An N-alkoxyacetamide compound, a pharmaceutical composition thereof, and use thereof

Abstract

This invention discloses an N-alkoxyacetamide compound, its pharmaceutical composition, and its applications. Specifically, this invention provides a compound as shown in formula (II), its pharmaceutically acceptable salt, or its stereoisomer, which can effectively treat or prevent chronic kidney disease, congestive heart failure, hypertension, hypertension complications, or primary aldosteronism; for example, hypertension or primary aldosteronism.

Description

[0001] This application claims priority to Chinese patent application 2024106853942, filed on May 30, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to an N-alkoxyacetamide compound, its pharmaceutical composition, and its application. Background Technology

[0003] Aldosterone is a key downstream regulatory molecule in the renin-angiotensin-aldosterone system (RAAS), a core regulatory system for blood flow, blood pressure, and water-electrolyte balance in the human body. Aldosterone is a steroid hormone (a member of the mineralocorticoid family) that promotes renal reabsorption of water and sodium by binding to and activating mineralocorticoid receptors (MR) in distal renal tubular and collecting duct epithelial cells, while simultaneously inducing potassium and hydrogen ion excretion, thus maintaining water and electrolyte balance and participating in maintaining appropriate blood pressure, vascular tone, and tissue perfusion. Furthermore, recent studies have shown that aldosterone can upregulate AT1R expression on vascular smooth muscle cells, altering vascular smooth muscle tone, response to vasoconstrictive signals, and arterial wall structure. It also increases the vascular pressor response to norepinephrine, leading to increased blood pressure, vascular smooth muscle cell proliferation, vascular wall thickening, and hyalinization.

[0004] Under normal circumstances, plasma aldosterone concentration is regulated by related stimulating factors such as the renin-angiotensin-aldosterone system (RAAS), serum potassium levels, and adrenocorticotropic hormone (ACTH). Elevated aldosterone levels can induce blood pressure abnormalities, cause inflammation, vascular remodeling, and tissue fibrosis associated with cardiometabolic diseases, ultimately leading to organ dysfunction, cardiovascular complications, advanced kidney disease, and an increased risk of death. Therefore, combating the harmful effects of excessive aldosterone in patients has been a targeted clinical strategy for many years.

[0005] For cardiovascular and renal diseases associated with aldosterone and its receptors, blocking the effects of aldosterone is an effective treatment. Aldosterone receptor antagonists (MRAs) and renin-angiotensin-aldosterone system antagonists (RAS inhibitors) are currently the clinical therapies used to antagonize aldosterone. MRAs (such as spironolactone) inhibit aldosterone action by competitively binding to mineralocorticoid receptors, while RAS inhibitors (such as angiotensin II receptor blockers) indirectly reduce aldosterone levels by blocking the upstream stimulation of angiotensin II. Clinically, on the one hand, MRAs can over-antagonize receptor effects (aldosterone receptors can also be stimulated by estrogen) and have off-target side effects from androgen receptor antagonism; on the other hand, RAS inhibitors do not completely inhibit excessive aldosterone, leading to drug resistance issues. Therefore, specific inhibitors (ASIs) that directly inhibit aldosterone synthase (AS) can completely reduce aldosterone production without producing additional effects and can serve as a superior alternative to MRAs and RAS inhibitors.

[0006] Aldosterone synthase (encoded by the CYP11B2 gene) controls aldosterone synthesis, catalyzing the final step in the conversion of cholesterol into aldosterone, and has been a pharmacological target for treating hypertension for decades. Potassium ions, angiotensin II, and leptin can all activate the production of CYP11B2, leading to aldosterone synthesis. Importantly, CYP11B2 is the only enzyme that catalyzes the final oxidation to aldosterone, and it is primarily expressed in the glomerular zona glomeruli of the adrenal glands; it is largely absent in other parts of the body, thus off-target effects are not expected.

[0007] Because the enzymes that produce aldosterone and cortisol are 93% identical (CYP11B1, or cortisol synthase, the final enzyme in the cortisol synthesis pathway), this high degree of similarity led to cross-reactivity and inhibition of cortisol synthesis by early aldosterone synthase inhibitors. Therefore, developing a drug that can inhibit aldosterone production without affecting cortisol is a current challenge and a major challenge.

[0008] LCI699 was the first orally active aldosterone synthase inhibitor to enter clinical trials for the treatment of essential aldosteronism. Oral administration of LCI699 resulted in decreased plasma aldosterone levels and lower blood pressure. However, LCI699 exhibited poor selectivity for CYP11B2 and CYP11B1, with a greater inhibitory effect on cortisol synthase, leading to side effects. Therefore, its poor tolerability prevented its development for hypertension. Subsequently, a new generation of highly selective ASI inhibitors were developed in clinical trials, with only a few products currently in clinical use.

[0009] Patent US20230365513 A1 discloses the specific structure of Lorundrostat. Lorundrostat (Mineralys) is a highly selective aldosterone synthase inhibitor that inhibits aldosterone synthase CYP11B2, reducing aldosterone levels in the body without inhibiting CYP11B1 (www.mineralystx.com). Another compound is Baxdrostat (CinCor Pharma / AstraZeneca). Preclinical studies of Baxdrostat showed that its inhibitory effect on aldosterone synthase is 100 times that on cortisol synthesis, classifying it as a highly selective aldosterone synthesis inhibitor that can dose-dependently reduce plasma aldosterone levels by >70% (New Eng.J.Med.2023, Vol 388, p395).

[0010] Although two clinical trial products exist, it remains unclear whether they will ultimately prove safe and effective in large-scale Phase III clinical trials. Therefore, highly selective aldosterone synthase inhibitors with better selectivity, safety, and efficacy remain in demand by patients. Summary of the Invention

[0011] The technical problem to be solved by this invention is to provide a highly selective aldosterone synthase inhibitor with a novel structure. This invention aims to provide a novel N-alkoxyacetamide compound, its pharmaceutical composition, and its applications. This class of compounds has a strong inhibitory effect on aldosterone synthase but almost no effect on cortisol synthase, exhibiting high selectivity and high safety, and shows great promise for the prevention and / or treatment of various aldosterone-related diseases.

[0012] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0013] This invention provides a compound of formula (II), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0014]

[0015] in,

[0016] R 1 It can be H, halogen, -CN, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy;

[0017] R 2 It is H, C1-C6 alkyl, and surrounded by 1, 2 or 3 Rs. 2-3 Substituted C1-C6 alkyl, C3-C7 cycloalkyl, with 1, 2 or 3 R2-1 Substituted C3-C7 cycloalkyl, "a 4-8 membered heterocycloalkyl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms", or surrounded by one, two, or three R... 2-2 The substituted "heteroatom is selected from one, two or three of N, O and S, and is a 4-8 membered heterocyclic alkyl group with one, two or three heteroatoms" and C6-C 10 aryl, with 1, 2 or 3 R 2-3 Replacement C6-C 10 aryl, "a 5-10 membered heteroaryl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms", or surrounded by one, two, or three R... 2-3 The substituted heteroatoms are selected from one, two, or three of N, O, and S, and are 5-10 membered heteroaryl groups with one, two, or three heteroatoms.

[0018] R 2-1 Independently for R 2-3 or -NR a -C(=O)-R 2-1-1 ;

[0019] R 2-1-1 It is a C1-C6 alkyl or C1-C6 alkoxy group;

[0020] R 2-2 Independently for R 2-3 or -C(=O)-C1-C6 alkyl;

[0021] R 2-3 Independent of halogen, -CN, -NO2, -OR a -、-NR b R c -C(=O)-OR a -C(=O)-NR b R c - C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C7 cycloalkyl, "4-8 membered heterocyclic alkyl with one, two or three heteroatoms selected from N, O and S, and one, two or three heteroatoms", C6-C 10 Aryl or "a 5-10 membered heteroaryl group selected from one, two or three of N, O and S, with one, two or three heteroatoms";

[0022] R a R b and R c It is independently H, C1-C6 alkyl, or C1-C6 haloalkyl.

[0023] R4 It can be hydrogen, deuterium, C1-C6 alkyl, C1-C6 deuterated alkyl, C1-C6 haloalkyl, C3-C7 cycloalkyl, "a 4-8 membered heterocyclic alkyl with one, two or three heteroatoms selected from N, O and S, and one, two or three heteroatoms", C6-C 10 Aryl or "a 5-10 membered heteroaryl group selected from one, two or three of N, O and S, with one, two or three heteroatoms";

[0024] Or; R 4 and R 2 Together with the intercalary atoms they are attached to, they form 4-10 membered heterocyclic alkyl groups; in addition to the attached N and O atoms, the heterocyclic alkyl groups also contain 0, 1, 2 or 3 heteroatoms selected from one, two or three of N, O, and S.

[0025] R 3 Independently, it is deuterium, oxoyl (=O), halogen, C1-C6 alkyl, C1-C6 deuterated alkyl, C1-C6 haloalkyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 deuterated alkoxy, C1-C6 haloalkoxy, C3-C7 cycloalkyl, "a 4-8 membered heterocyclic alkyl with one, two, or three heteroatoms selected from N, O, and S", C6-C 10 Aryl or "a 5-10 membered heteroaryl group selected from one, two or three of N, O and S, with one, two or three heteroatoms";

[0026] n is 0, 1, 2, or 3;

[0027] m is 0 or 2; when m is 0, R 5 It does not exist;

[0028] When m is 2, two R atoms attached to the same C atom 5 Together with the C atoms they are attached to, they form C3-C7 cycloalkyl groups or "4-8 membered heterocycloalkyl groups with one, two, or three heteroatoms selected from N, O, and S, and having one, two, or three heteroatoms"; or, two adjacent R atoms 5 Together with the C atoms they are attached to, they form C3-C7 cycloalkyl groups or "4-8 membered heterocycloalkyl groups with one, two or three heteroatoms selected from N, O and S, and having one, two or three heteroatoms";

[0029] X = CH or N;

[0030] Ring A is C6-C 10 Aryl or "a 5-10 membered heteroaryl group selected from one, two or three of N, O and S, with one, two or three heteroatoms".

[0031] This invention provides a compound of formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0032]

[0033] Among them, R 1 It can be H, halogen, -CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy;

[0034] R 2 It is H, C1-C6 alkyl, and surrounded by 1, 2 or 3 Rs. 2-3 Substituted C1-C6 alkyl, C3-C7 cycloalkyl, with 1, 2 or 3 R 2-1 Substituted C3-C7 cycloalkyl, "a 4-8 membered heterocycloalkyl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms", or surrounded by one, two, or three R... 2-2 The substituted "heteroatom is selected from one, two or three of N, O and S, and is a 4-8 membered heterocyclic alkyl group with one, two or three heteroatoms" and C6-C 10 aryl, with 1, 2 or 3 R 2-3 Replacement C6-C 10 aryl, "a 5-10 membered heteroaryl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms", or surrounded by one, two, or three R... 2-3 The substituted heteroatoms are selected from one, two, or three of N, O, and S, and are 5-10 membered heteroaryl groups with one, two, or three heteroatoms.

[0035] R 2- 1. Independently for R 2-3 or -NR a -C(=O)-R 2-1-1 ;

[0036] R 2-1-1 It is a C1-C6 alkyl or C1-C6 alkoxy group;

[0037] R 2-2 Independently for R 2-3 or -C(=O)-C1-C6 alkyl;

[0038] R 2-3 Independent of halogen, -CN, -NO2, -OR a -、-NR b R c -C(=O)-OR a -C(=O)-NR b R c- C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C7 cycloalkyl, "4-8 membered heterocyclic alkyl with one, two or three heteroatoms selected from N, O and S, and one, two or three heteroatoms", C6-C 10 Aryl or "a 5-10 membered heteroaryl group selected from one, two or three of N, O and S, with one, two or three heteroatoms";

[0039] R a R b and R c It is independently H, C1-C6 alkyl, or C1-C6 haloalkyl.

[0040] In certain preferred embodiments of the present invention, certain groups in the compounds (including those shown as in formulas (I) and (II)), their pharmaceutically acceptable salts, or their stereoisomers are defined as follows, and groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "in one embodiment of the present invention"). The halogen is independently fluorine, chlorine, bromine, or iodine, preferably fluorine.

[0041] In one embodiment of the present invention, the C1-C6 alkyl groups are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, preferably methyl, isopropyl, or tert-butyl.

[0042] In one embodiment of the present invention, the C3-C7 cycloalkyl groups are independently cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclobutyl.

[0043] In one embodiment of the present invention, the 4-8 membered heterocyclic alkyl groups are each independently a 5- or 6-membered heterocyclic alkyl group with N or O as the heteroatom and one heteroatom, preferably tetrahydropyrroleyl, piperidinyl, tetrahydrofuranyl, or tetrahydropyranyl, for example...

[0044] In one embodiment of the present invention, each of the C6-C 10 The aryl group can be phenyl or naphthyl independently; for example, phenyl.

[0045] In one embodiment of the invention, the 4-10 membered heterocyclic alkyl group is independently a heteroatom selected from one or both of N and O; a 6-8 membered bicyclic heterocyclic alkyl group having 3 heteroatoms; for example...

[0046] In one aspect of the present invention, R 1 It is a C1-C6 alkyl or C1-C6 haloalkoxy; for example, a C1-C6 haloalkoxy.

[0047] In one aspect of the present invention, R2 Independently selected from C1-C6 alkyl groups, surrounded by one, two, or three R groups. 2-1 Substituted C3-C7 membered cycloalkyl, "4-8 membered heterocycloalkyl with one, two or three heteroatoms selected from N, O and S, and one, two or three heteroatoms", or substituted with one, two or three R atoms. 2-2 The substituted heteroatom is a 4-8 membered heterocyclic alkyl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms. Examples include C1-C4 alkyl groups, substituted with one, two, or three R atoms. 2-1 Substituted C4-C6 cycloalkyl groups, "5- or 6-membered heterocycloalkyl groups with O as the heteroatom and one heteroatom", or those with one, two, or three R atoms 2-2 The substituted "5- or 6-membered heterocyclic alkyl group with N as the heteroatom and 1 heteroatom"; also, for example, C1-C4 alkyl groups, with 1, 2, or 3 R atoms. 2-1 The substituted C4-C6 cycloalkyl or "a 5- or 6-membered heterocycloalkyl with O as the heteroatom and 1 heteroatom"; further, for example, C1-C4 alkyl or "a 5- or 6-membered heterocycloalkyl with O as the heteroatom and 1 heteroatom".

[0048] In one aspect of the present invention, R 2-1 For -NR a -C(=O)-R 2-1-1 .

[0049] In one aspect of the present invention, R 2-1-1 It is a C1-C6 alkyl group.

[0050] In one aspect of the present invention, R 2-2 Independently for R 2-3 Or -C(=O)-C1-C6 alkyl.

[0051] In one aspect of the present invention, R a For H.

[0052] In one embodiment of this invention, n is 0.

[0053] In one aspect of the present invention, R 4 It is hydrogen.

[0054] In one embodiment of the present invention, X is N.

[0055] In one embodiment of the present invention, ring A is C6-C. 10 Aryl.

[0056] In one aspect of the present invention, R 1 -CH3 or Preferred

[0057] In one aspect of the present invention, R 2 for Preferred More preferably

[0058] In one aspect of the present invention, R 2 for Preferred It is also preferred Further preferred,

[0059] In one embodiment of the present invention, the 4-10 membered heterocyclic alkyl group is...

[0060] In one embodiment of the present invention, ring A is a phenyl group.

[0061] In one aspect of the present invention, the compound represented by formula (II) is the compound represented by formula (II-1).

[0062]

[0063] Among them, R 1 R 2 and R 4 The definition is as described in any one of the present invention.

[0064] In one aspect of the present invention, the compound represented by formula (I) is the compound represented by formula (I-1).

[0065]

[0066] Among them, R 1 and R 2 The definition is as described in any one of the present invention.

[0067] In one embodiment of the present invention, the compound is:

[0068]

[0069]

[0070] The present invention also provides a pharmaceutical composition comprising:

[0071] (1) The compound shown in formula (I) above, its pharmaceutically acceptable salt or its stereoisomer; and

[0072] (2) Pharmaceutically acceptable excipients.

[0073] The present invention also provides the use of the compound shown in formula (I), its pharmaceutically acceptable salt or stereoisomer, and the use of the above pharmaceutical composition, wherein the use is selected from:

[0074] (1) Preparation of aldosterone synthase inhibitors;

[0075] (2) Prepare a drug for treating and / or preventing diseases or conditions, wherein the diseases or conditions are chronic kidney disease, congestive heart failure, hypertension, hypertension complications or primary aldosteronism; preferably hypertension or primary aldosteronism; wherein the hypertension is preferably refractory hypertension;

[0076] (3) Prepare a treatment for and / or prevention of diseases or conditions related to aldosterone synthase; said disease or condition is chronic kidney disease, congestive heart failure, hypertension, hypertension complications or primary hyperaldosteronism; preferably hypertension or primary hyperaldosteronism; said hypertension is preferably refractory hypertension.

[0077] Terminology Explanation

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe this invention.

[0079] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0080] The term "alkyl" refers to a straight-chain or branched alkyl group having a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, etc.

[0081] The term "halogenated alkyl" refers to a halogen-substituted alkyl group, wherein the halogen and alkyl are the same as those defined above.

[0082] The term "deuterated alkyl" refers to a deuterated alkyl group, where the alkyl group is the same as the alkyl group defined above.

[0083] The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group (e.g., C2-C6 alkynyl) having at least one unsaturated position, i.e., a carbon-carbon sp triple bond.

[0084] The term "alkoxy" refers to the group R. Z -O-, where R Z It is an alkyl group as defined above.

[0085] The term "haloalkoxy" refers to a halogen-substituted alkoxy group, where the halogen and alkoxy group are the same as those defined above.

[0086] The term "deuterated alkoxy" refers to a deuterated alkoxy group, where the alkoxy group is the same as the alkoxy group defined above.

[0087] The term "cycloalkyl" refers to a saturated monocyclic cyclic group consisting only of carbon atoms and having a specified number of carbon atoms (e.g., C3-C7). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0088] The term "heterocyclic alkyl" refers to a cyclic, saturated monovalent group having a specified number of ring atoms (e.g., 4-8 or 4-10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S). Heterocyclic alkyl groups are attached to the remainder of the molecule via carbon atoms or heteroatoms. Heterocyclic alkyl groups include, but are not limited to: wait.

[0089] The term "aryl" refers to an aryl group having a specified number of carbon atoms (e.g., C6-C). 10 Aryl groups are cyclic, unsaturated hydrocarbon groups, which can be monocyclic or polycyclic (e.g., two or three). When polycyclic, the monocyclic rings share two atoms and one bond, and each ring is aromatic. Aryl groups include, but are not limited to, phenyl and naphthyl groups.

[0090] The term "heteroaryl" refers to a cyclic group having a specified number of ring atoms (e.g., 5-10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one, two, or three of N, O, and S), which can be monocyclic or polycyclic, and each ring is aromatic (conforming to Hückel's rule). Heteroaryl groups are attached to the rest of the molecule via carbon atoms or heteroatoms; they are attached to the rest of the molecule via rings with or without heteroatoms. Heteroaryl groups include, but are not limited to, furan rings, pyrrole rings, thiophene rings, pyrazole rings, imidazole rings, oxazole rings, thiazole rings, pyridine rings, pyrimidine rings, indole rings, benzopyrrole rings, etc.

[0091] The term "pharmaceutically acceptable salt" includes "pharmaceutically acceptable salt formed with organic or inorganic acids" and "pharmaceutically acceptable salt formed with organic or inorganic bases".

[0092] The term “stereoisomer” includes configurational isomers, which primarily include optical isomers, such as enantiomers, diastereomers or mixtures thereof.

[0093] The term "pharmaceuticalally acceptable excipient" refers to any formulation or carrier medium capable of delivering an effective amount of the active substance of the present invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient. Representative excipients include water, oil, vegetables and minerals, ointment bases, lotion bases, and ointment bases. These bases include suspending agents, thickeners, and transdermal penetration enhancers.

[0094] The term "pharmaceutical composition" refers to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and a pharmaceutically acceptable excipient, intended for administration to mammals, such as humans, in need of such treatment.

[0095] The term “treatment” refers to reversing, alleviating, inhibiting, or preventing the progression of an obstacle or condition to which the term applies, or one or more symptoms of such an obstacle or condition. As used herein, the term “treatment” refers to the action of the verb “to treat,” as previously defined.

[0096] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0097] The reagents and raw materials used in this invention are all commercially available.

[0098] The positive and progressive effects of this invention are as follows:

[0099] (1) This invention provides an aldosterone synthase inhibitor with a novel structure.

[0100] (2) The compound provided by the present invention has: ① superior inhibitory effect on hCYP11B2, with good inhibitory activity on hCYP11B2 and weak inhibitory activity on hCYP11B1, and excellent hCYP11B2 / hCYP11B1 selectivity; ② excellent pharmacokinetic properties and high safety: strong membrane permeability, good plasma protein binding rate and good metabolic stability in liver S9. Detailed Implementation

[0101] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0102] The raw materials or reagents used in this article are commercially available or prepared by synthetic methods commonly known in the art.

[0103] Example 1 (S)-N-((1-acetylpyrrolidone-3-yl)oxy)-2-(4-(5-(p-tolyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0104]

[0105] Step 1:

[0106] Intermediate 1 (5 g, 39.3 mmol) was dissolved in THF (150 mL), and intermediate 2 (1 M THF, 78.6 mL, 78.6 mmol) was added dropwise after cooling to 0 °C. After the addition was complete, the mixture was heated to room temperature and stirred overnight. After cooling to 0 °C, 1 N HCl was added to quench the reaction. The pH was adjusted to neutral by adding saturated sodium bicarbonate. After adding EA, the mixture was washed with water, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and then toluene (150 mL) and DDQ (11 g) were added. The mixture was stirred for 40 min. After adding 2 M NaOH and washing with sodium thiosulfate, the mixture was washed with water, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and then hexane (40 mL) and EA (20 mL) were added. The mixture was slurried and filtered to obtain a yellow solid intermediate 3 (6.07 g, yield: 71%).

[0107] LCMS(ESI) m / z: 218.1 [M+H] + .

[0108] 1 H NMR (400MHz, CDCl3) δ10.29 (s, 1H), 8.33 (d, J = 8.2Hz, 2H), 7.47 (d, J = 8.1Hz, 2H), 3.56 (s, 3H), 2.42 (s, 3H).

[0109] Step Two:

[0110] Intermediate 3 (6.07 g, 27.94 mmol) was added to DCM (120 mL), followed by a turbid DCM solution containing m-CPBA (14.2 g, 83.81 mmol). The mixture was stirred overnight at room temperature, quenched with sodium thiosulfate, and then extracted with saturated sodium bicarbonate / water. The DCM phase was combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and the EA was concentrated. ： n-Hexane = 2 ： 1. Pulping and filtering yielded intermediate 4 (3.22 g, yield: 46%), a yellow solid.

[0111] LCMS(ESI) m / z: 250.1 [M+H] + .

[0112] 1 H NMR (400MHz, DMSO) δ10.32(s,1H),8.35(d,J=8.3Hz,2H),7.49(d,J=8.0Hz,2H),3.59(s,3H),2.45(s,3H).

[0113] Step 3:

[0114] Intermediate 4 (3.22 g, 12.92 mmol) and intermediate 5 (4.64 g, 25.83 mmol) were added to ACN (100 mL), stirred overnight at room temperature, water was added, acetonitrile was distilled off, DCM extraction was performed, the solvent was evaporated, and the mixture was purified by reverse-phase column chromatography. The acetonitrile:water (0.1% NH4HCO3) ratio was 80%, yielding intermediate 6 (1.58 g, yield: 35%) as a yellow liquid.

[0115] LCMS(ESI) m / z: 342.2 [M+H] + .

[0116] 1 H NMR (400MHz, DMSO) δ9.34(s,1H),8.22(d,J=8.2Hz,2H),7.44(d,J=8.0Hz,2H),4.16(q,J=7.1H z,2H),4.02–3.91(m,4H),3.37(s,2H),2.75–2.68(m,4H),2.46(s,3H),1.26(t,J=7.1Hz,3H).

[0117] Step Four:

[0118] Intermediate 6 (1.58 g, 4.63 mmol) was dissolved in ethanol (20 mL) / THF (20 mL), and 2 M NaOH (3.0 mL) was added. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The solvent was removed by vacuum evaporation to obtain 1.62 g of crude intermediate 7, which was then directly used for the next reaction.

[0119] LCMS(ESI) m / z: 314.2 [M+H] + .

[0120] Step 5:

[0121] (S)-3-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 8, 4 g, 21.36 mmol) was dissolved in THF (200 mL), cooled to 0 °C, and intermediate 9 (14.95 g, 64.09 mmol) was added. Potassium tert-butoxide (7.2 g, 64.09 mmol) was added in portions, and the mixture was stirred overnight at 0 °C. Water (100 mL) and saturated sodium chloride (100 mL) were added, and the mixture was stirred at room temperature for 30 min. Extraction was performed using EA (100 mL * 3), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily intermediate 10 (3.0 g, yield: 69%).

[0122] Step Six:

[0123] Intermediate 10 (360 mg, 1.78 mmol), intermediate (597 mg, 1.78 mmol), and DIEA (575 mg, 4.45 mmol) were added to DMF (10 mL), followed by HATU (681 mg, 1.79 mmol). The mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The reaction solution was concentrated and purified by column chromatography (DCM:EtOH = 20:1) to obtain intermediate 11 (320 mg, yield: 36%) as a yellow solid product.

[0124] LCMS(ESI) m / z: 498.2 [M+H] + .

[0125] 1 H NMR (400MHz, DMSO) δ11.11(s,1H),9.28(s,1H),8.16(d,J=8.2Hz,2H),7.39(d,J=8.1Hz,2H),4.51(s,1H),3.92(s,4H),3.45(dd,J= 6.8, 5.3Hz, 1H), 3.29 (dd, J = 11.4, 7.5Hz, 3H), 3.03 (s, 2H), 2.59 (s, 4H), 2.40 (s, 3H), 2.04 (s, 1H), 1.96–1.85 (m, 1H), 1.40 (s, 9H).

[0126] Step Seven:

[0127] Intermediate 11 (320 mg, 0.64 mmol) was added to DCM (10 mL), followed by HCl / EA (10 mL). The mixture was stirred overnight at room temperature, and the solvent was evaporated to obtain intermediate 12 as a yellow crude solid. This crude solid was then directly used for the next reaction.

[0128] LCMS(ESI) m / z: 398.4 [M+H] + .

[0129] Step 8:

[0130] Intermediate 12 (400 mg, 1.01 mmol), acetic acid (78 mg, 1.31 mmol), and DIEA (390 mg, 3.02 mmol) were added to DMF (10 mL), followed by HATU (497 mg, 1.31 mmol). The mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The reaction solution was diluted with 30 mL of ethyl acetate, washed successively with water (10 mL * 2) and saturated sodium chloride solution (10 mL * 2), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The mixture was purified by reversed-phase C18 preparative chromatography (acetonitrile:water (0.1% NH4HCO3) = 72%) to obtain 53 mg of yellow solid (53 mg, yield: 12.1%) as described in Example 1.

[0131] LCMS(ESI) m / z: 440.2 [M+H] + .

[0132] 1 H NMR (400MHz, DMSO) δ11.12(s,1H),9.28(s,1H),8.16(d,J=8.2Hz,2H),7.39(d,J=8.0Hz,2H),4.56(d,J=18.4Hz,1H),3. 93(s,4H),3.53(ddt,J=26.5,19.9,10.8Hz,4H),3.05–2.99(m,2H),2.64–2.54(m,4H),2.40(s,3H),2.19–1.89(m,5H).

[0133] Following a similar experimental method, intermediate 8 in Example 1 was replaced with (1S,3S)-3-hydroxycyclobutylcarbamate and (1R,3R)-3-hydroxycyclobutylcarbamate, respectively, to synthesize Examples 2 and 2a. ：

[0134] Example 2: N-((1S,3S)-3-acetamidocyclobutoxy)-2-(4-(5-(p-tolyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0135]

[0136] LCMS(ESI) m / z: 440.2 [M+H] + .

[0137] 1H NMR (400MHz, DMSO) δ11.00(s,1H),9.28(s,1H),8.15(t,J=9.5Hz,3H),7.38(d,J=8.1Hz,2H),4.12(p,J=7.2Hz,1H),3.92(s,4H),3.75(dt ,J=16.3,7.9Hz,1H),2.99(s,2H),2.57(d,J=4.5Hz,4H),2.49–2.42(m,2H),2.40(s,3H),1.94(ddd,J=16.9,9.1,2.8Hz,2H),1.77(s,3H).

[0138] Example 2a N-((1R,3R)-3-acetamidocyclobutoxy)-2-(4-(5-(p-tolyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0139]

[0140] LCMS(ESI) m / z: 440.2 [M+H] + .

[0141] 1 H NMR (400MHz, DMSO) δ11.04(s,1H),9.28(s,1H),8.16(d,J=8.2Hz,3H),7.39(d,J=8.0Hz,2H),4.50(dq,J=9.8,3.3Hz,1H),4.28(dq ,J=14.3,7.2Hz,1H),3.92(s,4H),3.00(s,2H),2.64–2.54(m,4H),2.40(s,3H),2.36–2.27(m,2H),2.12–2.01(m,2H),1.78(s,3H).

[0142] Example 3: N-(tert-butoxy)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0143]

[0144] Step 1:

[0145] Intermediate 13 (35 g, 156.94 mmol) was dissolved in THF (100 mL), cooled to 0 °C, and isopropyl magnesium chloride-lithium chloride (1.3 M, 145 mL, 188.33 mmol) was added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred overnight. The reaction solution did not require further treatment and was directly used for the next step.

[0146] Step Two:

[0147] Intermediate 1 (10 g, 78.64 mmol) was dissolved in 50 mL of THF and added dropwise to the reaction solution from step one at 0 °C. After the addition was complete, the mixture was brought to room temperature and stirred overnight. The reaction solution was cooled to 0 °C, quenched with 100 mL of 1 N HCl, and the pH was adjusted to neutral with saturated sodium bicarbonate solution. 200 mL of ethyl acetate was added, and the mixture was washed with saturated sodium chloride solution (200 mL * 2), dried over anhydrous sodium sulfate, filtered, concentrated, and toluene (150 mL) and DDQ (11 g) were added to the residue. The mixture was stirred for 40 min. The reaction solution was washed with saturated sodium chloride solution (100 mL * 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was mixed with n-hexane (40 mL) and EA (20 mL) and filtered to obtain intermediate 15 as a yellow solid product (5.8 g, yield: 25%).

[0148] LCMS(ESI) m / z: 270.0 [M+H] + .

[0149] 1 H NMR (400MHz, DMSO) δ9.81 (s, 1H), 8.45–8.34 (m, 2H), 7.62 (s, 0.25H), 7.44 (s, 0.5H), 7.39 (d, J = 8.8Hz, 2H), 7.26 (s, 0.25H), 2.68 (s, 3H).

[0150] Step 3:

[0151] Intermediate 15 (2.9 g, 10.77 mmol) was dissolved in DCM (90 mL), and a turbid solution of m-CPBA (5.5 g, 32.31 mmol) in DCM was added. The mixture was stirred overnight at room temperature, quenched with 50 mL of 10% sodium thiosulfate solution, and then 50 mL of saturated sodium bicarbonate aqueous solution was added. The mixture was separated into layers, and the aqueous phase was extracted with DCM (50 mL * 2). The organic phases were combined, washed with 100 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was mixed with 10 mL of ethyl acetate and 5 mL of ethanol, and a solid precipitated out. The mixture was filtered and dried to obtain intermediate 16 (1.6 g, yield: 49%) as a yellow solid.

[0152] LCMS(ESI) m / z: 302.1 [M+H] + .

[0153] 1H NMR (400MHz, DMSO) δ10.36 (s, 1H), 8.56–8.50 (m, 2H), 7.67 (s, 0.25H), 7.49 (s, 0.5H), 7.46 (d, J = 8.9Hz, 2H), 7.31 (s, 0.25H), 3.60 (s, 3H).

[0154] Step Four:

[0155] Intermediate (1.6 g, 5.31 mmol) and 1-(ethoxycarbonylmethyl)piperazine (intermediate 5, 2.3 g, 13.28 mmol) were added to ACN (50 mL) and stirred overnight at room temperature. The reaction mixture was then diluted with 100 mL of water, extracted with DCM (50 mL x 3), and the organic phases were combined, concentrated, and purified by HPLC (acetonitrile:water (0.1% NH4HCO3) = 85%) to give intermediate 17 (1 g, yield: 48%).

[0156] LCMS(ESI) m / z: 394.2 [M+H] + .

[0157] 1 H NMR (400MHz, DMSO) δ9.32 (s, 1H), 8.37–8.30 (m, 2H), 7.60 (s, 0.25H), 7.42 (s, 0.5H), 7.36 (d, J = 8.8Hz, 2H), 7 .23(s,0.25H),4.11(q,J=7.1Hz,2H),3.91(d,J=4.7Hz,4H),3.32(s,2H),2.66(s,4H),1.21(t,J=7.1Hz,3H).

[0158] Step 5:

[0159] Intermediate 17 (1 g, 2.54 mmol) was dissolved in ethanol (20 mL) / THF (20 mL), 2 M NaOH (2 mL) was added, and the mixture was stirred overnight at room temperature. The solvent was evaporated to obtain crude intermediate 18, which was then directly used for the next reaction.

[0160] LCMS(ESI) m / z: 366.1 [M+H] + .

[0161] Step Six:

[0162] Intermediate 18 (200 mg, 0.52 mol), o-tert-butylhydroxylamine hydrochloride (98 mg, 0.77 mmol), and DIEA (200 mg, 1.55 mmol) were added to DMF (6 mL), followed by the addition of HATU (295 mg, 0.77 mmol). The mixture was stirred overnight at room temperature, diluted with 20 mL of ethyl acetate, washed with saturated sodium chloride solution (10 mL * 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by HPLC (acetonitrile:water (0.1% NH4HCO3) = 50%) to obtain the yellow solid of Example 3 (50 mg, yield 22%).

[0163] LCMS(ESI) m / z: 437.2 [M+H] + .

[0164] 1 H NMR (400MHz, DMSO) δ10.37(s,1H),9.32(s,1H),8.34(d,J=8.9Hz,2H),7.60(s,0.25H),7.42(s,0.5H ),7.36(d,J=8.8Hz,2H),7.24(s,0.25H),3.92(s,4H),3.03(s,2H),2.66–2.56(m,4H),1.17(s,9H).

[0165] Following a similar experimental method, Examples 4-11 were synthesized. ：

[0166] Example 4 2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)-N-isopropoxyacetamide

[0167]

[0168] LCMS(ESI) m / z: 423.1 [M+H] + .

[0169] 1 H NMR(400MHz,DMSO)δ10.82(s,1H),9.31(s,1H),8.36–8.30(m,2H),7.60(s,0.25H),7.42(s,0.5H),7.35(m,2H),7 .23(s,0.25H),4.02(dp,J=12.4,6.2Hz,1H),3.93(s,4H),3.00(s,2H),2.64–2.55(m,4H),1.15(d,J=6.2Hz,6H).

[0170] Example 5: N-((1S,3S)-3-acetamidocyclobutoxy)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0171]

[0172] LCMS(ESI) m / z: 491.7 [M+H] + .

[0173] 1 HNMR (400MHz, DMSO) δ11.02 (s, 1H), 9.32 (s, 1H), 8.40–8.29 (m, 2H), 8.15 (d, J = 7. 5Hz,1H),7.60(s,0.25H),7.42(s,0.5H),7.36(d,J=8.8Hz,2H),7.24(s,0.25H),4 .12(p,J=7.3Hz,1H),3.93(s,4H),3.76(dq,J=16.4,8.2Hz,1H),3.00(s,2H),2.63 –2.55(m,4H),2.50–2.41(m,2H),1.94(ddd,J=14.0,9.1,4.6Hz,2H),1.77(s,3H).

[0174] Example 5a N-((1R,3R)-3-acetamidocyclobutoxy)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0175]

[0176] LCMS(ESI) m / z: 491.7 [M+H] + .

[0177] 1H NMR (400MHz, DMSO) δ11.05(s,1H),9.31(s,1H),8.33(d,J=8.9Hz,2H),8.17(d,J=7 .2Hz,1H),7.60(s,0.25H),7.42(s,0.5H),7.36(d,J=8.8Hz,2H),7.23(s,0.25H), 4.55–4.44(m,1H),4.29(dq,J=14.4,7.3Hz,1H),3.93(s,4H),3.00(s,2H),2.58(s ,4H),2.33(ddd,J=13.3,8.2,3.2Hz,2H),2.10–2.02(m,2H),1.77(d,J=6.2Hz,3H).

[0178] Example 6 (S)-N-((1-acetylpyrrolidone-3-yl)oxy)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0179]

[0180] LCMS(ESI) m / z: 491.7 [M+H] + .

[0181] 1 H NMR (400MHz, DMSO) δ11.13(d,J=6.5Hz,1H),9.32(s,1H),8.34(d,J=8.7Hz,2H),7.60(s,0.25H),7.42(s,0.5H),7.36(d,J=8.6Hz,2H),7 .23(s,0.25H),4.56(d,J=18.2Hz,1H),3.93(s,4H),3.54(ddt,J=26.6,20.0,11.0Hz,4H),3.04(s,2H),2.59(s,4H),2.18–1.87(m,5H).

[0182] Example 6a(R)-N-((1-acetylpyrrolidone-3-yl)oxy)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0183]

[0184] LCMS(ESI) m / z: 492.2 [M+H] + .

[0185] 1H NMR (400MHz, DMSO) δ11.13(d,J=7.0Hz,1H),9.32(s,1H),8.34(d,J=8.8Hz,2H),7.60(s,0.25H),7.42(s,0.5H),7.36(d,J=8.8 Hz,2H),7.24(s,0.25H),4.63–4.49(m,1H),3.93(s,4H),3.70–3.38(m,4H),3.04(s,2H),2.64–2.55(m,4H),2.18–1.83(m,5H).

[0186] Example 7 2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)-N-((tetrahydro-2H-pyran-4-yl)oxy)acetamide

[0187]

[0188] LCMS(ESI) m / z: 464.9 [M+H] + .

[0189] 1 H NMR (400MHz, DMSO) δ10.95(s,1H),9.32(s,1H),8.34(d,J=8.9Hz,2H),7.60(s,0.25H),7.42(s,0.5H),7.36(d,J=8.8Hz,2H),7.23(s,0.25H),3 .99(td,J=8.5,4.2Hz,1H),3.93(s,4H),3.87–3.80(m,2H),3.41–3.35( m,2H),3.02(s,2H),2.59(s,4H),1.90–1.80(m,2H),1.55–1.44(m,2H).

[0190] Example 8 (S)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)-N-((tetrahydrofuran-3-yl)oxy)acetamide

[0191]

[0192] LCMS(ESI) m / z: 450.8 [M+H] + .

[0193] 1H NMR (400MHz, DMSO) δ11.11(s,1H),9.31(s,1H),8.40–8.29(m,2H),7.60(s,0.25H),7.42(s,0.5H),7.36(d,J=8.8Hz,2H),7.23(s ,0.25H),4.62(s,1H),3.93(s,4H),3.82(dt,J=15.3,9.0Hz,2H),3.72–3.59(m,2H),3.02(s,2H),2.59(s,4H),2.07–1.88(m,2H).

[0194] Example 8a(R)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)-N-((tetrahydrofuran-3-yl)oxy)acetamide

[0195]

[0196] LCMS(ESI) m / z: 451.0 [M+H] + .

[0197] 1 H NMR (400MHz, DMSO) δ11.10(s,1H),9.31(s,1H),8.36–8.30(m,2H),7.59(s,0.25H),7.41(s,0.5H),7.36(d,J=8.8Hz,2H),7.23(s,0. 25H),4.62(s,1H),3.93(s,4H),3.82(dt,J=15.3,9.0Hz,2H),3.73–3.60(m,2H),3.01(s,2H),2.65–2.56(m,4H),2.07–1.92(m,2H).

[0198] Example 9: N-((1-acetylpiperidin-4-yl)oxy)-2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)acetamide

[0199]

[0200] LCMS(ESI) m / z: 505.8 [M+H] + .

[0201] 1H NMR(400MHz,DMSO)δ10.98(s,1H),9.32(s,1H),8.41–8.31(m,2H),7.60(s ,0.25H),7.42(s,0.5H),7.36(d,J=8.8Hz,2H),7.23(s,0.25H),4.08–3.9 8(m,1H),3.93(s,4H),3.79–3.70(m,1H),3.70–3.59(m,1H),3.29–3.18(m ,2H),3.02(s,2H),2.65–2.57(m,4H),2.00(s,3H),1.92–1.72(m,2H),1.65

[0202] –1.37(m,2H).

[0203] Example 10a 2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)-N-((3aS,6aS)-oxacyclopentane[3,4-d]isoxazole-2(3H)-yl)acetamide

[0204]

[0205] LCMS(ESI) m / z: 463.2 [M+H] + .

[0206] 1 H NMR (400MHz, DMSO) δ7.80–7.73(m,2H),7.28-7.05(m,4H),7,4.08–3.60(m,5H),3.30-3.20(m,2H),3.15-3.08(m,6H),2.30–2.16(m,5H).

[0207] Example 11 2-(4-(5-(4-(difluoromethoxy)phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl)-N-cyclobutoxyacetamide

[0208]

[0209] LCMS(ESI) m / z: 435.2 [M+H] + .

[0210] 1H NMR (400MHz, DMSO) δ10.25(s,1H),9.30(s,1H),8.35-8.20(m,2H),7.60(s,0.25H),7.42(s,0.5H),7.36(d,J=8 .8Hz,2H),7.24(s,0.25H),4.02-3.95(m,1H),3.92(s,4H),3.03(s,2H),2.66–2.56(m,4H),2.50-2.20(m,6H).

[0211] Example 1: Inhibitory activity of compound 1 against hCYP11B2 / hCYP11B1

[0212] 1. Experimental system: G-402CYP11B2 or CYP11B1 high-expression stable transgenic strains

[0213] The above-mentioned high-expression stable transgenic strains were constructed based on the human adrenal leiomyosarcoma cell line G-402 (ATCC, CRL-1440), and artificial lentiviruses were introduced into human CYP11B2 (NM_000498.3) and CYP11B1 (NM_000497.4), respectively.

[0214] Maintenance medium: McCoy's 5A (modified, #16600082, GIBCO) + 10% FBS (GIBCO) + 1 μg / mL puromycin (A1113803, GIBCO).

[0215] Resuscitation and seeding medium: McCoy's 5A (modified, #16600082, GIBCO) + 10% FBS (GIBCO).

[0216] Reaction medium: DMEM / F12 (#11320033, GIBCO) + 2.5% activated carbon filtered FBS (S11695, R&D).

[0217] 2. Experimental steps:

[0218] Seeding plates: After cell resuscitation, culture the cells in maintenance medium until they reach a suitable state, and seed them in 96-well flat-bottom plates at a rate of 1×10⁴ / 100μL / well (uniform cell quantity).

[0219] Medium change: After the seed plate adheres to the wall overnight (>12 hours), the supernatant is aspirated and washed with 100-150 μL / well of serum-free medium. After aspiration, 50 μL / well of reaction medium is added for later use.

[0220] Preparation: Dilute the compound with reaction medium containing 0.4 μM substrate (final experimental concentration: 0.2 μM).

[0221] CYP11B2 substrate ： 11-Deoxycorticosterone (S4243, selleckchem) was used, with the final concentration of the reaction uniformly set at 0.2 μM.

[0222] CYP11B1 substrate ： 11-Deoxycortisol (S4775, selleckchem) was used, with the final concentration of the reaction uniformly set at 0.2 μM.

[0223] Sample loading: Add the above compound dilution to the cell plate at a rate of 50 μL / well, and set up background wells and control wells at the same time.

[0224] Sample processing and collection: After adding the sample, incubate in a cell culture incubator for 16 hours, then spin each cell plate at 450g for 2 minutes, take 75μL of supernatant and transfer it to a collection plate, freeze at -80℃ for later use (or directly detect).

[0225] Detection: The concentration of aldosterone or cortisol in the supernatant was determined using a homogeneous time-resolved fluorescence kit (Cisbio HTRF kit, Cat.64ALDPEG, Cat.62CRTPEG).

[0226] Analysis: The absolute IC50 values ​​for each compound were calculated using a four-parameter fitting method. 50 (Abs IC 50 ).

[0227] Table 1. Inhibitory activity of some compounds against hCYP11B2

[0228]

[0229]

[0230] Table 2 shows the inhibitory activity of some compounds against hCYP11B1 and their selectivity for hCYP11B1 / 2.

[0231]

[0232] The results in Tables 1 and 2 show that, compared with the reference Lorundrostat, the compounds of the present invention exhibit superior inhibitory activity against hCYP11B2 and weaker inhibitory activity against hCYP11B1. Specifically, Example 3 demonstrates 7 times the inhibitory activity against hCYP11B2 of Lorundrostat, with even better selectivity for hCYP11B2. This superior activity and selectivity are expected to result in high clinical safety and patient compliance, demonstrating significant clinical value and applicability.

[0233] Effect Experiment Example 2: Permeability Test

[0234] 1. Experimental Objective: To evaluate the permeability and efflux rate of the compounds in the examples using an MDR1-MDCKII monolayer cell model, and to determine the permeabilities of the compounds.

[0235] 2. Experimental Procedure: MDR1-MDCKII cells (from Piet Borst of the Netherlands Cancer Institute) were seeded in 96-well plates at a cell density of 3.33 x 10⁵ cells / mL and cultured for 4-7 days to form a copolymerized cell monolayer. Hank's balanced salt buffer (pH = 7.42) containing 10 mM 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was used as the transport buffer.

[0236] The test compounds were diluted to a concentration of 2.00 μM (DMSO < 1.0%) using transfer buffer and then spread onto the top (A) or basal (B) side of a cell monolayer. Detection was repeated in the A-to-B and B-to-A directions. Digoxin was tested at a concentration of 10.0 μM in both the A-to-B and B-to-A directions, while naldolol and metoprolol were tested at a concentration of 2.0 μM in the A-to-B direction. Plates were incubated at 37 °C, 5.0% CO2, and saturated humidity for 2.5 h without shaking. After 2.5 h, end samples were collected from both the donor and acceptor sides of each well and then analyzed by LC-MS / MS.

[0237] After the initial assay, a lucifer yellow rejection assay was performed to determine the integrity of the cell monolayer. Buffer was removed from the top and basal chambers, and then 75 μL of transfer buffer containing 100 μM lucifer yellow and 250 μL of transfer buffer were added to the top and basal chambers, respectively. The plates were incubated at 37°C in a CO2 incubator for 30 minutes at 5.0% CO2 concentration and saturated humidity without shaking. After 30 minutes of incubation, 20 μL of lucifer yellow sample was taken from the top, and then 60 μL of transfer buffer was added. Subsequently, 80 μL of lucifer yellow sample was extracted from the basal chamber. The relative fluorescence units (RFU) of lucifer yellow were measured using an Envision multi-plate reader at 425 / 528 nm wavelength (excitation / emission).

[0238] The apparent permeability coefficient (P) is calculated using the following formula. app (cm / s) and efflux ratio (ER).

[0239]

[0240] V R : Basal chamber volume (50 μL on top, 250 μL on bottom);

[0241] C R : Basal compartment concentration;

[0242] C0: Initial concentration in the top chamber;

[0243] Area ： Surface area of ​​a cell monolayer;

[0244] Time ： time;

[0245] P app (BA) ： From bottom to top;

[0246] P app (AB) ： From top to bottom;

[0247] The results are shown in the table below:

[0248] Table 3. Results of permeability tests on compounds from the examples.

[0249]

[0250] Conclusion: The compounds of this invention are highly permeable and low efflux compounds with good drug-like properties, and have better permeability than Lorundrostat.

[0251] Effect Experiment Example 3 ： Plasma protein binding rate test of the compound

[0252] 1. Experimental Objective: To evaluate the protein binding rate of the compounds in the examples in cynomolgus monkey and human plasma using the equilibrium dialysis method.

[0253] 2. Experimental Procedure:

[0254] 2.1 Dilution of test and control compounds

[0255] Dilute 4 μL of the test compound stock solution with 96 μL of DMSO to prepare a working solution (400 μM) of the test compound.

[0256] To prepare a working solution (400 μM), dilute 4 μL of the DMSO stock solution of the control compound (Lorundrostat) with 96 μL of DMSO.

[0257] Prepare a 2 μM loading matrix solution of the test compound by diluting and mixing 3 μL of the working solution of the test compound with 597 μL of blank matrix (Thermo, #28372).

[0258] Dilute 3 μL of the working solution with 597 μL of blank matrix and mix thoroughly to prepare a 2 μM supported matrix solution of the control compound.

[0259] 2.2 Transfer three 50 μL portions of the loading matrix containing the test compound or control compound to a sample collection plate. Prepare a final volume of 100 μL by mixing the sample with the corresponding blank PBS at a matrix-to-PBS volume ratio of 1:1 (v:v). Immediately add the matrix-to-PBS mixture to each well at a 1:1 (v:v) volume ratio. Add 500 μL of stop solution (acetonitrile containing 250 nM toluene-butyronitrile) to the T0 samples of the test and control compounds. Seal the plate and shake at 800 rpm for 10 minutes. Then store these T0 samples together with other post-dialysis samples at 2–8 °C for further processing.

[0260] 2.3. Assemble the dialysis unit according to the manufacturer's instructions. Transfer 100 μL of the loaded matrix solution containing the test compound or control compound to the sample side of each dialysis well, in triplicate. Then, load 100 μL of PBS onto the sample side of each dialysis well. Rotate the dialysis plate in a humidified incubator at 37°C and 5% CO2 at approximately 100 rpm for 4 hours. After dialysis, collect 50 μL of sample from both the PBS (recipient) and matrix (donor) sides of the dialysis unit and place them into a new 96-well plate (sample collection plate). Add an equal volume of the opposite blank PBS or matrix to each sample to bring the final volume to 100 μL. The matrix volume ratio is 1:1 (v:1), and the matrix to PBS volume ratio in each well is 1:1 (v:v). Add 500 μL of stop solution to these samples.

[0261] 2.4 After protein precipitation, LC / MS / MS analysis was performed, and the free compound concentration and plasma protein binding rate were calculated using the formulas: Free compound concentration (%) = 100 * Fc / T, Plasma protein binding rate (%) = 100% - Free compound concentration (%). Fc is the concentration of the compound at the buffer end of the dialysis plate; T is the concentration of the compound at the plasma end of the dialysis plate. The results are as follows:

[0262] Table 4. Results of plasma protein binding rates of the compounds in the examples.

[0263] Example Plasma protein binding rate in cynomolgus monkeys (%) Human plasma protein binding rate (%) Lorundrostat 84.1 85.6 Example 3 86.6 90 Example 5 78.8 84.4 Example 8a 75 86.2

[0264] Conclusion: The compounds in the examples showed moderate binding to plasma proteins of different species, with a moderate proportion of free drug in the plasma, indicating good drug-like properties.

[0265] Effect Experiment Example 4 ： In vitro study on the metabolic stability of liver S9

[0266] Objective: To evaluate the metabolic stability of the test compound in liver S9.

[0267] Experimental Procedure: Transfer 100 μL of S9 solution into reaction plates (blank, T0, T5, T15, T30, T45, T60, and NCF60). Add 2 μL of the test compound solution (final concentration 1 μM) to all 96-well reaction plates except for the blank (T0, T5, T15, T30, T45, T60, and NCF60). Incubate the reaction plates containing the compound and S9 solution mixture at 37°C for 10 minutes. Add 98 μL of 100 mM potassium dihydrogen phosphate buffer to the NCF60 reaction plate.

[0268] After pre-incubation, 98 μL of cofactor working solution (2.71 mM NADP (nicotinamide adenine dinucleotide phosphate), 6.88 mM G6P (glucose-6-phosphate), 0.83 unit / mL G6PDH (glucose-6-phosphate dehydrogenase), 5.21 mM UDPGA (uridine diphosphate glucuronide), 0.21 mM PAPS (3'-adenosine-5'-phosphate sulfate), 10.42 mM GSH (glutathione), and 6.88 mM MgCl2 in 100 mL potassium phosphate buffer) was added to each reaction plate except NCF60 (blank, T0, T5, T15, T30, T45, and T60), and the reaction plates were incubated at 37 °C.

[0269] At the end time point, add 600 μL of stop solution (pre-cooled acetonitrile containing 250 nM toluenesulfonate) to terminate the reaction. Shake for 10 minutes, then centrifuge at 3220 x g for 20 minutes at 4 °C.

[0270] 100 μL of supernatant was transferred to 300 μL of pure water, mixed with a plate shaker for 10 minutes, and then analyzed by LC-MS / MS. The results are shown in the table below:

[0271] Table 5. Stability results of the compounds in human liver S9.

[0272]

[0273] Conclusion: The compounds in this embodiment exhibited low clearance rates and good stability in the liver S9 cells of humans, rats, and cynomolgus monkeys.

[0274] Example 5: Pharmacokinetic Study of the Test Substance in Cynomolgus Monkeys

[0275] 1. Experimental objective: To study the pharmacokinetic behavior of the compounds of this invention when orally administered in cynomolgus monkeys, using cynomolgus monkeys as test animals.

[0276] 2. Experimental design: Three male adult cynomolgus monkeys of suitable weight and age were selected, with an acclimatization period of one week.

[0277] 3. Compound Preparation: Weigh 15 mg of the test compound and dissolve it directly in DMA (dimethylacetamide) to prepare a DMA stock solution of 10 mg / mL. Take 1.44 mL of the stock solution and add 7.2 mL of 30% Solutol and 57.6 mL of Saline. Vortex and mix until the DMA:30% Solutol:Saline ratio is 10:10:80 (v:v:v). Add an appropriate amount of 0.5 M HCl to adjust the pH to approximately 3-4 until the solution is completely dissolved, yielding a mixed drug solution with a final concentration of 0.2 mg / mL, for oral gavage administration.

[0278] 4. Administration: After fasting overnight, administer orally via gavage at a dose of 1 MPa.

[0279] 5. Sample collection:

[0280] Oral administration procedure: Crab-eating macaques were administered the medication via nasogastric feeding. After administration, the preparation was flushed with 3 mL (approximately three times the volume of the feeding tube). All tubes were of equal size and cut to the same length to control the flushing volume. On the day of administration, each group of cynomolgus macaques received a single oral dose of the mixed medication solution at a dose of 1 mg / kg. 0.5 mL of blood was collected venously at 0.25, 0.5, 1, 2, 4, 8, 10, and 24 hours post-administration and placed in EDTA-K2 anticoagulant tubes.

[0281] Sample collection: Place the collected whole blood in an EDTA-K2 anticoagulant tube, invert it several times to mix thoroughly, store it on wet ice, and centrifuge (1500-1600g) for 10 minutes within 30 minutes to separate the plasma. Store the obtained plasma sample in an environment of -90 to -60°C for biological sample analysis.

[0282] Plasma drug concentrations were analyzed using linear regression. The corresponding pharmacokinetic parameters were calculated using a non-compartmental model in Pharsight Phoenix 8.3. The results are as follows.

[0283] Table 6. Pharmacokinetic results of the compounds in the examples in cynomolgus monkeys.

[0284]

Claims

1. A compound of formula (II), its pharmaceutically acceptable salt, or its stereoisomer, in, R 1 is H, Ci-C6-alkyl, Ci-C6-haloalkyl, Ci-C6-alkoxy or Ci-C6-haloalkoxy; R 2 It is a C1-C6 alkyl group, with one, two or three R atoms. 2-3 Substituted C1-C6 alkyl, C3-C7 cycloalkyl, with 1, 2 or 3 R 2-1 Substituted C3-C7 cycloalkyl, "a 4-8 membered heterocycloalkyl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms", or substituted with one, two, or three R atoms. 2-2 The substituted heteroatom is selected from one, two, or three of N, O, and S, and is a 4-8 membered heterocyclic alkyl group with one, two, or three heteroatoms. R 2-1 Independently for -NR a -C(=O)-R 2-1-1 ; R 2-1-1 Ci-C6-alkyl or Ci-C6-alkoxy; R 2-2 Independently for R 2-3 or -C(=O)-C1-C6 alkyl; R 2-3 independently -C(=0)-OR a ; R a independently H or Ci-C6alkyl; R 4 is hydrogen or CrC6alkyl; Or; R 4 and R 2 Together with the intercalary atoms they are attached to, they form 4-10 membered heterocyclic alkyl groups; in addition to the attached N and O atoms, the heterocyclic alkyl groups also contain 0, 1, 2 or 3 heteroatoms selected from one, two or three of N, O, and S. n is 0, R 3 absent; m is 0, R 5 absent; X=N; Ring A is C6-C10aryl 10 aryl.

2. The compound of formula (II) as claimed in claim 1, its pharmaceutically acceptable salt, or its stereoisomer; characterized in that, The compound shown in formula (II) is the same as the compound shown in formula (I). ， Among them, R 1 It is H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy; R 2 It is a C1-C6 alkyl group, with one, two or three R atoms. 2-3 Substituted C1-C6 alkyl, C3-C7 cycloalkyl, with 1, 2 or 3 R 2-1 Substituted C3-C7 cycloalkyl, "a 4-8 membered heterocycloalkyl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms", or substituted with one, two, or three R atoms. 2-2 The substituted heteroatom is selected from one, two, or three of N, O, and S, and is a 4-8 membered heterocyclic alkyl group with one, two, or three heteroatoms. R 2-1 Independently for -NR a -C(=O)-R 2-1-1 ; R 2-1-1 Ci-C6-alkyl or Ci-C6-alkoxy; R 2-2 independently R 2-3 or -C(=0)-Ci-C6alkyl; R 2-3 independently -C(=0)-OR a ; R a independently H or C1-C6alkyl.

3. The compound of formula (II) as described in claim 1 or 2, its pharmaceutically acceptable salt, or its stereoisomer, characterized in that, It meets one or more of the following conditions: 1) The C1-C6 alkyl groups are, independently, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; 2) The C3-C7 cycloalkyl groups are each independently cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; and 3) The 4-8 member heterocyclic alkyl groups are each independently a 5- or 6-membered heterocyclic alkyl group with N or O heteroatom and 1 heteroatom.

4. The compound of formula (II) as claimed in claim 3, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, It meets one or more of the following conditions: 1) The C1-C6 alkyl groups are each independently methyl, isopropyl, or tert-butyl; 2) The C3-C7 cycloalkyl groups are each independently cyclobutyl; and 3) The 4-8 membered heterocyclic alkyl groups are independently tetrahydropyrrolidinyl, piperidinyl, tetrahydrofuranyl or tetrahydropyranyl.

5. The compound of formula (II) as claimed in claim 3, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, The 4-8 membered heterocyclic alkyl groups are each independently... , , , , or .

6. The compound of formula (II) as claimed in claim 1 or 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, It meets one or more of the following conditions: 1) R 1 Ci-C6alkyl or Ci-C6haloalkoxy; 2)R 2 Independently selected from C1-C6 alkyl groups, surrounded by one, two, or three R groups. 2-1 Substituted C3-C7 membered cycloalkyl, "4-8 membered heterocycloalkyl with one, two or three heteroatoms selected from N, O and S, and one, two or three heteroatoms", or substituted with one, two or three R atoms. 2-2 The substituted heteroatom is selected from one, two, or three of N, O, and S, and is a 4-8 membered heterocyclic alkyl group with one, two, or three heteroatoms. 3)R 2-1-1 It is a C1-C6 alkyl group; and 4)R a For H.

7. The compound of formula (II) as claimed in claim 6, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, R 2 Independently C1-C4 alkyl, surrounded by 1, 2 or 3 R 2-1 Substituted C4-C6 membered cycloalkyl, "5- or 6-membered heterocycloalkyl with O as the heteroatom and 1 heteroatom", or substituted with 1, 2, or 3 R 2-2 The substitution is a "5- or 6-membered heterocyclic alkyl group with N as the heteroatom and 1 heteroatom".

8. The compound of formula (II) as claimed in claim 1 or 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, It meets one or two of the following conditions: 1) R 1 is -CH3or ; and 2)R 2 for , , , , , or .

9. The compound of formula (II) as claimed in claim 8, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, R 2 for , , , , , , , , or .

10. The compound of formula (II) as claimed in claim 9, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, R 2 For , Or .

11. The compound of formula (II) as claimed in claim 1 or 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, The compound represented by formula (II) is the same as that represented by formula (I-1). 。 12. The compound of formula (II) as claimed in claim 1 or 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, It meets one or more of the following conditions: (1) Each of the C6-C 10 The aryl group can be phenyl or naphthyl independently; (2) The 4-10 membered heterocyclic alkyl group is independently selected from one or two of N and O as heteroatoms; or a 6-8 membered bicyclic heterocyclic alkyl group with 3 heteroatoms; (3) R 1 is Ci-C6haloalkyl; (4) R 2 It is a C1-C4 alkyl group, with one, two or three R atoms. 2-1 Substituted C4-C6 membered cycloalkyl groups or "5- or 6-membered heterocycloalkyl groups with O heteroatom and 1 heteroatom"; and (5) R 4 is hydrogen.

13. The compound of formula (II) as claimed in claim 12, its pharmaceutically acceptable salt, or its stereoisomer, characterized in that, It meets one or more of the following conditions: (1) Each of the C6-C 10 The aryl group is independently a phenyl group; (2) the 4-10 membered heterocycloalkyl is independently or ; (3) R 2 is CrC4alkyl or "5- or 6-membered heterocycloalkyl having 1 heteroatom which is O".

14. The compound of formula (II) as claimed in claim 1 or 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, It meets one or more of the following conditions: (1) R 1 To ; (2) R 2 for , , , , , , or ; (3) the 4-10 membered heterocycloalkyl is or ; and (4) Ring A is phenyl.

15. The compound of formula (II) as claimed in claim 14, its pharmaceutically acceptable salt, or its stereoisomer, characterized in that, R 2 for , , or .

16. The compound of formula (II) as claimed in claim 15, its pharmaceutically acceptable salt, or its stereoisomer, characterized in that, R 2 For , , or .

17. The compound of formula (II) as claimed in claim 16, its pharmaceutically acceptable salt, or its stereoisomer, characterized in that, R 2 For Or .

18. The compound of formula (II) as claimed in claim 1 or 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, The compound represented by formula (II) is the same as that represented by formula (II-1). 。 19. The compound of formula (II) as claimed in claim 1 or 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, The compound represented by formula (II) is any of the following compounds: 、 、 、 、 , , , , , , , , , or .

20. A pharmaceutical composition comprising, The pharmaceutical composition comprises: (1) The compound of formula (II) as claimed in any one of claims 1-19, its pharmaceutically acceptable salt or its stereoisomer; and (2) Pharmaceutically acceptable excipients.

21. Use in the preparation of a medicament of a compound of formula (II) as claimed in any one of claims 1-19, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, or a pharmaceutical composition as claimed in claim 20, wherein the use is selected from: (1) Preparation of aldosterone synthase inhibitors; and (2) Prepare medicines for treating and / or preventing diseases or conditions, said diseases or conditions being chronic kidney disease, congestive heart failure, hypertension, hypertension complications or primary aldosteronism.

22. The use of claim 21, wherein, The intended use is for the preparation of treatments and / or preventions of diseases or conditions associated with aldosterone synthase; the diseases or conditions associated with aldosterone synthase are chronic kidney disease, congestive heart failure, hypertension, hypertension complications, or primary hyperaldosteronism.

23. The use of claim 21, wherein, The disease or condition referred to is hypertension or primary aldosteronism.

24. The use as described in claim 22, characterized in that, The diseases or conditions associated with aldosterone synthase are hypertension or primary hyperaldosteronism.

25. The use of claim 21, wherein, The hypertension mentioned is refractory hypertension.

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