KR20020036817A - Pharmaceutical compositions containing tetrahydroisoquinoline compounds - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing tetrahydroisoquinoline compounds exhibiting an antiplatelet aggregation inhibiting action and iNOS inhibiting action except a cardiotonic action and blood pressure lowering action is provided which can be effectively as an agent for treatment of cardiac insufficiency. CONSTITUTION: This pharmaceutical composition contains as an effective component a compound selected from the group consisting of 1-α -naphtylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline represented by the formula 1 and 1-β-naphtylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline represented by the formula 2 and mixtures thereof.

Description

Pharmaceutical compositions containing tetrahydroisoquinoline compounds

The present invention relates to a novel use of the tetrahydroisoquinoline-based compounds represented by the following formula (1) and (2), specifically the use of the tetrahydroisoquinoline-based compound represented by the formula (1) and formula (2) as a therapeutic agent for heart failure It is about. In addition to cardiovascular and hypotensive effects, these compounds exhibit platelet aggregation and iNOS (induced NO synthase) inhibitory effects, which can lead to heart failure without any combination of other cardiovascular agents, vasodilators, antithrombotic agents, or iNOS tissue damage inhibitors. May be effective in treatment or prevention.

Formula 1

Formula 2

Heart failure, specifically congestive heart failure, is a syndrome caused by a number of conditions, including coronary artery disease, myocardial disease, and refractory disease, which is characterized by a decrease in cardiac output, congestion of various organs, and sympathetic nervous system excitement. It is characteristic. These heart failures include congestion of the liver and gastrointestinal liver and kidneys due to increased right ventricular pressure, reduction of these organ perfusions due to a decrease in cardiac output, retention of water and Na ⁺ due to a decrease in kidney blood flow, and consequent increase in fluid, swelling. In addition, the body's dynamics change due to redistribution of blood flow due to sympathetic nervous system excitation and changes in motility of the gastrointestinal tract. There is also strong evidence that lipid peroxide radicals persist in patients with heart failure.

Therefore, in order to effectively treat the heart failure, cardiac agents for improving contractility of weakened myocardium, vasodilators for relieving dyspnea by increasing cardiac output, antithrombotic agents for inhibiting thrombogenesis by inhibiting platelet aggregation. It was necessary to co-administer the tissue damage inhibitor by iNOS etc. which inhibits the tissue damage by this. Diuretics are also given to reduce swelling and congestion caused by salt and water retention.

Tetrahydroisoquinoline (hereinafter abbreviated as "THI") compounds are those in which N-alkylphenylethylamine is ring closed, especially 6,7-dihydroxytetrahydroisoquinolines It has a basic skeleton of catecholamines in its structure. That is, it has 3,4-dihydroxyphenylethylamine, which is a basic skeleton of catecholamines represented by epinephrine, norepinephrine, dopamine, and the like. Therefore, many THI-based compounds show affinity for various adrenergic receptors and act on α- or β-receptors depending on the type of substituents and the position of the substituents, resulting in an agonistic or antagonistic effect. It has been reported that it has various pharmacological actions.

In particular, it has been reported that THI series compounds having a benzyl group substituted with OH, OCH ₃ and halogen in the carbon at position 1 have strong actions such as bronchial relaxation, platelet aggregation inhibitory activity, and calcium channel inhibitory action (King, VF et al., J. Biol. Chem., 263, 2238-2244, 1988; Triggle, DJ et al., Med.Res. Rev., 9, 123-180, 1989; Lacorix, P. et al., Eur. J. Pharmacol., 192, 317-327, 1991; Chang, KC et al., Life. Sci., 51, 64-74, 1992; Chang, KC et al., Eur. J. Pharmacol., 238 , 51-60, 1993).

In addition, recently, substances such as bisbenzyl-tetrahydroisoquinoline-based compounds, such as tetrarandrine, isoterandrandrine and condocurine, are endotoxin lipopolysaccharides (lipopolysaccharide; It has been reported to strongly inhibit the production of nitrogen monoxide (NO) in large quantities by abbreviating "LPS" (Kondo, Y. et al., Biochem. Pharmacol., 46, 1861-). 1863, 1993).

Higenamine is a THI-based compound having 4-hydroxybenzyl on carbon 1 and two hydroxyl groups on positions 6 and 7, and structurally similar to dobutamine, which is used as a cardiovascular drug in clinical practice. Is very similar. This compound has been reported to increase myocardial contractility and heart rate in the isolated heart, in vitro effects such as inhibiting platelet aggregation, increased cardiac output, lowering blood pressure and inhibiting platelet aggregation in rats or rabbits. It has been. Hygenamine also reduced the expression of inducible NO synthase (hereinafter abbreviated "iNOS") by LPS in mouse peritoneal macrophages and rat isolated blood vessels, thereby reducing the production of large amounts of NO. It has been reported to suppress vascular reactivity and lower mortality caused by endotoxin shock. It has also been observed to have anti-inflammatory and analgesic effects in arthritis models (Park, CW et al., Arch. Int. Pharmacodyn., 267, 279-288, 1984; Chang, KC et al., Can. J.). Physiol.Pharmacol., 72, 327-334, 1994; Yun-Choi, HS et al., Yakhak Hoeju, 38, 191-196, 1994; Kang, YJ et al., Kor.J. Physio. Phamacol. 1 , 297-302, 1997; Shin, KH et al., Natural Products Sciences, 2, 24-28, 1996). However, in the case of hygenamine, the duration of its action is very short, and the effects of the above-mentioned actions are also insufficient.

The present inventors have tried to obtain a substance exhibiting excellent pharmacological effects by changing the structure of hygenamine, and thus synthesized the compounds of the formulas (1) and (2), which are novel substances having a very long duration of action, It has been found and patented (Korean Patent No. 148755). In addition, similar to hygenamine, the compounds of Formulas 1 and 2 exhibit potent myocardial contractility and heart rate in experiments using a rat-extracted heart, and have strong relaxation on the extracted blood vessels contracted with phenylephrine. It has also been reported to increase the heart rate and lower blood pressure when the compound of Formula 1 and Formula 2 is administered to rabbits (Lee, YH et al., Life Sciences, 55, PL415-420, 1994, Chong WS et al. Kor. J. Physiol. Pharmacol. 2, 323-330, 1998, Chang KC et al., Kor. J. Physiol. Pharmacol. 2, 461-469, 1998). However, the above patents and documents only disclose that the compounds of formulas (1) and (2) can play a role as cardiac agents and / or blood pressure enhancers, and therefore, in order to use them in the treatment of heart failure, other components, platelet aggregation inhibitory action, It has a problem that it should be administered in parallel with an antithrombotic agent having an anti-thrombotic agent and a tissue damage inhibitor by iNOS having an iNOS (induced NO synthase) inhibitory action.

Current agents for treating congestive heart failure include digitalis cardiac glycosides and dopamine. Digitalis cardiac glycosides have the advantage that oral administration is possible, but the safety zone is very narrow, resulting in risk of use and arrhythmia. In addition, sympathetic mimicry drugs such as dopamine and dobutamine are effective as a treatment for congestive heart failure, but have the disadvantage of intravenous injection and have problems such as down regulation of the sympathetic nervous system. In addition, the above-mentioned digitalis cardiac glycosides and dopamines should be administered in combination with vasodilators, antithrombotic agents, and tissue damage inhibitors by iNOS in order to be used for the treatment of heart failure as a cardiac agent. However, due to the complex administration of several drugs, there is a problem in that there are a lot of restrictions in use such as the increase in the risk of side effects due to the in vivo absorption of each drug and the drug metabolism due to the drug interaction by each drug. .

On the other hand, excessively generated oxygen free radicals, including NO, are recently caused by endotoxins caused by tissue damage or bacterial infection during reperfusion in inflammatory diseases such as arthritis, or myocardial infarction, stroke, or ischemic diseases. It has been found to be one of the major causes of various acute tissue / organ damage, such as complex organ damage. Therefore, a lot of attention has been focused on securing substances that inhibit the expression of iNOS or inhibit the production of large amounts of NO, and use them as therapeutic agents for various diseases caused by large amounts of NO. These substances are also expected to have the effect of inhibiting or treating the progression of heart failure by inhibiting myocardial damage caused by acute myocardial infarction, ischemic heart disease, and the like.

Accordingly, the present inventors have been working to solve the various problems described above, and the compounds of the formulas (1) and (2) simultaneously have a combination of cardiac action, vascular relaxation (blood pressure lowering), platelet aggregation, and iNOS inhibition. The present invention was completed by confirming that the present invention can be very usefully used as a therapeutic agent for heart failure without concurrent administration of other drugs.

It is an object of the present invention to provide new uses of compounds of formula 1 and compounds of formula 2.

Still another object of the present invention is to provide a pharmaceutical composition for treating heart failure, which contains only the compound of Formula 1 and the compound of Formula 2 as an active ingredient without concurrent administration of other drugs.

1 is a Northern blotting result showing that the compound of Formula 1 inhibits the expression of induced NO synthase (iNOS) mRNA by lipopolysaccharide (LPS) in an isolated blood vessel of a rat.

Lane 1: control group

Lane 2: LPS (300 ng / ml)

Lane 3: LPS (300 ng / ml) + compound of Formula 1 (10 μM)

Lane 4: LPS (300 ng / ml) + compound of Formula 1 (30 μM)

Lane 5: LPS (300 ng / ml) + compound of Formula 1 (100 μM)

2 is a Northern blot result showing that the compound of Formula 2 inhibits the expression of iNOS mRNA by LPS and interferon-kV (IFN-kV) in macrophages (RAW 264.7 cell),

Lane 1: control group

Lane 2: LPS (100 ng / ml) + IFN-γ (10 U / ml)

Lane 3: LPS (100 ng / ml) + IFN-γ (10 U / ml) + Compound of Formula 2 (1 μM)

Lane 4: LPS (100 ng / ml) + IFN-γ (10 U / ml) + Compound of Formula 2 (10 μM)

Lane 5: LPS (100 ng / ml) + IFN-γ (10 U / ml) + Compound of Formula 2 (100 μM)

Figure 3 shows the change in platelet count in blood when LPS (20 mg / kg) is administered to rats over 3 hours after oral administration of hygenamine, a compound of Formula 1 or a compound of Formula 2,

4 shows changes in prothrombin time (PT) when LPS (20 mg / kg) is administered to rats over 3 hours after oral administration of hygenamine, a compound of Formula 1 or a compound of Formula 2,

FIG. 5 shows the activated partial thromboplastin time (aPPT) when LPS (20 mg / kg) is administered to rats over 3 hours after oral administration of hygenamine, a compound of Formula 1 or a compound of Formula 2 Represents a change in,

FIG. 6 shows changes in blood fibrinogen concentrations when LPS (20 mg / kg) was administered to rats over 3 hours after oral administration of hygenamine, a compound of Formula 1, or a compound of Formula 2,

FIG. 7 shows the concentration of fibrin / fibrinogen degradation products (FDP) in blood when LPS (20 mg / kg) was administered to rats over 3 hours after oral administration of hygenamine, a compound of Formula 1 or a compound of Formula 2. Represents a change,

FIG. 8 shows the change in S-GOT when LPS (20 mg / kg) was administered to rats over 3 hours after oral administration of hygenamine, a compound of Formula 1 or a compound of Formula 2,

9 shows changes in S-GPT when oral administration of hygenamine, a compound of Formula 1 or a compound of Formula 2 to rats over 3 hours with LPS (20 mg / kg),

FIG. 10 shows changes in BUN concentration when LPS (20 mg / kg) was administered to rats over 3 hours after oral administration of hygenamine, a compound of Formula 1, or a compound of Formula 2,

Figure 11a shows the degree to which hygenamine lowers the mortality rate of mice caused by LPS,

-○-: LPS (20 mg / kg)

-△-: LPS (20 mg / kg) + Hygenamine (10 mg / kg)

-□-: LPS (20 mg / kg) + Hygenamine (20 mg / kg)

11b shows the degree to which the compound of formula 1 lowers the mortality rate of mice caused by LPS,

-○-: LPS (20 mg / kg)

-△-: LPS (20 mg / kg) + Compound of Formula 1 (10 mg / kg)

-□-: LPS (20 mg / kg) + Compound of Formula 1 (20 mg / kg)

Figure 11c shows the degree to which the compound of formula 2 reduces the mortality rate of mice caused by LPS.

-○-: LPS (20 mg / kg)

-△-: LPS (20 mg / kg) + Compound of Formula 2 (10 mg / kg)

-□-: LPS (20 mg / kg) + Compound of Formula 2 (20 mg / kg)

In order to achieve the above object, in the present invention, 1-α-naphthylmethyl-6,7-dihydride, which is a compound represented by the following Chemical Formula 1, which simultaneously shows the action of cardiac activity, blood pressure lowering, platelet aggregation, and iNOS At least one of oxy-1,2,3,4-tetrahydroisoquinoline and 1-β-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline of formula It provides a pharmaceutical composition for preventing or treating heart failure containing as an active ingredient.

Formula 1

Formula 2

Hereinafter, the present invention will be described in detail.

In the present invention, 1-α-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline represented by Chemical Formula 1, which is a THI compound, and 1-β represented by Chemical Formula 2 It provides a new use of -naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline.

In this case, the compounds of Formula 1 and Formula 2 include their tautomer isomers, and the compound corresponding to the present invention includes at least one asymmetric center, thereby including enantiomers. Also, if there are two or more asymmetric centers, diastereoisomers may be present. All such isomers and mixtures thereof are included within the scope of the present invention. Also included in the present invention are pharmaceutically acceptable salts of the compounds of Formula 1 and Formula 2 and their prodrugs.

Herein, salts of the formula (1) or (2) refer to conventional salts used in pharmaceuticals, and various acids used for preparing the salts include hydrochloric acid, bromic acid, sulfuric acid, methanesulfonic acid, propionic acid, succinic acid, glutaric acid, and citric acid. , Fumaric acid, maleic acid, tartaric acid, glutamic acid, gluconic acid, glucuronic acid, galacturonic acid, ascorbic acid, carbonic acid, phosphoric acid, nitric acid, acetic acid, L-aspartic acid, lactic acid, vanic acid and hydroiodic acid.

As described above, the present invention includes a prodrug of a compound of Formula 1 or Formula 2, which is generally a functional derivative of a compound of Formula 1 or Formula 2, and is easily changed to show its efficacy in entering a living body. It should be possible. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in the existing literature (Design of Prodrug, ed. H. Bundgaard, 1985).

1-α-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, a compound of Formula 1, and 1-β-naphthylmethyl-6,7, a compound of Formula 2 -Dihydroxy-1,2,3,4-tetrahydroisoquinoline is a pharmaceutical compound containing one or more of these as an active ingredient by simultaneously exhibiting a combination of cardiac action, hypotension, platelet aggregation, and iNOS inhibition The red composition may be very usefully used as a prophylactic or therapeutic agent for heart failure.

In order to treat heart failure, current clinical practice alone or in combination with the cardiac glycosides, digitalis agents, vasodilators, calcium antagonists, angiotensin converting enzyme (ACE) inhibitors, etc., to improve myocardial contractility and reduce the burden on the heart. It is used as a combination therapy because the hemodynamic improvement is expected by co-administration of these drugs for the purpose of improving circulatory function and showing cardiac action by vasodilation. On the other hand, the compounds of the formula (1) and (2) of the present invention satisfies both requirements in that it is a cardiac-releasing agent (inodilator) that can simultaneously exhibit a cardiac action and vascular relaxation action.

In addition, cardiac insufficiency causes severe symptoms such as atherosclerosis, hypertension, and circulatory diseases such as prolonged periods of time, increasing the burden on the heart, or ischemic heart disease such as coronary artery disease or myocardial infarction caused by blood clots. Higher people are strongly encouraged to continue to administer platelet aggregation inhibitors for the purpose of preventing, recurring or preventing the progression of various heart and circulatory diseases. Accordingly, the compounds of formulas (1) and (2) of the present invention have antithrombotic action by inhibiting platelet aggregation, thereby fulfilling another requirement as a therapeutic agent for heart failure. In addition, patients with chronic heart failure have increased immune activity and increased expression of iNOS in the intestine, blood vessels, etc. At this time, a large amount of NO is produced to inhibit the contractile force of the myocardium and damage tissues. The compound also has the effect of inhibiting the production of iNOS by inhibiting NO production, thereby inhibiting tissue damage caused by oxygen free radicals, and reducing the occurrence of NO, one of the causes of the reduction of myocardial contractility in patients with chronic heart failure. . That is, the compounds of the formulas (1) and (2) of the present invention can be used as a therapeutic agent for heart failure as it shows the various actions simultaneously.

The treatment for heart failure is due to myocardial contraction due to acute myocardial infarction, myocardial contraction due to increased immune activity such as chronic inflammation, myocardial contraction due to ischemic heart disease, or persistent hypertension, arteriosclerosis, coronary artery disease. It is to suppress or treat the progression of heart failure caused by congestive heart failure due to myocardial contractile function.

The pharmaceutical compositions of the present invention may be administered orally, intravenously, parenterally or rectally, and may be in the form of injections, capsules, tablets, dragees, suppositories, granules, solutions, suspensions, emulsions, suppositories, and the like.

Pharmaceutical compositions can be used as an active ingredient in admixture with pharmaceutically acceptable carriers, such as organic or inorganic, solid, semisolid or liquid excipients, if necessary, auxiliary substances, stabilizers, wetting or emulsifying agents, buffers. Other commonly used additives may be included in these pharmaceutical compositions.

In the present invention, it was found through experiments that the compound of the present invention maintains blood levels for a considerable time even when administered orally. Accordingly, the dosage of the compound of the present invention is 0.01-5 mg / kg in the case of injection, oral In the case of a dosage, it is 2-200 mg / kg.

Toxicity experiments during oral and intraperitoneal administration of a compound of the present invention showed that 50% lethal dose (LD ₅₀ ) by oral toxicity test was at least 1000 mg / kg or more. In addition, in the intraperitoneal toxicity test, the LD ₅₀ value of 1-α-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline of formula 1 was 290.0 mg / kg, 95% The confidence limit was 235.1 to 359.9 mg / kg, and the LD ₅₀ value was 438.6 mg for 1-β-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline of formula (2). / kg, 95% confidence limit was 350.9 ~ 548.2 mg / kg.

In the present invention, 1-α-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline of formula 1 and 1-β-naphthylmethyl-6,7- of formula 2 To confirm that dihydroxy-1,2,3,4-tetrahydroisoquinoline exhibits a variety of actions as described above, and to compare the effects with hygenamine, which is expected to have a similar action, The experiment was performed. As a result, the compounds of the formulas (1) and (2) have platelet aggregation inhibitory activity, and in particular, epinephrine rather than adenosine diphosphate (abbreviated as "ADP") or collagen (collagen) rather than platelet aggregation Platelet aggregation was strongly inhibited. It is also thought to have an affinity for adrenergic α-receptors, and therefore these substances relax the blood vessels and inhibit platelet aggregation through the α-receptor inhibitory action.

In addition, the compounds of the formulas (1) and (2) inhibit the expression of iNOS by endotoxins, inhibit the production of NO, and in particular exhibit a concentration dependent inhibitory effect.

Compounds of formulas (1) and (2) significantly reduce mortality in mice due to acute thrombus formation, even in in vivo experiments, and for tubes for arterio-venous shunts (hereinafter abbreviated as "AV shunts") It exhibited excellent antithrombotic action, such as suppressing the generation of thrombus generated in the (AVS tube).

On the other hand, endotoxins such as LPS cause various disseminated vascular coagulation. Indicators of these disseminated intravascular coagulation (DIC) include decreased platelet counts in blood, decreased fibrinogen levels, increased concentrations of fibrin / fibrinogen degradation products (FDP), and prothrombin time (prothrombin). PT; and extended partial thromboplastin time (aPTT). The compounds of the present invention inhibit the decrease of blood platelet count and fibrinogen concentration by LPS, increase the concentration of fibrin / fibrinogen breakdown products, and prolong prothrombin time and activated partial thromboplastin time. Various indicators of disseminated intravascular coagulation induced by LPS, such as inhibition and shortening the time, were improved. Meanwhile, it is known that endotoxins increase serum glutamic-oxaloacetic transaminase (S-GOT), serum glutamic-pyruvic transaminase (S-GPT), and blood urea nitrogen (BUN). Various indicators of multiple organ failure (MOF) -induced symptoms induced by LPS were also improved, such as decreasing the increase in GOT, S-GPT and BUN. In addition, since the compounds of the present invention significantly lower the mortality caused by LPS, it can be seen that the effect of protecting against shock caused by endotoxin.

Hereinafter, the present invention will be described in detail by experimental examples. However, the following experimental examples are only illustrative of the present invention, and the present invention is not limited thereto.

Experimental Example 1 Effect of Inhibiting Platelet Aggregation

Human platelet concentrates purchased from the Seoul National University Hospital blood bank were measured using a platelet analyzer (Platelet Analyzer, PLT-4, Texas Instruments) to measure platelet count, and phosphate buffered saline (abbreviated as "PBS"). Platelet-rich plasma (hereinafter, abbreviated as "PRP") was prepared by adjusting the number of platelets to 300-400 × 10 ⁶ / ml by diluting with, and using the same in the experiment. After incubating PRP for 3 minutes at 37 ° C, adenosine diphosphate (ADP), collagen or epinephrine were added as platelet aggregation-inducing substances, and then platelet aggregation was performed using a platelet aggregometer (Chrono-Log Corp. USA, 500VS). Cohesion was measured as a change in turbidity with.

The degree of aggregation inhibition by the test substance was calculated by calculating the inhibition rate by the following equation (1).

A: platelet aggregation degree by aggregation inducing substance

B: Platelet aggregation degree when agglutination inducer and sample were added at the same time

Table 1 shows the aggregation inhibitory effect of the compound of formula 1 and the compound of formula 2 measured using human platelets. On the other hand, hygenamine, which is similar in chemical structure to the compound of Formula 1 and compound of Formula 2 and has antiplatelet aggregation and antithrombotic action, was used as a comparative material.

Inhibition of Platelet Aggregation by Compounds of Formulas 1 and 2 and Hygenamine IC ₅₀ (M) ADP Collagen Epinephrine Hygenamine > 1 × 10 ^-3 7.0 × 10 ^-5 1.9 × 10 ^-5 Compound of Formula 1 7.3 × 10 ^-4 9.2 × 10 ^-5 3.4 × 10 ^-6 Compound of formula (2) 8.6 × 10 ^-4 9.6 × 10 ^-6 6.0 × 10 ^-6 ADP; 1 × 10 ^-5 M, collagen; 2 to 4 x 10 ^-6 g / ml, epinephrine: 2 x 10 ^-6 M

As a result, hygenamine has no inhibitory effect on platelet aggregation by ADP even at a high concentration of 1 × 10 ⁻³ M, whereas the compound of Formula 1 and Formula 2 exhibited IC ₅₀ of 7.3 × 10 ^{−4 to} 8.6 × It was weak as 10 ^-4 M but showed inhibitory effect. For platelet aggregation by collagen, the compound of formula 1 exhibited an inhibitory effect similar to that of hygenamine with IC ₅₀ of 9.2 × 10 ^-5 M, and the compound of formula 2 was higher than that of hygenamine or the compound of formula 1. About 10-fold platelet aggregation inhibitory effect was shown. For platelet aggregation by epinephrine, the IC ₅₀ values of the compounds of Formula 1 and Formula 2 were 3.4 × 10 ⁻⁶ M and 6.0 × 10 ⁻⁶ M, respectively, with hygenamine (IC ₅₀ 19 × 10 ^-6 M). It more strongly inhibited platelet aggregation by epinephrine. As such, the compounds of Formula 1 and Formula 2 showed a stronger inhibitory effect than hygenamine against platelet aggregation induced by ADP or epinephrine, and were similar or more potent than hygenamine for platelet aggregation by collagen. It showed an inhibitory effect.

Experimental Example 2 Inhibition of α-Receptor Binding

To examine whether the test substances have the above-described platelet aggregation inhibitory effect by competitively binding to the adrenergic α-adrenoceptor or have vascular relaxation as previously reported, the rat brain rich in α-receptor A cerebral cortical membrane was prepared to investigate α-receptor binding inhibition.

First, rats (Sprague-Dawley; 250 ± 20 g, unless otherwise noted, use ground rats). Brain tissue (cerebral cortex) was 20 times buffered (50 mM Tris, 5 mM MgSO ₄ , 1 mM EDTA, 1 mM ascorbic acid, pH 7.7), and then uniformly mixed and centrifuged three times for 15 minutes at 35,000 × g to obtain a precipitate and stored at -70 ℃. Dissolve the precipitate 5 mg in the buffer solution 1 ㎖, ^[3 H] prazosin ^{([3 H] prazosin; 200pM} ) and after the experimental samples were incubated at 25 ℃ 30 bungan buffer solution of 10 ㎖ (50 mM Tris, pH 7.7) was added to stop the reaction, which was filtered using Whatman GF / C glass microfiber filter paper. The filter paper was shaken for 2 hours in a scintillation reaction solution (scintillation cocktail) and then measured the radioactivity (radioactivity) with a scintillation counter (Scintillation Counter, Beckman, LS6500).

Comparison of Dissociation Constants (Ki) and IC ₅₀ Values in the Rat Aorta of the Compounds of Formula 1, Compound 2 and Hygenamine for α-receptor in Rat Brain compound Ki (M) IC ₅₀ (M) Hygenamine 1.26 × 10 ^-6 1.02 × 10 ^-6 Compound of Formula 1 0.27 × 10 ^-6 2.75 × 10 ^-6 Compound of formula (2) 0.15 × 10 ^-6 2.81 × 10 ^-6

The coupling reaction of prazosin resulted in a Kd value of 133.5 ± 8.91 pM and a Bmax of 15.15 ± 0.64 fmol / mg. The dissociation constant (Ki) of the test materials was 0.15 to 1.26 μM, indicating that the affinity for the α-receptor was found (see Table 2 ). In addition, these dissociation constants are similar to IC ₅₀ values (1.02 to 2.81 μM) and platelet aggregation inhibition by epinephrine (IC ₅₀ ; 3.4 to 19 μM) in rat blood vessels contracted with phenylephrine. It was found that blocking receptors relaxes blood vessels and inhibits platelet aggregation.

Experimental Example 3 Effect of Inhibiting iNOS mRNA Expression by LPS in Rat Blood Vessels

Rats were anesthetized with Pentobarbital sodium 50 mg / kg and the aorta was removed to remove endothelial cells. After incubating for 8 hours in a Krebs solution containing 300 ng / ml LPS at 37 ° C, total RNA was extracted using Trizol solution (GibcoBRL; Life Technologies). Total RNA was quantified with an ultraviolet spectrophotometer (Shimadzu, UV-1201), electrophoresed on formamide-formaldehyde agarose gel and then transferred to a nylon membrane. Then, the amount of mRNA of iNOS was observed by hybridizing with cDNA of iNOS (induced NO synthase) as a probe. At this time, the cDNA of iNOS was used by labeling with ³² P-dCTP by random-primer method, and the nylon membrane was exposed to X-ray film to increase the size of photosensitive GAPDH (Glyceraldehyde-3-phosphate). The amount of mRNA expressed was quantified compared to the expression of dehydrogenase).

When blood vessels were cultured with LPS in vitro , iNOS mRNA expression was induced in a large amount. When the compound of Formula 1 was added and cultured together, iNOS mRNA expression was suppressed. That is, iNOS mRNA was strongly expressed in the LPS (300 ng / ml) administration group, but iNOS mRNA expression by LPS was inhibited when cultured with 10 μM or 30 μM of the compound of Formula 1 (see FIG. 1 ).

Experimental Example 4 Effects of Inhibiting iNOS mRNA Expression by LPS and IFN-VII in Macrophages

In DMEM medium containing 10% fetal calf serum, 100 U / ml penicillin and 100 mg / ml streptomycin, heat-produce macrophage cells (RAW 264.7 cells). ) Was incubated in a CO ₂ incubator. Cultured cells were transferred to serum-free DMEM medium and incubated for another 24 hours, followed by LPS 100 ng / ml, interferon-VII (hereinafter abbreviated as "IFN-VII") 10 U / Incubated for 18 hours in the presence of ml and compounds of formula 2 (0 μM, 1 μM, 10 μM and 100 μM) and total RNA was extracted using Trizol solution. Total RNA was quantified spectrophotometrically and electrophoresed on formamide-formaldehyde agarose gel and then transferred to nylon membrane. Then, the amount of mRNA of NO synthase was expressed by hybridizing with cDNA of iNOS as a probe. At this time, the cDNA of iNOS was labeled with ³² P-dCTP by the random-primer method, and the nylon membrane was sensitized on the X-ray film to quantify the amount of mRNA expressed by comparing the size of the photosensitive with that of GADPH. .

Expression of iNOS mRNA was confirmed when macrophages were incubated with LPS (100 ng / ml) and IFN-γ (10 U / ml) for 18 hours. In this case, to determine whether the compound of Formula 2 inhibits the expression of iNOS mRNA in a concentration-dependent manner, the compound of Formula 2 was treated simultaneously with LPS and IFN-γ by 1 μM, 10 μM and 100 μM, respectively. As a result, it was confirmed that the compound of Formula 2 inhibits the expression of iNOS mRNA in a concentration-dependent manner (see FIG. 2 ).

Experimental Example 5 Effect of Inhibiting NO Production by LPS and IFN-γ in Macrophages

In DMEM medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin, heat-treated macrophages (RAW 264.7 cells) were incubated to grow sufficiently in a CO ₂ incubator. The cultured cells were further incubated for 24 hours in serum-free DMEM medium, followed by LPS 100 ng / ml, IFN-γ 10 U / ml and compounds of Formula 1 or Formula 2 (0 μM, 1 μM, 10 μM and 100). culture continued for 18 hours in the presence of μM). The NO produced at this time was measured indirectly by the amount of nitrite (NO oxide), and the nitrite was colored with grease reagent [0.1% naphthylethylenediamine dihydrochloride, 1% sulphanilamide, 5% phosphoric acid solution] at 550 nm. The absorbance of was measured with a spectrophotometer to quantify the amount. The amount of nitrite was calculated based on NaNO ₂ and the results are shown in Table 3 .

Effects of Compounds of Formulas 1 and 2 on NO Production by LPS and IFN-γ in Macrophages Nitrite Concentration (μM) Formula 1 Formula 2 Control 8 ± 0.1 8 ± 1.1 LPS + IFN-γ 57 ± 3.3 57 ± 4.2 Compound 1μM + LPS + IFN-γ 30 ± 2.9 34 ± 3.1 Compound 10 μM + LPS + IFN-γ 25 ± 3.4 22 ± 2.8 Compound 100μM + LPS + IFN-γ 9 ± 3.0 11 ± 2.0

As shown in Table 3 , when the macrophages were incubated with endotoxin, a large amount of nitrite was detected, indicating that NO was produced in large quantities. That is, 8 μM of nitrite was detected in the control group to which endotoxin and IFN-γ were not added, but 57 μM of nitrite was detected in the culture medium to which LPS and IFN-γ were added. When the compounds of Formula 1 and Formula 2 were added to the culture with LPS and IFN-γ, the concentration of nitrite in the culture was 30 μM and 34 μM, respectively. The concentration of nitrite was higher than that of only LPS and IFN-γ. Decreased. In addition, as the addition amount of the compound of Formula 1 and Formula 2 increased, the concentration of nitrite was detected to be low, indicating that the compounds of Formula 1 and Formula 2 according to the present invention strongly inhibit NO production by endotoxin in a concentration-dependent manner. there was.

Experimental Example 6 Effect of Inhibiting Mouse Death by Acute Thrombosis

A mixed solution of collagen (300 μg / kg) and epinephrine (30 μg / kg) was injected into the tail vein of the mouse (ICR; 20 ± 20 g, using the same type of mouse in all experiments below) in a short time. By inducing a large amount of thrombus to form, an acute thrombus model was induced in which 1-3 minutes after injection of the mixed solution, general paralysis occurred and the majority died within 15 minutes. The sample material was orally administered for 1 day or 3 days, and a mixed solution of collagen and epinephrine was injected 1 hour after the last oral administration. After that, the degree of mortality was reduced and the degree of recovery from paralysis was observed. The results are shown in Tables 4 and 5 .

Recovery rate from paralysis due to thrombus formation when the compound of Formula 1, Compound 2 or Hygenamine is administered orally to mice compound Dose (mg / kg) The total number of mice used in the experiment Mice recovered from thrombosis within 15 minutes Number % Control - 60 13 17 aspirin 50 53 32 52 Hygenamine 50 30 11 37 100 27 13 48 Compound of Formula 1 50 17 8 47 100 20 9 45 Compound of formula (2) 50 28 14 50 100 24 12 50

Recovery rate from paralysis due to thrombus formation when oral administration of a compound of Formula 1, compound of Formula 2, or hygenamine to mice for 3 consecutive days compound Dose (mg / kg) The total number of mice used in the experiment Mice recovered from thrombosis within 15 minutes Number % Control - 20 4 20 aspirin 50 20 11 55 Hygenamine 10 12 2 17 50 11 5 45 Compound of Formula 1 10 10 3 30 50 12 5 42 Compound of formula (2) 10 10 7 70 50 11 7 64

Intravenous injection of collagen and epinephrine mixed solution in mice produces a large amount of blood clots in the pulmonary artery in a short time, causing most of them to become paralyzed due to general paralysis within one minute after injection, resulting in enlarged pupils, dyspnea and convulsions. About 70-80% of the mice die within minutes and most of the remaining deaths occur within 15 minutes or paralysis lasts for more than 15 minutes. As shown in Table 4 and Table 5 , only 17-20% of the mice in the control group administered with the collagen and epinephrine mixed solution without the test substance recovered from paralysis within 15 minutes and moved freely. Aspirin or hygenamine used as a positive control drug showed 52% and 37% recovery rate after 50 mg / kg oral dose and 48% recovery rate when 100 mg / kg dose of hygenamine was administered. . When the compound of Formula 1 and the compound of Formula 2, which were the compounds of the present invention, were administered once at 50 mg / kg or 100 mg / kg, the recovery rate was about 50%, similar to that of aspirin. In addition, the recovery rate of the compound of Formula 1 did not increase significantly even when administered continuously for 3 days at 50 mg / kg for 3 days, but the recovery rate was 64 to 70% when the compound of Formula 2 was administered at 10 mg / kg or 50 mg / kg for 3 days. Increased to.

Experimental Example 7 Effects of Inhibiting Thrombus Formation in Arterial-Vous Shunt (A-V Shunt) of Rat

An AV shunt tube (AVS tube) was prepared as follows. One end of the scalp vein set was cut to 18 cm in length, and 18G needles were connected at both ends. And the center of the tube was fixed 5 cm 100% cotton thread. Rats were anesthetized by injection of ketamine (250 mg / kg) ( im ), incised abdomen, filled with saline into the AVS tube prepared above, one needle was inserted into the abdominal aorta and the other into the renal vein. It was allowed to flow so that blood clots were generated in the cotton chamber. After 15 minutes, it was measured the amount of pull out the yarn from the tube generated put the weight of thrombus to a change in the weight, by observing clot formation inhibitory effect by oral administration of the test substance The results are shown in Table 6 and Table 7 It was.

Inhibition of thrombus formation in A-V shunt when oral administration of a compound of Formula 1, a compound of Formula 2, or hygenamine to rats compound Dose (mg / kg) The total number of rats used for the experiment Weight (mg) Suppression% Control - 4 33.61 ± 7.620 aspirin 50 6 19.89 ± 7.213 ^* 40.8 Hygenamine 25 8 22.98 ± 11.271 31.6 Compound of Formula 1 25 8 20.27 ± 5.366 ^** 39.7 Compound of formula (2) 25 8 22.68 ± 10.127 32.5 Control - 9 39.54 ± 5.625 aspirin 50 7 25.18 ± 6.725 ^*** 36.3 Hygenamine 50 8 29.61 ± 5.059 ^** 25.1 Compound of Formula 1 50 8 29.72 ± 5.975 ^** 24.8 Compound of formula (2) 50 6 28.58 ± 4.234 ^*** 27.7 *; p <0.05, **; p <0.01, *** ; p <0.001

Inhibition of thrombus formation in A-V shunt when oral administration of a compound of Formula 1, compound of Formula 2, or hygenamine to rats for 3 consecutive days compound Dose (mg / kg) The total number of rats used for the experiment Weight (mg) Suppression% Control - 8 42.67 ± 5.381 aspirin 50 9 37.37 ± 7.369 12.4 Hygenamine 10 12 36.76 ± 7.964 13.9 50 12 34.68 ± 8.513 ^* 18.7 Compound of Formula 1 10 11 31.78 ± 8.385 ** 25.5 50 11 30.72 ± 6.334 ^*** 28.0 Compound of formula (2) 10 11 32.24 ± 4.836 ^*** 24.4 50 9 30.18 ± 6.571 ^*** 29.3 *; p <0.05, **; p <0.01, *** ; p <0.001

The weight of the generated thrombi was significantly different depending on the climate (air pressure, humidity, etc.) on the day of the experiment, and compared with the aspirin administration group administered 50 mg / kg of aspirin, an antithrombotic drug. 33-42 mg of thrombus was generated in the control group not administered the test substance. One dose of 50 mg / kg of aspirin, a positive control, showed 36-40% inhibition of thrombus formation, and 25-31% of 25 mg / kg or 50 mg / kg of hygenamine. It had a thrombus inhibitory effect. On the other hand, the compound of Formula 1 and the compound of Formula 2 showed a blood clotting inhibitory effect of 24 to 39% when administered once at 25 mg / kg or 50 mg / kg, from which the compound of the present invention was associated with hygenamine. It can be seen that there is a similar or stronger inhibitory effect on thrombus formation.

In the case of continuous administration of the test substance for 3 days, the thrombus inhibitory effect was weak as 12% in the experimental group in which 50 mg / kg of aspirin was administered three times. In the case of more than average blood clots produced in the control AV shunt according to the climatic conditions on the day of the experiment, the inhibitory effect of thrombosis formation by the sample drug was relatively low, and thus a positive control group in the same experimental period (aspirin group in this experiment) Relative comparison with was essential.

When hygenamine was administered continuously for 10 days or 50 mg for 3 days, it showed a 13 to 18% thrombus inhibitory effect, similar to that of the aspirin group. However, when the compound of Formula 1 and the compound of Formula 2 were administered at 10 mg / kg for 3 days, they showed antithrombotic activity of about 24% and 50 mg / kg of aspirin or Haige. Compared with the namin-administered group, it was found that the antithrombotic effect was improved.

Experimental Example 8 Effect of Improving Various Indices in DIC and MOF-induced Rats Induced by Bacterial Endotoxin (LPS)

Rats (250 ± 50 g) were anesthetized by injection of ketamine (250 mg / kg) (i.m.) and LPS (20 mg / kg) was infused into the tail vein over 3 hours after 30 minutes. Thereafter, blood was collected from the aorta and the concentrations of platelet count, prothrombin time, aPPT time, fibrinogen, fibrin / fibrinogen degradation products, S-GOT, S-GPT and BUN were measured as follows. Test substances were administered orally for 2 days and the experiment was started 1 hour after the second dose.

First, sodium citrate was used as an anticoagulant, and the platelet count of blood was measured using a platelet meter. On the other hand, the anticoagulant blood was centrifuged at 1,500 × g for 10 minutes and then plasma was used in the following experiment.

The prothrombin time was obtained by incubating 100 ul of the plasma obtained above at 37 ° C for 3 minutes, and then adding 50 ul of thromboplastin reagent (Thromboplastin reagent, Sigma, USA) incubated at 37 ° C for 5 minutes in advance. The clotting time was measured using a meter (Fibrometer, Becton Dickinson Co., Canada). Activated partial thromboplastin time, after culturing 100 ul of the plasma for 3 minutes at 37 ℃, pre-incubated for 1 minute at 37 ℃ (Activated Partial Thromboplastin Time Reagent, Sigma, USA) 100 ul and 100ul of 0.02 M CaCl ₂ preheated at 37 ° C. were added, and the thrombus formation time was measured using a fibrometer.

Fibrinogen concentration was measured by adding 180 ul of buffer to 20 ul of the plasma, incubating at 37 ° C. for 2 minutes, and then adding 100 ul of thrombin reagent (Sigma, USA) to measure thrombus formation time. The fibrinogen reference was used to obtain a calibration curve drawn in advance.

In addition, the concentration of fibrin / fibrinogen degradation products was determined by the following method using the Trombo-Welcotest Kit (Murex Biotech Limit, UK). First, blood (non-citrated) was coagulated by mixing with soybean trypsin inhibitor and Bothrops Atrox venom, and then serum was obtained by centrifuging twice at 1,500 × g for 10 minutes, and glycine saline buffer solution ( Dilution with glycine saline buffer) was performed. 50 ul of the diluted serum thus obtained was added to the slide glass, and a drop of latex suspension was added to the mixture, and the mixture was well spread. The mixture was shaken in a shaker for 2 minutes, and then observed for aggregation. The concentration of FDP was determined semisemiquantitatively. Namely, at 1: 0 dilution, (-) 0 µg / ml, (+) 1 µg / ml, (++) 2 µg / ml; At 1: 1 dilution, (+) 3 μg / ml, (++) 4 μg / ml; In the case of 1: 2 dilution, it was determined as (+) 5 µg / ml, (++) 6 µg / ml and the like.

Concentrations of S-GOT, S-GPT and BUN were commissioned by the Green Cross Reference Lab. Measured using. An AST kit was used for S-GOT measurement, an ALT kit for S-GPT measurement, and an urea kit for BUN measurement.

Various indicators of DIC and MOF symptoms, which occurred when LPS was injected over a long period of time and caused septic shock in experimental animals, were ameliorated by oral administration of a compound of Formula 1 or Formula 2. That is, in the control group administered with LPS only, the platelet count and fibrinogen concentration in blood were drastically decreased, the prothrombin time and activated partial thromboplastin time were prolonged, and the concentration of fibrin / fibrinogen degradation product in blood was rapidly increased. In addition, as the liver and kidney function deteriorated, S-GOT, S-GPT, BUN, and the like increased. Various indicators of such DIC and MOF symptoms were confirmed to be improved when oral administration of the compound of Formula 1 or Formula 2.

Specifically, when oral administration of hygenamine, a compound of Formula 1 or Formula 2, LPS 20 mg / kg in rats over 3 hours to induce DIC and MOF status, compared to the control group administered only LPS and DIC and It was observed whether the indicators of MOF symptoms improved.

In the control group, which received only LPS platelet count 296 × 10 ³ / ul was a significantly reduced to half or less than 747 × 10 ³ / ul of the control group (see Fig. 3), prothrombin time and partial thromboplastin time enabling each 19 and 33 seconds, about 50% more than the 14 and 20 seconds of the normal group (see FIGS . 4 and 5 ). On the other hand, in the control group, fibrinogen was 104 mg / ml and fibrin / fibrinogen degradation products were 168 ug / ml, whereas in the normal group, 202 mg / ml and 1 ug / ml, respectively. Fibrin / fibrinogen degradation products showed a sharp increase, indicating a significant improvement in DIC symptoms (see FIGS . 6 and 7 ). In addition, the concentrations of S-GOT, S-GPT and BUN were 231.0 U / L, 60.8 U / L and 25.6 mg / dL, respectively, compared to 167 U / L, 49.3 U / l and 15.0 mg / dL of normal group. In addition, the symptoms of MOF also progressed.

However, when LPS was injected orally after 10 mg / kg or 50 mg / kg of hygenamine, a compound of Formula 1, or a compound of Formula 2, respectively, platelet count was increased compared to the control group that received only LPS, and prothrombin time and The activated partial thromboplastin time was shortened, the concentration of fibrinogen increased and the concentration of fibrin / fibrinogen degradation products decreased (see FIGS . 3-7 ). Therefore, these substances were found to be effective in improving DIC symptoms. In addition, the concentrations of S-GOT, S-GPT and BUN also decreased compared to the control group administered with only LPS (see FIGS . 8 to 10 ), whereby the compounds of the present invention are MOF such as liver or kidney damage caused by LPS. It was confirmed that the symptoms could be alleviated.

Experimental Example 9 Effect of Inhibiting Death by Bacterial Endotoxin (LPS)

Injecting large amounts of LPS into animals results in various symptoms of disseminated intravascular coagulation and most often die of septic shock in a short time. In this experiment, 20 mg / kg of LPS was injected intraperitoneally in mice and the number of surviving mice for 48 hours was counted.

Mice were injected with LPS 20 mg / kg intraperitoneally ( ip ) and then counted the surviving mice every 6 hours for 48 hours. In addition, the test substances were injected intraperitoneally 30 minutes before the LPS injection to observe the effect of inhibiting the death of mice by these test substances.

Survival after 48 hours with LPS alone was about 20%, but before administration of LPS, hygenamine (see FIG. 11A ), a compound of Formula 1 (see FIG. 11B ) or a compound of Formula 2 (see FIG. 11C ), respectively When 20 mg / kg was administered, no mice died until 30 hours, and more than 90% of mice survived after 48 hours. Hygenamine and the compound of Formula 2 had a survival rate of 80% or more after 48 hours when 10 mg / kg was administered. When the compound of Formula 1 was administered at 10 mg / kg, the survival rate after 48 hours was about 60%, which was lower than that of hygenamine or the compound of Formula 2, but was higher than that of the control group. Therefore, the compound of Formula 1 or compound of Formula 2 was found to have the effect of protecting the shock by LPS.

As described above, the compounds of the formulas (1) and (2) simultaneously exhibit a combination of the cardiac action and the blood pressure lowering action as well as the platelet aggregation inhibitory action and the iNOS inhibitory action, which are already known by the present inventors, without requiring parallel administration with other drugs. It can be usefully used as a treatment for heart failure. In addition, since it is useful for treating heart failure without using it in combination with other drugs, problems that may occur when multiple drugs are administered in combination for effective treatment of heart failure, for example, due to interactions between drugs to be administered in combination It has the advantage of eliminating side effects that may occur by affecting in vivo absorption and drug metabolism of each drug. Furthermore, the pharmaceutical composition comprising the compound of the present invention has the advantage of wide safety zone and long duration of action.

Claims (2)

A pharmaceutical composition for treating heart failure, wherein the pharmaceutical composition is 1-α-naphthylmethyl-6,7 represented by the following Chemical Formula 1 without being used in combination with a cardiac agent, a vasodilator, an antithrombotic agent, and an iNOS tissue damage inhibitor. -Dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1-β-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoiso represented by formula (2) A pharmaceutical composition for treating heart failure, characterized in that it contains a compound selected from the group consisting of quinoline and mixtures thereof as an active ingredient. Formula 1 Formula 2 The treatment of heart failure according to claim 1, wherein the treatment of heart failure is caused by acute myocardial infarction, decreased myocardial contraction function, decreased myocardial contraction function due to increased immune activity such as chronic inflammation, decreased myocardial contraction function due to ischemic heart disease or persistent hypertension, arteriosclerosis, coronary artery. Pharmaceutical composition, characterized in that to suppress or treat the progression of heart failure caused by congestive heart failure due to arterial disease, myocardial contractility.