HK1074775B - Remedy for hypertension - Google Patents

Description

Therapeutic agent for hypertension

Technical Field

The present invention relates to a novel therapeutic agent for hypertension.

Background

Intracellular free Ca²⁺Has important effects in controlling myocardial or various smooth muscle contraction, neurotransmitter release, and gene expression, and can regulate Ca in cell membrane and muscle vesicle membrane²⁺Pump, Ca²⁺Channels and Na⁺/Ca²⁺Exchange transporter (NCX) to regulate Ca²⁺The concentration of (c). In particular Na therein⁺/Ca²⁺The exchanger transporter plays an important role in contraction and relaxation of cardiac and vascular smooth muscle (ann. rev. physiol. 52, pp.467 (1990)). Currently, 3 NCX genes are isolated and identified from mammals. And it was found that the NCX1 protein is expressed in a large amount in the brain, heart, kidney, that the NCX2 protein is mainly expressed in the brain and expressed in a small amount in the smooth muscle of the internal organ, and that the NCX3 protein is mainly expressed in the brain and expressed in a small amount in the skeletal muscle (blood vessel, Vol.24, No.3, p.101 (2001), am. J. physiol., 272, C1250-C1261 (1997)).

In addition, the effectiveness of K-BR7943 in acute myocardial infarction models and ischemia reperfusion models of the brain and kidney was confirmed in reports on isothiourea derivatives such as 2- [2- [4[ nitrobenzyloxy ] phenyl ] ethyl ] isothiourea methanesulfonate (KB-R7943) or phenoxyaniline derivatives such as 2- [4- [ (2, 5-difluorophenyl) methoxy ] phenoxy ] -5-ethoxyaniline (SEA0400) as NCX inhibitors (J.Pharmacol. exp. ther. 296, page 412 (2001)). However, there have been no reports on the use of NCX inhibitors in the treatment of hypertension.

Disclosure of Invention

The present inventors used Na prepared from brain, heart and kidney⁺/Ca²⁺Transporter protein (NCX) inhibition by K-BR7943, SEA0400, etc. was determined. The results showed that phenoxyaniline represented by SEA0400 was derived from NCX derived from brain tissueThe substance and the phenoxypyridine derivative have selective inhibitory effects on NCX from heart and kidney.

In addition, studies so far have shown that the above-mentioned compounds hardly have any effect on other receptors, channels and transporters at concentrations that inhibit NCX (j. pharmacol. exp. ther. 298, page 249 (2001)).

The above results show that phenoxyaniline derivatives and the like have high selectivity to NCX 1.

In addition, the present inventors have conducted studies on the use of a NCX1 selective inhibitor in various disease models (diabetic rats, salt-sensitive hypertension models, and the like) in order to reveal the relationship between NCX1 and diseases and treatments, and as a result, have found that the inhibition of NCX1 in the salt-sensitive hypertension model can effectively lower blood pressure, and have completed the present invention.

Namely, the present invention relates to the inhibition of Na⁺/Ca²⁺A hypertension therapeutic agent comprising a compound that exchanges transporter 1(NCX1) as an active ingredient.

The therapeutic agent for hypertension of the present invention comprises a 2-phenoxyaniline derivative represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient,

in the formula, R¹Represents a hydrogen atom or C₁-C₆Alkoxy radical, R²Represents a halogen atom or a nitro group,

R³represents a hydrogen atom or a halogen atom.

Detailed Description

The NCX 1-inhibiting compound of the present invention is not particularly limited as long as it has an inhibitory effect on kidney-derived NCX1, and is preferably a compound that inhibits at least 50% at a concentration of 3 μ M in the test (reference example 3) described later. Furthermore, for the purpose of preventing side effects, a compound having a specific inhibitory action on NCX1 is preferable.

The compound having a specific inhibitory activity against NCX1 means a compound which hardly exhibits an inhibitory activity against other receptors, channels and transport cross-over proteins at a concentration at which NCX1 is inhibited, and particularly preferably, for example, Ca at a concentration of 3. mu.M²⁺Channel, Na⁺Channel, K⁺Channel, Na⁺/H⁺Transporter, norepinephrine transporter, Na⁺、K⁺-ATPase, Ca²⁺-ATPase, phospholipase A₂Compounds that inhibit less than 50% of phospholipase C, 5-lipoxygenase, inducible nitric oxide synthase, constitutive nitric oxide synthase, adenosine receptors, adrenergic receptors, glutamate receptors, bradykinin receptors, LTB4 receptors, PAF receptors, and the like. The measurement method using each ion channel, enzyme, and receptor was performed according to the method described in j.pharmacol.exp.ther. 298, 249 page (2001) and the documents cited therein.

Examples of the compound specifically inhibiting NCX1 include phenoxyaniline derivatives and phenoxypyridine derivatives.

Preferably a compound represented by the formula (2) or a pharmaceutically acceptable salt thereof,

in the formula, R⁴、R⁵And R⁶The same or different, represent a hydrogen atom or a halogen atom. X represents

R⁷Represents a hydrogen atom, a substituted or unsubstituted C₁-C₆Alkyl or substituted or unsubstitutedC₁-C₆An alkoxy group. Z represents nitro, amino or NHC (O) CH₂R⁸。R⁸Represents a hydrogen atom, a substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₁-C₆Alkoxy group, halogen atom, hydroxy group, C₂-C₇Acyloxy group, NR⁹R¹⁰Or

R⁹And R¹⁰The same or different, represent a hydrogen atom, a substituted or unsubstituted C₁-C₆Alkyl, N-methyl-4-piperidinyl, R¹¹Represents a hydrogen atom, a hydroxyl group or C₂-C₇Alkoxycarbonyl, Y represents methylene, epoxy, thio or NR¹²N represents an integer of 1 to 4, R¹²Represents a hydrogen atom, a substituted or unsubstituted C₁-C₆Alkyl or substituted or unsubstituted phenyl.

From the viewpoint of the inhibitory activity against NCX1, a 2-phenoxyaniline derivative represented by formula (1) or a pharmaceutically acceptable salt thereof is preferable,

in the formula, R¹Represents a hydrogen atom or C₁-C₆Alkoxy radical, R²Represents a halogen atom or a nitro group, R³Represents a hydrogen atom or a halogen atom.

In the formulae (1) and (2), C₁-C₆The alkoxy group means a straight-chain or branched alkoxy group having 1 to 6 carbon atoms, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy, tert-pentyloxy, 1-methylbutyloxy, 2-methylbutyloxy, 1, 2-dimethylpropoxy, hexyloxy, isohexyloxy and the like.

As a substituent C₁-C₆Substituents for alkoxy groups, for example, chlorine atom, fluorine atom, nitro group, amino group, dimethylamino group, carboxyl group, methoxycarbonyl group, ethoxycarbonyl group, phenyl group, hydroxyl group, cyano group, carbamoyl group and the like.

The halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

As C₁-C₆Alkyl means a straight-chain or branched alkyl group having 1 to 6 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl, 1, 2-dimethylpropyl, hexyl, isohexyl and the like.

As a substituent C₁-C₆Examples of the substituent for the alkyl group include a chlorine atom, a fluorine atom, a nitro group, an amino group, a dimethylamino group, a carboxyl group, a methoxycarbonyl group, an ethoxycarbonyl group, a phenyl group, a methoxy group, an ethoxy group, a hydroxyl group, a cyano group, a carbamoyl group and the like.

As C₂-C₇The acyloxy group means a linear or branched acyloxy group having 2 to 7 carbon atoms, and the acyl moiety may be cyclic or may contain an aromatic group. For example, acetoxy, propionyloxy, isopropionyloxy, cyclohexanoyloxy, benzoyloxy and the like.

As C₂-C₇The alkoxycarbonyl group means a linear or branched alkoxycarbonyl group having 2 to 7 carbon atoms, and the alkoxy moiety may be cyclic or may contain an aromatic group. Specific examples thereof include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, isopentyloxycarbonyl, neopentyloxycarbonyl, tert-pentyloxycarbonyl, 1-methylbutoxycarbonyl, 2-methylbutoxycarbonyl, 1, 2-dimethylpropoxycarbonyl, hexyloxycarbonyl and isohexyloxycarbonyl.

Examples of the substituent for the substituted phenyl group include a chlorine atom, a fluorine atom, a nitro group, an amino group, a dimethylamino group, a carboxyl group, a methoxycarbonyl group, an ethoxycarbonyl group, a methyl group, an ethyl group, a methoxy group, an ethoxy group, a hydroxyl group, a cyano group, and a carbamoyl group.

Specific examples of the compounds having an excellent antihypertensive effect include compounds represented by the following formula (SEA0400)

And formula

The compound (SEA 0064).

For the purpose of preventing side effects, compounds having stronger inhibitory activity against NCX1 than NCX2 and NCX3 are preferable. For example, Na described later⁺/Ca²⁺Among the methods for measuring the transport protein exchange, the method is preferred

IC₅₀(from the renal cortex)/IC₅₀(from the brain)

IC₅₀(from the renal cortex)/IC₅₀(from myocardium)

Is less than the calculated value when SEA0400 is used.

The compounds represented by the formulae (1) and (2) can be synthesized by the production methods described in WO98/43943, WO99/20598, JP-A-10-265460, JP-A-10-218844, JP-A-11-49752, and JP-A-11-92454.

The invention relates to a medicament for treating hypertension, which is a medicament for treating salt-sensitive hypertension, renal hypertension, primary hypertension, gestational hypertension and primary aldosteronism.

The therapeutic agent of the present invention can be formulated into appropriate pharmaceutical compositions (tablets, pills, capsules, granules, dry syrups, injections, etc.) with appropriate known carriers, diluents, etc., and administered orally or parenterally.

In the case of producing a solid preparation, various additives such as an excipient, a disintegrant, a binder, a lubricant, and a coating base can be used, and the preparation can be carried out by a stirring granulation method, a fluidized bed granulation method, and a crushing granulation method.

An antioxidant, a coating agent, a coloring agent, a flavoring agent, a surfactant, a plasticizer, and the like may be added as necessary.

The dosage of the active ingredients of the medicine of the invention varies according to age, weight, administration form and the like, and adults usually take the medicine once or several times a day at a dose of 0.1-1000 mg/day.

The present invention will be described below by way of formulation examples and test examples, but the present invention is not limited to these test examples.

Formulation example 1

SEA0400 50mg

Lactose 40mg

Corn starch 49.75mg

Microcrystalline cellulose 17mg

Carboxymethylcellulose calcium 17mg

Hydroxypropyl cellulose 5.25mg

Magnesium stearate 1mg

Total amount of 180mg

Mixing SEA0400, lactose, corn starch, microcrystalline cellulose and carboxymethylcellulose calcium uniformly, adding 10% hydroxypropyl cellulose water solution, mixing, drying, sieving with 30 mesh sieve to obtain uniform granule, adding magnesium stearate, and tabletting.

Preparation example 2

SEA0464 50mg

Lactose 40mg

Corn starch 49.75mg

Microcrystalline cellulose 17mg

Carboxymethylcellulose calcium 17mg

Hydroxypropyl cellulose 5.25mg

Magnesium stearate 1mg

Total amount of 180mg

Mixing SEA0464, lactose, corn starch, microcrystalline cellulose, and carboxymethylcellulose calcium uniformly, adding 10% hydroxypropyl cellulose water solution, mixing, drying, sieving with 30 mesh sieve to obtain uniform granule, adding magnesium stearate, and tabletting.

Reference example 1 brain microsome Na⁺/Ca²⁺Method for determining cross-over transporters

Microsomes (1.5mg/ml) obtained from the brain of a rat (8 weeks old) were pretreated with a buffer containing 160mM NaCl, and Na was loaded into vesicles (membrane derived dve)sicles). The suspension was diluted with a solution containing 20. mu.M⁴⁵CaCl₂50-fold dilution of buffer (2) induced⁴⁵After uptake of Ca, the reaction was stopped by dilution with a buffer (0 ℃ C.), and the vesicles on the nitrocellulose membrane were recovered quickly. Then the liquid scintillation method is used to treat the vesicle⁴⁵Ca was quantified. Na in the above-mentioned brain microsomes can be treated according to the method of J.biol.chem.257, page 5111 (1982)⁺/Ca²⁺The exchange activity was measured.

Reference example 2 dog myocardial vesicle Na⁺/Ca²⁺Method for determining cross-over transporters

Dog myocardial vesicles (0.5mg/ml) were prepared by centrifugal fractionation as described in Methods Enzymology, volume 157, page 85 (1988), suspended in solution A (20mM MOPS-Tris (pH7.4), 160mM NaCl or KCl), and left to stand at room temperature for about 1 hour to load Na or K into the vesicles. The suspension was diluted with a solution containing 20. mu.M⁴⁵CaCl₂50-fold dilution of buffer (2) induced⁴⁵After uptake of Ca, the reaction was stopped by dilution with a buffer (0 ℃ C.), and the vesicles on the nitrocellulose membrane were recovered quickly. Then the liquid scintillation method is used to treat the vesicle⁴⁵Ca was quantified. Evaluation of Na was made by subtracting the value obtained when K was loaded from the value obtained when Na was loaded⁺/Ca²⁺Exchange activity. The Na in the dog myocardial membrane vesicles described above was treated according to the method of J.biol.chem.257, page 5111 (1982)⁺/Ca²⁺The exchange activity was measured.

Reference example 3 preparation method of BLMV (basal membranes) derived from the renal cortex of white rat and Na⁺/Ca²⁺Method for measuring exchange activity

(method of modulating BLMV)

Preparation of BLMV from rat Kidney cortex and Na⁺/Ca²⁺The exchange activity can be determined according to the method described in am.J.Physiol.266, page F785 (1994).

After the kidney of the rat was removed, the rat was placed in an ice-cooled sucrose buffer (0.25mM sucrose, 0.1mM PMSF, 10mM Tris-HCl (pH7.6)) to remove the coating, and the separated cortex was finely cut in the sucrose buffer. The slurry was homogenized by a Dounce type homogenizer and then homogenized by a Polytron type homogenizer. Then, 2500g of the supernatant was centrifuged for 15 minutes to recover the supernatant. The pellet was centrifuged at 24000g for 20 minutes to recover a white loose fraction. Sucrose buffer was added thereto, the mixture was homogenized by a Dounce type homogenizer, Percoll (trade name) was added thereto, and then the mixture was centrifuged at 30000g for 35 minutes to collect an intermediate layer. After further addition of a buffer (100mM KCl, 100mM mannitol, 5mM HEPES-Tris (pH7.4)), 34000g was centrifuged for 30 minutes to recover a loose white pellet (BLMV). After suspending again in KCl-mannitol buffer, the pellet was recovered by centrifugation at 34000g for 30 minutes and used for the activity measurement.

(Na⁺/Ca²⁺Transporter (Na-dependent)⁴⁵Uptake of Ca) measurement method

The BLMV was pre-equilibrated buffer (100mM NaCl, 40mM KCl, 1mM MgSO)₄10mM glucose, 5mM HEPES-Tris (pH7.4)) was equilibrated (37 ℃ for 10 minutes), centrifuged (20000g, 5min), and the precipitate was recovered and resuspended in a pre-equilibration buffer. After the pellet was recovered by centrifugation again, it was resuspended in the pre-equilibration buffer. The BLMV suspension was applied to external media (100mM choline hydrochloride, 40mM KCl, 1mM MgSO)₄10mM glucose, 5mM HEPES-Tris (pH7.4), 25. mu.M valinomycin, 10. mu.M CaCl₂，1mCi/l ⁴⁵CaCl₂) Ingestion was started by dilution 20 times. After a reaction at 25 ℃ for a certain period of time, 2ml of a solution (stop solution) (150 mM KCl cooled with ice) for terminating the reaction was added thereto, and after the reaction was stopped, the reaction mixture was rapidly filtered through an ultrafiltration membrane (0.45. mu.M nitrocellulose filter), and BLMV was recovered from the filter. Then washing with 2ml of the termination reaction solution 2 times, and quantitatively adding to BLMV by liquid scintillation⁴⁵Ca。

Table 1: na (Na)⁺/Ca²⁺Exchange inhibition Activity (Na dependence)⁴⁵Intake of Ca)

(IC₅₀The value: mu M)

	Brain	Myocardium	Renal cortex
				K-BP7943	11.0	7.0	6.8
SEA0400	0.2	0.09	0.02

In addition, Na is present in the renal cortex⁺/Ca²⁺In the measurement of the exchange activity, the inhibitory activity when SEA0064 was used was found to be about 0.07 times that of SEA 0400.

Test example 1

(method)

Production of Dah1 salt-sensitive hypertension rat model

A Dah1 salt-sensitive hypertensive rat (7 weeks old) was selected for the experiment, and given a high-salt feed (0.6% in normal feed) formulated with 4% NaCl, water was freely taken. After 2 weeks of high salt load, hypertension induction was confirmed, and the test was conducted in 4 groups by the following method.

(Experimental animal group)

Group I: solvent (20% fat emulsion)

Group II: SEA04003mg/kg

Group III: SEA 040010 mg/kg

Group IV: SEA040030mg/kg

(Experimental procedure)

After 2 weeks of high-salt feeding using the above method, acute experiments were performed. Systolic blood pressure (pre-dose value) was measured on the day of the experiment with a non-invasive indirect blood pressure measuring apparatus under a non-anesthesia condition. Then, the solvent and SEA0400(3, 10, 30mg/kg) were orally administered, respectively, and the systolic blood pressure over time was measured.

(results)

The systolic blood pressures of groups I, II and III before administration were 140. + -.3, 148. + -.4, 143. + -.3 and 142. + -. 4mmHg, respectively. The rate of change of blood pressure 1 hour after administration was: the blood pressure values of the solvent group were-3.8. + -. 1.0%, the SEA04003mg/kg group was-9.5. + -. 1.8%, the SEA 040010 mg/kg group was-12.0. + -. 0.9%, and the SEA040030mg/kg group was-12.7. + -. 3.4%, which were significantly lower in the groups administered with 10 and 30mg/kg than in the solvent group.

Application in industry

The present invention provides a novel mechanism of action-based therapeutic agent for hypertension, which is a therapeutic and prophylactic agent for hypertension with fewer side effects.

Claims (1)

1. Use of a compound represented by the formula (1) for producing a salt-sensitive therapeutic agent for hypertension,