyl - propan - 2 - amine DMF N,N - dimethylformamide DMSO Dimethyl sulfoxide DPPA Diphenylphosphoryl azide DTBBPY 4,4'-Di-tert-butyl-2,2'-dipyridyl EDC 3-(Ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine; hydrochloride ESI Electrospray ionization Et Ethyl Et2O Diethyl ether EtOAc Ethyl acetate EtOH Ethanol h / hr Hour HATU (Dimethylamino)-N,N-dimethyl(3-oxide-1H-[1,2,3]triazolo[4,5-b]pyridinyl)methaniminium hexafluorophosphate HOBt 1-Hydroxybenzotriazole; hydrate HPLC High performance liquid chromatography HRMS High resolution mass spectrometry IPA Isopropyl alcohol IPAc Isopropyl acetate [Ir(COD)OMe]2 Bis(1,5-cyclooctadiene)di-μ-methoxydiiridium(I) L Liter Me Methyl MeCN Acetonitrile mL Milliliter MeOH Methanol 2-Me-THF 2-Methyltetrahydrofuran Min Minute MS Mass spectrometry MTBE Methyl tert-butyl ether NMR Nuclear magnetic resonance OAc Acetate PE Petroleum ether Pd / C Palladium on carbon Rt Room temperature Sat Saturated SFC Supercritical fluid chromatography T3P 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinan 2,4,6-trioxide TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography 【0085】 Intermediate Intermediate 1: Ethyl 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylate 【Chemical formula】 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 hexane, 1.09 kg, 3.92 mol) was added over 3 hours while maintaining the temperature at -50 to -40 °C. The solution was stirred at -50 to -40 °C for 3 hours, and then ethyl 1,4-dioxaspiro[4.5]decane-8-carboxylate (700 g, 3.27 mol, 2.34 M in THF) was added over 2 hours while maintaining the temperature at -50 to -40 °C. The reaction mixture was stirred at -50 to -40 °C for 4 hours, and then methyl iodide (603 g, 4.25 mol, 3.04 M in THF) was added over 3 hours while maintaining the temperature at -50 to -30 °C. The temperature was maintained at -50 to -30 °C. The reaction mixture was further stirred at -50 to -30 °C for 2 hours, and then an aqueous NH4Cl solution (3.50 L, 20% weight / weight in H2O) was added over 1 hour while maintaining the temperature below 0 °C. The solution was warmed to 15 to 25 °C, held for 0.5 hour, and then the layers were separated. The organic layer was washed with an aqueous NH4Cl solution (2 × 3.50 L, 20% weight / weight in H2O). By exchanging the organic reaction solvent from THF to EtOH under reduced pressure while maintaining the temperature below 45 °C, the title compound was obtained as a 27% weight / weight solution in EtOH (2.51 kg, 2.91 mol, 89%). For the purified compound 1 H NMR (400 MHz CDCl 3) δ 1.18 (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。 【0086】 Intermediate 2: 8-Methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid 【Chem.】 To a solution of Intermediate 1 (1.13 kg, 1.31 mol, 27% w / w in EtOH), EtOH (900 mL) was added followed by aqueous NaOH solution (2.63 L, 5.26 mol, 2 M in H2O) while maintaining the temperature at 15 - 30 °C. The solution was heated to 50 - 60 °C and then held for 6 hours, cooled to 15 - 30 °C, and concentrated under reduced pressure to 1.8 - 2.4 L. Hexane (1.50 L) was added and the layers were separated. The aqueous layer was recovered and the pH was adjusted to 3 - 4 by adding aqueous HCl solution (1.30 L, 5.2 mol, 4 M in H2O) while maintaining the temperature below 20 °C. This aqueous solution was extracted with DCM (2 × 1.50 L) and the combined organic phases were concentrated under reduced pressure while maintaining the temperature below 30 °C to give the title compound as a 17% w / w solution in DCM (1.43 kg, 1.24 mol, 94%). For the purified compound, 1 1H NMR (500 MHz CDCl 3) δ 1.25 (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。 【0087】 Intermediate 3: 1-Methyl-4-oxocyclohexane-1-carboxylic acid Method A 【Chem.】 To a solution of Intermediate 2 (367 g, 250 mmol, 14% w / w in DCM), TFA (95.3 mL, 142 g, 1.25 mol) was added. After maintaining the reaction temperature at 25 - 35 °C for 20 h, it was cooled to 0 - 10 °C. H2O (250 mL) was added to the reaction solution, and the pH of the aqueous phase was adjusted to 9 - 10 by adding aqueous NaOH solution (440 mL, 1.76 mol, 4 M in H2O). The layers were separated, the aqueous layer was retained and cooled to 0 - 10 °C. The pH was adjusted to 2 - 3 by adding aqueous HCl solution (73.5 mL, 294 mmol, 4 M in H2O), then extracted with DCM (3 × 250 mL), and the combined DCM solution was concentrated to 150 - 200 mL under reduced pressure. By exchanging the organic reaction solvent from DCM to MeCN under reduced pressure while maintaining the temperature below 40 °C, the title compound was obtained as a 30% w / w solution in MeCN (119 g, 227 mmol, 91%). For the purified compound 1 1H NMR (400 MHz CDCl3) δ 1.39 (3H, s), 1.73 (2H, td), 2.43 (6H, m). MS (ESI): m / z [M + H] + 157.1。 【0088】 Method B To a solution of Intermediate 2 (6.17 kg, 3.83 mol, 12.4% in DCM), TFA (1.42 L, 2.18 kg, 19.13 mol) was added. After maintaining the reaction temperature at 25 - 35 °C for 20 h, it was cooled to 0 - 10 °C. Aqueous NaOH solution (918 g, dissolved 22.96 mol in 7.66 L of H2O) was added to the reaction solution, and the pH of the aqueous phase was adjusted to 9 - 11. The layers were separated, the aqueous layer was retained and cooled to 0 - 10 °C. When aqueous HCl solution (1.52 L, 6.08 mol, 4 M in H2O) was added following DCM (3.83 L), the pH was adjusted to 3 - 4. The organic layer was retained, the aqueous layer was extracted with DCM (2 × 3.83 L), and the combined organic phase was washed with brine (2.3 L, 15% w / w NaCl). The organic phase was concentrated under reduced pressure from 2.3 L to 3.1 L. By exchanging the organic reaction solvent from DCM to MeCN under reduced pressure while maintaining the temperature below 45 °C, the title compound was obtained as an 18% solution in MeCN (2.85 kg, 3.32 mol, 87%). For the purified compound1 1H NMR (400 MHz, CDCl3) δ 1.39 (3H, s), 1.73 (2H, td), 2.43 (6H, m). MS (ESI): m / z [M+H] + 157.1。 【0089】 Intermediate 4: Naphthalen-1-ylmethyl 1-methyl-4-oxocyclohexane-1-carboxylate Method A 【Chemical formula】 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 at 50 - 60 °C for 8 h and then cooled to 0 - 10 °C. H2O (240 mL) was added and the pH of the reaction mixture was adjusted to 3 - 4 by the addition of aqueous HCl solution (55.0 mL, 220 mmol, 4 M in H2O). The reaction mixture was extracted with MTBE (2 × 150 mL) and the combined organic phases were washed with aqueous NaHCO3 solution (150 mL, 144 mmol, 8% w / w in H2O). The organic reaction solvent was exchanged from MTBE to IPA under reduced pressure while maintaining the temperature below 40 °C. The temperature of the reaction solution was lowered to -10 - 3 °C and the solution was stirred for 2 h, whereupon a solid precipitate formed. The solid was filtered and dried under N2 for 15 h to give the title compound as a white solid (42.8 g, 144 mmol, 74%); 1 1H NMR (500 MHz, CDCl 3) 1.30 (3H, s), 1.65 (2H, td), 2.16 - 2.47 (6H, m), 5.66 (2H, s), 7.46 (1H, dd), 7.51 - 7.63 (3H, m), 7.78 - 7.93 (2H, m), 7.93 - 8.05 (1H, m). MS (ESI): m / z [M+Na] + 319.1。 【0090】 Method B To a solution of Intermediate 3 (2.66 kg, 3.09 mol, 18.2% in MeCN), 1-chloromethylnaphthalene (535 g, 2.94 mol) was added, followed by potassium carbonate (513 g, 3.71 mol) and a further portion of fresh MeCN (714 mL). The suspension was heated at 50 - 60 °C for 17 h and then cooled to 25 - 30 °C. The solid was removed by filtration through a Celite pad and washed with MeCN (2×967 mL). Under reduced pressure, the filtrate was concentrated to 1.45 - 1.93 L. The MeCN was exchanged with isopropanol under reduced pressure while maintaining the temperature below 50 °C. When the temperature of the mixture was lowered to 20 - 25 °C, a solid precipitate formed. The mixture was further cooled to -10 - 0 °C and then the solid was filtered, washed with isopropanol and dried under N2 to give the title compound as a white solid (752.6 g, 2.49 mol, 80.5%); 1 H NMR (500 MHz, CDCl 3) 1.30 (3H, s), 1.65 (2H, td), 2.16 - 2.47 (6H, m), 5.66 (2H, s), 7.46 (1H, dd), 7.51 - 7.63 (3H, m), 7.78 - 7.93 (2H, m), 7.93 - 8.05 (1H, m). MS (ESI): m / z [M+Na] + 319.1。 【0091】 Intermediate 5: Methyl 5-(1,3,6,2-dioxazaborinan-2-yl)-4-fluoro-2-methoxybenzoate Method A 【Chemical formula】 B2Pin2 (362 g, 1.43 mol) was added to 2-Me-THF (1.75 L) degassed with N2 to less than 1% oxygen. The solution was maintained at 20 - 30 °C, and methyl 4-fluoro-2-methoxybenzoate (250 g, 1.36 mol) was added. DTBBPY (1.09 g, 4.10 mmol) was added, the reaction vessel was evacuated, and refilled with N2 until the oxygen level was less than 0.5%. [Ir(COD)OMe]2 (1.35 g, 2.04 mmol) was added, the reaction vessel was evacuated, and refilled with N2 until the oxygen level was less than 0.5%. The reaction mixture was heated to 80 - 85 °C and held at that temperature for an additional 2 hours. The reaction mixture was cooled to 0 - 5 °C, and then diethanolamine (428 g, 4.07 mol, 10.9 M in IPA) was slowly added over 2.5 hours, during which H2 gas was simultaneously evolved. The reaction mixture was stirred at 0 - 5 °C for 2.5 hours, then filtered, and the solid was washed with 2-Me-THF (3 × 750 mL). The solid was dried under N2 for 10 hours to obtain the title compound as a white solid (356 g, 1.20 mol, 88%); 1 H NMR (500 MHz, DMSO-d6) δ 2.81 - 2.89 (2H, m), 3.14 (2H, dq), 3.71 (2H, ddd), 3.74 (3H, s), 3.78 (3H, s), 3.84 (2H, td), 6.77 (1H, d), 7.10 (1H, s), 7.83 (1H, d). MS (ESI): m / z [M + H] + 297.1。 【0092】 Method B B2Pin2 (29.0 g, 114 mmol) and methyl 4-fluoro-2-methoxybenzoate (20.6 g, 109 mmol) were added to 2-Me-THF (140 mL) degassed with N2 to less than 1% oxygen. The solution was maintained at 20 - 30 °C, then DTBBPY (88 mg, 0.33 mmol) and [Ir(COD)OMe]2 (108 mg, 0.16 mmol) were added, the reaction vessel was evacuated and refilled with N2 until the oxygen level was less than 0.5%. The reaction mixture was heated to 80 - 85 °C and held at that temperature for an additional 3 hours. The reaction mixture was cooled to 0 - 10 °C, and then isopropanol (12.4 mL, 218 mmol) was slowly added, whereupon H2 gas was evolved simultaneously. A seed (100 mg of intermediate 5) was added, followed by diethanolamine (22.84 g, 218 mmol) dissolved in IPA (20 mL) to obtain a mobile slurry. The slurry was warmed to 20 - 30 °C and the solid was recovered by filtration. It was then washed with 2-Me-THF (160 ml) and the solid was dried under N2 for 10 hours to obtain the title compound as a white solid (29.1 g, 96 mol, 88%); 1 H NMR (500 MHz, DMSO-d6) δ 2.81 - 2.89 (2H, m), 3.14 (2H, dq), 3.71 (2H, ddd), 3.74 (3H, s), 3.78 (3H, s), 3.84 (2H, td), 6.77 (1H, d), 7.10 (1H, s), 7.83 (1H, d). MS (ESI): m / z [M+H] + 297.1。 【0093】 Intermediate 6: Methyl 4-fluoro-5-hydroxy-2-methoxybenzoate Method A 【Chemical Structure】 THF (1.75 L) was added to a suspension of intermediate 5 (350 g, 1.18 mol) in H2O (1.05 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 0 - 10 °C. NaBO3·4H2O (217 g, 1.41 mol) was added in 10 equal portions over 2 hours while maintaining the reaction temperature at 0 - 30 °C. The reaction temperature was adjusted to 20 - 30 °C and held for 1 hour. An aqueous solution of NaHSO3 (1.96 L, 942 mmol, 0.48 M in H2O) was added over 3 hours, and the reaction mixture was stirred for an additional 0.5 hour. The reaction mixture was filtered, and the solid was washed with ethyl acetate (700 mL). The filtrate and washings were combined to give a two-phase solution. The solution was separated, and the retained organic phase solvent was exchanged from THF / ethyl acetate to MeOH under reduced pressure while maintaining a temperature below 40 °C. H2O (3.50 L) was added dropwise over 4 hours, the reaction mixture was cooled to 0 - 5 °C, and held for 2 hours. The reaction mixture was filtered, the recovered solid was washed with H2O (3 × 350 mL), and dried under warm air below 40 °C to give the title compound as a white solid (195 g, 974 mmol, 83% yield); 1 H NMR (500 MHz, CDCl 3) δ 3.82 (3H, s), 3.86 (3H, s), 6.72 (1H, d), 7.54 (1H, d). MS (ESI): m / z [M + H] + 201.0。 【0094】 Method B Intermediate 5 (32.41 g, 67.3 mmol) was dissolved in 2-Me-THF (100 mL) together with acetic acid (12.13 g, 202 mmol) and cooled to 0 - 10 °C. Hydrogen peroxide solution (30% weight / weight, 9.16 g, 80.8 mmol) was added over 2 hours, then the reaction temperature was adjusted to 20 - 30 °C and maintained for 18 hours. The mixture was quenched with an aqueous solution of Na2S2O3·5H2O (20% weight / weight, 50 mL) to obtain phase separation. The aqueous phase was discarded and the organic phase was washed twice with an aqueous solution of Na2S2O3·5H2O (5% weight / weight, 100 mL). The organic phase was concentrated to 60 mL under reduced pressure and then further vacuum distilled twice with 2-Me-THF (100 mL) to obtain a dissolved solution at 35 - 45 °C. Nucleation was controlled by the addition of a seed (100 mg of Intermediate 6), followed by the slow addition of 300 mL of n-heptane over 5 hours. The resulting slurry was adjusted to 20 - 30 °C, stirred overnight, and then filtered. The recovered solid was washed with n-heptane (2 × 60 mL) and dried to obtain the title compound as a white solid (12.5 g, 62.5 mmol, 93% yield); 1 H NMR (500 MHz, CDCl 3) δ 3.82 (3H, s), 3.86 (3H, s), 6.72 (1H, d), 7.54 (1H, d). MS (ESI): m / z [M + H] + 201.0。 【0095】 Intermediate 7: (1R,2R,3S,4S)-3-(Methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid 【Chemical formula】 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 -10 to -5 °C, and MeOH (227 g, 286 mL, 7.08 mol) was added dropwise over 1.5 h, and then maintained at -10 to -5 °C for 14 h. The reaction mixture was warmed to -5 to 5 °C, held for 2 h, and then filtered. The solid was washed with toluene (3 × 387 mL), and the filtrate and washings were combined and cooled to 0 to 10 °C. Separately, aqueous HCl solution (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 while maintaining the reaction solution below 10 °C. The reaction mixture was warmed to 10 to 20 °C, held for 0.5 h, and then filtered. The solid was washed with toluene (1.94 L), and the filtrate and washings were combined and the biphasic solution was separated. The organic phase was washed with aqueous NaCl solution (3.87 L, 20% w / w in H2O) and stored below 5 °C to obtain the title compound as a 5.9% w / w solution in toluene (6.19 kg, 1.83 mol, 78%); 1 1H NMR (400 MHz DMSO-d6) δ 1.25 - 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. 【0096】 Intermediate 8: Methyl (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylate hydrochloride [Chemical formula] To a solution of Intermediate 7 (6.19 kg, 5.9% w / w, 1.85 mol) in toluene at -5 to 5 °C, while maintaining the reaction solution below 5 °C, TEA (307 mL, 223 g, 2.22 mol) was added, followed by DPPA (538 g, 1.94 mol). The reaction mixture was stirred at -5 to 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 at -5 to 5 °C for 6 h, then while maintaining the reaction solution below 10 °C, H2O (3.6 L) was added. 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 an aqueous NaCl solution (3.6 L, 15% w / w in H2O), and stored at 2 - 8 °C to obtain methyl (1S,2S,3R,4R)-3-(azidocarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylate (Intermediate 9) as a solution in toluene, which was used directly in the next step. Intermediate 9 as a solution in toluene at 2 - 8 °C was added to a reactor charged with toluene (1.80 L) at 70 - 80 °C over 2 h while maintaining the reaction temperature below 80 °C. The resulting solution was stirred for 1 h and then cooled to 20 - 30 °C. By exchanging the organic reaction solvent from toluene to 1,4-dioxane under reduced pressure while maintaining the temperature below 50 °C, methyl (1S,2S,3R,4R)-3-isocyanatobicyclo[2.2.1]hept-5-ene-2-carboxylate (Intermediate 10) was obtained 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 at 10 - 20 °C, HCl (420 mL, 1.68 mol, 4 M in 1,4-dioxane) was added, followed by H2O (360 mL, 1.68 mol, 4.67 M in 1,4-dioxane). The reaction mixture was warmed to 25 - 35 °C and held for 16 h. MTBE (1.65 L) was added dropwise, the reaction mixture was filtered, washed with MTBE / 1,4-dioxane (1:1, 660 mL) and MTBE (660 mL), and then dried under vacuum at 30 - 40 °C to obtain the title compound as a white solid (258 g, 1.27 mol, >99% ee, 75%); 11H NMR (400 MHz, DMSO-d6) δ 1.45 (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 【0097】 Intermediate 11: Naphthalen-1-ylmethyl (1r,4r)-4-hydroxy-1-methylcyclohexane-1-carboxylate 【Chemical formula】 Route A 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 - 30 °C, Intermediate 4 (50.0 g, 169 mmol) was added as a solution in IPA (450 mL). The pH of the reaction solution was adjusted to 7.3 - 7.8 using 6 M HCl, 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) available from Johnson Matthey PLC, UK. The reaction mixture was then held at 33 - 37 °C for 18 h, after which it was concentrated to 300 - 400 mL under reduced pressure while maintaining the temperature below 45 °C. NaCl (150 g), Celite® (20.0 g, 0.4 wt%) and MTBE (500 mL) were added and the reaction was held for 0.5 h. The mixture was filtered and the filter cake was washed with MTBE (250 mL). The combined filtrates were separated and the aqueous phase was extracted with MTBE (500 mL). The combined organic phases were washed with H2O (250 mL) and then the solvent was exchanged to THF under reduced pressure while maintaining the temperature below 45 °C to obtain the title compound as a solution in THF (138 g, 33% w / w, >99:1 trans:cis, <0.1% IPA, 92% yield), which was used directly in the next step. For the purified compound 11H NMR (500 MHz, CDCl 3) δ 1.21 (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. 【0098】 Route B A solution of lithium tri-sec-butylborohydride (1.06 g, 5.6 mmol) in THF (5 mL) was added dropwise over 1 minute under nitrogen to a stirred solution of Intermediate 4 (1.00 g, 3.37 mmol) in THF (10 mL) cooled to -78 °C. The resulting solution was stirred at -78 °C for 2 hours. The reaction mixture was quenched with 0.1 M HCl (10 mL) at -78 °C and then extracted with EtOAc (3 × 50 mL). The organic layers were pooled, dried over Na2SO4, filtered, and evaporated. The residue was purified by preparative TLC (EtOAc / PE, 1:3) to afford the title compound (0.488 g, 48.5%) as a pale yellow gum. The isolated material had a cis / trans ratio of 3:100. 1 1H NMR (400 MHz, CDCl 3) δ 1.21 - 1.25 (s, 3H), 1.37 - 1.49 (m, 1H), 1.49 - 1.61 (m, 2H), 1.61 - 1.74 (m, 4H), 1.83 - 1.95 (m, 2H), 3.74 - 3.83 (dq, 1H), 5.57 - 5.61 (s, 2H), 7.43 - 7.54 (dd, 1H), 7.50 - 7.61 (m, 3H), 7.84 - 7.94 (m, 2H), 7.97 - 8.04 (m, 1H.). MS (ESI): m / z [M+Na] + 321. 【0099】 Intermediate 12: Methyl 4-fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate 【Chemical Structure】 To a solution of Intermediate 11 (736 g, 34% w / w, 839 mmol) in THF, THF (156 mL), PPh3 (248 g, 944 mmol), and Intermediate 6 (140 g, 699 mmol) were added. After heating the solution to 30 °C, DIAD (184 g, 909 mmol) was added dropwise over 1 hour while maintaining the reaction temperature below 40 °C. After holding the solution at 30 - 40 °C for 1 hour, it was cooled to 20 - 30 °C, and then an aqueous HCl solution (700 mL, 20% w / w in H2O) was added. The layers were separated, and the crude solution of the title compound in THF was used directly in the next step. For the purified compound 1 H NMR (500 MHz, CDCl 3) δ 1.17 (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. 【0100】 Intermediate 13: 4-Fluoro-2-methoxy-5-(((1s,4s)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid 【Chemical Structure】 To the crude solution of Intermediate 12 directly used from the previous step at 0 - 5 °C, an aqueous solution of LiOH·2H₂O (88.0 g, 2.10 mol, in 525 mL of H₂O) was added over 1 hour while maintaining the reaction temperature below 10 °C. The solution was warmed to 15 - 30 °C and stirred vigorously for 16 hours. IPAC (1.68 L) was added, the solution was cooled to 0 - 10 °C, and then H₃PO₄ (1.26 L, 2.52 M, 2 M in H₂O) was added dropwise while maintaining the reaction temperature below 10 °C to obtain a solution with a pH of 4.0 - 5.0. The organic layer was separated and washed with an aqueous solution of NaCl (700 mL, 20% weight / weight in H₂O). While maintaining the temperature below 50 °C, THF was removed under reduced pressure, IPAC (4.20 L) was added, and the title compound was obtained in IPAC and used directly in the next step. For the purified compound 1 H NMR (500 MHz, CDCl 3) δ 1.18 (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). 【0101】 Intermediate 14: 4 - Fluoro - 2 - methoxy - 5 - (((1s,4s) - 4 - methyl - 4 - ((naphthalen - 1 - ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate cyclohexanaminium salt 【Chemical formula】 To a crude solution of Intermediate 13 in IPAC directly used from the previous step at 50 - 55 °C, a solution of cyclohexylamine (280 mL, 699 mmol, 2.5 M in IPAC) was added dropwise over 3 hours. The heterogeneous slurry was stirred at 50 - 55 °C for 0.5 hour, and then stirred at 40 - 45 °C for an additional 1 hour. The reaction mixture was filtered, and the solid was washed with IPAC (3 × 0.98 L) pre-warmed to 40 - 45 °C and dried at 45 °C for 16 hours under a N2 stream. The dried and recovered solid was added with MeOH (3.64 L), and the mixture was heated to 55 - 56 °C. H2O (1.58 L) was added dropwise over 1 hour, then the mixture was stirred for 1 hour and then cooled to 0 - 5 °C over 3 hours. The heterogeneous slurry was held for an additional 1 hour and then filtered, washed with 5:3 MeOH:H2O (2 × 750 mL) at 0 °C, and the solid was dried at 45 °C for 16 hours under N2 to obtain the title compound as a white solid (332 g, 85% from methyl 4-fluoro-5-hydroxy-2-methoxybenzoate); 1 H NMR(500MHz,CDCl 3) δ 0.96(1H,ddt),1.03 - 1.36(6H,m), overlapping 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. 【0102】 Intermediate 15: Methyl (1S,2S,3R,4R)-3-(4-fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamide)bicyclo[2.2.1]hepta-5-ene-2-carboxylate 【Chemical Structure】 To a solution of Intermediate 14 (149 g, 264 mmol) in DCM (750 mL) at 15 - 30 °C was added H2O (450 mL), followed by slow addition of HCl (300 mL, 1 M in H2O). The biphasic solution was stirred for 0.5 h, then separated, and the organic phase was washed with HCl (750 mL, 0.2 M in H2O), then H2O (3 × 750 mL). The organic solution was concentrated under reduced pressure while maintaining the temperature below 30 °C and dried to less than 0.1% H2O. The solution was diluted with DCM (450 mL) to a total volume of 750 mL, then Intermediate 8 (59.3 g, 291 mmol) was added to obtain 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 0 - 10 °C, followed by addition of H2O (750 mL), and then stirred for an additional 0.5 h. The biphasic solution was separated, and the organic phase was washed with H2O (2 × 750 mL), then the solvent was exchanged to THF under reduced pressure to obtain the title compound in THF, which was used directly in the next step. For the purified compound 1 H NMR (500 MHz, CDCl 3) δ1.16 (3H, s), 1.25 (2H, td), 1.49 - 1.69 (3H, m), 1.92 - 2.01 (2H, m), 2.04 - 2.1 (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。 【0103】 Intermediate 16: (1S,2S,3R,4R)-3-(4-Fluoro-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamide)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid [Chem.] The crude solution of Intermediate 15 in THF (750 mL) from the previous step was cooled to 0 - 5 °C. An aqueous solution of LiOH·2H₂O (27.7 g, 661 mmol, in 150 mL of H₂O) was added and the solution was held for 36 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 held at 0 - 5 °C for 1 h. The heterogeneous slurry was filtered and the solid was washed at 0 °C with 1:3 MeOH:H₂O (600 mL), and the solid was dried under N₂ at 45 °C for 16 h to obtain the title crude compound as a white solid (158 g, 99%). The crude product (150 g) was slurried in IPAC (1.13 L) at 60 - 65 °C for 0.5 h. The heterogeneous mixture was cooled to 0 - 5 °C over 3 h, then stirred for 1 h and filtered. The recovered solid was treated with IPAC (2 × 300 mL) at 0 - 5 °C and then dried under N₂ at 45 °C for 12 h to obtain the title compound as a white solid (127 g, 82% from Intermediate 14); 1 H NMR (500 MHz, CDCl 3) δ 1.16 (3H, s), 1.2 - 1.35 (2H, m), 1.50 - 1.69 (3H, m), 1.89 - 2.08 (3H, m), 2.27 (2H, ddd), 2.72 (1H, dd), 2.80 (1H, s), 3.06 (1H, s), 3.75 (3H, s), 4.15 (1H, tt), 4.43 - 4.54 (1H, m), 5.59 (2H, s), 6.24 (2H, ddd), 6.53 (1H, d), 7.45 (1H, dd), 7.47 - 7.58 (3H, m), 7.8 - 7.9 (3H, m), 7.94 - 8.05 (1H, m), 8.59 (1H, d). MS (ESI): m / z [M + H] + 602.3. 【0104】 Intermediate 17: Naphthalen-1-ylmethyl (1S,4S)-4-(2-fluoro-4-methoxy-5-(((1R,2R,3S,4S)-3-(((1-methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-5-en-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylate [Chemical formula] To a solution of DIPEA (6.45 g, 49.9 mmol) in DCM (300 mL) at 0 - 5 °C, Intermediate 16 (30.6 g, 49.9 mmol) was added, followed by (1-methylcyclobutyl)methanamine hydrochloride (8.63 g, 62.4 mmol). DIPEA (25.8 g, 200 mmol) was added dropwise while maintaining the temperature at 0 - 5 °C, and then T3P (50.8 g, 79.8 mmol, 50% w / w in EtAOc) was added over 0.5 h. The solution was warmed to 15 - 25 °C, stirred for 1 h, and then H2O (150 mL) was added dropwise while maintaining the temperature below 30 °C. The biphasic solution was separated, the organic phase was washed with H2O (2 × 150 mL), and then the solvent was exchanged to EtOH under reduced pressure to obtain the title compound as a crude solution in EtOH (128 g, 26% w / w, 96% yield), which was used directly in the next step. For the purified compound 1 H NMR (500 MHz, CDCl 3) δ 0.98 (3H, s), 1.16 (3H, s), 1.21 - 1.29 (2H, m), 1.51 - 1.66 (5H, m), 1.66 - 1.76 (3H, m), 1.76 - 1.82 (1H, m), 1.88 - 2.02 (2H, m), 2.26 (3H, dd), 2.40 (1H, dd), 2.80 (1H, s), 3.00 (1H, s), 3.05 (1H, dd), 3.21 (1H, dd), 3.93 (3H, s), 4.06 - 4.2 (1H, m), 4.39 (1H, td), 5.60 (2H, s), 5.64 (1H, t), 6.19 - 6.38 (2H, m), 6.70 (1H, d), 7.46 (1H, dd), 7.49 - 7.62 (3H, m), 7.75 - 7.93 (3H, m), 8.00 (1H, d), 8.66 (1H, d). MS (ESI): m / z [M + H] +683.3。 【0105】 Intermediate 19: (1R,2S,3R,4S)-3-(5-(((1S,4S)-4-carboxy-4-methylcyclohexyl)oxy)-4-cyano-2-methoxybenzamide)bicyclo[2.2.1]heptane-2-carboxylic acid 【Chem.】 Step A. Intermediate 20: Methyl 4-cyano-2-methoxy-5-(((1S,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoate 【Chem.】 A solution of methyl 4-cyano-5-hydroxy-2-methoxybenzoate (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. DIAD (1.97 mL, 10.1 mmol) was added slowly, and then the reaction mixture was stirred at 60 °C for 14 h. Then the solvent was removed under reduced pressure, and the residue was redissolved in EtOAc (150 mL) and washed successively with NaHCO3 (saturated, 200 mL), NH4Cl (saturated, 250 mL) and brine (saturated, 250 mL). The organic layer was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography using a concentration gradient of 0 - 18% EtOAc in PE as the mobile phase to give the title compound as a white solid (3.25 g, 99%). MS (ESI): m / z [M+Na] + 510.3。 【0106】 Step B. Intermediate 21: 4-Cyano-2-methoxy-5-(((1S,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzoic acid 【Chem.】 LiOH (1.6 g, 66.7 mmol) was added portionwise to a stirred solution of intermediate 20 (3.25 g, 6.7 mmol) in H2O:THF 1:3 (80 mL) at 10 °C, 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 (2 M). The reaction mixture was diluted with EtOAc (350 mL) and washed successively with brine (saturated, 350 mL), H2O (350 mL), and brine (350 mL). 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 give the title compound as a white solid (3.16 g, 100%). MS (ESI): m / z [M+Na] + 496.3。 【0107】 Step C. Intermediate 22: Methyl (1S,2S,3R,4R)-3-(4-cyano-2-methoxy-5-(((1s,4S)-4-methyl-4-((naphthalen-1-ylmethoxy)carbonyl)cyclohexyl)oxy)benzamide)bicyclo[2.2.1]hepta-5-ene-2-carboxylate 【Chemical formula】 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 h. 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 concentration gradient of 0 - 20% EtOAc in PE as the mobile phase to give the title compound as a white solid (2.6 g, 62%). MS (ESI): m / z [M+H] + 623.4。 【0108】 Process D. Intermediate 23: (1S,4S)-4-(2-Cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(methoxycarbonyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid 【Chem.】 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 for 14 h under a hydrogen atmosphere (1.5 atm). The mixture was filtered through a Celite® pad 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。 【0109】 Process E. (1R,2S,3R,4S)-3-(5-(((1S,4S)-4-Carboxy-4-methylcyclohexyl)oxy)-4-cyano-2-methoxybenzamide)bicyclo[2.2.1]heptane-2-carboxylic acid A solution of LiOH (50 mL, 52.6 mmol, 1.05 M in H2O) was added to a stirred solution of Intermediate 23 (5.1 g, 10.5 mmol) in THF (100 mL) at 10 °C. The reaction mixture was warmed to room temperature and stirred for 14 h, then acidified to pH 2 using HCl (1 M aqueous solution). The reaction mixture was diluted with EtOAc (350 mL) and washed successively with brine (300 mL, saturated), H2O (300 mL) and brine (300 mL, saturated). The organic phase was separated, dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude product was purified by precipitation from EtOAc / Et2O followed by reverse-phase flash chromatography on a C18 column using a concentration gradient of 0 - 50% MeCN in HCl (0.4% aqueous solution) as the mobile phase to give the title compound as a white solid (4.00 g, 82%); 11H NMR (400 MHz, DMSO-d6) δ 1.13 (s, 3H), 1.20 (s, 1H), 1.23 (s, 2H), 1.33 (t, 2H), 1.46 (q, 4H), 1.84 (d, 1H), 1.92 (d, 2H), 2.03 - 2.14 (m, 4H), 2.38 (d, 1H), 2.67 (d, 1H), 3.89 (s, 3H), 4.23 (t, 1H), 4.43 (dt, 1H), 7.54 (s, 1H), 7.59 (s, 1H), 8.67 (d, 1H), 12.30 (s, 2H). MS (ESI): m / z [M + H] + 471.3。 【0110】 Example 1: (1S,4S)-4-(2-Fluoro-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-methoxycyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid (Form A) 【Chemical Structure】 To a solution of Intermediate 17 in EtOH (206 g, 19% w / w, 57.9 mmol) was added EtOH (385 mL), followed by 10% w / w Pd / C (3.96 g, 5% w / w). The vessel was purged 6 times with N2 and then an additional 6 times with H2. The vessel was pressurized with H2 to 0.4 MPa and the reaction solution was stirred at 20 - 30 °C for 20 h. After completely replacing the H2 atmosphere with N2, the reaction mixture was filtered and the solid was washed with EtOH (3 × 80 mL). A second identical batch was carried out and the recovered EtOH solutions were combined to give a single solution. The solvent was exchanged to EtOAc under reduced pressure while maintaining a temperature below 45 °C. The EtOAc solution (280 mL) was heated at 70 - 75 °C for 0.5 h, then cooled to 40 - 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 20 - 25 °C over 2 h and then held for an additional 2 h. The heterogeneous slurry was filtered and the solid was washed twice with 1:2 EtOAc / n-heptane (160 mL) and then dried at a temperature below 45 °C for 20 h to obtain the crude title compound as a white solid (55.7 g, 87%). Part 1: The crude title compound (2.50 g, 4.59 mmol) was dissolved in EtOH (15.0 mL). During the dropwise addition of water (7.50 mL), the temperature of the solution was maintained at 25.0 ± 2.0 °C, during which a precipitate formed. The heterogeneous slurry was stirred for an additional 1.0 hour and then recovered by filtration. The solid was washed with a (2:3) mixture of EtOH / water (2 × 5.00 mL), recovered, and dried under N2 to give the title compound as a white solid (1.80 g, 72%). This material was characterized as Form A and used as a seed for the following method described in Part 2. 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 the vessel and then passed through the filter to obtain a combined EtOH solution. During the slow addition of H2O (150 mL) over 0.5 hour, the temperature of the solution was maintained at 25.0 ± 2.0 °C. After the solution was stirred for an additional 0.5 hour, the seed material from Part 1 (0.005 g, 0.1% w / w) was added. The solution was held for 6 hours and then cooled to 20.0 ± 0.5 °C over 2 hours and then held for an additional 6 hours. H2O (150 mL) was slowly added over 6 hours and the mixture was held for an additional 2 hours and then filtered. EtOH (45 mL) and H2O (30 mL) were added to the vessel and used to wash the filter cake. The solid was recovered and dried under N2 at less than 45 °C for 12 hours to give the title compound Form A as a white solid (42.2 g, 85%); 1 H NMR (500 MHz, CDCl 3) δ 0.97 (3H, s), 1.12 - 1.42 (5H, m), overlapping 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). HRMS (ESI) m / z [M + H] + Calculated for C30H42FN2O6: 545.3022 Found: 545.3019. 【0111】 Example 2: (1S,4S)-4-(5-(((1S,2R,3S,4R)-3-((Cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid 【Chem.】 Step A Intermediate 18: Naphthalen-1-ylmethyl (1S,4S)-4-(5-(((1R,2R,3S,4S)-3-((Cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]hepta-5-en-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylate To a solution of DIPEA (1.07 g, 8.31 mmol) in DCM (50.5 mL) at 0 - 5 °C was added Intermediate 16 (5.00 g, 8.31 mmol), followed by cyclobutylmethanamine hydrochloride (1.26 g, 10.4 mmol). While maintaining the temperature at 0 - 5 °C, DIPEA (4.21 g, 32.6 mmol) was added dropwise, followed by T3P (8.46 g, 13.3 mmol, 50% w / w in EtOAc) added over 0.5 h. The solution was warmed to 15 - 25 °C, stirred for 2 h, and then water (25.0 mL) was added dropwise while maintaining the temperature below 30 °C. The biphasic solution was separated, and the organic phase was washed with water (2 × 25.0 mL), then the solvent was exchanged to EtOH (75.0 mL) under reduced pressure to obtain the title compound as a solution in EtOH, which was used directly in the next step. 【0112】 Step B (1S,4S)-4-(5-(((1S,2R,3S,4R)-3-((Cyclobutylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-2-fluoro-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid To a solution of Intermediate 18 in EtOH, 10 wt% Pd / C (290 mg, 5% wt / wt) was added. The vessel was purged 6 times with N2 and then an additional 6 times with H2. The vessel was pressurized to 0.4 MPa with H2 and the reaction solution was stirred at 20 - 30 °C for 20 h. After completely replacing the H2 atmosphere with N2, the reaction mixture was filtered and washed with EtOH (2 × 12.2 mL). While maintaining the temperature below 45 °C, the solvent was exchanged to EtAOc under reduced pressure. The EtAOc solution (41.0 mL) was heated to 70 - 75 °C for 0.5 h, then cooled to 40 - 45 °C, and n-heptane (34.8 mL) was added dropwise over 0.5 h. The mixture was stirred for 0.5 h, then cooled to 20 - 25 °C over 2 h and then held for an additional 2 h. The heterogeneous slurry was filtered and the solid was then washed twice with 1:2 EtOAc / n-heptane (11.6 mL) and dried at below 45 °C for 20 h to obtain the title compound as a white solid (3.28 g, 74%). 1 H NMR (500 MHz, DMSO-d6) δ 1.04 - 1.30 (6H, m), overlapping 1.10 (3H, s), 1.35 - 1.58 (5H, m), 1.59 - 1.69 (2H, m), 1.71 - 1.82 (2H, m), 1.83 - 1.92 (2H, m), 1.95 - 2.02 (1H, m), 2.01 - 2.10 (3H, d), 2.18 - 2.31 (2H, m), 2.50 - 2.55 (1H, d), 2.92 - 3.00 (1H, m), 3.06 - 3.14 (1H, m), 3.89 (3H, s), 4.07 - 4.17 (2H, m), 7.11 (1H, d), 7.67 (1H, d), 7.94 (1H, t), 8.83 (1H, d). MS (ESI): m / z [M+H] + 531.3。 【0113】 Example 3. (1S,4s)-4-(2-Cyano-5-(((1S,2R,3S,4R)-3-((Cyclopropylmethyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid 【Chem.】 A solution of Intermediate 19 (500 μL, 0.024 g, 0.05 mmol, 0.1 M in DMF), a solution of cyclopropylmethanamine (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 at room temperature. A solution of HATU (500 μL, 0.15 mmol, 0.3 M in DMF) was added, and the reaction mixture was stirred at 40 °C overnight. The crude mixture was washed with DMSO (3 × 500 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 250×30IDmm) using 20 mM NH3 in MeOH / CO2 as the mobile phase to give the title compound (10.3 mg, 39%). HRMS (ESI) m / z [M+H] + C 29 H 38 Calculated for C21H27N3O6: 524.2756; Found: 524.2752. 【0114】 Example 4: (1S,4S)-4-(2-Cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-Methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid 【Chemical Structure】 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)benzamide)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid 【Chemical Structure】 A 2 M aqueous solution of 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 then the reaction mixture was stirred at room temperature for 12 h. A 10% aqueous solution of citric acid was added to the reaction mixture until pH < 3, and then the reaction mixture was extracted twice with EtOAc. The combined organic layers were concentrated under vacuum to give the title compound (2.5 g, 97%). MS (ESI) m / z 609.3 [M+H] + 。 【0115】 Step B: Intermediate 25: Naphthalen-1-ylmethyl (1S,4s)-4-(5-(((1R,2R,3S,4S)-3-(((1-Methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]hept-5-en-2-yl)carbamoyl)-2-cyano-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylate 【Chem.】 HATU (344 mg, 0.904 mmol) and DIPEA (0.43 mL, 2.46 mmol) were added to a solution of intermediate 24 (500 mg, 0.821 mmol) and (1-Methylcyclobutyl)methylamine hydrochloride (134 mg, 0.986 mmol) in DMF (4 mL), and then 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 then the residue was dried under a vacuum pump to give the title compound (611 mg, 100%). MS (ESI) m / z 690.4 [M+H] + 。 【0116】 Step C: (1S,4s)-4-(2-Cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(((1-Methylcyclobutyl)methyl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid 【Chem.】 Palladium (10% Pd / C, moistened with 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 h under a hydrogen atmosphere at 1 atm. The hydrogen in the reaction vessel was replaced with argon, and the reaction mixture was filtered through Celite®. The filtrate was concentrated in vacuo and the crude product was purified by flash chromatography using a concentration gradient of 0 - 5% MeOH in CHCl3 as the mobile phase to afford the title compound (389 mg, 80%). 1 H NMR (400 MHz, DMSO-d6) δ 0.99 (s, 3H), 1.21 (s, 3H), 1.23 - 1.41 (m, 5H), 1.45 - 1.85 (m, 10H), 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). HRMS (ESI) m / z [M + H] + Calculated for C31H42N3O6: 552.3068; Found: 552.3064. 【0117】 Example 5: (1S,4S)-4-(2-Cyano-4-methoxy-5-(((1S,2R,3S,4R)-3-(neopentylcarbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)phenoxy)-1-methylcyclohexane-1-carboxylic acid 【Chemical Structure】 The title compound was prepared in the same manner as in Example 3 using 2,2-dimethylpropan-1-amine instead of cyclopropylmethanamine. HRMS (ESI) m / z [M + H] + Calculated for C30H42N3O6: 540.3068; Found: 540.3076. 【0118】 Example 6: (1S,4S)-4-(2-Cyano-5-(((1S,2R,3S,4R)-3-((3-Fluorobicyclo[1.1.1]pentan-1-yl)carbamoyl)bicyclo[2.2.1]heptan-2-yl)carbamoyl)-4-methoxyphenoxy)-1-methylcyclohexane-1-carboxylic acid 【Chemical formula】 The title compound was prepared in the same manner as in Example 3, using 3-fluorobicyclo[1.1.1]pentan-1-amine instead of cyclopropylmethanamine. HRMS (ESI) m / z [M+H] + Calculated for C30H37FN3O6: 554.2666; Found: 554.2670. 【0119】 Biological and physicochemical data RXFP1 Hu cAMP (Test A) To screen for modulators of hRXFP1, an assay was used to identify compounds that stimulate cAMP production via the Gs-coupled hRXFP1 receptor. The cAMP HiRange HTRF kit (available from CisBio Bioassays, France; catalog number 62AM6PEJ) was used extensively according to the manufacturer's recommendations for the detection of cAMP. The HTRF method is a competitive immunoassay between native cAMP produced by cells and cAMP labeled with the dye d2. The tracer binding is visualized with a cryptate-labeled antibody against cAMP, and thus the signal is inversely proportional to the amount of cAMP produced. 【0120】 Preparation of assay reagents: Assay buffer: HBSS (ThermoFisher, 14065) with 5 mM Hepes (ThermoFisher, 15630), pH 7.4, containing 0.1% BSA (Sigma, A8806) Cells: Jump-In™ T-REx™ CHO-K1 cells (ThermoFisher) stably transfected with human RXFP1 were used. The cells were induced to express human RXFP1 by treatment with 10 ng / ml doxycycline for 24 hours. Subsequently, the cells were cryopreserved for long-term storage. At the start of each experiment, the cells were thawed, washed with PBS, and resuspended in assay buffer to 1.875 * 10^5 cells / ml. cAMP standard: The stock standard cAMP provided in the CisBio kit was diluted in assay buffer to a maximum final concentration of 2.8 μM in the assay. HTRF detection reagent: cAMP-d2 and anti-cAMP cryptate reconstituted according to the CisBio instructions were diluted 1:40 in lysis buffer with the HTRF kit. 【0121】 Step-by-step procedure for performing the assay: 1. 40 nL of the test compound dissolved in DMSO was acoustically dispensed (Labcyte Echo) into a white 384-well plate (Greiner; 784075), sealed, and stored at room temperature until the assay. 2. 40 nL of 200 nM relaxin-2 in DMSO (final concentration 1 nM) was added to 100% control wells, and 40 nL of DMSO was added to 0% wells using an Echo acoustic dispenser on the assay day. 3. 4 μL of assay buffer containing 1 mM IBMX (final concentration 0.5 mM) to block phosphodiesterase was added using a Multidrop Combi (ThermoFisher). 4. 4 μL of the cell solution at 1.875 * 10^5 cells / ml was added using a Multidrop Combi to obtain 750 cells / well. 5. Incubated at room temperature for 45 minutes. 6. 4 μL of cAMP-d2 in lysis buffer was added using a Multidrop Combi. 7. 4 μL of anti-cAMP cryptate in the lysis buffer was added using a Multidrop Combi. 8. Incubated for 2 hours at room temperature. 9. The homogeneous time-resolved fluorescence (HTRF) signal was detected using an Envision (PerkinElmer) or Pherastar (BMG Labtech) reader (λex = 340 nm, λem = 665 and 615 nm). 【0122】 The HTRF data was converted to the amount of cAMP produced in the sample using a cAMP calibration curve and subsequently used for the calculation of the concentration response. The concentration response data was fitted using a four-parameter logistic fit, Hill equation. The results from the assay were reported in Table 1 as EC 50 (μM) and S inf (%). 【0123】 EC 50 is defined as the concentration at which the stimulatory activity reaches 50% of its maximum level. When the assay was performed multiple times for the same compound, the geometric mean was reported. 【0124】 S inf is the fitted activity level, efficacy at an infinite concentration of the test compound. To facilitate comparison of the efficacy data, the efficacy was normalized to the % effect of the response stimulated by a saturating concentration (1 nM) of relaxin. When the assay was performed multiple times for the same compound, the arithmetic mean was reported. 【0125】 Human plasma protein binding (Test B) The assay was performed according to the human plasma protein binding assay described on pages 167 - 170 of Wernevik, J. et al., “A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling”, Assay and Drug Development Technologies, 2020, 18(4), 157 - 179. The data was reported in Table 1 as the unbound fraction (fu )(as % free) and report it. When the assay is performed multiple times for the same compound, report the arithmetic mean value. 【0126】 Human liver microsome stability (Test C) The assay was performed according to the human liver microsome stability assay described on pages 170 - 174 of Wernevik, J. et al., “A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling”, Assay and Drug Development Technologies, 2020, 18(4), 157 - 179. Report the data in Table 1 as CL int (μl / min / mg protein) and report it. When the assay is performed multiple times for the same compound, report the arithmetic mean value. 【0127】 Human hepatocyte stability (Test D) The metabolic stability of the compound in human hepatocytes was evaluated using the following protocol: 1. Prepare 10 mM stock solutions of the compound and the control compound in a suitable solvent (DMSO). Place the incubation medium (L - 15 medium) in a water bath at 37 °C and warm it for at least 15 minutes before use. 2. Add 80 μL of acetonitrile to each well of a 96 - well deep - well plate (“reaction stop plate”). 3. In a new 96 - well plate, dilute the 10 mM test compound and the control compound to 100 μM by mixing 198 μL of acetonitrile and 2 μL of the 10 mM stock solution. 4. Remove a vial of cryopreserved (-below -150 °C) human hepatocytes (LiverPool™ 10-donor human hepatocytes obtained from Bioreclamation IVT (product number S01205)) from the storage and ensure that the vial remains at cryogenic temperature until the thawing process is carried out. Thaw the cells as rapidly as possible by placing the vial in a 37 °C water bath and gently agitating. The vial should be left in the water bath until all ice crystals have dissolved and are no longer visible. After thawing is complete, spray 70% ethanol on the vial and transfer the vial to a biosafety cabinet. 5. Open the vial and pour the contents into a 50 mL conical tube containing thawing medium. Place the 50 mL conical tube in a centrifuge and spin at 100 g for 10 minutes (room temperature). After spinning is complete, aspirate the thawing medium and resuspend the hepatocytes in sufficient incubation medium to yield approximately 1.5×10 6 cells / mL. 6. Use a Cellometer® Vision to count the cells and determine the viable cell density. Cells with insufficient viability (<80% viability) are not acceptable for use. Dilute the cells in incubation medium to a research cell density of 1.0×10 6 viable cells / mL. 7. Transfer 247.5 μL of the hepatocytes to each well of a 96-well cell incubation plate. Place this plate on an Eppendorf Thermomixer Comfort plate shaker and warm the hepatocytes for 10 minutes. 8. Add 2.5 μL of a 100 μM test compound or control compound to the incubation wells containing the cells to initiate the reaction. 9. Incubate this plate on an Eppendorf Thermomixer Comfort plate shaker at 37 °C and 900 rpm. At 0.5, 5, 15, 30, 45, 60, 80, 100, and 120 minutes, transfer 20 μL of the incubated mixture to a separate "reaction stop plate" and then mix the sample by vortexing for 2 minutes. 10. Centrifuge the reaction stop plate at 4,000 rpm for 20 minutes. Transfer 30 μL of the supernatant of each compound to a 96-well analysis plate. Pool four compounds together in one cassette. Then dilute the pooled sample by adding 180 μl of pure water. All incubations are carried out separately. 【0128】 All calculations were performed using Microsoft Excel. Peak areas were determined from the extracted ion chromatograms. The in vitro intrinsic clearance (in vitro Cl int , μL / min / 10 6 cells per unit) of the parent compound was determined by regression analysis of the disappearance rate (%) Ln of the parent against the time curve. The in vitro intrinsic clearance (in vitro Cl int , μL / min / 10 6 cells per unit) was reported in Table 1 and determined from the gradient value using the following equation: In vitro Cl int =kV / N V = incubation volume (0.25 mL); N = number of hepatocytes per well (0.25 × 10 6 cells). When the assay is performed multiple times for the same compound, the geometric mean value is reported. 【0129】 Rat hepatocyte stability (Test E) The assay was performed according to the rat hepatocyte stability assay described on pages 170 - 174 of Wernevik, J. et al., “A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling”, Assay and Drug Development Technologies, 2020, 18(4), 157 - 179. The data was reported in Table 1 as mean Cl int (μL / min / 10 6 cells). When the assay is performed multiple times for the same compound, the geometric mean value is reported. 【0130】 Solubility (Test F) The assay was performed according to the solubility assay described on pages 164 - 167 of Wernevik, J. et al., “A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling”, Assay and Drug Development Technologies, 2020, 18(4), 157 - 179. The data is reported as solubility (μM) in Table 1. When the assay is performed multiple times for the same compound, the arithmetic mean value is reported. 【0131】 [Table 1] 【0132】 Human RXFP1 cGMP Production Assay (Test G) To profile compounds for RXFP1 agonist activity related to cGMP production, the Green GENIe cGMP assay (Montana Molecular; catalog number D800G) was used. This assay is based on an mNeonGreen fusion protein fluorescent biosensor delivered to mammalian cells with a BacMaM vector. When cGMP binds to the biosensor, fluorescence decreases. 【0133】 Preparation of assay reagents: Assay buffer: DPBS (Gibco; 14040133) containing 0.1% BSA (Sigma; A8806) Cells: HEK293s cells stably transfected with human RXFP1 in pIRESneo3 were used. The cells were cultured in DMEM medium (Gibco; 31966) supplemented with 10% FBS at 0.8 mg / mL to maintain RXFP1 expression. 【0134】 Step - by - step protocol for performing the assay: Day 1 1. The cells were split one day before transduction and seeded at 63,000 cells / cm2 in DMEM medium containing 10% FBS without antibiotics in a tissue culture flask. Day 2 2. After washing with PBS, the cells were detached using Accutase (Gibco; 1737341), resuspended in the medium, and collected in a 50 mL tube. 3. The cells were counted using CEDEX (Innovatis) and diluted to 267,000 cells / mL with the medium. 4. The viral transduction master mix was prepared by mixing the reagents at the following ratios for a single well: 6 μL of GENIe BacMAM vector 0.2 μL of 500 mM sodium butyrate 13.8 μL of DMEM medium containing 10% FBS Total volume 20 μL 5. The cells and the transduction master mix were mixed at a ratio of 30 μL of cells and 20 μL of master mix for a single well. 6. 50 μL of the cell transduction mix from the above was aliquoted per well into a μclear 384-well plate (Greiner; 781946) coated with black poly-D-lysine. 7. The plate was incubated at 37 °C and 5% CO2 in the dark for 24 hours. Day 3 8. The medium was removed from the plate using a Bluewasher (BluCatBio). 9. 20 μL of assay buffer was added using a Multidrop Combi (ThermoFisher). 10. The plate was incubated at room temperature in the dark for 30 minutes before the assay. 11. The plate was assayed using a FLIPR Tetra (Molecular Devices): 10 μL of the compound diluted in assay buffer was added to each well by the FLIPR Tetra, and the green fluorescence was measured over time for up to 3 hours. 【0135】 The data was processed using Screener software (Genedata AG). After subtraction of the background fluorescence (before the compound point), the area under the curve values from 0 to 90 minutes after compound addition were used for the calculation of the reaction. The concentration - response data was fitted with a four - parameter logistic fit, and the EC 50 value (nM) was reported in Table 2. 【0136】 Human RXFP1 phospho - ERK assay (Test H) To profile compounds for RXFP1 agonist activity regarding ERK phosphorylation, an advanced phospho - ERK (Thr202 / Tyr204) cell kit (CisBio; 64AERPEH) was used. The assay uses two antibodies. One was labeled with a donor fluorophore (Eu cryptate) and the second with an acceptor (d2). The first antibody binds specifically to phosphorylated ERK, and the second antibody binds to another motif of ERK and is independent of its phosphorylation state. ERK phosphorylation enables the formation of an immune complex with the two antibodies, thereby generating a FRET signal. Its intensity is proportional to the concentration of phosphorylated ERK in the sample. The assay was performed according to the manufacturer's recommendations. 【0137】 Preparation of assay reagents: Cells: HEK293s cells stably transfected with human RXFP1 in pIRESneo3 were used. The cells were cultured in DMEM medium (Gibco; 31966) supplemented with 10% FBS at 0.8 mg / mL to maintain RXFP1 expression. The assay was performed on cells maintained in continuous culture. Dilution of test compounds: Compounds were diluted to the desired concentration with serum - free DMEM without phenol red (Gibco; 31053 - 038). The DMSO concentration was adjusted to 0.4%. Antibody mixture: The EU - and d2 - labeled anti - ERK1 / 2 antibodies were separately diluted 20 - fold with the detection buffer provided in the kit. Shortly before the experiment, equal volumes of each diluted antibody solution were combined to form the antibody mix. 【0138】 Step-by-step protocol for performing the assay: Day 1 1. Detach the cells from the culture flask using Accutase (Gibco; 1737341), resuspend them in phenol red-free DMEM medium containing 10% FBS, and collect them in a 50 mL tube. 2. Count the cells using CEDEX (Innovatis) and dilute them to 320,000 cells / mL in the above medium. 3. Dispense 100 μL of the cell suspension per well into a μclear 96-well plate (Greiner; 655946) coated with Black μclear poly-D-lysine. 4. Incubate the plate at 37 °C and 5% CO2 for 24 hours. Day 2 5. Serum starvation: Remove the medium and replace it with 50 μL of serum-free DMEM without phenol red. Incubate the plate at 37 °C and 5% CO2 for 5 hours. 6. Add 50 μL of the test compound solution per well. 7. Incubate the plate at room temperature for 5 minutes. 8. Quickly remove the medium and stop the stimulation by adding 50 μL of lysis buffer (diluted to 1× final concentration before addition) per well. 9. Transfer the plate to -80 °C and freeze the lysates. Day 3 10. Thaw the plate and shake it at room temperature for 30 minutes. 11. Homogenize the cell lysates by pipetting. 12. Transfer 16 μL of the homogenate per well to a white low-volume 384-well plate (Greiner; 784075). 13. Add 4 μL of the antibody mix per well. 14. Incubate the plate in the dark at room temperature for 4 hours. 15. The homogeneous time-resolved fluorescence (HTRF) signal was detected using a Pherastar (BMG Labtech) reader (λex = 340 nm, λem = 665 and 615 nm). 【0139】 The HTRF ratio data was processed using Screener software (Genedata AG). The concentration-response data was fitted to a four-parameter logistic fit and reported as EC 50 values (nM) in Table 2. 【0140】 【Table 2】 【0141】 One of ordinary skill in the art will understand that the biological assays described above can be performed using minor variations of alternative devices and protocols without significantly affecting the results. 【0142】 This description and its specific examples are illustrative only and are intended to show particular embodiments. Accordingly, the present disclosure is not limited to the exemplary embodiments described herein and can be variously modified. Additionally, it should be recognized that various embodiments described in relation to separate embodiments for clarity may be combined to form a single embodiment. Conversely, various embodiments described in relation to a single embodiment for brevity may be combined to form these partial combinations. 【0143】 All publications disclosed herein are hereby incorporated by reference into this specification.

Claims

[Claim 1] For use in the treatment of heart failure accompanied by resistant hypertension or pulmonary hypertension, 【Chemistry 1】 【Chemistry 2】 A pharmaceutical composition comprising an RXFP1 modulator selected from or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. [Claim 2] The pharmaceutical composition according to claim 1, wherein the RXFP1 regulator is compound 1 or a pharmaceutically acceptable salt thereof. [Claim 3] The pharmaceutical composition according to claim 1 or 2, wherein the aforementioned pathological condition is heart failure accompanied by pulmonary hypertension. [Claim 4] The pharmaceutical composition according to claim 1 or 2, wherein the heart failure is heart failure with reduced ejection fraction, heart failure with moderate ejection fraction, or heart failure with preserved ejection fraction. [Claim 5] The pharmaceutical composition according to claim 1 or 2, wherein the aforementioned pathological condition is resistant hypertension. [Claim 6] The pharmaceutical composition according to claim 1 or 2, wherein the subject has an average pulmonary artery pressure of about 25 mmHg or more and / or a right ventricular systolic pressure of about 40 mmHg or more. [Claim 7] The pharmaceutical composition according to claim 1 or 2, wherein the subject has a pulmonary vascular resistance of less than 3.0 Wood units. [Claim 8] The pharmaceutical composition according to claim 1 or 2, wherein the target has a pulmonary vascular resistance of 3.0 Wood units or more. [Claim 9] The pharmaceutical composition according to claim 1 or 2, wherein the target is fitted with a blood pressure monitoring device, and optionally a pulmonary artery pressure monitoring device. [Claim 10] The pharmaceutical composition according to claim 9, wherein the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

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