HK1139942B - Atropisomer of pyrrole derivative - Google Patents

Description

Atropisomers of pyrrole derivatives

Technical Field

The present invention relates to an atropisomer of a pyrrole derivative having an excellent mineralocorticoid receptor antagonistic action and a preventive or therapeutic agent for hypertension, angina pectoris, acute coronary syndrome (acute coronary syndrome), congestive heart failure, nephropathy, arteriosclerosis, cerebral infarction, fibrosis or primary aldosteronism, which comprises the same.

Background

Mineralocorticoid Receptor (MR) (aldosterone receptor) is known to play an important role in the regulation of body electrolyte balance and blood pressure (see, for example, non-patent document 1), and mineralocorticoid receptor antagonists such as spironolactone and eplerenone (eplerenone) having a steroid structure are known to be useful in the treatment of hypertension and heart failure.

Further, as a mineralocorticoid receptor antagonist having a nonsteroidal skeleton, an azole derivative described in WO2006/012642 (patent document 1) is known. However, atropisomers of the compounds of the invention having the general formula (I) are not known.

[ non-patent document 1 ]: advances in Physiology edition, 26 (1): 8-20(2002)

[ patent document 1 ]: WO2006/012642

Disclosure of Invention

The present inventors have made an effort to develop an excellent preventive or therapeutic drug for cardiovascular diseases, and have conducted intensive studies on the pharmacological activities of various pyrrole derivatives. As a result, they found that the compounds having the general formula (I) have atropisomers, and one of them has extremely excellent mineralocorticoid receptor antagonistic action (in vitro and in vivo activities) and persistence of drug effect, and also has excellent properties of solubility, oral absorbability, blood concentration, metabolic stability and safety, and is useful as a medicament, preferably as a prophylactic or therapeutic medicament (particularly a therapeutic medicament) for diseases such as hypertension, angina pectoris, acute coronary syndrome, congestive heart failure, nephropathy, arteriosclerosis, cerebral infarction, fibrosis, primary aldosteronism or heart disease, more preferably for congestive heart failure, nephropathy, hypertension and the like, particularly preferably for hypertension, thereby completing the present invention.

The present invention provides an atropisomer of a compound having the general formula (I) having an excellent mineralocorticoid receptor antagonistic action and a medicament (prophylactic or therapeutic medicament (particularly therapeutic medicament)) containing the same for hypertension, angina pectoris, acute coronary syndrome, congestive heart failure, nephropathy, arteriosclerosis, cerebral infarction, fibrosis, primary aldosteronism or heart disease (more preferably for congestive heart failure, nephropathy and hypertension; particularly preferably for hypertension).

Namely, the present invention relates to:

(1) atropisomers of compounds of the following general formula (I):

[ solution 1]

(wherein R is¹Is C1-C3 alkyl or hydroxy-C1-C3 alkyl; and R is²Is a hydrogen atom or a C1-C3 alkoxy group).

Further, the present invention relates to the following aspects:

(2) among a pair of atropisomers of each compound represented by the general formula (I), an atropisomer which exhibits a stronger mineralocorticoid receptor antagonistic action;

(3) atropisomers according to (1) or (2), wherein R¹Is methyl or 2-hydroxyethyl;

(4) the atropisomer according to any one of (1) to (3), wherein R²Is a hydrogen atom or a methoxy group;

(5) atropisomers according to (1) or (2), wherein R¹Is 2-hydroxyethyl and R²Is a hydrogen atom;

(6) atropisomers according to (1) or (2), wherein R¹Is methyl and R²Is a hydrogen atom;

(7) atropisomers according to (1) or (2), wherein R¹Is 2-hydroxyethyl and R²Is a methoxy group;

(8) (-) -1- (2-hydroxyethyl) -4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide;

(9) (+) -1, 4-dimethyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide;

(10) (-) -1- (2-hydroxyethyl) -5- [ 4-methoxy-2- (trifluoromethyl) phenyl ] -4-methyl-N- [4- (methylsulfonyl) phenyl ] -1H-pyrrole-3-carboxamide;

(11) a medicament containing an atropisomer according to any one of (1) to (10) as an active ingredient;

(12) a prophylactic or therapeutic agent for cardiovascular diseases, which comprises the atropisomer according to any one of (1) to (10) as an active ingredient;

(13) a prophylactic or therapeutic agent for hypertension, which contains the atropisomer according to any one of (1) to (10) as an active ingredient;

(14) a pharmaceutical composition comprising an atropisomer according to any one of (1) to (10) and a pharmaceutically acceptable carrier.

Examples of the "C1-C3 alkyl group" in the above general formula (I) include straight or branched alkyl groups having 1 to 3 carbon atoms such as methyl, ethyl, n-propyl, isopropyl and the like, preferably methyl.

The "hydroxy-C1-C3 alkyl group" in the above general formula (I) means a group obtained by substituting the above-mentioned "C1-C3 alkyl group" with one hydroxy group, and examples thereof include 2-hydroxyethyl, 2-hydroxy-1-methylethyl, 2-hydroxypropyl, 3-hydroxypropyl and the like, preferably 2-hydroxyethyl.

The "C1-C3 alkoxy group" in the general formula (I) means a C1-C3 alkyloxy group formed from the aforementioned "C1-C3 alkyl group", and is a straight-chain or branched-chain alkyloxy group having 1 to 3 carbon atoms, for example, methoxy, ethoxy, n-propoxy or isopropoxy, preferably methoxy.

"atropisomers" refer to structural isomers based on axial (axial) or planar chirality (planar chirality) resulting from restricted rotation within a molecule. The compounds of the general formula (I) according to the invention have two atropisomers, which result from the axial chirality resulting from the restricted rotation of the bond between the phenyl group and the substituted pyrrole ring, which is substituted at the ortho position by trifluoromethyl, due to steric hindrance. The "atropisomer" of the present invention is any one of the two atropisomers of the compound having the general formula (I). However, atropisomers having more excellent pharmacological activity, stability, in vivo kinetic properties, safety and the like, and thus having advantageous properties as a drug, are preferable.

The atropisomers of the compound of the general formula (I) of the present invention exhibit excellent mineralocorticoid receptor antagonistic action and high plasma concentration and retention in blood, are excellent in pharmacological action and in vivo kinetic properties such as oral absorbability, in vivo distribution and retention in blood, and are highly safe for organs such as kidney and liver. Further, the atropisomers of the compounds of the present invention having the general formula (I) are very stable. For example, no racemization was observed even when the atropisomer was treated in methanol at room temperature for 7 days or in acetonitrile-phthalic acid buffer at 60 ℃ for 4 hours.

Thus, the atropisomers of the compounds of the general formula (I) according to the invention are useful, for example, as medicaments, in particular as medicaments for the treatment or prophylaxis of various cardiovascular diseases, preferably hypertension, angina pectoris, acute coronary syndrome, congestive heart failure, renal diseases, arteriosclerosis, cerebral infarction, fibrosis, primary aldosteronism or cardiac diseases.

Detailed Description

Atropisomers of the compounds of the general formula (I) according to the invention can be obtained by optical resolution of the racemic compounds prepared using the method described in WO 2006/012642. The optical resolution of atropisomers is essentially the same as that of enantiomers due to sp3 asymmetric carbon atom and the like, and examples thereof include (1) a method by crystallization; (2) a method by an enzymatic reaction; and (3) methods by chromatography. However, it is not limited to these methods. Hereinafter, a representative optical resolution method will be described in detail.

(1) Optical resolution by crystallization

(a) Preferential crystallisation (preferential crystallisation)

This is an optical resolution method using the nature of natural crystallization of a racemic mixture, which can achieve optical resolution without asymmetric factors.

(b) Diastereomer method

In this method, a chiral compound called an optical resolving agent is allowed to act on a racemic compound to derive the compound into two diastereomers, and separation of the diastereomers is achieved by fractional crystallization using the difference in solubility between these diastereomers. The optical purity can be improved by repeated recrystallization. The target enantiomer can be obtained by removing the resolving agent from the individual diastereomer obtained. In the present invention, a method of deriving a covalently bonded crystalline diastereomer and then performing fractional crystallization is preferable. For example, in the thermal resolution of racemic alcohols, which are derivatized to diastereomeric esters by chiral carboxylic acids, the sparingly soluble diastereomers are removed therefrom by recrystallization, and the resulting individual diastereomeric esters are hydrolyzed, thereby obtaining optically active alcohols.

(c) Embedding of complex compounds

This is an optical resolution method which utilizes a chiral host molecule and an enantiomer of a racemic compound as a guest molecule to form an embedded complex diastereoselectively and to improve optical purity by recrystallization.

(d) Preferential enrichment (preferential enrichment)

This process is characterized in that the concentration of one enantiomer is caused in the mother liquor by recrystallization of the racemic crystals. At the same time, crystals of low optical purity having the opposite chirality to that of the mother liquor are deposited.

(2) Enzyme reaction

In the case of addition reactions of racemic compounds using enzymes such as lipases, only one optically active compound reacts in the case of certain substrates. This is a method of utilizing such a property, in which after the enzymatic reaction, the obtained product is isolated and purified by recrystallization or chromatography, and then the attached functional group is removed under appropriate conditions to obtain the objective optically active compound. On the other hand, there is also a method in which a racemic compound is specifically modified in advance, the modified racemic compound is subjected to an enzymatic decomposition reaction, and then only one optically active compound is obtained in the same manner as described above.

(3) Direct optical resolution by chromatography

When a stationary phase incorporating an asymmetric factor of a derivative to which a sugar or the like is bonded is used as a carrier, a difference in retention time in chromatography can be made, and thus resolution can be achieved. By utilizing this property, direct resolution can be performed by high performance liquid chromatography using a chiral column. Examples of the chiral column include CHIRALPAK AD-H, CHIRALCEL OJ-RH (DAICEL) and the like.

When the atropisomers of the present invention are used as a medicament, the atropisomers of the compounds having the above general formula (I) may be administered orally in the form of tablets, capsules, granules, powders, syrups or the like, or parenterally in the form of injections, suppositories, patches, external preparations or the like, alone or in admixture with suitable pharmaceutically acceptable excipients, diluents or the like.

These preparations are prepared by a known method using additives such as excipients, lubricants, binders, disintegrants, emulsifiers, stabilizers, flavoring agents or diluents.

The amount of atropisomers used varies depending on the symptoms, age, etc. However, the preferred dose is a dose of 0.02mg/kg (preferably 0.1mg/kg) as the lower limit and a dose of 100mg/kg (preferably 10mg/kg) as the upper limit in the case of oral administration to an adult, and a dose of 0.002mg/kg (preferably 0.01mg/kg) and a dose of 10mg/kg (preferably 1mg/kg) per time as the lower limit and a dose of 10mg/kg (preferably 1mg/kg) as the upper limit in the case of parenteral administration, and is administered 1 to 6 times per day depending on the symptoms.

Hereinafter, the present invention is described in more detail by examples, test examples and formulation examples. However, the scope of the present invention is not limited to these.

Examples

(example 1)

(+/-) -1, 4-dimethyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide

This compound was synthesized by the method described in example 16 of WO 2006/012642.

¹H-NMR(400MHz，CDCl₃)δ：7.90(2H，d，J＝8.6Hz)，7.83-7.80(3H，m)，7.70-7.58(3H，m)，7.34(1H，d，J＝7.4Hz)，7.30(1H，s)，3.32(3H，s)，3.05(3H，s)，2.09(3H，s).

HR-MS (ESI) as C₂₁H₂₀F₃N₂O₃S[M+H]⁺Calculation, theoretical value: m/z: 437.1147, found: 437.1157.

(example 2)

Optical resolution of the Compound of example 1

Using (+/-) -1, 4-dimethyl-N- [4- (methylsulfonyl) phenyl]-5- [2- (trifluoromethyl) phenyl]5mL of an ethanol solution (4-6 mg/mL) of (E) -1H-pyrrole-3-carboxamide was resolved by 9 times under the following HPLC conditions, and the fraction (t) containing the isomer A was isolated_R11min) to give isomer a 86mg in solid form, from fraction containing isomer B (t)_R18min) to yield the isomer B87 mg in solid form.

The separation by HPLC using a chiral column employed the following conditions:

the device comprises the following steps: shimadzu Class-VP system (LC-8/SCL-10AVP/SPD-10 AVP); a chromatographic column: CHIRALPAK AD-H (2 cm. times.25 cm) semifractionated column (semi-fractionation column); flow rate: 8.0 mL/min; eluting solvent: ethanol (100%, isocratic elution); and (3) detection: UV (254nm)

Isomer a: (-) -1, 4-dimethyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide

[α]_D ²¹：-18°(c＝1.0，EtOH).

¹H-NMR(500MHz，CDCl₃)δ：7.92(2H，d，J＝8.3Hz)，7.85-7.80(3H，m)，7.69-7.64(2H，m)，7.61(1H，t，J＝7.3Hz)，7.35(1H，d，J＝7.3Hz)，7.31(1H，s)3.33(3H，s)，3.06(3H，s)，2.10(3H，s).

HR-MS (ESI) as C₂₁H₂₀F₃N₂O₃S[M+H]⁺Calculation, theoretical value: m/z: 437.1147, found: 437.1138.

retention time: 4.1min.

The analysis by HPLC using a chiral column employed the following conditions. (hereinafter, analysis was performed using the same conditions, retention time was measured using chiral HPLC.)

An analysis device: shimadzu Class-VP system (LC-10ADVP/SCL-10AVP/SPD-M10AVP/CTO10ACVP/DGU 12A); a chromatographic column: CHIRALPAKAD-H (0.46 cm. times.25 cm); flow rate: 1.0 mL/min; eluting solvent: ethanol (100%, isocratic elution); and (3) detection: UV (254nm)

Isomer B: (+) -1, 4-dimethyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide

[α]_D ²²：+18°(c＝1.2，EtOH).

¹H-NMR(500MHz，CDCl₃)δ：7.91(2H，d，J＝8.8Hz)，7.85-7.80(3H，m)，7.68-7.64(2H，m)，7.61(1H，t，J＝7.3Hz)，7.35(1H，d，J＝7.3Hz)，7.31(1H，s)，3.33(3H，s)，3.06(3H，s)，2.10(3H，s).

HR-MS (ESI) as C₂₁H₂₀F₃N₂O₃S[M+H]⁺Calculation, theoretical value: m/z: 437.1147, found: 437.1153.

retention time: 6.3min.

(example 3)

(+/-) -1- (2-hydroxyethyl) -4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide

After 4-methyl-5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxylic acid methyl ester was obtained by the method described in example 16 of WO2006/012642, the following reaction was carried out using this compound as a starting material.

Methyl 4-methyl-5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxylate (1.4g, 4.9mmol) was dissolved in methanol (12mL), 5M aqueous sodium hydroxide solution (10mL) was added thereto, and the resulting mixture was heated under reflux for 3 hours. After the mixture was cooled to room temperature, formic acid (5mL) was added thereto to stop the reaction. After the mixture was concentrated under reduced pressure, water (10mL) was added thereto to suspend the resulting residue. The solid was collected by filtration and washed 3 times with water. The obtained solid was dried under reduced pressure, whereby 4-methyl-5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxylic acid (1.1g, 83%) was obtained as a solid. The resulting solid was suspended in dichloromethane (10mL), oxalyl chloride (0.86mL, 10mmol) was added thereto, and the resulting mixture was stirred at room temperature for 2 hours. After the mixture was concentrated under reduced pressure, the residue was dissolved in tetrahydrofuran (10mL), 4- (methanesulfonyl) aniline hydrochloride (1.0g, 4.9mmol) and N, N-diisopropylethylamine (2.8mL, 16mmol) were sequentially added to the solution, and the resulting mixture was heated under reflux for 18 hours. After the mixture was cooled to room temperature, the solvent was distilled off under reduced pressure, and acetonitrile (10mL) and 3M hydrochloric acid (100mL) were added to the residue. The precipitated solid was pulverized, collected by filtration and washed with water, followed by drying under reduced pressure, whereby 4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide (1.4g, 89%) was obtained as a solid.

¹H-NMR(400MHz，DMSO-d6)δ11.34(1H，brs，)，9.89(1H，s)，7.97(2H，d，J＝6.6Hz)，7.87-7.81(3H，m)，7.73(1H，t，J＝7.4Hz)，7.65-7.61(2H，m)，7.44(1H，d，J＝7.8Hz)，3.15(3H，s)，2.01(3H，s).

Sodium hydride (0.12g, 3mmol, 60% dispersion in mineral oil) was dissolved in N, N-dimethylformamide (1.5mL), 4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide (0.47g, 1.1mmol) was added thereto, and the resulting mixture was stirred at room temperature for 30 min. Then, 1, 3, 2-dioxathiolane-2, 2-dioxide (1, 3, 2-dioxathioolane-2, 2-dioxide) (0.14g, 1.2mmol) was added thereto, and the resulting mixture was stirred at room temperature. After 1 hour, sodium hydride (40mg, 1.0mmol, oily, 60%) was added again thereto, and the resulting mixture was stirred at room temperature for 30 minutes. Then, 1, 3, 2-dioxathiolane-2, 2-dioxide (12mg, 0.11mmol) was added thereto, and the resulting mixture was stirred at room temperature for 1 hour. After the mixture was concentrated under reduced pressure, methanol (5mL) was added to the residue, insoluble materials were removed by filtration, and the filtrate was concentrated again. Tetrahydrofuran (2mL) and 6M hydrochloric acid (2mL) were added to the residue, and the resulting mixture was stirred at 60 ℃ for 16 hours. The reaction was cooled to room temperature, then dissolved in ethyl acetate, and washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and filtered. Then, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate), whereby the objective compound (0.25g, 48%) was obtained.

¹H-NMR(400MHz，CDCl₃)δ：7.89-7.79(m，6H)，7.66-7.58(m，2H)，7.49(s，1H)，7.36(d，1H，J＝7.4Hz)，3.81-3.63(m，4H)，3.05(s，3H)，2.08(s，3H).

HR-MS (ESI) as C₂₂H₂₂F₃N₂O₄S[M+H]⁺Calculation, theoretical value: m/z: 467.1252, found: 467.1246.

according to C₂₂H₂₁F₃N₂O₄S, analysis and calculation: c, 56.65; h, 4.54; n, 6.01; f, 12.22; s, 6.87, found: c, 56.39; h, 4.58; n, 5.99; f, 12.72; and S, 6.92.

(example 4)

Optical resolution of the Compound of example 3

The resolution was carried out 4 times in the same manner as in example 2, thereby obtaining a fraction (t) containing isomer C_R10min) to give isomer C74 mg in solid form, from fraction containing isomer D (t)_R11min) to yield isomer D71 mg as a solid.

Isomer C: (+) -1- (2-hydroxyethyl) -4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide

[α]_D ²¹：+7.1°(c＝1.0，EtOH).

¹H-NMR(400MHz，CDCl₃)δ：7.91(s，1H)，7.87-7.79(m，5H)，7.67-7.58(m，2H)，7.51(s，1H)，7.35(d，1H，J＝7.0Hz)，3.78-3.65(m，4H)，3.05(s，3H)，2.07(s，3H).

HR-MS (ESI) as C₂₂H₂₂F₃N₂O₄S[M+H]⁺Calculation, theoretical value: m/z: 467.1252, found: 467.1260.

retention time: 4.0min.

Isomer D: (-) -1- (2-hydroxyethyl) -4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide

[α]_D ²¹：-7.2°(c＝1.1，EtOH).

¹H-NMR(400MHz，CDCl₃)δ：7.88-7.79(m，6H)，7.67-7.58(m，2H)，7.50(s，1H)，7.36(d，1H，J＝7.5Hz)，3.79-3.65(m，4H)，3.05(s，3H)，2.08(s，3H).

HR-MS (ESI) as C₂₂H₂₂F₃N₂O₄S[M+H]⁺Calculation, theoretical value: m/z: 467.1252, found: 467.1257.

retention time: 4.5min.

(example 5)

(+/-) -1- (2-hydroxyethyl) -5- [ 4-methoxy-2- (trifluoromethyl) phenyl ] -4-methyl-N- [4- (methylsulfonyl) phenyl ] -1H-pyrrole-3-carboxamide

This compound was synthesized by the method described in WO 2006/012642.

¹H-NMR(500MHz，CDCl₃) δ: 7.91(2H, d, J ═ 8.3Hz), 7.82(2H, d, J ═ 8.3Hz), 7.69(1H, s), 7.46(1H, s), 7.32(1H, d, J ═ 2.0Hz), 7.28-7.27(1H, m), 7.14(1H, dd, J ═ 8.3 and 2.0Hz), 3.92(3H, s), 3.82-3.66(4H, m), 3.06(3H, s), 2.10(3H, s).

HR-MS (ESI) as C₂₃H₂₄F₃N₂O₅S[M+H]⁺Calculation, theoretical value: m/z: 497.1358, found: 497.1361.

(example 6)

Optical resolution of the Compound of example 5

The resolution was carried out 7 times in the same manner as in example 2, thereby obtaining a fraction (t) containing isomer E_R11min) to give isomer E50 mg in solid form, from fraction containing isomer F (t)_R14min) gave isomer F41 mg as a solid.

Isomer E: (-) -1- (2-hydroxyethyl) -5- [ 4-methoxy-2- (trifluoromethyl) phenyl ] -4-methyl-N- [4- (methylsulfonyl) phenyl ] -1H-pyrrole-3-carboxamide

[α]_D ²²：-1.3°(c＝1.0，EtOH).

¹H-NMR(500MHz，CDCl₃) δ: 7.90(2H, d, J ═ 8.3Hz), 7.83-7.79(3H, m), 7.48(1H, s), 7.32(1H, d, J ═ 2.4Hz), 7.28-7.25(1H, m), 7.14(1H, dd, J ═ 8.3 and 2.4Hz), 3.92(3H, s), 3.81-3.65(4H, m), 3.06(3H, s), 2.09(3H, s), 1.82(1H, brs).

HR-MS (ESI) as C₂₃H₂₄F₃N₂O₅S[M+H]⁺Calculation, theoretical value: m/z: 497.1358, found: 497.1359.

retention time: 4.1min.

Isomer F: (+) -1- (2-hydroxyethyl) -5- [ 4-methoxy-2- (trifluoromethyl) phenyl ] -4-methyl-N- [4- (methylsulfonyl) phenyl ] -1H-pyrrole-3-carboxamide

[α]_D ²³：+1.6°(c＝0.8，EtOH).

¹H-NMR(500MHz，CDCl₃) δ: 7.91(2H, d, J ═ 8.8Hz), 7.81(2H, d, J ═ 8.8Hz), 7.69(1H, s), 7.46(1H, s), 7.32(1H, d, J ═ 2.4Hz), 7.28-7.25(1H, m), 7.14(1H, dd, J ═ 8.3 and 2.4Hz), 3.92(3H, s), 3.82-3.66(4H, m), 3.05(3H, s), 2.09(3H, s).

HR-MS (ESI) as C₂₃H₂₄F₃N₂O₅S[M+H]⁺Calculation, theoretical value: m/z: 497.1358, found: 497.1340.

retention time: 4.7min.

(test example 1)

A plasmid pM-bMR-LBD obtained by linking the ligand binding domain (LBD, corresponding to a region of about 308 amino acids at the carboxy terminus) of the human mineralocorticoid receptor (bMR, NM-000901) and the DNA binding domain (corresponding to a region of 147 amino acids at the amino terminus) of the yeast transcription factor GAL4 and expressing the GAL4-hMR receptor was constructed. Reporter assays were performed using a reporter plasmid (e.g., plasmid pFR-Luc from Stratagene Cloning System, Inc.) having a sequence (UAS sequence) that binds to the DNA binding domain of GAL4 and containing the luciferase gene.

The previously obtained plasmid pM-hMR-LBD and reporter plasmid were transfected into human fetal-derived kidney cell line HEK293 by lipofection. On the next day, cells were treated and collected with trypsin, white 96-well plates (manufactured by Costar, inc.) were prepared, and cells were dispensed into each well in an amount of 95 μ L wells using DMEM medium containing 5% FBS (fetal bovine serum) that had been treated with activated carbon.

For each test compound, a solution obtained by dissolving the test compound in dimethyl sulfoxide at a prescribed concentration was used, and the solution was diluted with an appropriate medium and added to the cells in a white 96-well plate to reach a final concentration of 0.1%. 1nM aldosterone was allowed to co-exist when the test compound was added. One group of wells to which dimethylsulfoxide was added was labeled as control 1 group; one set of wells to which 1nM aldosterone was added was labeled control 2. After the addition was complete, the culture was allowed to stand overnight.

On the next day, the medium was removed, and luciferase substrates (Wako pure chemical Industries, Ltd.) were prepared according to the package insert and added to each well in an amount of 50. mu.L/well, followed by stirring for about 30 minutes. The luminescence intensity of each well was measured using Analyst (manufactured by Molecular Devices Corporation) and was taken as luciferase activity. When the luciferase activity value of the control 1 group was 0% and the luciferase activity value of the control 2 group was 100%, the relative luciferase activity values at the respective amounts of the test compound in the group to which the test compound was added were plotted to prepare a curve. From the curve, the maximum value was calculated as Imax (%), and the concentration of the test compound showing the value of Imax/2 was calculated as ICmax50 (M). Table 1 shows ICmax50 values.

(results) as shown in the following table 1, the atropisomers of the present invention showed significant mineralocorticoid receptor antagonistic activity compared to the corresponding racemic compound.

(Table 1)

Test compound	ICmax50(nM)	Imax(％)
			Compound of example 1	13	95
Isomer A	＞1000	ND.1
			Isomer B	2.6	123
Compound of example 3	5.3	105
			Isomer C	＞1000	N.D.1
Isomer D	2.4	99
			Compound of example 5	5.3	97
Isomer E	1.8	115
			Isomer F	＞1000	N.D.1

¹: not determined

(test example 2)

Monkeys were fasted the day before administration of test compounds using cynomolgus monkey (male). To the test compound, a 0.5% MC (methyl cellulose) solution was added to prepare a drug administration sample so that the dose was 3mg/2 mL/kg. Each administration sample was perfused into the stomach of a cynomolgus monkey using a tube. After administration, about 5mL more water was given. Three cynomolgus monkeys were given as a group per administration sample.

For blood sampling, approximately 1mL was sampled from the femoral vein using heparin-treated syringes prior to dosing, 30 minutes and 1, 2, 4, 6, 8, 24 and 48 hours post-dose. The collected blood samples were centrifuged (15000 Xg, 3min, 4 ℃) to obtain plasma. The resulting plasma was stored in a refrigerator (-20 ℃) until pre-treatment.

Preparation of standard and Internal Standard (IS) solutions: each test compound was dissolved in acetonitrile to obtain a solution of 1mg/mL of each test compound. And diluting each solution of the compounds to be detected with acetonitrile to prepare a standard solution. In addition, warfarin sodium (Wako Pure Chemical Industries, Ltd.) was dissolved in acetonitrile to make a 500ng/mL IS solution.

Pretreatment of plasma samples: a50. mu.L sample of plasma was taken and 50. mu.L of acetonitrile was added thereto. As a calibration curve, 50. mu.L of each standard solution (acetonitrile solution) was added to 50. mu.L of blank plasma. For all samples, 150. mu.L of IS in acetonitrile was added, and the resulting mixture was stirred and then centrifuged (about 1800 Xg, 30min, 4 ℃). After filtration using a Sirocco protein precipitation plate (Waters Corporation), the filtrate was diluted appropriately with a mobile phase as a sample for LC-MS/MS analysis.

Quantification of test compounds: the plasma concentration of each test compound was analyzed by LC-MS/MS method.

[ conditions for HPLC analysis ]

HPLC: LC-10Avp series, conference (Shimadzu corporation)

A chromatographic column: X-Bridge RP18, 2.0mm I.D. X50 mm, 2.5 μm (Waters corporation)

Mobile phase: a10 mM ammonium formate in water and B acetonitrile

[ MS/MS analysis conditions ]

MS：API 4000(AB/MDS SCIEX，Inc.)

The ionization method comprises the following steps: turbo ion spray (positive or negative ion)

Ionization mode: atmospheric Pressure Chemical Ionization (APCI)

Detection mode: MRM

And (3) analysis: pharmacokinetic parameters were calculated based on the plasma concentrations of each drug using WinNonlin Professional (version 4.0.1, Pharsight Corporation). Incidentally, a non-compartmental model (Noncompartment model) was used as a model for calculating the parameters.

(results) evaluation was made on the compounds of example 1, isomer B of example 2, isomer D of example 3, example 4, isomer E of example 5 and example 6. As a result, as shown in Table 2, isomer B, isomer D and isomer E are atropisomers which were confirmed to have high activity in test example 1, and the plasma concentration was greatly increased as compared with the corresponding racemic compounds of example 1, example 3 and example 5.

(Table 2)

Test compound	AUC1(ng·h/mL)	Cmax2(ng/mL)
			Example 1	860	42
Isomer B	3446	184
			Example 3	33	2
Isomer D	7187	681
			Example 5	7667	380
Isomer E	26390	1330

¹: AUC (ng. h/mL): area under the plasma concentration (determined by LC-MS/MS) versus time (0-48 hours);

²: cmax (ng/mL): maximum concentration

(preparation example 1) capsules

Mixing the above powders, sieving with 60 mesh sieve, and encapsulating with No. 3 gelatin capsule to obtain capsule containing 250mg of the above powders.

(formulation example 2) tablet

Isomer D50.0 mg

Lactose 124.0

Corn starch 25.0

Magnesium stearate 1.0

200mg

The powders of the above formula were mixed and compressed by a tablet press to prepare tablets (200 mg/tablet).

[ Industrial Applicability ]

The atropisomers of the compound having the general formula (I) of the present invention exhibit particularly excellent pharmacological activities such as mineralocorticoid receptor antagonistic action, antihypertensive action ], vasodilatory action, cardioprotective action, kidney disease inhibitory action, anti-arteriosclerosis action, diuretic action and the like, and are also high in safety, and therefore, can be used as a preventive or therapeutic drug for hypertension, angina pectoris, acute coronary syndrome, congestive heart failure, kidney disease, arteriosclerosis, cerebral infarction, fibrosis or primary aldosteronism.

Claims (8)

1. Atropisomers selected from the group consisting of:

(-) -1- (2-hydroxyethyl) -4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide;

(+) -1, 4-dimethyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide; and

(-) -1- (2-hydroxyethyl) -5- [ 4-methoxy-2- (trifluoromethyl) phenyl ] -4-methyl-N- [4- (methylsulfonyl) phenyl ] -1H-pyrrole-3-carboxamide.

2. An atropisomer according to claim 1, which is (-) -1- (2-hydroxyethyl) -4-methyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide.

3. An atropisomer according to claim 1, which is (+) -1, 4-dimethyl-N- [4- (methylsulfonyl) phenyl ] -5- [2- (trifluoromethyl) phenyl ] -1H-pyrrole-3-carboxamide.

4. An atropisomer according to claim 1, which is (-) -1- (2-hydroxyethyl) -5- [ 4-methoxy-2- (trifluoromethyl) phenyl ] -4-methyl-N- [4- (methylsulfonyl) phenyl ] -1H-pyrrole-3-carboxamide.

5. A medicament containing an atropisomer according to any one of claims 1 to 4 as an active ingredient.

6. A preventive or therapeutic agent for cardiovascular diseases, which comprises the atropisomer according to any one of claims 1 to 4 as an active ingredient.

7. A prophylactic or therapeutic agent for hypertension, which comprises the atropisomer according to any one of claims 1 to 4 as an active ingredient.

8. A pharmaceutical composition comprising an atropisomer according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.