RU2717310C1 - Aldosterone synthase inhibitors based on 2-amino-4h-pyran-3-carbonitrile derivatives - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and to pharmacy. Disclosed is the use of an inhibitor of aldosterone synthase CYP11B2, a compound of formula I or a salt thereof, solvate, hydrate (version: use of a pharmaceutical composition containing an effective amount of at least one said compound) for treating and/or preventing a disease/pathological condition caused by aldosterone hyper-synthesis.

EFFECT: technical result: compounds of formula I inhibit activity of CYP11B2 in the absence or weak binding with steroid enzymes involved in the metabolism of drug compounds, not inhibiting steroidogenic enzymes of family P450 and are not cytotoxic, use thereof enables reducing the level of aldosterone and eliminating disorders caused by its excess, without side effects caused by inhibition of synthesis of glucocorticoid hormones.

8 cl, 1 tbl

Description

Technical field

This invention relates to the chemistry of organic compounds, pharmacology, medicine, and can be used to treat diseases and pathological conditions caused by and / or accompanied by hypersynthesis of aldosterone, including tumors of the adrenal cortex, rare hereditary diseases (primary hyperaldosteronism), chronic heart failure, cirrhosis, arterial hypertension, chronic kidney disease, diabetic nephropathy and metabolic syndrome.

State of the art

The mineralocorticoid hormone aldosterone is an important regulator of blood pressure, circulating blood volume and electrolyte balance in the body to maintain vascular pressure and tissue perfusion. Normally, the regulation of aldosterone production is regulated by the renin-angiotensin-aldosterone system, the concentration of potassium in the blood, as well as adrenocorticotropic hormone. Aldosterone acts on the body through mineralocorticoid and non-mineralocorticoid pathways / receptors. An excess of aldosterone, in addition to increasing blood pressure, has a pathological effect on organs (especially the kidneys) and tissues through the mechanism of vascular remodeling and fibrotic changes, and is also accompanied by cardio-metabolic disorders.

At present, mineral corticoid (MK) receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, and angiotensin receptor blockers are used to reduce the negative effects of aldosterone excess. Nevertheless, direct blockade can disrupt the electrolyte balance, leading to hyperkalemia, and, in addition, it does not lead to a decrease in the level of aldosterone, which can act through non-MK receptors, while the use of ACE inhibitors and angiotensin receptor blockers in 30-40 % of cases have a temporary effect and are accompanied by a “breakthrough of aldosterone”.

An alternative approach to eliminating excess aldosterone is to directly reduce synthesis through selective inhibition of aldosterone synthase (AS, cytochrome P450 11B2, CYP11B2). In clinical practice, an AC inhibitor (AAS) can eliminate or reduce the pathological effect of aldosterone through the MK and non-MK pathways, providing a more effective therapeutic effect than antagonists of MK receptors. IAS will have a better pharmacodynamic profile due to the maintenance of MK receptors, which will maintain the electrolyte balance and minimize the risk of hyperkalemia. However, the development of selective and effective IAS is particularly challenging due to its high homology with the isoform of the CYP11B1 enzyme (93% amino acid sequence identity) and other steroidogenic enzymes.

Disclosure of Invention

The objective of the present invention is to develop new effective aldosterone synthase inhibitors (IAS), which are promising for the treatment of diseases caused and / or accompanied by hypersynthesis of aldosterone.

The technical result of the invention is the development of chemical compounds characterized by high efficiency in the inhibition of aldesterone synthase, as well as characterized by high selectivity for this key enzyme biosynthesis of the steroid hormone aldosterone. These compounds are promising for the prevention and treatment of diseases or pathological conditions caused by hypersynthesis of aldosterone, in particular a tumor of the adrenal cortex, rare hereditary diseases (e.g. primary hyperaldosteronism), chronic heart failure, cirrhosis, arterial hypertension, chronic kidney disease, diabetic nephropathy and / or metabolic syndrome.

The specified technical result is achieved by using compounds of the general formula I or their pharmaceutically acceptable salts, hydrates or solvates as inhibitors of aldosterone synthase CYP11B2:

формула I,

Formula I

Where:

R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C ( = O) OC _1-10 -alkyl, partially or fully halogenated -С (= О) ОС _1-10- alkyl, -С (= О) ОС _2-10 -alkenyl, partially or fully halogenated -С (= О) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl;

R ₂ is independently selected and is H, —C _1-4 alkyl, —C _2-4 alkenyl, —C _2-4 alkynyl, halogen, or —O — C _1-4 alkyl;

R ₃ is independently selected and is 6-10 membered aryl, 5-7 membered heteroaryl containing 1-2 S atoms, wherein R ₃ may optionally be substituted with 1-3 R ₄ substituents;

R ₄ is independently selected and is —O — C _1-4 alkyl.

A separate subclass of compounds of interest includes compounds of formula (I) in which:

R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C ( = O) OC _1-10 -alkyl, partially or fully halogenated -С (= О) ОС _1-10- alkyl, -С (= О) ОС _2-10 -alkenyl, partially or fully halogenated -С (= О) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl;

R ₂ is independently selected and is methyl or —O — CH ₃ ;

R ₃ is independently selected and is phenyl, naphthyl, thiophenyl, wherein R ₃ may optionally be substituted with 1 to 3 R ₄ substituents;

R ₄ is independently selected and is —O — C _1-4 alkyl.

In particular embodiments of the invention, the compounds of interest may be selected from the following compounds of general formula (I):

allyl-6-amino-5-cyano-2-methyl-4- (thiophen-2-yl) -4H-pyran-3-carboxylate;

allyl-6-amino-5-cyano-4- (3,4-dimethoxyphenyl) -2-methyl-4H-pyran-3-carboxylate;

allyl-6-amino-5-cyano-2-methyl-4- (naphthalen-1-yl) -4H-pyran-3-carboxylate.

The present invention also relates to the use of the subject compounds for the treatment and / or prevention of a disease caused by hypersynthesis of aldosterone and / or to a pharmaceutical composition for the treatment and / or prevention of a disease caused by hypersynthesis of aldosterone. In particular embodiments, the disease is an adrenal cortex tumor, primary hyperaldosteronism, chronic heart failure, cirrhosis of the liver, arterial hypertension, chronic kidney disease, diabetic nephropathy and / or metabolic syndrome.

The invention also relates to the use of a pharmaceutical composition for treating and / or preventing a disease caused by hypersynthesis of aldosterone, comprising an effective amount of at least one compound of general formula I:

формула I,

Formula I

or a pharmaceutically acceptable salt, solvate or hydrate thereof,

Where:

R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C ( = O) OC _1-10 -alkyl, partially or fully halogenated -С (= О) ОС _1-10- alkyl, -С (= О) ОС _2-10 -alkenyl, partially or fully halogenated -С (= О) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl;

R ₂ is independently selected and is H, —C _1-4 alkyl, —C _2-4 alkenyl, —C _2-4 alkynyl, halogen, or —O — C _1-4 alkyl;

R ₃ is independently selected and is 6-10 membered aryl, 5-7 membered heteroaryl containing 1-2 S atoms, wherein R ₃ may optionally be substituted with 1-3 R ₄ substituents;

R ₄ is independently selected and is —O — C _1-4 alkyl;

and at least one pharmaceutically acceptable excipient.

A separate subclass of famraceutical compositions of interest include compounds of formula (I) in which:

R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C ( = O) OC _1-10 -alkyl, partially or fully halogenated -С (= О) ОС _1-10- alkyl, -С (= О) ОС _2-10 -alkenyl, partially or fully halogenated -С (= О) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl;

R ₂ is independently selected and is methyl or —O — CH ₃ ;

R ₃ is independently selected and is phenyl, naphthyl, thiophenyl, wherein R ₃ may optionally be substituted with 1 to 3 R ₄ substituents;

R ₄ is independently selected and is —O — C _1-4 alkyl.

In particular embodiments of the invention, compositions of interest may include the following compounds of general formula (I):

allyl-6-amino-5-cyano-2-methyl-4- (thiophen-2-yl) -4H-pyran-3-carboxylate;

allyl-6-amino-5-cyano-4- (3,4-dimethoxyphenyl) -2-methyl-4H-pyran-3-carboxylate;

allyl-6-amino-5-cyano-2-methyl-4- (naphthalen-1-yl) -4H-pyran-3-carboxylate.

In particular embodiments of the invention, the pharmaceutical compositions of the invention can be used to treat an adrenal cortical tumor, primary hyperaldosteronism, chronic heart failure, cirrhosis, arterial hypertension, chronic kidney disease, diabetic nephropathy and / or metabolic syndrome.

In particular embodiments, the pharmaceutically acceptable excipient is a carrier, excipient and / or solvent.

The present invention also relates to a method of treating and / or preventing a disease caused by hypersynthesis of aldosterone, comprising administering (as monotherapy or in combination with one or more agents) a therapeutically effective amount of a compound of the invention or a pharmaceutical composition of the invention to a human body, in need of treatment and / or prevention of such diseases. The term “administration” to a body of a compound of the present invention includes the delivery to a recipient of a compound of the present invention, a prodrug, or other pharmacologically acceptable derivative of such a compound, using any suitable preparation or route of administration to the body well known to those skilled in the art.

The invention also includes the preparation of compounds of general formula (I).

Definitions (terms)

For a better understanding of the present invention, the following are some of the terms used in the present description of the invention.

In the description of the present invention, the terms “includes” and “including” are interpreted as meaning “includes, among other things.” These terms are not intended to be construed as “consists of only”.

The term “alkyl” as used herein also refers to groups typically having one to ten carbon atoms. For example, the term —C _1-10 -alkyl means methyl, ethyl, propyl, isobutyl, etc. Similar conventions apply to another general term, such as “alkenyl,” where the position of one or more double bonds is determined by any C _2-10 carbon atom. Similar conventions apply to another general term, such as “alkynyl,” where the position of one or more triple bonds is defined for any C _2-10 carbon atom.

The term "partially or fully halogenated" includes branched or unbranched hydrocarbon chains in which one or more hydrogen atoms are replaced by halogen. Examples of haloalkyl groups include, but are not limited to, the following groups: trifluoromethyl, trichloromethyl, —C (CF ₃ ) ₂ CH _3, and the like.

The term "halogen" by itself or in part of another term refers to an atom of fluorine, chlorine, bromine or iodine.

The term “aryl” as used herein means groups containing an aromatic ring having from 6 to 10 carbon atoms. An example of aryl ring groups is phenyl, naphthyl and the like.

The term “heteroaryl,” as used herein, means a stable heterocyclic aromatic moiety having 5-7 atoms in a ring. A heteroaryl group may be substituted or unsubstituted. Possible substituents include, but are not limited to, any of the previously mentioned substituents. Examples of typical heteroaryl rings are five- and six-membered monocyclic groups such as thiophenyl and the like. The term “heteroaryl” also includes rings with possible substituents. The term “heteroaryl” may be used equivalently with the terms “heteroaryl ring” or “heteroaromatic”.

This invention contains only those combinations of substituents and derivatives that form a stable or chemically possible compound. A stable or chemically feasible compound is a compound whose stability is sufficient for its synthesis and analytical detection. Preferred compounds of this invention are sufficiently stable and do not decompose at temperatures up to 40 ° C in the absence of chemically active conditions, for at least one week.

The term “solvate” refers to an association or complex of one or more solvent molecules and a compound of the invention. Examples of solvate forming solvents include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The term "hydrate" refers to a complex wherein the solvent molecule is water.

The compounds of the present invention may exist in free form or, if desired, in the form of a pharmaceutically acceptable salt or other derivative. As used herein, the term “pharmaceutically acceptable salt” refers to those salts which, within the framework of a medical opinion, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, etc., and correspond to a reasonable balance of benefits and risk. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates and other types of compounds are well known in medicine. Salts can be prepared in situ during the isolation or purification of the compounds of the invention, and can also be prepared separately by reacting the free base of the compound of the invention with a suitable acid. Examples of pharmaceutically acceptable, non-toxic acid salts include amino salts formed by inorganic acids such as hydrochloric, hydrobromic, phosphoric and sulfuric acids, or organic acids such as acetic, oxalic, maleic, tartaric, succinic or malonic acids, or obtained by other methods used in this field, for example, by ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, hemeflucetate, glucose gluconate, formate glucose phosphate heptane, hexanate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pepate Ulfat, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undekanat, valerate, and the like.

The term “patient” encompasses all types of mammals, preferably humans, and also includes birds.

The terms “treatment”, “therapy” cover the treatment of pathological conditions in mammals, preferably in humans, and include: a) blocking (stopping) the course of the disease, b) alleviating the severity of the disease, i.e. induction of disease regression.

The term "prevention", "prevention", "preventive therapy" covers the elimination of risk factors, as well as the preventive treatment of subclinical stages of the disease in humans, aimed at reducing the likelihood of clinical stages of the disease. Patients for prophylactic therapy are selected on the basis of factors that, based on known data, entail an increase in the risk of clinical stages of the disease compared with the general population. Preventive therapy includes a) primary prevention and b) secondary prevention. Primary prophylaxis is defined as prophylactic treatment in patients whose clinical stage of the disease has not yet begun. Secondary prevention is the prevention of the re-occurrence of the same or similar clinical condition of the disease.

The term “risk reduction” encompasses therapy that reduces the incidence of the clinical stage of the disease. Examples of reducing the risk of disease are primary and secondary disease prevention.

Detailed description of the invention

Excess aldosterone leads, in particular, to the development of various cardiovascular diseases, as well as to impaired renal function. Existing drugs on the world market that are inhibitors of mineralocorticoid receptors have a number of side effects, but most importantly, they cause the accumulation of aldosterone, which can have a negative effect, bypassing mineralocorticoid receptors, showing a negative negative effect. The development of effective chemical compounds aimed directly at the main enzyme for the synthesis of aldosterone, aldostenronsynthase, can reduce the level of the hormone and eliminate the imbalance / disturbances caused by its excess. The compounds of the invention are able to selectively inhibit aldostenronsynthase, minimally inhibiting the closely related enzyme 11β-hydroxylase - CYP11B1 (this level of inhibition cannot lead to any side effects due to the suppression of the synthesis of glucocorticoid hormones), as well as not inhibiting the steroidogenic enzymes of the cytochrome P450 family ( ) and CYP enzymes involved in the metabolism of xenobiotics. Moreover, the compounds of the invention are not cytotoxic at a concentration of 50 μM.

A study of the biological activity of the compounds of the invention was carried out using a purified CYP11B2 enzyme preparation (an expression and purification system was previously developed (PMID: 23322723). The selectivity of the claimed compounds was tested in an in vitro reconstructed system containing redox partner proteins, adrenodoxine reductase and adrenodoxin as well as NADPH-regenerating system. Analysis of the activity of the resulting steroids was carried out on reverse-phase HPLC. At a concentration of compounds according to the invention of 1–0.5 μM CYP11B2 activity was inhibited by 93% (CYP11B1 only 25%).

The claimed compounds did not bind to the enzymes CYP17A1, CYP21A2, CYP19A1, as well as to the CYP enzyme (CYP3A4) involved in the metabolism of many drug compounds CYP3A4, as evidenced by spectrophotometric titration.

In addition, an MTT test was carried out at concentrations of the studied compounds from 1 to 50 μM on a HEK293T cell culture, which showed the absence of cytotoxic effects in the claimed series of compounds (in particular, in examples of compounds 1-3), as well as for the fadrozole comparison compound.

Examples of the preparation of compounds of the invention.

The general scheme for the synthesis of compounds according to the invention is given below:

Scheme 1. General scheme for the synthesis of compounds according to the invention.

General method for the synthesis of substituted 4 H- pyranes according to the invention. 1.5 ml of piperidine was added to an aqueous deionized solution of aldehyde III (6 mmol), allyl acetoacetate II (6 mmol) and dinitrile malonic acid I (6 mmol) and the mixture was stirred for 15 min. The precipitate formed was filtered off, washed with water and ethyl acetate: petroleum ether = 1: 4. Crystallization from ethanol provides the final product. Scheme 2 below shows examples of specific examples of the preparation of compounds of the invention, the yield of final products 1, 2 and 3 was 50-80%.

Scheme for the synthesis of compounds according to the invention

NMR data confirming the structure of the compounds of the invention are given below.

Compound 1: ¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 2.37 (3H, s, Me), 4.51 (2H, br s, NH ₂ ), 4.59 (2H, m, 6'-H), 4.83 ( 1H, s, 4-H), 5.18 (1H m, 8 ' _cis- H), 5.22 (1H m, 8' _trans- H), 5.84 (1H, m, 7'-H), 6.91 (2H, m , 3'-H and 5'-H), 7.16 (1H m, 4'-H).

¹³ C NMR (500 MHz, CDCl ₃ ) δ ppm: 165.44 (CO), 158.03 (s), 157.56 (s), 148.25 (s), 131.86 (C-7 '), 127.09 (d), 124.76 (2d) , 118.71 (CN), 118.60 (C-8 '), 108.26 (C-3), 65.67 (C-6'), 62.56 (C-5), 33.84 (C-4), 18.71 (Me).

Compound 2: ¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 2.37 (3H, s, Me), 3.85 and 3.86 (each 3H, s, OMe), 4.45 (2H, br s, NH ₂ ), 4.43 ( 1H, s, 4-H), 4.50 (2H, m, 7'-H), 5.13 (1H, m, 9 ' _trans- H), 5.16 (1H, br s, 9' _cis- H), 5.75 ( 1H, m, 8'-H), 6.73 (2H, m, 2'-H and 6'-H), 6.79 (1H, d, J = 8.5 Hz, 5'-H)

¹³ C NMR (500 MHz, CDCl ₃ ) δ ppm: 165.80 (CO), 157.40 (s), 156.88 (s), 149.09 (s), 148.36 (s), 136.53 (s), 131.92 (C-8 ') , 119.73 (d), 119.00 (CN), 118.39 (C-9 '), 111.51 (d), 111.13 (d), 108.14 (C-3), 65.48 (C-7'), 62.90 (C-5) 56.06 (2OMe), 38.47 (C-4), 18.63 (Me).

Compound 3: ¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 2.46 (3H, s, Me), 4.28 (2H, m, 9'-H), 4.43 (2H, br s, NH ₂ ), 4.82 ( 1H, dd, J = 17.0, 1.0 Hz, 11 ' _trans- H), 4.89 (1H, dd, J = 10.5, 1.0 Hz, 11' _cis- H), 5.35 (1H, m, 10'-H), 5.41 (1H, s, 4-H), 7.30 (1H, d, J = 7.0 Hz), 7.42 (1H, t, J = 7.5 Hz), 7.48 (1H, t, J = 7.5 Hz), 7.57-7.52 (1H, m), 7.73 (1H, d, J = 8.0 Hz), 7.84 (1H, d, J = 8.0 Hz).

¹³ C NMR (500 MHz, CDCl ₃ ) δ ppm: 165.67 (CO), 157.57 (s), 157.48 (s), 140.67 (s), 133.99 (s), 131.53 (d), 131.26 (s), 128.79 ( d), 128.02 (d), 126.45 (d), 126.07 (d), 125.89 (d), 125.68 (d), 123.26 (d), 118.89 (CN), 118.22 (C-11 '), 108.48 (C- 3), 65.39 (C-9 '), 63.04 (C-5), 33.43 (C-4), 18.60 (Me).

The use of chemical compounds according to the invention

The use of compounds for medical reasons

The compounds described in this invention can be used to treat diseases or pathological conditions caused and / or accompanied by hypersynthesis of aldosterone, including tumors of the adrenal cortex, rare hereditary diseases (primary hyperaldosteronism), chronic heart failure, cirrhosis, arterial hypertension, chronic kidney disease , diabetic nephropathy and metabolic syndrome.

Method for the therapeutic use of compounds

The subject of the invention also includes the administration to a subject in need of appropriate treatment of a therapeutically effective amount of a compound of general formula (I).

By a therapeutically effective amount is meant an amount of a compound administered or delivered to a patient in which the patient is most likely to exhibit the desired response to treatment (prophylaxis). The exact amount required may vary from subject to subject, depending on the age, body weight and general condition of the patient, the severity of the disease, the method of drug administration, combined treatment with other drugs, etc.

A compound of the invention or a pharmaceutical composition containing the compound can be administered to a patient in any amount and by any route of administration effective for treating or preventing a disease.

After mixing the drug with a specific suitable pharmaceutically acceptable carrier in the desired dosage, the compositions constituting the essence of the invention can be administered orally, parenterally, topically, and the like to the human body or other animals.

In the case where the compound of the invention is used as part of a combination therapy regimen, a dose of each of the components of the combination therapy is administered during the required treatment period. The compounds that make up the combination therapy can be administered into the patient's body both at a time, in the form of a dosage containing all the components, and in the form of individual dosages of the components.

Pharmaceutical Compositions

The invention also relates to pharmaceutical compositions that contain a compound of general formula (I) (or a prodrug, a pharmaceutically acceptable salt or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, solvents and / or excipients, such as may be introduced into the patient’s body together with the compound that is the essence of the present invention, and which do not destroy the pharmacological activity of this compound, and are non-toxic administered in doses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions of this invention comprise the compounds of this invention together with pharmaceutically acceptable carriers, which may include any solvents, diluents, dispersions or suspensions, surfactants, isotonic agents, thickeners and emulsifiers, preservatives, astringents, lubricants materials, etc., suitable for a particular dosage form. Materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, mono- and oligosaccharides, as well as their derivatives; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut, cottonseed, safrole, sesame, olive, corn and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic solution, Ringer's solution; ethyl alcohol and phosphate buffers. Also in the composition of the composition may be other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as dyes, release fluids, film-forming agents, sweeteners, flavors and fragrances, preservatives and antioxidants.

The subject of the present invention is also dosage forms — a class of pharmaceutical compositions whose composition is optimized for a particular route of administration into the body in a therapeutically effective dose, for example, for administration to the body orally, topically, by the intraocular route, pulmonary, for example, as an inhalation spray, or intravascular by the method, intranasally, subcutaneously, intramuscularly, as well as by the infusion method, in the recommended dosages.

Dosage forms of the present invention may contain formulations prepared using liposome methods, microencapsulation methods, methods for preparing nanoforms of the preparation, or other methods known in the pharmaceutical art.

In preparing the composition, for example, in tablet form, the active principle is mixed with one or more pharmaceutical excipients, such as gelatin, starch, lactose, magnesium stearate, talc, silica, gum arabic, mannitol, microcrystalline cellulose, hypromellose or the like.

Tablets may be coated with sucrose, a cellulosic derivative, or other suitable coating materials. Tablets can be prepared in various ways, such as direct compression, dry or wet granulation, or hot fusion.

A pharmaceutical composition in the form of a gelatin capsule can be obtained by mixing the active principle with a solvent and filling the mixture with soft or hard capsules.

For parenteral administration, aqueous suspensions, isotonic saline solutions or sterile injectable solutions are used that contain pharmacologically compatible agents, for example propylene glycol or butylene glycol.

Analysis of the biological activity of the compounds of the invention

Determination of the affinity of the compounds of the invention for the active site of aldosterone synthase

Studies have been conducted to determine the binding constant of the compounds of the invention to the target, in particular, examples of the compounds of the invention shown in Table 1 have been investigated.

Table 1. Examples of compounds of the invention.

Connection No. Title Structure 1 allyl-6-amino-5-cyano-2-methyl-4- (thiophen-2-yl) -4H-pyran-3-carboxylate

2 allyl-6-amino-5-cyano-4- (3,4-dimethoxyphenyl) -2-methyl-4H-pyran-3-carboxylate

3 allyl-6-amino-5-cyano-2-methyl-4- (naphthalen-1-yl) -4H-pyran-3-carboxylate

The authors of the present invention used the features of AS to change the spectral properties when binding substrates and inhibitors during in vitro analysis in assessing the affinity of aldosterone synthase inhibitors (compounds of the invention) using a double-beam spectrophotometer ..

A protein solution (recombinant human CYP11B1 or CYP11B2) at a final concentration of 1 μM in 50 mM in potassium phosphate buffer, pH 7.4, was placed in two glass cuvettes (l = 1 cm). A solution of a compound of the invention dissolved in DMSO was added to the test cuvette, an equal volume of solvent was added to the control cuvette. The difference absorption spectrum of the protein was recorded at a wavelength of 350–500 nm. The ligand concentration range (compounds of the invention) used was 0.1-100 μM. During measurements, the baseline was monitored, a change in which indicated an increase in light scattering and a decrease in the solubility of the added ligand.

Determination of the binding constant of the substrate-ligand complex

As a parameter characterizing the binding of the compounds of the invention in the active site of the enzyme, the dissociation constant of the enzyme-ligand complex (K _d ) was used. K _{d was} calculated by the formula:

dA = (dA _max / 2 · P ₀ ) (B ₀ + P ₀ + K _d ) - (√ ((B ₀ + P ₀ + K _d ) ² - (4 · B ₀ · P ₀ )))

where B ₀ is the ligand concentration, P ₀ is the cytochrome P450 concentration, A is the observed absorption value, A _max is the maximum absorption change, K _d is the dissociation constant for the enzyme-ligand complex.

When the compounds of the invention are added to the purified CYP11B2 enzyme preparation, a peak shift is observed in the Cora region from 420 nm, which is typical for the binding of the compounds to the active center.

The compounds of the invention showed high affinity for CYP11B2. The dissociation constant of the enzyme-ligand complex for compounds 1, 2, and 3 was Kd-1.12 μM, Kd, respectively, 12.48 and 2.45 μM.

With respect to CYP11B1, these compounds had low affinity (Kd> 50 μM). The comparison compound, in contrast, has a higher affinity for CYP11B1.

Investigation of the inhibitory effect of the compounds of the invention in relation to the CYP11B2 enzyme

The enzymatic activity of the CYP11B2 enzyme in an in vitro reconstructed system is effectively inhibited by the compounds of the invention, in particular, the activity of the CYP11B2 enzyme is inhibited by 67%, 69% and 72% for the compounds of the invention 1, 2, 3 (respectively). The concentration of the test compounds of the invention in the reaction medium is 2 μM. Evaluation of the inhibitory effect of the compounds of the invention on enzymatic activity was carried out in a reconstructed system using CYP11B2 and redox partners, adrenodoxin and adrenodoxine reductase, as well as the NADPH regenerating system and a deoxycorticosterone substrate.

Enzyme activity was determined in an in vitro reconstructed system using redox partners in the ratio Adrenodoxine reductase: Adrenodoxin: CYP11B2 = 1: 8: 4 in 25 mM Hepes buffer, pH 7.2, containing 0.1 mM EDTA (ethylenediaminetetraacetic acid), 0.1 mM DTT ( dithiothreitol), 0.15% sodium cholate, at 37 ^° C. A mixture of concentrated proteins was preincubated at room temperature for 5 min and then diluted with buffer to a final concentration of 1.0 μM CYP11B, 2.0 μM adrenodoxin and 0.25 μM adrenodoxine reductase. As a substrate, 11-deoxycorticosterone is used in a final concentration of 100 μM (10 mM stock solution in ethanol). The reaction was initiated by adding 100 μM NADPH and NADPH-regenerating system. The reaction products were extracted with dichloromethane and evaporated in a stream of argon. The analysis of steroids was carried out on reverse phase HPLC using 60% methanol for elution, using a computerized HPLC system with spectrophotometric detection. As a result of the studies, it was found that the compounds of the invention lead to effective inhibition of human CYP11B2, in particular, the activity of CYP11B2 was inhibited by 67%, 69% and 72% for compounds 1, 2 and 3, respectively.

The study of the selectivity of the compounds of the invention in relation to the human P450 cytochrome enzyme involved in the biosynthesis of sex hormones, corticoid hormone precursors and the drug metabolism enzyme CYP3A4.

The selectivity of the claimed compounds was tested using purified preparations of the steroidogenic enzymes CYP17A1, CYP19A1, CYP21A2, and the CYP3A4 enzyme involved in the metabolism of drug compounds. To evaluate the binding of compounds to the active site of P450 cytochrome data, the same method was used (spectrophotometric titration), which was used to evaluate the binding of compounds to CYP11B2 (see the description of the method above). The compounds of the invention, and in particular the compounds of Examples 1, 2, and 3, showed the absence or weak binding of the enzymes studied. Comparison compound (fadrozole) showed high affinity for human CYP19A1 ..

Assessment of the cytotoxicity of the compounds of the invention in cell cultures.

The following study was performed to evaluate the cytotoxicity of the compounds of the invention. HEK 293T cells are seeded in a 96-well plate at a concentration of 100,000 cells / ml, 100 μl per well. After 24 hours, after the cells are attached, the test compound is added to the medium. The concentration of compounds: 50, 35, 20 and 5 μm in 2 agility. At the same time, wells for control samples are left — wells without cells passing through all stages of the experiment (Blank); wells with cells passing all stages of the experiment. Instead of the test compound, a solvent (DMSO) is added to the control wells. After incubation with the test compound, the medium is removed from the wells, 90 μl PBS + 10 μl MTT (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide - formazan) (5 mg / ml). The culture plate is returned to the CO ₂ incubator for 2 hours, 80 μl of PBS with MTT is removed. Add 100 μl of DMSO to dissolve the crystals of formazan. Read optical absorption at 570 nm. The results of the study - the percentage of surviving cells in comparison with the control when adding compounds of the invention 1-3 to the medium was 87-106%.

Compared with the already described CYP11B2 inhibitors and known inhibitors of corticoid biosynthesis (fadrozole) or steroid biosynthesis (ketoconazole), the claimed compounds are more selective with respect to CYP11B2. Kd (human CYP19A1) for fadrozole - 16 nM. Kd (human CYP17A1) for ketoconazole is <0.1 μM. Kd (human CYP2C9) for ketoconazole is 0.14 μM. The Kd for the compounds of the invention with respect to these enzymes (CYP19A1, CYP17A1 and CYP2C19) exceeds 10 μM. The presented class of compounds is first used to inhibit steroidogenic cytochromes P450.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as in any way limiting the scope of the invention. It should be understood that various modifications are possible without departing from the gist of the present invention.

Claims (36)

1. The use of compounds that are inhibitors of aldosterone synthase CYP11B2, General formula I

or a pharmaceutically acceptable salt, solvate or hydrate thereof, where R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C (= O) OC _1-10 -alkyl, partially or fully halogenated -C (= O) OC _1-10 -alkyl, -C (= O) OC _2-10- alkenyl, partially or fully halogenated -C (= O ) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl; R ₂ is independently selected and is H, —C _1-4 alkyl, —C _2-4 alkenyl, —C _2-4 alkynyl, halogen, or —OC _1-4 alkyl; R ₃ is independently selected and is a 6-10 membered aryl, 5-7 membered heteroaryl containing 1-2 S atoms, wherein the R ₃ substituent may optionally be substituted with 1-3 R ₄ substituents; R ₄ is independently selected and is —O — C _1-4 alkyl; for the treatment and / or prevention of a disease or pathological condition caused by hypersynthesis of aldosterone. 2. The use of claim 1, wherein R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C ( = O) OC _1-10 -alkyl, partially or fully halogenated -C (= O) OC _1-10 -alkyl, -C (= O) OC _2-10- alkenyl, partially or fully halogenated -C (= O) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl; R ₂ is independently selected and is methyl or —O — CH ₃ ; R ₃ is independently selected and is phenyl, naphthyl, thiophenyl, wherein R ₃ may optionally be substituted with 1 to 3 R ₄ substituents; R ₄ is independently selected and is —O — C _1-4 alkyl. 3. The use of claim 1, wherein the compound of general formula I is selected from the group: allyl-6-amino-5-cyano-2-methyl-4- (thiophen-2-yl) -4H-pyran-3-carboxylate; allyl-6-amino-5-cyano-4- (3,4-dimethoxyphenyl) -2-methyl-4H-pyran-3-carboxylate; allyl-6-amino-5-cyano-2-methyl-4- (naphthalen-1-yl) -4H-pyran-3-carboxylate. 4. The use of a pharmaceutical composition for the treatment and / or prevention of a disease or pathological condition caused by hypersynthesis of aldosterone, comprising an effective amount of at least one compound that is an inhibitor of aldosterone synthase CYP11B2, General formula I

or a pharmaceutically acceptable salt, solvate or hydrate thereof, where R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C (= O) OC _1-10 -alkyl, partially or fully halogenated -C (= O) OC _1-10 -alkyl, -C (= O) OC _2-10- alkenyl, partially or fully halogenated -C (= O ) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl; R ₂ is independently selected and is H, —C _1-4 alkyl, —C _2-4 alkenyl, —C _2-4 alkynyl, halogen, or —OC _1-4 alkyl; R ₃ is independently selected and is a 6-10 membered aryl, 5-7 membered heteroaryl containing 1-2 S atoms, wherein the R ₃ substituent may optionally be substituted with 1-3 R ₄ substituents; R ₄ is independently selected and is —OC _1-4 alkyl; and  at least one pharmaceutically acceptable excipient. 5. The use of claim 4, wherein R ₁ is independently selected and is —C _1-10 alkyl, partially or fully halogenated —C _1-10 alkyl, —C _2-10 alkenyl, partially or fully halogenated —C _2-10 alkenyl, —C ( = O) OC _1-10 -alkyl, partially or fully halogenated -C (= O) OC _1-10 -alkyl, -C (= O) OC _2-10- alkenyl, partially or fully halogenated -C (= O) OC _2-10 alkenyl, -C _2-10 alkynyl or partially or fully halogenated C _2-10 alkynyl; R ₂ is independently selected and is methyl or —O — CH ₃ ; R ₃ is independently selected and is phenyl, naphthyl, thiophenyl, wherein R ₃ may optionally be substituted with 1 to 3 R ₄ substituents; R ₄ is independently selected and is —O — C _1-4 alkyl. 6. The use of claim 4, wherein the compound of general formula I is selected from the group: allyl-6-amino-5-cyano-2-methyl-4- (thiophen-2-yl) -4H-pyran-3-carboxylate; allyl-6-amino-5-cyano-4- (3,4-dimethoxyphenyl) -2-methyl-4H-pyran-3-carboxylate; allyl-6-amino-5-cyano-2-methyl-4- (naphthalen-1-yl) -4H-pyran-3-carboxylate. 7. The use according to claim 4, in which the disease is a tumor of the adrenal cortex, primary hyperaldosteronism, chronic heart failure, cirrhosis, arterial hypertension, chronic kidney disease, diabetic nephropathy and / or metabolic syndrome. 8. The use of claim 4, wherein the pharmaceutically acceptable excipient is a carrier, excipient and / or solvent.