KR100614203B1 - Chromene-6-carbonylguanidine derivatives, a process for the preparation thereof and agent for preventing and treating of ischemic heart disease comprising the same - Google Patents

Abstract

The present invention relates to a (chromen-6-carbonyl) guanidine derivative, a preparation method thereof, and an agent for preventing and treating ischemic heart disease including the same. The (chromen-6-carbonyl) guanidine derivatives of the present invention enhance cardiac function recovery against ischemia / reperfusion injury and reduce myocardial infarction rate through a potent inhibitory action on sodium / hydrogen exchange pathway NHE-1. By protecting the cells, it can be used as an agent for the prevention and treatment of ischemic heart diseases such as myocardial infarction, heart failure, angina pectoris, and cardioprotection during reperfusion therapy such as coronary artery bypass surgery, coronary percutaneous plastic surgery, or drug therapy using thrombolytics. Can be used zero.

Description

(Chromene-6-carbonyl) guanidine derivatives, a process for the preparation according to and preparation for agent and preventing and treating of ischemic heart disease ischemic heart disease comprising the same}

The present invention relates to a (chromen-6-carbonyl) guanidine derivative, a preparation method thereof, and an agent for preventing and treating ischemic heart disease including the same.

Myocardial ischemia is a phenomenon in which the blood supply to the heart is blocked and the oxygen supply of the myocardium is markedly short of the required amount. Such myocardial ischemia ultimately leads to irreversible damage of the myocardium, that is, necrosis of cells and tissues. . In the early stages of reversible damage, irreversible damage can be prevented by surgical treatments such as coronary percutaneous grafting and coronary artery bypass surgery, or by reperfusion therapy such as drug therapy using thrombolytics. It is known that reperfusion injury occurs at a high rate, such as deterioration, arrhythmia, and neurocognitive decline [Robert, M. (2003) Ann. Thorac. Surg. 75: S700-708. Ischemic heart diseases such as myocardial infarction, arrhythmia, and heart failure caused by myocardial cell damage and cardiac function during ischemia / reperfusion are high in morbidity, mortality, and hard to cure. [Wang, QD. et al., (2002) Cardiovasc. Res. 55: 25-37. Ischemia / reperfusion injury involves various physiological mechanisms such as metabolism, immune response and ionic constant change, oxygen free radicals, etc. Therefore, research is being conducted in various fields such as immunomodulators, apoptosis-related substances and ion channel regulators [Hearse, DJ. (1998) Prog. Cariovasc. Dis. 30: 381-402. In addition to the mechanism research, the development of therapeutic agents and surgical procedures by the new point of action has been actively conducted, but the technology for protecting cardiomyocytes from ischemia / reperfusion has not been commercialized clinically. Therefore, there is a need for development of a prophylactic or therapeutic agent for ischemic heart disease or a cardioprotective agent that can slow the progression of cardiomyocyte damage due to ischemia and alleviate reperfusion injury.

Meanwhile, intracellular pH changes play an important role in the pathophysiology of essential hypertension, ischemic myocardial disease, dysfunction due to ischemia / reperfusion and cell death. Cells have a mechanism for regulating intracellular pH, which is activated to correct intracellular acidification by ischemia and reperfusion. One of the major ion channels present in cardiomyocytes and alkalizing acidified intracellular pH is the sodium-hydrogen exchanger (NHE), which is essential for normalizing intracellular pH. However, sodium overload can cause cell damage.

NHE is expressed in a variety of cell species as a membrane protein that plays an important role in regulating intracellular pH while maintaining neutrality electrostatically by sending one hydrogen ion out of the cell and bringing one Na ⁺ into the cell. To date, seven subtypes have been identified and NHE-1, the main subtype of cardiomyocytes, is known to play a major role in ischemia / reperfusion injury [Avkiran, M. et . al ., (2002) J. Am. Coll. Cardiol. 39 : 747-753. At normal physiological pH (≒ 7.2), NHE-1 hardly works. In the ischemic state lacking oxygen, energy production depends on glycolysis, so the concentration of hydrogen ions in the cells increases and acidifies (pH 6.4). NHE-1 with a proton sensor is activated to activate hydrogen ions. The concentration of sodium ions in the cells increases because of the release of sodium ions into the cells. In ischemia, Na ⁺ / K ⁺ ATPase is inhibited due to the reduction of ATP energy production, so sodium ions accumulated in cells by the sodium pump cannot be exported. Therefore, in normal state, NCX (Na ⁺ / Ca ²⁺ exchanger), which is a sodium / calcium channel that exports calcium and imports sodium, works in reverse direction to release sodium ions, import calcium ions, and bring calcium ions into cells. The concentration of becomes high. Increasing intracellular calcium concentration causes cell damage by activating enzymes such as proteases, phospholipases and endonucleases, causing various protein breakdowns, increased free radicals due to lipometabolic disorders, and DNA modification. Therefore, by inhibiting the activity of NHE-1 and inhibiting the increase of intracellular sodium ion concentration, it is possible to suppress the reverse operation of NCX, and to suppress the increase of intracellular calcium ion concentration to prevent cell damage by ischemia / reperfusion. Can be. Increased hydrogen ions are regulated by other ion channels, so that it is reported that intracellular acidification by inhibition of NHE-1 does not occur. The diuretic pyrazine derivative amylolide is known for the first time as an NHE inhibitor [Benos, DJ. (1982) AJ Physiol. 242 : C131]. Amylolide has an inhibitory effect on NHE-1, and rat extraction cardiac experiments have been shown to enhance the recovery of cardiac function after ischemia / reperfusion, but in addition to NHE-1, it inhibits NHE-2 and sodium channels. There was a problem as a protective agent. Subsequently, studies on drug development with selectivity for NHE-1 were carried out and cariporide (HOE), a benzoylguanidine derivative having high selectivity for NHE-1 by Hoechst Marion Roussel (now Aventis). -694) have been developed [Scholz, W. et . al ., (1993) Br. J. Pharmacol. 109 : 562]. Carriporide showed an excellent cardioprotective effect in animal models and clinically showed significant protective effect in patients with coronary artery bypass surgery. Most of the NHE-1 inhibitors known to date are acylguanidine derivatives, and many drugs such as eniporide, zoniporide, SM-20220, and BMS-284640 are being developed as selective NHE-1 inhibitors. They have been shown to have protective effects against ischemia / reperfusion injury by improving myocardial contractility, reducing arrhythmia, reducing apoptosis and necrosis, improving metabolic status and overloading sodium and calcium ions [Karmazyn, M ( 2002) Science & Medicine : 18-26 ].

Therefore, the present inventors have synthesized (chromen-6-carbonyl) guanidine derivatives while trying to develop a safe and effective drug that can slow the progression of cardiomyocyte damage caused by ischemia and alleviate reperfusion injury. They found that the selective inhibitory effect on NHE-1 was excellent and completed the present invention.

The present invention seeks to provide a (chromen-6-carbonyl) guanidine derivative.

The present invention also provides a method for preparing a (chromen-6-carbonyl) guanidine derivative.

In addition, the present invention is to provide an agent for the prevention and treatment of ischemic heart disease, comprising (chromen-6-carbonyl) guanidine derivatives and pharmaceutically acceptable salts thereof as an active ingredient.

The present invention provides a (chromen-6-carbonyl) guanidine derivative represented by the following formula (1).

또는 R^a이며, R³는 H, CH₃, Br, NO₂, NH₂, Cl, NMe₂ 또는 NH-SO₂Me이다. 이때, R^a는 C₁~C₄의 직쇄 또는 측쇄 알킬기이고,

은 단일결합 또는 이중결합을 나타낸다.)Wherein R ¹ is H or OH, R ² is H, OH, OR ^a , OCH ₂ Ph, Br,

Or R ^a and R ³ is H, CH ₃ , Br, NO ₂ , NH ₂ , Cl, NMe ₂ or NH-SO ₂ Me. In this case, R ^a is a C ₁ ~ C ₄ straight or branched alkyl group,

Represents a single bond or a double bond.)

The (chromen-6-carbonyl) guanidine derivative of the present invention represented by Chemical Formula 1 may be used in the form of a pharmaceutically acceptable salt, and the salt may be formed by a pharmaceutically acceptable free acid. Acid addition salts are useful. Inorganic and organic acids can be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. can be used as the inorganic acid, citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfur Phonic acid, acetic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid, and the like can be used. Preferably, hydrochloric acid may be used as the inorganic acid, and methanesulfonic acid may be used as the organic acid.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of Formula 1 in a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile and adding an excess of organic acid or It can be prepared by adding an acidic aqueous solution of precipitated and then crystallizing. The solvent or excess acid may then be evaporated and dried in this mixture to obtain an addition salt or the precipitated salt may be prepared by suction filtration.

In addition, the (chromen-6-carbonyl) guanidine derivative represented by Chemical Formula 1 may include all salts, hydrates, and solvates that may be prepared by conventional methods, as well as pharmaceutically acceptable salts. And all possible stereoisomers.

Preferred compounds of the (chromen-6-carbonyl) guanidine derivative represented by Chemical Formula 1 of the present invention are specifically as follows, and the structural formulas are shown in Table 1.

1) (4-hydroxy-2,2-dimethylchroman-6-carbonyl) guanidine,

2) (4-methoxy-2,2-dimethylchroman-6-carbonyl) guanidine,

3) (4-benzyloxy-2,2-dimethylchroman-6-carbonyl) guanidine,

4) (2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine,

5) (8-bromo-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine,

6) (8-nitro-2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine,

7) (7-chloro-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine,

8) (2,2,7-trimethyl- 2H -chromen-6-carbonyl) guanidine,

9) (8-dimethylamino-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine,

10) (8-methanesulfonylamino-2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine,

11) (2,2-dimethylchroman-6-carbonyl) guanidine,

12) (8-bromo-2,2-dimethylchroman-6-carbonyl) guanidine,

13) (8-nitro-2,2-dimethylchroman-6-carbonyl) guanidine,

14) (8-amino-2,2-dimethylchroman-6-carbonyl) guanidine,

15) (4-bromo-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine,

16) (4,8-dibromo-2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine,

17) (4-bromo-8-nitro-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine,

18) (4-bromo-7-chloro-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine,

19) (4-bromo-2,2,7-trimethyl-2 H -chromen-6-carbonyl) guanidine,

20) [(3R, 4S) -3-hydroxy-4-methoxy-2,2-dimethylchroman-6-carbonyl] guanidine,

21) [(3R, 4S) -3-hydroxy-4-imidazol-1-yl-2,2-dimethylchroman-6-carbonyl] guanidine,

22) [(3R, 4S) -3-hydroxy-4-pyrrole-1-yl-2,2-dimethylchroman-6-carbonyl] guanidine,

23) (2,2,4-trimethyl- 2H -chromen-6-carbonyl) guanidine,

24) (8-bromo-2,2,4-trimethyl-2 H -chromen-6-carbonyl) guanidine,

25) (7-chloro-2,2,4-trimethyl-2 H -chromen-6-carbonyl) guanidine, and

26) (2,2,4,7-tetramethyl- 2H -chromen-6-carbonyl) guanidine.

In addition, the present invention provides a method for preparing a (chromen-6-carbonyl) guanidine derivative of Formula 1 represented by the following Scheme 1.

(Wherein R ¹ , R ² and R ³ are as defined in Formula 1, and L is a leaving group that can be easily released by guanidine.)

Leaving groups include C ₁ -C ₃ alkoxy groups, halide groups, carboxylate groups, hydroxy groups, p-nitrophenoxy groups, N-hydroxysuccinimide groups, pentafluorophenoxy groups and the like.

In Scheme 1, the carboxylic acid derivative (II) may be reacted with an excess of guanidine or with an equivalent amount of guanidine in the presence of a base to prepare a (chromen-6-carbonyl) guanidine derivative of Formula 1.

Examples of the carboxylic acid derivative (II) include esters, acyl halides, carboxylic acid anhydride derivatives, and the like. The ester derivative is a general alkyl ester (eg methyl ester, ethyl ester) or an active ester derivative (eg p-nitrophenyl ester, N-hydroxysuccinimide ester, pentafluorophenyl ester). Such carboxylic acid derivatives can be readily prepared from carboxylic acids by conventional known methods. In the present invention, a methyl ester compound (L = OMe) is mainly used as the carboxylic acid derivative (II).

When the leaving group of the carboxylic acid derivative (II) is methyl ester, compound (I) is prepared by reacting with an equivalent or excess guanidine using a suitable solvent.

The reaction solvent may be an alcohol solvent such as methanol, ethanol or isopropanol, an ether solvent such as tetrahydrofuran, dioxane or 1,2-dimethoxyethane, dimethylformamide (DMF), or a mixed solvent of such solvents. Can be. The reaction temperature is from room temperature to the boiling point of the solvent.

Compound (II-1) represented by the following Chemical Formula 2, wherein the leaving group of the carboxylic acid derivative (II) used as the starting material in Scheme 1 is methyl ester, is prepared by the following method.

(Wherein R ¹ , R ² , and R ³ are as defined in Formula 1 above)

1) In the compound (II-1) represented by the formula (2) , the compound (II-3) wherein R ¹ is H and R ² is OH or OR ^a is 2,2-dimethyl-4-oxochromen-6 -Carboxylic acid methyl ester (III) was prepared as a starting material, and is shown in Scheme 2 below.

(Wherein R ³ and R ^a are as defined in Formula 1, and X is a halogen group.)

In Scheme 2, compound (III) is used by using a commercially available compound or by a known method.

In the reduction reaction, most reducing agents capable of reducing ketone groups to alcohol groups may be used, and sodium borohydride is generally used in an alcohol solvent such as methanol. Equivalent or excess reducing agent may be used and the reaction temperature is from 0 ° C. to the boiling point of the solvent.

In the alkylation reaction, the alkyl halogen compound (R ^a X) is reacted with the compound (II-2) in the presence of a base to produce the compound (II-3). As the base to be used, inorganic bases such as sodium hydride, potassium t -butoxide and sodium methoxide may be used, or organic bases such as N , N -diisopropylamine and DBU may be used. The reaction solvent may be used alone or in combination with an ether solvent such as tetrahydrofuran, dioxane, 1,2-dimethoxyethane, DMF, dimethyl sulfoxide and the like. The reaction temperature is from 0 ° C. to the boiling point of the solvent.

2) In the compound (II-1) represented by the formula (2), R ¹ and R ² are H and C ₃ -C ₄ are double bonds, and a chroman compound (II) having a single bond with a chromen compound (II-4) The preparation method of -5) is shown in Scheme 3 below.

(Wherein R ³ is as defined in Formula 1 above)

Dehydration of the hydroxy group of the alcohol compound (II-2) prepared in Scheme 2 yields chromium compound (II-4), and reduction of compound (II-4) to give a chroman compound (II-5). Manufacture.

In the dehydration reaction, alcohol compound (II-2) is first reacted with mesyl chloride to be converted to a mesylate compound, followed by removal in the presence of a base to prepare compound (II-4). In this case, 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), potassium t -butoxide or sodium ethoxide may be used as the base, and as a solvent, benzene, Toluene, t -butanol, ethanol and the like can be used. The reaction temperature can be selected between room temperature and the boiling point of the solvent.

In the reduction reaction, it is preferable to hydrogenate using a hydrogen gas in the presence of a palladium catalyst (Pd / C).

3) In compound (II-1) represented by the formula (2) , compound (II-7) wherein C ₃ -C ₄ is a double bond, R ¹ is H and R ² is Br, represents compound (II-4) Compound (II-6) is prepared by dibromination using bromine, and prepared by monodibromination using base, and is shown in Scheme 4 below.

(Wherein R ³ is as defined in Formula 1 above)

In the reaction scheme 4, the bromination reaction is carried out using an equivalent amount of bromine in a dichloromethane or chloroform solvent. The base and reaction conditions used in the monodibromination reaction are the same as those used in the dehydration reaction of Scheme 3.

4) In the compound (II-1) represented by the formula (2), the compound (II-9) wherein R ¹ is OH and R ² is OR ^a , imidazole or pyrrole , epoxy compound (II-4) as an oxidizing agent. After the polymerization reaction, an epoxy ring-opening reaction is prepared using an alcohol compound R ^a -OH, imidazole or pyrrole, respectively, and is shown in Scheme 5 below.

이다. 이때 R^a는 화학식 1에서 정의한 바와 같다.)Wherein R ³ is as defined in Formula 1, and R ^b is OR ^a ,

to be. Wherein R ^a is as defined in Formula 1.)

In the scheme 5, the epoxidation reaction is carried out using m -chloroperbenzoic acid as an oxidizing agent in a dichloromethane or chloroform solvent, or a known method for chiral preparation of an oxirane compound (II-8). Sharpless epoxidation or Jacobsen epoxidation can be used.

In the reaction scheme 5, the epoxy ring opening reaction is reacted with the alcohol compound R ^a OH in the presence of an acid catalyst when R ^b is OR ^a . The acid catalyst may use anhydrous HCl or Lewis acid. The reaction solvent is preferably dichloromethane or chloroform, the reaction temperature is from room temperature to the boiling point of the solvent.

When R ^b is an imidazole or pyrazole group, the oxirane compound (II-8) and 1 to 5 equivalents of imidazole or pyrazole are mixed and prepared by heating to 50 ° C. to 100 ° C.

5) In the compound (II-1) represented by the formula (2), R ¹ is H, R ² is R ^a, and C ₃ -C ₄ is a double bond. It is shown in Scheme 6.

(Wherein R ³ and R ^a are as defined in Formula 1 above)

In the Scheme 6, the alkyl addition reaction reacts an alkyl grignard agent with a ketone group of compound (III) to prepare a tertiary alcohol compound (II-10). The reaction solvent is preferably an ether solvent such as tetrahydrofuran or diethyl ether, and the reaction temperature is from room temperature to the boiling point of the solvent.

When preparing the compound (II-11) by dehydrating the tertiary alcohol compound (II-10), a catalytic amount of p -toluenesulfonic acid is used in a benzene or toluene solvent. The reaction temperature is from room temperature to the boiling point of the solvent.

The present invention also provides a prophylactic and therapeutic agent for ischemic heart disease using the (chromene-6-carbonyl) guanidine derivative represented by Chemical Formula 1 and a pharmaceutically acceptable salt thereof as an active ingredient.

The (chromen-6-carbonyl) guanidine derivatives of the present invention and pharmaceutically acceptable salts thereof have a potent inhibitory effect on NHE-1 in cells expressing human NHE-1 and ischemia in an isolated ischemic heart model. Promote the recovery of myocardial damage by reperfusion and significantly reduce the size of myocardial infarction in the ischemic animal model in vivo and show excellent cardioprotective effect. Therefore, the (chromen-6-carbonyl) guanidine derivatives and pharmaceutically acceptable salts thereof of the present invention can be used as a prophylactic and therapeutic agent for ischemic heart diseases such as myocardial infarction, heart failure, angina pectoris, coronary artery bypass surgery, coronary percutaneous plastic surgery It may be used as a cardioprotectant during reperfusion therapy procedures such as surgical therapy or drug therapy using thrombolytics.

The compound of the present invention may be administered in various oral and parenteral dosage forms for clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. Are manufactured. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, uthepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

In addition, the dosage of the compound of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally based on an adult patient having a weight of 70 kg. It is 0.1-1000 mg / day, Preferably it is 1-500 mg / day, It can also divide and administer once a day to several times at regular time intervals according to a decision of a doctor or a pharmacist.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

In the present invention, the molecular structure was confirmed by comparing infrared spectroscopy, nuclear magnetic resonance spectra, mass spectroscopy, liquid chromatography, X-ray structure determination, photoluminescence measurement and elemental analysis calculations and actual measurements of representative compounds.

Production Example 1 4-hydroxy-2,2-dimethylchroman-6-carboxylic acid methyl ester

800 mg (3.42 mmol) of 2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester was dissolved in 10 ml of methol, and 194 mg (5.13 mmol) of NaBH ₄ were slowly added at 0 ° C. After stirring for 30 minutes at room temperature, 50 ml of water were added. Extracted with ethyl acetate (50 mL x 2), the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to give 772 mg (yield 96%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.35 (s, 3H), 1.46 (s, 3H), 1.98 (dd, 1H), 2.11 (dd, 1H), 3.87 (s, 3H), 4.43 (t, 1H), 6.80 (d, 1H), 7.85 (dd, 1H), 8.13 (d, 1H)

Production Example 2 4-methoxy-2,2-dimethylchroman-6-carboxylic acid methyl ester

110 mg (0.47 mmole) of the compound obtained in Preparation Example 1 was dissolved in 3 ml of DMF, and 34 mg (0.94 mmol) of 60% sodium hydride was slowly added at 0 ° C., followed by stirring for 20 minutes. 0.2 ml (1.41 mmol) of iodomethane were added and the temperature was gradually raised to room temperature and stirred for 1 hour. The reaction solution was diluted with 15 mL of water and extracted with ethyl acetate (30 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated to obtain a residue obtained by silica gel column chromatography (hexane: ethyl acetate = 6: 1), to obtain 85 mg (yield 72%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.35 (s, 3H), 1.46 (s, 3H), 1.97 (dd, 1H), 2.10 (dd, 1H), 3.49 (s, 3H), 3.88 (s, 3H), 4.43 (t, 1H), 6.79 (d, 1H), 7.84 (dd, 1H), 8.11 (d, 1H)

Production Example 3 4-benzyloxy-2,2-dimethylchroman-6-carboxylic acid methyl ester

140 mg (0.59 mmol) of the compound obtained in Preparation Example 1 was reacted with benzyl bromide in the same manner as in Preparation Example 2 to obtain 125 mg (yield 64%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.34 (s, 3H), 1.48 (s, 3H), 2.06 (m, 2H), 3.87 (s, 3H), 4.62 (t, 1H), 4.64 (q, 2H), 6.79 (d, 1H), 7.40 (m, 5H), 7.83 (dd, 1H), 8.12 (d, 1H)

Production Example 4 2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

800 mg (3.42 mmol) of the compound obtained in Preparation Example 1 were dissolved in 10 mL of dichloromethane, and 0.81 mL (4.64 mmol) of N , N -diisopropylethylamine and 0.27 mL (3.48 mmol) of methane sulfonyl chloride were added thereto, and then at room temperature. Stir for 24 hours. The reaction solution was diluted with 50 mL of water and extracted with dichloromethane (50 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give 440 mg of sulfonate compound. The sulfonate compound was dissolved in 10 ml of toluene, 0.69 ml (4.64 mmol) of 1,8-diazabicyclo (5,4,0) undec-7-ene was added, and the mixture was heated to reflux for 5 hours. The reaction solution was cooled and purified by silica gel column chromatography (hexane: ethyl acetate = 20: 1) to give 300 mg (yield 79%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.42 (s, 6H), 3.88 (s, 3H), 5.66 (d, 1H), 6.43 (d, 1H), 6.78 (d, 1H), 7.86 (dd, 1H), 8.08 (d, 1H)

Production Example 5 8-Bromo-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

8-bromo-2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester compound was used in the same manner as in Preparation Example 4 to obtain 192 mg (yield 66%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.50 (s, 3H), 1.54 (s, 3H), 3.88 (s, 3H), 5.67 (d, 1H), 6.30 (d, 1H), 7.61 (d, 1H), 8.04 (d, 1H)

Production Example 6 8-nitro-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

300 mg (1.08 mmol) of 8-nitro-2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester compound were reacted in the same manner as in Preparation Example 1 to obtain 300 mg of alcohol compound, which was dissolved in 10 ml of toluene. . 20 mg (0.11 mmol) of p- TsOH were added, followed by heating to reflux for 3 hours. After cooling the reaction solution, the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 6: 1) to obtain 205 mg (yield 73%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.54 (s, 3H), 1.57 (s, 3H), 3.92 (s, 3H), 5.81 (d, 1H), 6.39 (d, 1H), 7.84 (s, 1H), 8.37 (s, 1H)

Preparation Example 7 7-chloro-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

480 mg (1.78 mmol) of 7-chloro-2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester was reacted in the same manner as in Preparation Example 6 to obtain 324 mg (yield 72%) of the title compound. .

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.44 (s, 6H), 3.89 (s, 3H), 5.64 (d, 1H), 6.28 (d, 1H), 6.85 (s, 1H), 7.56 (d, 1H)

Preparation Example 8 2,2,7-trimethyl-2 H -Cromen-6-carboxylic acid methyl ester

300 mg (1.21 mmol) of 2,2,7-trimethyl-4-oxochroman-6-carboxylic acid methyl ester compound was reacted in the same manner as in Preparation Example 6 to obtain 225 mg (yield 80%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.43 (s, 6H), 2.54 (s, 3H), 3.85 (s, 3H), 5.58 (d, 1H), 6.30 (d, 1H), 6.63 (s, 1H), 7.62 (s, 1H)

MS ( m / z ) M ⁺ = 232 (M ⁺ )

Production Example 9 8-dimethylamino-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

233 mg (1.00 mmole) of 8-amino-2,2-dimethyl- 2H -chromen-6-carboxylic acid methyl ester compound was dissolved in 4 mL of DMF, 52 mg (1.3 mmole) of sodium hydride was added at 0 ° C, and 30 Stir for minutes. Iodomethane 0.17 ml (1.1 mmol) was added to the reaction mixture, the temperature was gradually raised, and the mixture was stirred at room temperature for 24 hours. The reaction solution was diluted with 20 mL of water and extracted with ethyl acetate (20 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (hexane: ethyl acetate = 10: 1) to give 119 mg (yield 46%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.51 (s, 6H), 2.83 (s, 6H), 3.87 (s, 3H), 5.61 (d, 1H), 6.31 (d, 1H), 7.37 (d, 1H), 7.48 (d, 1H)

Preparation Example 10 8-methanesulfonylamino-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

186 mg (0.80 mmol) of 8-amino-2,2-dimethyl- 2H -chromen-6-carboxylic acid methyl ester compound was dissolved in 10 ml of dichloromethane, and 0.17 ml (1.20 mmol) of triethylamine was added thereto. 0.068 mL (0.88 mmol) of methanesulfonyl chloride was slowly added thereto at 0 ° C, and the temperature was raised to room temperature, followed by stirring for 1 hour. The reaction solution was diluted with 50 mL of water, extracted with CH ₂ Cl ₂ (50 mL × 2), and the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give 154 mg (yield 62%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.51 (s, 6H), 3.01 (s, 3H), 3.88 (s, 3H), 5.68 (d, 1H), 6.36 (d, 1H), 6.70 (s, 1H), 7.56 (d, 1H), 8.03 (d, 1H)

Production Example 11 2,2-dimethylchroman-6-carboxylic acid methyl ester

105 mg (0.38 mmol) of the compound obtained in Preparation Example 4 was dissolved in 3 ml of methanol, and then 10% Pd / C was added thereto, followed by stirring for 1 hour under 1 atmosphere of hydrogen gas. The reaction solution was filtered through celite and washed with 50 ml of ethyl acetate to remove the black carbon solid, and the solvent was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography (hexane: ethyl acetate = 10: 1). 100 mg (yield 95%) of the title compound were obtained.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.29 (s, 3H), 1.82 (m, 1H), 2.03 (m, 1H), 2.78 (t, 2H), 3.51 (s, 3H), 3.55 (s, 3H), 3.87 (s, 3H), 4.19 (s, 1H), 6.81 (d, 1H), 7.77 (m, 2H)

Production Example 12 8-Bromo-2,2-dimethylchroman-6-carboxylic acid methyl ester

205 mg (0.93 mmol) of the compound obtained in Preparation Example 11 was dissolved in 5 mL of chloroform at 0 ° C., and then 0.1 mL (1.86 mmol) of bromine was slowly added thereto. After stirring for 1 hour, it was diluted in 30 ml of water and extracted with ethyl acetate (50 ml x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (hexane: ethyl acetate = 10: 1) to give 254 mg (yield 91%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.40 (s, 6H), 1.84 (t, 2H), 2.83 (t, 2H), 3.87 (s, 2H), 7.73 (s, 1H), 8.04 (d, 1H)

Production Example 13 8-nitro-2,2-dimethylchroman-6-carboxylic acid methyl ester

200 mg (0.91 mmol) of the compound obtained in Preparation Example 11 was dissolved in 1 ml of acetic anhydride, and 0.1 ml (1.4 mmol) of 63% nitric acid was mixed with 0.8 ml of acetic acid and added slowly at room temperature. After stirring for 24 hours, the mixture was extracted with ethyl acetate (20 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (hexane: ethyl acetate = 1: 2) to give 200 mg (yield 82%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.42 (s, 6H), 1.92 (t, 2H), 2.90 (t, 2H), 3.91 (s, 3H), 7.96 (d, 1H), 8.30 (d, 1H)

Production Example 14 8-amino-2,2-dimethylchroman-6-carboxylic acid methyl ester

120 mg (0.45 mmole) of the compound obtained in Preparation Example 13 was dissolved in 6 ml of methanol, 10% Pd / C 20 mg was added, and the mixture was stirred for 1 hour and 30 minutes at a hydrogen pressure of 50 psi. After the reaction, the reaction solution was filtered through celite and washed with 50 ml of ethyl acetate to remove the black carbon solid, the solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol: dichloromethane = 1: 9). ) To give 81 mg (yield 76%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.42 (s, 6H), 1.90 (t, 2H), 2.84 (t, 2H), 3.90 (s, 3H), 3.98 (br, 2H), 6.80 (d, 1H), 7.53 (d, 1H)

Production Example 15 4-Bromo-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

80 mg (0.37 mmol) of the compound obtained in Preparation Example 4 was dissolved in 5 mL of chloroform at 0 ° C., and then 0.019 mL (0.37 mmole) of bromine was slowly added thereto. After stirring for 1 hour, it was diluted in 15 mL of water and extracted with ethyl acetate (20 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (hexane: ethyl acetate = 10: 1) to give 121 mg (yield 86%) of a dibromo compound. .

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.70 (s, 3H), 3.90 (s, 3H), 4.47 (d, 1H), 5.53 (d, 1H), 6.82 (d, 1H), 7.86 (dd, 1H), 8.21 (d, 1H)

MS ( m / z ) M ⁺ = 378 (M ⁺ )

100 mg (0.36 mmol) of the dibromo compound obtained above was dissolved in 3 ml of toluene, and 0.072 ml (0.48 mmol) of 1,8-diazabicyclo (5,4,0) undec-7-ene was added. Heat reflux for a time. The reaction solution was cooled and then purified by silica gel column chromatography (hexane: ethyl acetate = 10: 1) to give 99 mg (yield 93%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.47 (s, 6H), 3.88 (s, 3H), 6.05 (s, 1H), 6.78 (d, 1H), 7.86 (dd, 1H), 8.08 (d, 1H)

Production Example 16 4,8-Dibromo-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

294 mg (0.99 mmol) of the compound obtained in Preparation Example 5 were reacted in the same manner as in Example 15, to obtain 227 mg (yield 61%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.50 (s, 3H), 1.54 (s, 3H), 3.88 (s, 3H), 6.08 (s, 1H), 8.02 (d, 1H), 8.11 (d, 1H)

MS ( m / z ) M ⁺ = 376 (M ⁺ ), 363

Production Example 17 4-Bromo-8-nitro-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

136 mg (0.52 mmole) of the compound obtained in Preparation Example 6 was reacted in the same manner as in Example 15 to obtain 123 mg (yield 69%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.56 (s, 6H), 3.95 (s, 3H), 6.22 (s, 1H), 8.29 (d, 1H), 8.42 (d, 1H)

Production Example 18 4-Bromo-7-chloro-2,2-dimethyl-2 H -Cromen-6-carboxylic acid methyl ester

420 mg (1.55 mmol) of the compound obtained in Preparation Example 7 were reacted in the same manner as in Example 15, to obtain 441 mg (yield 86%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.47 (s, 6H), 3.92 (s, 3H), 6.05 (s, 1H), 6.88 (s, 1H), 7.95 (s, 1H)

Production Example 19 4-Bromo-2,2,7-trimethyl-2 H -Cromen-6-carboxylic acid methyl ester

120 mg (0.52 mmol) of the compound obtained in Preparation Example 8 were reacted in the same manner as in Example 15 to obtain 99 mg (yield 61%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.46 (s, 6H), 2.54 (s, 3H), 3.85 (s, 3H), 5.99 (s, 1H), 6.64 (s, 1H), 7.99 (s, 1H)

Production Example 20 (3R, 4S) -3-hydroxy-4-methoxy-2,2-dimethylchroman-6-carboxylic acid methyl ester

120 mg (0.55 mmol) of the compound obtained in Preparation Example 4 and 17 mg of the (R, R) Jacobson's reagent were dissolved in 1.5 ml of dichloromethane, and then slowly added to 1.6 ml of 0.55 M NaCl and 1.6 ml of 0.05 M Na ₂ HPO _4. . After reacting for 24 hours at room temperature, the mixture was filtered through celite and extracted with 50 ml of dichloromethane. The organic layer was dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 106 mg of epoxide compound (yield 82%).

100 mg (0.43 mmole) of (3R, 4R) epoxide compound was dissolved in 2 ml of methanol, 1 ml of 5% HCl / MeOH solution was added, followed by stirring at room temperature for 30 minutes. The reaction solution was diluted with 10 mL of water and extracted with ethyl acetate (25 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to give 97 mg (yield 84%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.30 (s, 1H), 1.48 (s, 3H), 2.43 (d, 1H), 3.57 (s, 3H), 3.88 (m, 4H), 4.34 (d, 1H), 6.80 (d, 1H), 7.27 (dd, 1H), 8.07 (s, 1H)

Production Example 21 (3R, 4S) -3-hydroxy-4-imidazol-1-yl-2,2-dimethylchroman-6-carboxylic acid methyl ester

120 mg (0.51 mmol) of the (3R, 4R) epoxide compound obtained in Preparation Example 20 and 104 mg (1.53 mmol) of imidazole were heated at 70 ° C. for 1 hour, and the reaction solution was diluted with 15 ml of water and ethyl acetate. Extracted with (20 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated, and the residue was purified by silica gel column chromatography (methanol: dichloromethane = 1: 9) to give 100 mg (yield 65%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.32 (s, 3H), 1.54 (s, 3H), 3.80 (s, 3H), 3.94 (d, 1H), 5.32 (d, 1H), 6.93 (d, 1H), 7.03 (d, 2H), 7.41 (s, 1H), 7.89 (m, 2H)

MS ( m / z ) M ⁺ = 302, 271, 235

Preparation Example 22 (3R, 4S) -3-hydroxy-4-pyrrole-1-yl-2,2-dimethylchroman-6-carboxylic acid methyl ester

128 mg (0.55 mmol) of the (3R, 4R) epoxide compound obtained in Preparation Example 20 and 1.52 ml (2.20 mmol) of pyrrole were heated at 100 ° C. for 2 hours. The reaction solution was diluted with 15 mL of water and extracted with ethyl acetate (20 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to give 91 mg (yield 55%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.27 (s, 3H), 1.53 (s, 3H), 2.12 (d, 1H), 3.65 (dd, 1H), 3.84 (s, 3H), 3.96 (d, 1H), 6.22 (m, 2H), 6.78 (d, 1H), 6.86 (t, 1H), 7.60 (d, 1H), 7.81 (d, 1H), 7.97 (s, 1H)

MS ( m / z ) M ⁺ = 301 (M ⁺ ), 270, 228

Production Example 23 2,2,4-trimethyl-2 H -Cromen-6-carboxylic acid methyl ester

293 mg (1.25 mmol) of 2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester was added 5 ml of diethyl ether, and 0.8 ml (2.5 mmol) of 3M methyl magnesium bromide was slowly added at 0 ° C. The mixture was gradually heated to 50 ° C. for 2 hours, and 15 ml of saturated NH ₄ Cl aqueous solution was added thereto, followed by extraction with ethyl acetate (20 ml × 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to give 156 mg (yield 50%) of tertiary alcohol compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.42 (s, 6H), 1.63 (s, 3H), 1.85 (s, 1H), 2.04 (dd, 2H), 3.89 (s, 3H), 6.83 (d, 1H), 7.84 (dd, 1H), 8.21 (d, 1H)

142 mg (0.57 mmol) of tertiary alcohol compound was dissolved in 5 ml of toluene, and 11 mg (0.057 mmol) of p -toluenesulfonic acid was added and heated to reflux for 10 minutes. The reaction solution was cooled and purified by silica gel column chromatography (hexane: ethyl acetate = 10: 1) to give 100 mg (yield 75%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.42 (s, 6H), 2.05 (s, 3H), 3.88 (s, 3H), 5.46 (s, 1H), 6.78 (dd, 1H), 7.81 (m, 2H)

MS ( m / z ) M ⁺ = 232 (M ⁺ ), 217, 201

Production Example 24 8-Bromo-2,2,4-trimethyl-2 H -Cromen-6-carboxylic acid methyl ester

248 mg (0.79 mmole) of 8-bromo-2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester compound was reacted in the same manner as in Preparation Example 147 mg (yield 60%) Got.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.45 (s, 3H), 1.50 (s, 3H), 1.63 (s, 3H), 1.88 (s, 1H), 2.07 (m, 2H), 3.89 (s, 3H), 8.15 (s, 2H)

Production Example 25 7-Chloro-2,2,4-trimethyl-2 H -Cromen-6-carboxylic acid methyl ester

6-Chloro-2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester compound was reacted in the same manner as in Preparation Example 23 using 405 mg (1.51 mmol) of 233 mg (yield 58%) of the target compound. Got it.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.41 (s, 6H), 2.01 (s, 3H), 3.90 (s, 3H), 5.46 (s, 1H), 6.86 (s, 1H), 7.68 (s, 1H)

MS ( m / z ) M ⁺ = 266 (M ⁺ -1)

Production Example 26  2,2,4,7-tetramethyl-2 H -Cromen-6-carboxylic acid methyl ester

314 mg (1.27 mmol) of 6-methyl-2,2-dimethyl-4-oxochroman-6-carboxylic acid methyl ester compound was reacted in the same manner as in Preparation Example 23 to obtain 196 mg of the target compound (yield 63%). Got it.

¹ H NMR (300 MHz, CDCl ₃ ) δ 2.02 (s, 3H), 2.54 (s, 3H), 3.86 (s, 3H), 5.40 (s, 1H), 6.64 (s, 1H), 7.76 (s, 1H)

Example 1 Preparation of (4-hydroxy-2,2-dimethylchroman-6-carbonyl) guanidine

285 mg (1.21 mmole) of the compound obtained in Preparation Example 1 was dissolved in 5 mL of DMF, and 3.6 mL (7.26 mmol) of 2.0 M guanidine methanol solution was added thereto. After stirring at room temperature for 24 hours, the reaction solution was diluted with 25 mL of water and extracted with ethyl acetate (25 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (methanol: dichloromethane = 1: 9) to give 174 mg (yield 56%) of the title compound.

¹ H NMR (200 MHz, CDCl ₃ ) δ 1.32 (s, 3H), 1.45 (s, 3H), 1.90 (m, 1H), 2.15 (m, 1H), 4.81 (m, 1H), 5.23 (br s , 2H), 7.78 (dd, 1H), 7.83 (dd, 1H), 8.26 (s, 1H)

Example 2 Preparation of (4-methoxy-2,2-dimethylchroman-6-carbonyl) guanidine

80 mg (0.32 mmole) of the compound obtained in Preparation Example 2 was dissolved in 3 ml of DMF, and then 1.0 ml of 2.0 M guanidine methanol solution was added thereto. After stirring for 24 hours at room temperature the reaction solution was diluted with 25 mL of water and extracted with ethyl acetate (25 mL x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (methanol: dichloromethane = 1: 9) to obtain 77 mg (yield 64%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.34 (s, 3H), 1.45 (s, 3H), 1.94 (dd, 1H), 2.08 (dd, 1H), 3.48 (s, 3H), 4.45 (t, 1H), 6.31 (br, 3H), 6.77 (d, 1H), 8.01 (d, 1H), 8.23 (s, 1H)

MS ( m / z ) M ⁺ = 277 (M ⁺ ), 244, 221

Example 3 Preparation of (4-benzyloxy-2,2-dimethylchroman-6-carbonyl) guanidine

20 mg (0.37 mmol) of the compound obtained in Preparation Example 3 was reacted in the same manner as in Example 2 to obtain 104 mg (yield 80%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.34 (s, 3H), 1.48 (s, 3H), 2.10 (m, 2H), 4.70 (m, 3H), 6.50 (br, 3H), 6.77 (d, 1H), 7.26 (m, 5H), 7.92 (dd, 1H), 8.14 (s, 1H)

MS ( m / z ) M ⁺ = 353 (M ⁺ ), 311

Example 4 (2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

200 mg (0.98 mmol) of the compound obtained in Preparation Example 4 were reacted in the same manner as in Example 2 to obtain 108 mg (yield 45%) of the title compound.

¹ H NMR (200 MHz, CDCl ₃ ) δ 1.44 (s, 3H), 1.45 (s, 3H), 5.64 (d, 1H, J = 10 Hz), 6.33 (d, 1H, J = 10 Hz), 6.78 (d, 1H, J = 8.6 Hz), 7.91 (d, 1H, J = 1.6 Hz), 7.99 (dd, 1H, J = 8.6, 1.6 Hz), 8.53 (br s, 2H), 8.92 (br s, 2H), 11.27 (br s, 1H)

Example 5 (8-Bromo-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

150 mg (0.51 mmol) of the compound obtained in Preparation Example 5 were reacted in the same manner as in Example 2 to obtain 124 mg (yield 75%) of the title compound.

¹ H NMR (300MHz, CDCl ₃ ) δ 1.46 (s, 6H), 5.75 (d, 1H), 6.37 (d, 1H), 7.68 (d, 1H), 8.07 (d, 1H)

MS ( m / z ) M ⁺ = 323, 325 (M ⁺ )

Example 6 (8-nitro-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

70 mg (0.27 mmol) of the compound obtained in Preparation Example 6 was reacted in the same manner as in Example 2 to obtain 70 mg (yield 92%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.52 (s, 6H), 5.75 (d, 1H), 6.32 (br, 2H), 6.40 (d, 1H), 7.98 (d, 1H), 8.56 (d, 1H)

MS ( m / z ) M ⁺ = 290 (M ⁺ ), 275, 258

Example 7 (7-Chloro-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

100 mg (0.40 mmol) of the compound obtained in Preparation Example 7 was reacted in the same manner as in Example 2 to obtain 50 mg (yield 45%) of the title compound.

¹ H NMR (300MHz, CDCl ₃ ) δ 1.32 (s, 6H), 5.64 (d, 1H), 6.26 (d, 1H), 6.66 (s, 1H), 7.16 (s, 1H)

MS ( m / z ) M ⁺ = 280 (M ⁺ )

Example 8 (2,2,7-trimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

110 mg (0.40 mmol) of the compound obtained in Preparation Example 8 was reacted in the same manner as in Example 2 to obtain 41 mg (yield 37%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.42 (s, 6H), 2.44 (s, 3H), 5.72 (d, 1H), 6.38 (d, 1H), 6.67 (s, 1H), 7.34 (s, 1H)

MS ( m / z ) M ⁺ = 259, 244, 227

Example 9 (8-dimethylamino-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

110 mg (0.42 mmol) of the compound obtained in Preparation Example 9 were reacted in the same manner as in Example 2 to obtain 60 mg (yield 68%) of the title compound.

¹ H NMR (300MHz, DMSO- d ₆ ) δ 1.40 (s, 6H) 2.70 (s, 6H), 5.73 (d, 1H), 6.36 (d, 1H), 7.37 (d, 1H), 7.44 (d , 1H)

MS ( m / z ) M ⁺ = 288 (M ⁺ ), 271, 256

Example 10 (8-methanesulfonylamino-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

Reaction was carried out in the same manner as in Example 2 using 91 mg (0.29 mmol) of the compound obtained in Preparation Example 10 to obtain 29 mg (yield 30%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.49 (s, 6H), 2.99 (s, 3H), 4.59 (s, 3H), 5.77 (d, 1H), 6.41 (d, 1H), 7.61 (s , 1H), 7.98 (s, 1H)

MS ( m / z ) M ⁺ = 338, 323, 306

Example 11 Preparation of (2,2-dimethylchroman-6-carbonyl) guanidine

148 mg (0.72 mmol) of the compound obtained in Preparation Example 11 were reacted in the same manner as in Example 2, obtaining 110 mg of the target compound (yield 62%).

¹ H NMR (200 MHz, CD ₃ OD) δ 1.33 (s, 6H), 1.80 (t, 2H), 2.79 (t, 2H), 5.29 (br s, 4H), 6.75 (d, 1H), 7.91 ( m, 2H)

Example 12 Preparation of (8-Bromo-2,2-dimethylchroman-6-carbonyl) guanidine

242 mg (0.81 mmol) of the compound obtained in Preparation Example 12 were reacted in the same manner as in Example 2 to obtain 235 mg (yield 89%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.39 (s, 6H), 1.87 (t, 2H), 2.87 (t, 2H), 7.80 (d, 1H), 8.08 (d, 1H)

MS ( m / z ) M ⁺ = 327 (M ⁺ ), 270

Example 13 Preparation of (8-nitro-2,2-dimethylchroman-6-carbonyl) guanidine

200 mg (0.75 mmol) of the compound obtained in Preparation Example 13 were reacted in the same manner as in Example 2 to obtain 195 mg (yield 89%) of the target compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.40 (s, 6H), 1.90 (t, 2H), 2.89 (t, 2H), 6.28 (br, 3H), 8.09 (d, 1H), 8.50 (d, 1H)

MS ( m / z ) M ⁺ = 292 (M ⁺ ), 275, 259, 237

Example 14 Preparation of (8-amino-2,2-dimethylchroman-6-carbonyl) guanidine

120 mg (0.41 mmol) of the compound obtained in Preparation Example 14 were reacted in the same manner as in Example 2 to obtain 80 mg (yield 76%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.40 (s, 6H), 1.88 (t, 2H), 2.83 (t, 2H), 7.17 (m, 2H)

MS ( m / z ) M ⁺ = 262 (M ⁺ ), 245, 206

Example 15 (4-Bromo-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

90 mg (0.30 mmol) of the compound obtained in Preparation Example 15 was reacted in the same manner as in Example 2 to obtain 89 mg (yield 92%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.46 (s, 6H), 6.02 (s, 1H), 6.35 (br, 2H), 6.76 (d, 1H), 8.02 (dd, 1H), 8.23 (d , 1H)

MS ( m / z ) M ⁺ = 323, 325 (M ⁺ )

Example 16 (4,8-Dibromo-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

Using 100 mg (0.27 mmol) of the compound obtained in Preparation Example 16, the reaction was carried out in the same manner as in Example 2 to obtain 87 mg (yield 80%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.50 (s, 6H), 6.27 (s, 1H), 8.14 (d, 1H), 8.21 (d, 1H)

MS ( m / z ) M ⁺ = 405, 403 (M ⁺ )

Example 17 (4-Bromo-8-nitro-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

105 mg (0.31 mmol) of the compound obtained in Preparation Example 17 were reacted in the same manner as in Example 2 to obtain 100 mg (yield 87%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.55 (s, 6H), 6.42 (s, 1H), 8.43 (d, 1H), 8.50 (d, 1H)

MS ( m / z ) M ⁺ = 370 (Br81), 368 (Br79), 355, 336

Example 18 (4-Bromo-7-chloro-2,2-dimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

105 mg (0.32 mmol) of the compound obtained in Preparation Example 18 were reacted in the same manner as in Example 2 to obtain 64 mg (yield 56%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.46 (s, 6H), 6.23 (s, 1H), 6.84 (s, 1H), 7.63 (s, 1H)

MS ( m / z ) M ⁺ = 359, 357 (M ⁺ .Br81 / Br79)

Example 19 (4-Bromo-2,2,7-trimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

112 mg (0.36 mmol) of the compound obtained in Preparation Example 19 was reacted in the same manner as in Example 2 to obtain 73 mg (yield 60%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.42 (s, 6H), 2.43 (s, 3H), 6.14 (s, 1H), 6.65 (s, 1H), 7.64 (s, 1H)

MS ( m / z ) M ⁺ = 339 (Br81), 337 (Br79)

Example 20 Preparation of [(3R, 4S) -3-hydroxy-4-methoxy-2,2-dimethylchroman-6-carbonyl] guanidine

90 mg (0.34 mmol) of the compound obtained in Preparation Example 20 was reacted in the same manner as in Example 2 to obtain 40 mg (yield 40%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.27 (s, 3H), 1.43 (s, 3H), 3.64 (s, 3H), 3.75 (d, 1H), 4.32 (d, 1H), 6.74 (d , 1H), 7.89 (dd, 1H), 8.14 (d, 1H)

MS ( m / z ) M ⁺ = 293 (M ⁺ )

Example 21 Preparation of [(3R, 4S) -3-hydroxy-4-imidazol-1-yl-2,2-dimethylchroman-6-carbonyl] guanidine

100 mg (0.33 mmole) of Compound 35 obtained in Preparation Example 21 was dissolved in 2 ml of DMF, and 1.0 ml of 2.0 M guanidine methanol solution was added thereto. After heating at 80 ° C. for 24 hours, the reaction solution was diluted with 20 mL of water and extracted with ethyl acetate (20 mL × 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a residue obtained by silica gel column chromatography (methanol: dichloromethane = 1: 9) to give 43 mg (40%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.27 (s, 3H), 1.49 (s, 3H), 3.83 (d, 1H), 5.25 (d, 1H), 6.82 (d, 1H), 6.97 (d , 2H), 7.52 (d, 1H), 7.86 (s, 1H), 7.94 (m, 1H)

MS ( m / z ) M ⁺ = 329 (M ⁺ ), 296

Example 22 Preparation of [(3R, 4S) -3-hydroxy-4-pyrrole-1-yl-2,2-dimethylchroman-6-carbonyl] guanidine

130 mg (0.43 mmol) of the compound obtained in Preparation Example 22 were reacted in the same manner as in Example 21 to obtain 59 mg (yield 42%) of the title compound.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.22 (s, 3H), 1.46 (s, 3H), 3.77 (d, 1H), 3.94 (d, 1H), 6.04 (m, 3H), 6.65 (m , 1H), 6.74 (m, 1H), 7.63 (d, 1H), 7.78 (m, 1H)

MS ( m / z ) M ⁺ = 328 (M ⁺ ), 286

Example 23 (2,2,4-trimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

Reaction was carried out in the same manner as in Example 2 using 91 mg (0.39 mmol) of the compound obtained in Preparation Example 23 to obtain 87 mg (yield 87%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.40 (s, 6H), 2.04 (s, 3H), 5.43 (s, 1H), 6.77 (d, 1H), 7.96 (m, 2H)

MS ( m / z ) M ⁺ = 259 (M ⁺ ), 238, 227

Example 24 (8-Bromo-2,2,4-trimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

100 mg (0.32 mmol) of the compound obtained in Preparation Example 24 were reacted in the same manner as in Example 2 to obtain 82 mg (yield 75%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.45 (s, 6H), 2.05 (s, 2H), 5.47 (s, 1H), 6.30 (br, 3H), 7.92 (s, 1H), 8.26 (s, 1H)

MS ( m / z ) M ⁺ = 339, 337 (M ⁺ , Br81, Br79), 322, 305

Example 25 (7-Chloro-2,2,4-trimethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

122 mg (0.46 mmol) of the compound obtained in Preparation Example 25 was reacted in the same manner as in Example 2 to obtain 90 mg (yield 67%) of the title compound.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.38 (s, 6H), 2.01 (s, 3H), 4.59 (s, 1H), 5.56 (s, 1H), 6.76 (s, 1H), 7.41 (s, 1H)

MS ( m / z ) M ⁺ = 293 (M ⁺ -1)

Example 26 (2,2,4,7-Tetramethyl-2 H Preparation of -Cromen-6-carbonyl) guanidine

160 mg (0.65 mmol) of the compound obtained in Preparation Example 26 were reacted in the same manner as in Example 2 to obtain 59 mg (yield 33%) of the title compound.

¹ H NMR (300 MHz, DMSO) δ 1.36 (s, 6H), 2.01 (S, 3H), 2.41 (s, 3H), 5.57 (s, 1H), 6.67 (s, 1H), 7.61 (s, 1H )

MS ( m / z ) M ⁺ = 273, 258

Various pharmacological actions were investigated by performing the following experiments on the compounds of Formula 1 according to the present invention.

Experimental Example 1 Inhibitory Effect of NHE-1

In order to measure the NHE-1 inhibitory effect of the (chromen-6-carbonyl) guanidine derivative of the present invention at the cellular stage, the following experiment was performed.

Human NHE-1 was expressed in CCL39-derived PS120 cells, and DMEM (Dulbecco's modified) supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin (100X solution), and 1% L-glutamine (200 mM solution) The cells were cultured in Eagle's medium). After treatment with trypsin, PS120 / NHE-1 cells grown about 80-90% in a 100 mm diameter dish, and once with PBS (phosphate buffer saline), Na-free buffer (138.2 mM Choline chloride, 4.9 mM) KCl, 1.5 mM CaCl ₂ · washed once in _{2H 2 O, 1.2 mM MgSO 4} · 7H 2 O, 1.2mM KH 2 PO 4, 15mM D- glucose, 20mM HEPES, at pH 7.4) . This was centrifuged to precipitate the precipitate in sodium free buffer containing 20 mM NH ₄ Cl and 10 uM BCECF-AM [2 ', 7'-bis (2-carboxyethyl) -5,6-carboxy-fluorescein acetoxymethyl ester]. , 37 ℃, incubated for 30 minutes in a CO ₂ incubator. In order to remove NH ₄ Cl and simultaneously wash the remaining BCECF-AM, PS120 / NHE-1 cells were centrifuged and washed once with sodium-free buffer, so that the number of cells was 2.5 × 10 ⁴ cells / 10 μl. A suspension was made and stored in the dark at 4 ° C. 180 μl HBS buffer (137 mM NaCl, 4.9 mM KCl, 1.5 mM CaCl ₂ · 2H ₂ O, 1.2 mM MgSO ₄ · 7H ₂ O, 1.2 mM KH ₂ PO ₄ , 15 mM D-glucose, 20 mM HEPES, in 96 well plates at pH7.4) and 10 μl each of the compound dissolved in DMSO or DMSO (0.03˜10 uM), and mixed well. Finally, 10 μl of PS120 / NHE-1 cells, which induced intracellular acidification, were added and stirred. Four minutes after the addition of the cells, fluorescence (excitation: 485/444 nm, emission: 535 nm) was measured using a fluorescence spectrophotometer (XEMINI-XS; Molecular Device) for 96 well plates. The measured fluorescence value was converted to pH using high-K ⁺ / nigericin technique. Cells that induced intracellular acidification with NH ₄ Cl prepulse are restored to normal intracellular acidification by the operation of NHE-1. At this time, a concentration of a compound that inhibits the recovery of intracellular acidification by 50% is obtained (IC _50). Value) The inhibitory effect on NHE-1 was measured. As a control material was tested using a cariporide (cariporide).

The results are shown in Table 2.

Inhibitory Effect of the Compound of Formula 1 on NHE-1 compound IC ₅₀ (uM) compound IC ₅₀ (uM) compound IC ₅₀ (uM) Cariporide 0.68 Example 9 9.41 Example 18 0.31 Example 1 > 30 Example 10 8.68 Example 19 0.08 Example 2 > 30 Example 11 2.84 Example 20 > 30 Example 3 7.40 Example 12 1.65 Example 21 > 30 Example 4 1.50 Example 13 5.53 Example 22 > 30 Example 5 1.36 Example 14 35.26 Example 23 0.82 Example 6 2.01 Example 15 0.72 Example 24 0.46 Example 7 1.53 Example 16 0.67 Example 25 0.66 Example 8 0.25 Example 17 0.49 Example 26 0.33

As shown in Table 2, the control material, Carporide, exhibited an IC ₅₀ of 0.68 uM, indicating an excellent inhibitory effect on NHE-1. Among the compounds of the present invention, the compounds of Examples 4-8, 11-12, 15-19 and 23-26 showed IC ₅₀ of 3.0 uM or less, which showed a similar or superior NHE-1 inhibitory effect to carporide. Among them, the compounds of Examples 8, 15-19, 23-26 exhibited IC ₅₀ of 1.0 uM or less, and thus the NHE-1 inhibitory effect was very excellent, especially the compounds of Examples 8, 17, 18, 19, 24, and 26. Silver IC ₅₀ was 0.5 uM or less, superior to carporide. In Example 19, the IC ₅₀ was 0.08 uM, and NHE-1 inhibitory effect was 8 times stronger than that of the carporide.

Therefore, the (chromen-6-carbonyl) guanidine derivatives of the present invention show a strong inhibitory effect on NHE-1, and thus can be usefully used as a protective agent against ischemia / reperfusion injury through NHE-1 inhibition.

Experimental Example 2 Cardioprotective Effect on Extracted Ischemic Heart in Rats

In order to find out whether the (chromen-6-carbonyl) guanidine derivatives of the present invention show a cardioprotective effect by enhancing the function recovery of the ischemic heart in the extracted heart, the following extraction heart experiment was conducted in rats.

Male rats (300-450 g, Korea Research Institute of Chemical Technology) were anesthetized by intraperitoneal injection of 100 mg / kg of sodium pentobarbital, and heparin 1000 U / kg was administered intravenously and the heart was extracted. Specifically, a cannula (PE 240) is inserted into the trachea, and artificial respiration is performed using a rodent ventilator. In this state, the aortic cannula is inserted into the aorta, and the heart is extracted under retrograde perfusion, and the Langendorff Apparatus Hung quickly on the heart and removing unnecessary tissue attached to the heart, and then modified Krebs-Henseleit bicarbonate buffer (modified Krebs-Henseleit bicarbonate buffer; composition <mM / L) saturated with 95% O ₂ /5% CO ₂ under constant pressure perfusion (85 mmHg). >: 116 NaCl, 4.7 KCl, 1.1 MgSO ₄ , 1.17 KH ₂ PO ₄ , 24.9 NaHCO ₃ , 2.52 CaCl ₂ , 8.32 glucose, 2.0 pyruvate). A metal cannula connected with a latex balloon filled with a mixture of ethanol and distilled water (1: 1 vol / vol) is inserted through the pulmonary vein into the left ventricle and the left ventricular pressure delivered to the balloon is equilibrated through a pressure transducer. (isovolumetric) magnification (Plugsys bridge amplifier) was processed and recorded on a recorder (Linearcorder mark 8 WR 3500). After the heart was stabilized for 15 minutes, the left ventricular enddiastolic pressure (LVEDP) was set at 5 mmHg, and the balloon volume was maintained for the entire experimental period.

Baseline heart contractile function, heart rate (HR) and coronary flow (CF) were measured. Left ventricular developed pressure (LVDP), an indicator of cardiac contractile function, is the difference between left ventricular peak systolic pressure (LVSP) and left ventricular end diastolic ptrssure (LVEDP). Calculated. Unlike the in vivo heart, the heart rate-pressure product, an important indicator of cardiac performance indirectly in Langendorff heart, in which cardiac output cannot be measured, the double product RPP ( rate-pressure product] was calculated by multiplying the heart rate (HR) by the left ventricular development pressure (LVDP). The temperature of the heart was kept constant by soaking the heart in a physiological fluid at 37 ° C. continuously supplied with 95% O ₂ /5% CO ₂ over the entire period of the experiment. After stabilization, the heart is perfused for 10 minutes with a solvent (0.04% dimethylsulfoxide, DMSO) or a solution containing a certain concentration of the compound of the present invention or a control drug, and then the heart contraction function and heart rate (HR) and Coronary flow (CF) was measured again and the perfusion solution was completely blocked to induce global ischemia for 30 minutes. After perfusion for 30 minutes, each indicator (LVDP, HR, LVEDP, CF) was measured again. The negative control group was administered with only solvent, and the carrier was used as a carrier.

The results are shown in Table 3.

Protective Effect of the Compound of Formula 1 on Extracted Ischemia / Reperfusion Heart in Rats compound Concentration (uM) RPP ¹ (%) LVEDP ² (mmHg) Negative Control - 15.5 55.3 Cariporide 10 69.8 22.4 Example 4 10 43.3 21.3 Example 5 10 45.7 28.0 Example 7 10 25.9 33.0 Example 8 10 17.4 51.7 Example 11 10 24.2 40.3 Example 12 10 11.0 23.7 Example 15 10 44.4 27.7 Example 16 10 8.9 51.5 Example 17 10 64.9 14.0 Example 18 10 32.3 25.3 Example 19 10 23.1 39.0 Example 23 10 50.8 15.7 Example 24 10 4.2 3.7 Example 25 10 24.0 29.0 Example 26 10 14.9 50.5 1: product of heart rate-pressure; RPP (rate pressure product), LVDP (left ventricular pressure) × HR (heart rate)% of pre-ischemic induction value 2: left vetricualr end diastolic pressure

As shown in Table 3, in the isolated ischemic heart experiment using the extracted heart of the rat, the product of the left ventricular development pressure (LVDP) and the heart rate (Double Product parameter, LVDP x HR) in the negative control group well reflects the contractile function of the heart. It was found that the contractile function of the heart was markedly decreased by 15.5% before the ischemic induction, and the systolic contraction caused by ischemia / reperfusion was shown. Significantly increased.

Carriporide 10uM treated group, myocardial contractile function (LVDP x HR) after reperfusion was 69.8% before ischemia induction, significantly increased compared to negative control group, and left ventricular diastolic pressure (LVEDP) was significantly lower than negative control group. It showed a protective effect on the heart. Compounds of Examples 4, 5, 15, 17 and 23, which had excellent inhibitory effects on NHE-1 in cell experiments, induced ischemia-induced myocardial contractile function (LVDP x HR) decreased by ischemia / reperfusion by 10 uM treatment. He recovered to more than 40% of the previous time and also significantly reduced left ventricular diastolic pressure. In particular, the compound of Example 17 restored the cardiac contractility to 64.9%, and lowered to 14.0 mmHg of the left ventricular diastolic pressure, showing a protective effect on the ischemia / reperfusion heart similar to that of the carporide.

Therefore, the (chromen-6-carbonyl) guanidine derivatives of the present invention have an excellent protective effect on the ischemic heart by promoting recovery of heart function by ischemia / reperfusion injury, thus preventing and treating diseases related to ischemic heart disease. It can be usefully used.

Experimental Example 3: In Vivo of Rats in vivo ) Cardioprotection against ischemic heart model

In order to find out whether the (chromen-6-carbonyl) guanidine derivatives of the present invention have a protective effect on the ischemic heart in vivo, the following experiments were carried out on the anti-ischemic effect in rats. It was investigated through.

Male rats (350-450 g, Korea Research Institute of Chemistry) were anesthetized with pentobarbital at 75 mg / kg intraperitoneally. After tracheotomy, artificial respiration was performed at a stroke volume of 10 ml / kg and 60 heart rate per minute. The cannula was inserted into the femoral vein and the femoral artery and used for drug administration and blood pressure measurement, respectively. On the other hand, in the ischemic myocardial injury model, the body temperature has a significant effect on the results. Therefore, the temperature of the rat was constant at 37 ° C. using a probe inserted in the rectum and a homeothermic blanket control unit. Maintained. Since then, the mean arterial blood pressure and heart rate (HR) of the rat were continuously measured. At this time, the Statham P23XL pressure transducer (Statham P23XL pressure transducer, Grass Ins., MA, USA) was used, and the ECG / RATE Coupler, Hugo Sachs Electronic (Germany) was used to measure the heart rate. . In addition, all changes were recorded continuously using a Graphtec Linearcorder WR 3310, Hugo Sachs Electronic.

The left coronary artery was ligated by the method of Selye H. as follows. In other words, a part of the chest of the rat is opened by left thoracotomy, the pressure is applied to the right chest of the rat anesthetized with the left hand of the anesthesia, and the heart is pushed to the outside to lighten the heart with the thumb and index finger of the left hand. Fixed. After that, the surgeon (5-0 silk ligature) attached suture needle carefully lifted the part containing the left anterior desending coronary artery (LAD), and quickly thoracic the heart. repositioned in the cavity and both ends of the surgeon were positioned externally. Both ends of the surgeon were allowed to pass through a PE tube (PE100, 2.5 cm) and left for 20 minutes to stabilize. Thereafter, a vehicle or a drug was administered through the cannula inserted into the femoral vein, and left for 30 minutes to fully exhibit the effect of the drug. At this time, carrieporide was used as a control drug.

Then, the PE tube inserted into the thread was pushed into the heart, and the end thread of the tube was pulled with hemostatic tweezers, and the PE tube was pressed vertically to the coronary artery, and the pressure was left to remain for 45 minutes. After occlusion, hemostatic tweezers were removed and reperfused for 90 minutes.

The coronary artery was religated by this method and 2 ml of 1% Evans blue solution was administered intravenously. Pentobarbital was then intravenously administered to slaughter rats and the heart was removed to remove the right ventricle and both atria. The left ventricle was horizontally cut into 5-6 slices from the apex and the weight of each section was measured. The surface of each cardiac segment is input to a computer using a Hi-scope, a compact micro vision system, and an image pro plus, from which the blue Areas of normal blood flow tissue colored and uncolored areas were measured. The area ratio of the uncolored area was calculated for the total area of each section, and the weight of each section was multiplied to calculate the area at risk (AAR). The AAR for each section thus obtained was summed and divided by the total left ventricular weight to obtain AAR (%) by Equation 1 below.

AAR (%) = [(sum of AAR for each section) / (total left ventricular weight)] × 100

In addition, cardiac sections were incubated for 15 minutes in 1% 2,3,5-triphenyltetrazolium chloride phosphate buffer solution [2,3,5-triphenyltetrazolium chloride (TTC) phosphate buffer, 37 ° C, pH 7.4) and 10% It was fixed in formalin solution for 20-24 hours. This reduces 2,3,5-triphenyltetrazolium chloride by dehydrogenase of the myocardium and NADH, a cofactor, to formazan dye, so that the normal part of the tissue is red brick. (brick-red color). On the other hand, since there are no dehydrogenases and cofactors in the infarcts of tissues, 2,3,5-triphenyltetrazolium chloride is not reduced and thus does not have a red brick color.

As described above, the normal area and the infarct size of each section were determined by the same method as that of the AAR by whether or not the tissue site was stained by 2,3,5-triphenyltetrazolium chloride . The infarct regions for each of the sections thus obtained were summed and divided by the total AAR weight or the total left ventricular weight to obtain IS (%) by the following equation (2). In this experimental model, the lower the IS (%), the smaller the infarct area, and therefore, the anti-ischemic effect of the test substance was determined to be strong.

The results are shown in Table 4.

IS (%) = [(sum of infarct size for each section) / (weight of total left ventricle or total AAR)] × 100

Protective Effect of the Compound of Formula 1 on Ischemia / Reperfusion Heart in Rats compound Myocardial infarction rate (IS / AAR ¹ ,%) Negative Control 58.6 Cariporide 0.1 mg / kg 40.5 0.3 mg / kg 37.9 Example 4 0.1 mg / kg 45.8 Example 5 1.0 mg / kg 30.2 Example 7 1.0 mg / kg 36.6 Example 12 0.1 mg / kg 45.9 Example 16 1.0 mg / kg 54.2 Example 17 0.1 mg / kg 42.8 1.0 mg / kg 35.1 Example 18 1.0 mg / kg 31.0 Example 23 1.0 mg / kg 48.8 1: IS / AAR (infacrt size / area at risk)

As shown in Table 4, the (chromen-6-carbonyl) guanidine derivatives of the present invention also showed a significantly reduced myocardial infarction rate in the risk region in the in vivo ischemic myocardial injury model using rats. Specifically, the negative control group (solvent-administered group) had a myocardial infarction rate (IS / AAR,%) of 58.6% for the risk area (AAR), indicating that myocardial damage caused by ischemia is very severe. When administered, myocardial infarction was 40.5% and 37.9% by 0.1 and 0.3 mg / kg, respectively, and showed significant anti-ischemic action. Compounds of Examples 4 and 12 showed a 45.8%, 45.9% myocardial infarction rate at 0.1 mg / kg administration, showing a significant decrease over the negative control group. Compounds of Examples 5, 7, 17, and 18 showed myocardial infarction rates of 30.2%, 36.6%, 35.1% and 31.0%, respectively, by 1.0 mg / kg administration, indicating that the ischemic cardioprotective effect was similar to or superior to that of carporide. . In particular, the inhibitory effect on NHE-1 was superior to that of carporide, and the compound of Example 17, which showed excellent cardiac function restoring effect in the extracted ischemia / reperfusion heart model of rats, was administered 42.8% by 0.1 and 1.0 mg / kg. Myocardial infarction was 35.1%, showing a similar protective effect of ischemic heart as compared to the carporide.

Thus, the (chromen-6-carbonyl) guanidine derivatives of the present invention are in vivo In the ischemic heart model, the myocardial infarction rate is reduced and the protective effect of the ischemic heart is excellent.It can be used as a preventive and therapeutic agent for ischemic heart disease such as myocardial infarction, arrhythmia and angina pectoris. It can be used as a cardioprotectant.

Experimental Example 4  : Acute Toxicity of Oral Administration in Rats

In order to determine the acute toxicity of the (chromen-6-carbonyl) guanidine derivatives of the present invention, the following experiment was performed.

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally administered at a dose of 10 mg / kg / 15 ml, each of the compounds obtained in Examples 1-26, suspended in 0.5% methylcellulose solution.

After administration of the test substance, mortality, clinical symptoms, and changes in body weight were examined. Hematological and hematological examinations were performed. Necropsy was performed to visually observe abdominal and thoracic organ abnormalities.

As a result, all animals treated with test substance showed no clinical symptoms and no dead animals, and no toxic changes were observed in weight change, blood test, blood biochemical test, autopsy findings. As a result, all of the tested compounds did not show toxicological changes up to 10 mg / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of at least 10 mg / kg or more.

On the other hand, the compound according to the present invention can be formulated in various forms according to the purpose. Some formulation methods are described below in which the compound according to the present invention is contained as an active ingredient, but the present invention is not limited thereto.

Preparation Example 1 Tablet (Direct Pressurization)

After sifting 5.0 mg of the active ingredient, 14.1 mg of lactose, 0.8 mg of crospovidone USNF, and 0.1 mg of magnesium stearate were mixed and pressed to prepare a tablet.

Preparation Example 2 Tablet (Wet Assembly)

After sifting 5.0 mg of the active ingredient, 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of polysorbate 80 was dissolved in pure water and then an appropriate amount of this solution was added and then atomized. After drying, the fine particles were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were pressed to make tablets.

Preparation Example 3 Powder and Capsule

After sifting 5.0 mg of the active ingredient, it was mixed with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of magnesium stearate. The mixture was prepared using a suitable apparatus. Filled in 5 gelatin capsules.

<Example 4> Injection

Injectables were prepared by containing 100 mg as the active ingredient, as well as 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ .12H ₂ O and 2974 mg of distilled water.

The (chromen-6-carbonyl) guanidine derivative of the present invention exhibits potent inhibitory action against NHE-1, a sodium / hydrogen exchange pathway, and enhances recovery of cardiac function impairment by ischemia / reperfusion in an isolated ischemic heart model. In vivo, ischemic animal model significantly reduces the size of myocardial infarction and shows excellent cardioprotective effect.

Therefore, the composition of the present invention can be used as an agent for the prevention and treatment of ischemic heart disease such as myocardial infarction, heart failure, angina pectoris, and at the time of reperfusion therapy, such as pharmacotherapy using thrombolytic agents or surgical therapy such as coronary artery bypass surgery, coronary percutaneous plastic surgery, etc. It can be used as a cardioprotectant.

Claims (7)

A (chromen-6-carbonyl) guanidine derivative represented by the following formula (1). <Formula 1>

Wherein R ¹ is H or OH, R ² is H, OH, OR ^a , OCH ₂ Ph, Br,

Or R ^a and R ³ is H, CH ₃ , Br, NO ₂ , NH ₂ , Cl, NMe ₂ or NH-SO ₂ Me. In this case, R ^a is a C ₁ ~ C ₄ straight or branched alkyl group,

Represents a single bond or a double bond.) According to claim 1, wherein the compound of Formula 1 1) (4-hydroxy-2,2-dimethylchroman-6-carbonyl) guanidine, 2) (4-methoxy-2,2-dimethylchroman-6-carbonyl) guanidine, 3) (4-benzyloxy-2,2-dimethylchroman-6-carbonyl) guanidine, 4) (2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine, 5) (8-bromo-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine, 6) (8-nitro-2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine, 7) (7-chloro-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine, 8) (2,2,7-trimethyl- 2H -chromen-6-carbonyl) guanidine, 9) (8-dimethylamino-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine, 10) (8-methanesulfonylamino-2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine, 11) (2,2-dimethylchroman-6-carbonyl) guanidine, 12) (8-bromo-2,2-dimethylchroman-6-carbonyl) guanidine, 13) (8-nitro-2,2-dimethylchroman-6-carbonyl) guanidine, 14) (8-amino-2,2-dimethylchroman-6-carbonyl) guanidine, 15) (4-bromo-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine, 16) (4,8-dibromo-2,2-dimethyl- 2H -chromen-6-carbonyl) guanidine, 17) (4-bromo-8-nitro-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine, 18) (4-bromo-7-chloro-2,2-dimethyl-2 H -chromen-6-carbonyl) guanidine, 19) (4-bromo-2,2,7-trimethyl-2 H -chromen-6-carbonyl) guanidine, 20) [(3R, 4S) -3-hydroxy-4-methoxy-2,2-dimethylchroman-6-carbonyl] guanidine, 21) [(3R, 4S) -3-hydroxy-4-imidazol-1-yl-2,2-dimethylchroman-6-carbonyl] guanidine, 22) [(3R, 4S) -3-hydroxy-4-pyrrole-1-yl-2,2-dimethylchroman-6-carbonyl] guanidine, 23) (2,2,4-trimethyl- 2H -chromen-6-carbonyl) guanidine, 24) (8-bromo-2,2,4-trimethyl-2 H -chromen-6-carbonyl) guanidine, 25) (7-chloro-2,2,4-trimethyl-2 H -chromen-6-carbonyl) guanidine, and 26) A (chromen-6-carbonyl) guanidine derivative characterized in that it is (2,2,4,7-tetramethyl- 2H -chromen-6-carbonyl) guanidine. A method for producing the (chromen-6-carbonyl) guanidine derivative according to claim 1, wherein the carboxylic acid derivative (II) is reacted with guanidine to obtain compound (I). <Scheme 1>

(Wherein R ¹ , R ² and R ³ are as defined in Formula 1, and L is a leaving group that can be easily released by guanidine.) An agent for preventing and treating ischemic heart disease, comprising the (chromen-6-carbonyl) guanidine derivative of claim 1 as an active ingredient thereof. The method of claim 4, wherein the ischemic heart disease is myocardial infarction, arrhythmia or angina pectoris prevention and treatment of ischemic heart disease. The method of claim 5, wherein the prevention and treatment of ischemic heart disease comprises protecting the heart against ischemia / reperfusion injury during reperfusion therapy. 7. The agent for preventing and treating ischemic heart disease according to claim 6, wherein the reperfusion therapy is a surgical therapy such as coronary artery bypass surgery, coronary percutaneous plastic surgery, or a drug therapy such as thrombolytics.