Use of irdenafil or its salt in the preparation of a medicament for preventing or treating ischemic brain injury

Abstract

The present invention relates to the use of the compound 1-[4-ethoxy-3-[5-(6,7-dihydro-1-methyl-7-oxy-3-n-propyl-1H-pyrazolo[4,3-d]pyrimidine)]benzenesulfonyl]-3,5-dimethylpiperazine, in particular the use of the compound in the preparation of a drug for the prevention and treatment of diseases caused by cerebral ischemia, in particular ischemic cranial nerve damage and necrosis.

Description

[Technical Field]

[0001] The present invention relates to the pharmaceutical field, in particular to the use of the compound 1-[4-ethoxy-3-[5-(6,7-dihydro-1-methyl-7-oxy-3-n-propyl-1H-pyrazolo[4,3-d]pyrimidine)]benzenesulfonyl]-3,5-dimethylpiperazine, in particular to the use of the compound in the preparation of drugs for the prevention and treatment of diseases caused by cerebral ischemia, in particular to the compound for use in the treatment of ischemic cranial nerve damage and necrosis. [Background technology]

[0002] To date, the number of drugs officially approved for clinical use to treat diseases caused by cerebral ischemia is very limited. t-PA is the first and only thrombolytic drug approved by the US FDA for the treatment of acute cerebral ischemia. It was approved for sale in 1987, but this drug is only effective within three hours of onset. Beyond this time window, its effectiveness is unclear and may result in higher incidence of bleeding. Edaravone, approved for sale in Japan, protects brain cells by scavenging oxygen free radicals and inhibiting lipid peroxidation. This product has been reported to have significant adverse reactions, causing severe death. Butylbenzene peptide, approved for sale in China in 2004, can only be used for acute cerebral ischemia. The drugs mentioned above are only used for acute cerebral ischemia and have limited therapeutic efficacy.

[0003] The compound involved in the present invention is disclosed in Patent No. CN1127506C, and its use in the treatment of erectile dysfunction is also disclosed. Up to now, there has been no report on the prevention and treatment of cerebral ischemia-related diseases. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] CN1127506C Summary of the Invention [Problem to be solved by the invention]

[0005] Currently, the only commercially available product of airdenafil citrate is a tablet. However, oral administration of tablets has disadvantages, such as the time required for the disintegration and dissolution process, the time required for blood drug concentrations to reach their peak, the slow onset of effect, and the difficulty of administration to patients with serious illnesses (e.g., stroke, coma, or inability to eat). Therefore, it is necessary to develop an injectable and oral liquid formulation of airdenafil salt to address these shortcomings in clinical use. However, the solubility of airdenafil citrate in water at room temperature is only about 2 mg / ml. When airdenafil citrate is used as an injectable formulation in water, the dosage concentration is low, the total dosage volume is large, and it is difficult to reconstitute. This limits the rate at which airdenafil blood drug concentrations can increase in vivo and the method of administration of airdenafil injections, such as intravenous infusion or large-volume injection.

[0006] In the prior art, no injectable or oral liquid formulation of irdenafil has been developed, nor has any high-concentration drug composition for injection or oral liquid been developed.High-concentration injectable irdenafil can be administered directly and rapidly, reaching peak blood concentration in the body, producing rapid effects, and avoiding large-volume and long-term administration.Irdenafil injection and oral liquid have important medical value for patients with acute, severe, or swallowing dysfunction (such as stroke, acute thrombosis, confusion, inability to eat, etc.). [Means for solving the problem]

[0007] The present invention provides a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the preparation of a medicament for the treatment or prevention of a human disease caused by cerebral ischemia.

[0008] [ka]

[0009] The compounds of formula (I) have cis- or trans-isomers at the 3,5-dimethyl of the piperazine ring, and the structures of the cis- and trans-isomers are as follows:

[0010] [ka]

[0011] The compound of formula (I) having a cis-structure is airdenafil, whose chemical name is 1-[3-(6,7-dihydro-1-methyl-7-oxy-3-n-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxybenzenesulfonyl]-3,5-dimethylpiperazine.

[0012] In one embodiment, the present invention provides the use of a pharmaceutically acceptable salt or pharmaceutical composition comprising irdenafil in the preparation of a medicament for the treatment or prevention of a human disease caused by cerebral ischemia.

[0013] In one embodiment, the present invention provides a pharmaceutically acceptable salt or a pharmaceutical composition thereof for preparing a medicament for the treatment or prevention of human focal ischemic cerebral infarction, human ischemic stroke, human focal ischemic memory impairment, and senile dementia, energy metabolic depletion due to global cerebral ischemia in humans, or cerebral ischemic neurological symptoms caused by human cerebral trauma.

[0014] In one embodiment, the pharmaceutically acceptable salts of irdenafil of the present invention include citrate, sulfate, hydrochloride, phosphate, tartrate or hemi-tartrate, nicotinate, lactate, gluconate, maleate, or aspartate, preferably citrate, sulfate, hydrochloride, phosphate, tartrate, or hemi-tartrate.

[0015] In one embodiment, the airdenafil tartrate or hemi-tartrate used in the present invention is D-tartrate or hemi-D-tartrate, L-tartrate or hemi-L-tartrate, DL-tartrate or hemi-DL-tartrate, meso-tartrate or hemi-meso-tartrate, preferably airdenafil D-tartrate or hemi-D-tartrate, or airdenafil L-tartrate or hemi-L-tartrate.

[0016] As an illustrative example in the present invention, the half-L-tartrate salt (2:1) of 1-[4-ethoxy-3-[5-(6,7-dihydro-1-methyl-7-oxy-3-n-propyl-1H-pyrazolo[4,3-d]pyrimidine)]benzenesulfonyl]-cis-3,5-dimethylpiperazine (airdenafil) is as follows:

[0017] [ka]

[0018] The L-tartrate salt (1:1) of 1-[4-ethoxy-3-[5-(6,7-dihydro-1-methyl-7-oxy-3-n-propyl-1H-pyrazolo[4,3-d]pyrimidine)]benzenesulfonyl]-cis-3,5-dimethylpiperazine (airdenafil) has the following structure:

[0019] [ka]

[0020] The pharmaceutical compositions containing airdenafil or its pharmaceutically acceptable salts in the present invention may be in a variety of drug formulations known in the art, including, but not limited to, oral formulations, suppositories, topical formulations, injections, or compound formulations. The drug may include airdenafil or its pharmaceutically acceptable salts, or a pharmaceutical composition containing any one of them and a pharmaceutically acceptable carrier. A preferred effective dosage range for humans is 0.1 to 20 mg / kg / day, administered once or multiple times per day. Preferably, oral dosages for humans are 0.2 mg / kg / day to 10 mg / kg / day, and parenteral dosages for humans are 0.1 mg / kg / day to 5 mg / kg / day. Illustrative examples of oral dosages include 0.25 mg / kg / day, 1.0 mg / kg / day, or 2.0 mg / kg / day. The dosage for injection administration can be 0.2 mg / kg / day, 0.5 mg / kg / day, or 1.0 mg / kg / day.

[0021] In one embodiment of the present invention, the oral preparations include tablets, granules, capsules, or oral liquids, and the injectable preparations include injection solutions and freeze-dried powders.In one embodiment, the pharmaceutical composition comprising airdenafil or its pharmaceutically acceptable salt according to the present invention is an oral or injectable preparation, preferably a tablet, oral liquid, or injectable liquid.

[0022] In one embodiment of the use of the present invention, the pharmaceutically acceptable salt of the airdenafil compound is airdenafil citrate.

[0023] In the use of the present invention, as one embodiment, when the drug combination is in tablet form, it may contain airdenafil citrate, microcrystalline cellulose, anhydrous calcium hydrogen phosphate, cross-linked sodium carboxymethylcellulose, and magnesium stearate, and preferably contains 420 g of airdenafil citrate, a suitable amount of microcrystalline cellulose, a suitable amount of anhydrous calcium hydrogen phosphate, a suitable amount of cross-linked sodium carboxymethylcellulose, and a suitable amount of magnesium stearate.

[0024] The present invention also provides a pharmaceutically acceptable salt of airdenafil, which is airdenafil phosphate or airdenafil tartrate.

[0025] In one embodiment, the irdenafil tartrate used in the present invention is D-tartrate, L-tartrate, DL-tartrate, or meso-tartrate, preferably D-tartrate or L-tartrate.

[0026] To solve the problem of airdenafil solubility, the inventors accidentally discovered during experiments that airdenafil could be converted into phosphate or tartrate salts, which greatly increased solubility. The solubility of airdenafil phosphate in water at room temperature reaches approximately 50 mg / ml, with the aqueous solution having a pH of 5-6, which is close to neutral. The solubility of airdenafil L-tartrate in water at room temperature is approximately 50 mg / ml, while the solubility of airdenafil gluconate in water at room temperature is actually less than 0.2 mg / ml. In other words, the solubility of airdenafil L-tartrate and phosphate in water at room temperature is more than 200 times higher than that of airdenafil gluconate. Furthermore, the dissolution of airdenafil phosphate and L-tartrate in water is very rapid, and the aqueous solutions are very stable. No impurities were produced after 30 minutes of high-temperature, high-pressure sterilization at 121°C. Thus, the solubility problem of high concentration injectable and oral liquid formulations of irdenafil has been unexpectedly solved.

[0027] On the other hand, the present invention provides high solubility and high stability of irdenafil salts in water-irdenafil phosphate and tartrate.Irdenafil and tartaric acid can form salts in a molar ratio of 1:1 or 2:1.Tartaric acid can be D-tartaric acid, L-tartaric acid, mesotartaric acid, or DL-tartaric acid.

[0028] On the other hand, the present invention also provides the use of airdenafil phosphate and tartrate in the preparation of airdenafil injections and solutions. The solubility of airdenafil phosphate is close to 50 mg / ml, and the pH value of the aqueous solution is close to neutral. The solution has good stability and is very suitable for injection use. The solubility of airdenafil tartrate in water at room temperature is greater than 50 mg / ml, making it very suitable for preparing high-concentration airdenafil injections and solutions. After preparing a freeze-dried powder injection containing airdenafil phosphate and tartrate, the high-concentration reconstitution time is very short and the solution is clear, which cannot be achieved with other salts or other solubilization methods. This is the best choice for preparing freeze-dried powder injections of airdenafil.

[0029] Compared with the prior art, the phosphate and tartrate salts of airdenafil provided by the present invention have the following advantageous effects:

[0030] 1. The irdenafil phosphate and tartrate salts of the present invention are easily and rapidly soluble in water at room temperature, and their solubility is surprisingly increased by more than 200 times compared with other salts such as gluconate. Therefore, this allows for rapid intravenous administration of irdenafil injections in small volumes, greatly improving the rate at which blood drug concentration increases and shortening the time required for drug onset. They are particularly suitable for patients who require rapid administration (such as those with stroke, acute embolism, etc.) and patients with dysphagia.

[0031] 2. The irdenafil phosphate and tartrate salts of the present invention have high solubility in water, which allows for the preparation of highly concentrated injections. This allows for rapid intravenous administration in a small volume, avoiding the need for long-term or multiple injections in a large volume, and reducing the side effects caused by large volume or multiple injections, such as the burden on the body caused by high volume and the toxicity caused by the injection of excessive osmotic agents such as sodium chloride and glucose.

[0032] 3. The irdenafil phosphate and tartrate salts of the present invention have good solubility, low degradation impurities, and fast reconstitution speed when used to prepare freeze-dried powder needles, and there is no precipitation of the drug at high concentrations.

[0033] 4. The synthesis process for irdenafil phosphate and tartrate of the present invention is simple, and the yield of the salt is nearly 100%. The process is stable, has good reproducibility, is easy to operate, has strong controllability, and is suitable for industrial production.

[0034] The present invention also provides a process for preparing airdenafil or a pharmaceutically acceptable salt thereof, comprising cyclization of 4-[2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)benzamido]-1-methyl-3-n-propylpyrazole-5-formamide to synthesize airdenafil, wherein the base catalyst is sodium tert-butoxide. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows HNMR of airdenafil L-tartrate in Example 3. [Figure 2] FIG. 10 shows blank water in Example 18. [Figure 3] FIG. 10 shows citric acid in Example 18. [Figure 4] FIG. 1 shows airdenafil citrate in Example 18. [Figure 5] FIG. 1 shows the airdenafil citrate sample after 1.5 hours of sterilization in Example 18. [Figure 6] FIG. 1 shows airdenafil phosphate in Example 18. [Figure 7] FIG. 1 shows the airdenafil phosphate sample after 1.5 hours of sterilization in Example 18. [Figure 8] FIG. 1 shows L-tartaric acid in Example 18. [Figure 9]FIG. 1 shows irdenafil L-tartrate in Example 18. [Figure 10] FIG. 1 shows the airdenafil L-tartrate sample after 1.5 hours of sterilization in Example 18. [Figure 11] FIG. 10 shows the results of monitoring rat body weight in Example 21. [Figure 12] FIG. 1 shows a typical legend for ADLS1026 treatment of cerebral infarction area for acute ischemic stroke in rats in Example 24, including A: model control group, B: low dose group, C: medium dose group, and D: high dose group. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be further illustrated by the following examples. The examples of the present invention are not intended to limit the present invention, and it should be understood that any simple improvement made to the present invention under the premise that the present invention is within the scope of protection claimed by the present invention. [Example]

[0037] Example 1: Preparation of airdenafil and airdenafil citrate Step 1: Preparation of 5-chlorosulfonyl-2-ethoxybenzoic acid 23 ml of sulfoxide chloride was added to a 250 ml four-neck bottle under nitrogen protection at a freezer bath temperature of -5°C, and 81 ml of chlorosulfonyl chloride was added dropwise. 2-Ethoxybenzoic acid was dissolved in a warm water bath to form a solution, and 52.5 g of 2-ethoxybenzoic acid was weighed and added dropwise while releasing heat. After the addition of 2-ethoxybenzoic acid, the mixture was stirred and allowed to react overnight. The reaction solution was brown and viscous. The reaction solution was removed and left at room temperature.

[0038] A 1000 ml three-neck bottle was placed in the frozen solution, and 750 ml of water was added and frozen to 3°C. The reaction solution was added dropwise at a temperature below 10°C. After the dropwise addition of the reaction solution, a white solid precipitated. At the beginning, the solid slightly adhered to the wall, but could then be stirred and dispersed. After the addition, stirring was continued at room temperature for at least 1 hour. The solution was filtered, washed with 50 ml x 3 water at room temperature, and dried by suction to obtain 68.8 g of wet white crystalline solid. The white crystals were dried at room temperature overnight. The melting point was measured to be 113-116°C.

[0039] Step 2: Preparation of 2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)benzoic acid (III).

[0040] [ka]

[0041] 170 ml of water was added to a 1000 ml three-neck bottle and cooled to 5°C. 60 g of the wet mass of the product obtained in step 1, 5-chlorosulfonyl-2-ethoxybenzoic acid, was added and dispersed. cis-2,6-dimethylpiperazine was added in one portion under stirring to release heat from the reaction. The reaction temperature was controlled below 10°C. After the addition, freezing was stopped, and the solution was stirred overnight. The pH was measured to be 10. The solution was stirred for 2 hours for crystallization, filtered, washed three times with ice water, and dried by suction to obtain 95.0 g of wet white product. The white product was air-dried at 50°C, sampled, and tested. The pH of the filtrate was adjusted to 6-7 using 85% phosphoric acid to precipitate the crystals. After adjustment, the solution was stirred for 1 hour, filtered, and washed with water to obtain approximately 30 g of wet white crystals. The white crystals were air-dried at 50°C to obtain a total of 67.0g. The mixture was crushed, 200ml of acetone was added, and the mixture was refluxed for 1 hour. When the mixture was almost completely dissolved, it was cooled to room temperature, allowed to stand for 1 hour, filtered, rinsed with 50ml x 3 of acetone, dried by suction, and air-dried at 50°C for 2 hours to obtain 67.2g of almost white crystals. The melting point was 258.1-258.7°C.

[0042] Step 3: Preparation of 2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)benzoyl chloride

[0043] [ka]

[0044] 67.2 g of the product from Step 2 was placed in a 500 ml bottle, 150 ml of sulfur dioxide was added, and the mixture was dissolved and allowed to effervescent. The mixture was heated to 50°C to form a clear solution, and then heated and refluxed for 3 hours. The released hydrogen chloride gas was absorbed with alkaline water, spin-dried, and 300 ml of ethyl acetate was added. The mixture was shaken to form a homogeneous solution, rapidly precipitating yellow crystals. The yellow crystals were left to crystallize overnight, filtered, rinsed with ethyl acetate, dried by suction and at room temperature for 1 hour, and then air-dried at 50°C for 2 hours to yield 65.0 g of yellow crystalline product.

[0045] Step 4: Preparation of 4-[2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)benzamido]-1-methyl-3-n-propylpyrazole-5-formamide

[0046] [ka]

[0047] 1.0 g of DMPA, 30.0 g of pyrazoleformamide, and 320 ml of dichloromethane were added to a 1000 ml three-neck bottle and dissolved to clarify. 34.0 g of triethylamine was added and the mixture was cooled to 0°C under stirring. The reaction solution temperature was -3°C. A total of 63.5 g of the product from Step 3 was added in one portion, and heat was released. The temperature of the reaction solution was controlled below 5°C. After the addition, freezing was stopped, the temperature was allowed to rise to room temperature, and the reaction was stirred for over 2 hours. The solution was evaporated to dryness under reduced pressure, 300 ml of water was added, and the mixture was stirred to crystallize. The mixture was filtered to give a reddish-brown filtrate and a yellow product. The product was washed with 100 ml of water three times and 100 ml of ethyl acetate three times. The red color was washed away with ethyl acetate, and the essentially colorless filtrate was obtained. The filtrate was dried by suction to give 88.6 g of loose, off-white crystals with a wet weight. The crystals were first air-dried at room temperature and then blow-dried at 50°C for 3 hours to give 69.0 g, with a measured melting point of 194.3-195.1°C.

[0048] 69 g of the crude product obtained above was placed in a 1000 ml bottle, and 560 ml of water and 280 ml of methanol were added. The mixture was heated to reflux. It was found that the product was not completely dissolved. 100 ml of methanol was added, and the mixture was refluxed to dissolve the product until clear. The white product was filtered while still hot, and white crystals rapidly precipitated from the filtrate. The filtrate was allowed to stand for 5 hours, filtered, washed with 150 ml of a 1:2 ratio of methanol:water, dried by suction, and air-dried at 50°C to a constant mass, yielding 60.0 g. The melting point was measured to be 199.4-199.9°C.

[0049] Detection method: Use octadecylsilane-bonded silica gel as the filler, 0.05 mol / L triethylamine phosphate (7 ml of triethylamine is diluted to 1000 ml with water and the pH is adjusted to 3.0 with phosphoric acid)-methanol acetonitrile (58:25:17) as the mobile phase, and a detection wavelength of 290 nm.

[0050] Step 5: Preparation of cyclization of 5-[2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)phenyl]-1-methyl-3-n-propyl-7,6-dihydro-1H-pyrazolo[4,3-d]pyrimidine-7-ketone

[0051] [ka]

[0052] 300 ml of tert-butanol was added to a 1000 ml dry single-mouth bottle, and 3.0 g of metallic sodium was added in one portion at room temperature with magnetic stirring. After the addition of metallic sodium, the bottle was heated to 70 °C and stirred magnetically until the metallic sodium disappeared (sodium tert-butoxide was generated in situ, or commercially available sodium tert-butoxide products could be added directly). 57 g of the product obtained in Step 4 was added in one portion with stirring. After the addition, the mixture was stirred and refluxed for at least 8 hours. The reaction solution was cooled to room temperature, and 500 ml of water was added dropwise at room temperature and stirred uniformly. The tert-butanol was removed by vacuum evaporation, and the solution was stirred at room temperature for more than 2 hours, filtered, washed with water until neutral, and air-dried at 50 °C to obtain 40 g of crude product. 1000 ml of ethanol was added to the crude product, refluxed to obtain a clear solution, and cooled to crystallize. The solution was filtered and the crystals were dried under air at 50°C to give 35.1 g of airdenafil product.

[0053] Step 6: Preparation of Irdenafil Citrate

[0054] [ka]

[0055] 870 ml of absolute ethanol, 34.8 g of irdenafil, and 17.4 g of citric acid monohydrate were added to a 1000 ml single-bottle flask and heated and refluxed for 1.5 hours. Heating was stopped. The solution was stirred for over 5 hours and allowed to cool to room temperature for crystallization. The solution was filtered, and the crystals were rinsed with ethanol, dried by suction, and air-dried at 50°C to a constant mass. The product, 47.4 g of irdenafil citrate, was white crystals with a 97.7% yield, a melting point of 210.2-211.9°C (melt decomposition), and a purity of over 99.9%. The product was crushed through an 80-mesh sieve to obtain a white powder product. Mass spectrometry [M+H]+ was 489.50.

[0056] Example 2: Preparation of Irdenafil Citrate

[0057] [ka]

[0058] 60 ml of absolute ethanol and 2.45 g of irdenafil were added to a 250 ml single-bottle bottle and heated to reflux to dissolve and clarify, followed by the addition of a solution containing 1.1 g of citric acid monohydrate and 5 ml of absolute ethanol. The heating was stopped, and the solution was stirred for more than 5 hours and allowed to cool to room temperature for crystallization. The solution was filtered to obtain crystals, which were rinsed with ethanol, dried by suction, and dried by air blowing at 60°C to a constant mass to obtain 3.33 g of product, which was a white crystal with a yield of 97.7%, a melting point of 216.5-217.0°C (melting decomposition), and a purity of 100%.

[0059] Example 3: Preparation of Irdenafil L-tartrate

[0060] [ka]

[0061] 2.45 g of irdenafil white crystalline powder was added to a 250 ml bottle, and 60 ml of absolute ethanol was added. The solution was heated to reflux for dissolution and clarification. A clear solution of 0.77 g of L-tartaric acid and 5 ml of absolute ethanol was added to the solution to precipitate a solid. This was then refluxed for 30 minutes. The solution was allowed to cool and stirred overnight at room temperature for crystallization. The solution was suction filtered, and the crystals were rinsed twice with 5 ml of absolute ethanol and then dried by suction to obtain a white solid. The crystals were air-dried at 60°C to a constant mass. The crystals were oven-dried to a constant mass to obtain 3.2 g of white crystals with a 100% yield and a melting point of 205.8-207.4°C. The H NMR image is shown in Figure 1.

[0062] Example 4: Preparation of irdenafil phosphate

[0063] [ka]

[0064] 4.89 g of irdenafil white crystalline powder was added to a 250 ml bottle, 120 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. 1.15 g of 85% phosphoric acid was added to obtain a cloudy reaction solution and precipitate a solid. After the addition, reflux was continued for 30 minutes. The mixture was allowed to cool and crystallize, and the solution was stirred at room temperature overnight. The solution was filtered under suction, and the crystals were rinsed twice with 5 ml of absolute ethanol and then dried by suction to obtain a white solid. The white solid was air-dried at 80°C to a constant mass. This was then oven-dried to a constant mass to obtain 5.8 g of white crystals with a yield of 98.8% and a melting point of 225.3-227.0°C.

[0065] Example 5: Preparation of airdenafil maleate

[0066] [ka]

[0067] 2.45 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 60 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve. 0.59 g of maleic acid was added to precipitate the solid. After the addition, the mixture was refluxed for 30 minutes. The mixture was allowed to cool and crystallize, and the solution was stirred at room temperature overnight. The solution was filtered under suction, and the crystals were rinsed twice with 5 ml of absolute ethanol and then dried by suction to obtain a white solid. The white solid was dried by blowing air at 60°C until a constant mass was obtained. This was then dried to a constant mass to obtain 2.8 g of white crystals with a yield of 92.4% and a melting point of 221.0-222.3°C.

[0068] Example 6: Preparation of Irdenafil Gluconate

[0069] [ka]

[0070] 1.00 g of irdenafil white crystalline powder was added to a 250 ml single-bottle, 26 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify the powder. 0.81 g of gluconic acid solution (49-53%) was added dropwise. After the addition, the mixture was refluxed for 30 minutes. The mixture was allowed to cool and crystallize, resulting in a cloudy solution due to the precipitation of crystals at approximately 45°C. The solution was stirred overnight at room temperature. The mixture was filtered with suction, rinsed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. The solid was dried by blowing air at 60°C to a constant weight. The solid was oven-dried to a constant weight to obtain 0.6 g of white crystals with a yield of 42.8% and a melting point of 195.0-195.8°C.

[0071] Example 7: Preparation of irdenafil lactate

[0072] [ka]

[0073] 2.45 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 60 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. 0.51 g of lactic acid was then added. After addition, the mixture rapidly dissolved and refluxed for 30 minutes. The mixture was allowed to cool and crystallize, starting at 40°C. The solution was stirred overnight at room temperature, filtered with suction, washed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. The solid was blown dry at 60°C to a constant mass. The solid was oven-dried to a constant mass to obtain 2.2 g of white crystals with a yield of 75.9% and a melting point of 180.6-181.8°C.

[0074] Example 8: Preparation of irdenafil aspartate

[0075] [ka]

[0076] 1.00 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 26 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. 0.31 g of aspartic acid was then added. After addition, the mixture rapidly dissolved and cleared, and refluxed for 30 minutes. The solution was allowed to cool and crystallize. The solution was stirred overnight at room temperature, suction filtered, washed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. The solid was blown dry at 60°C to a constant mass. The solid was oven-dried to a constant mass to obtain 1.0 g of white crystals with a yield of 78.9% and a melting point of 193.1-194.4°C.

[0077] Example 9: Preparation of irdenafil nicotinate

[0078] [ka]

[0079] 2.45 g of irdenafil white crystalline powder was added to a 250 ml bottle, 60 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify, followed by the addition of 0.62 g of niacin. After the addition, the mixture rapidly dissolved and cleared, and refluxed for 30 minutes. The mixture was allowed to cool and crystallize, starting at around 45°C, and stirred overnight at room temperature. The mixture was then suction filtered, washed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. The solid was air-dried at 60°C to a constant mass. The solid was oven-dried to a constant mass to obtain 2.8 g of white crystals with a yield of 91.5% and a melting point of 204.0°C to 205.0°C.

[0080] Example 10: Preparation of irdenafil hydrochloride

[0081] [ka]

[0082] 4.89g of irdenafil white crystalline powder was added to a 250ml single-bottle, 120ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. 1.01g of 36.5% hydrochloric acid was added to precipitate solids, and the mixture was refluxed for 30 minutes after addition. After cooling naturally to crystallize, the solution was stirred overnight at room temperature, suction filtered, washed twice with 5ml of absolute ethanol, and then dried by suction to obtain a white solid. This was blown dry at 60°C until constant weight. This was oven-dried until constant weight, obtaining 5.2g of white crystals with a yield of 99.0% and a melting point of over 250°C.

[0083] Example 11: Preparation of airdenafil sulfate

[0084] [ka]

[0085] 4.89 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 120 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. 0.52 g of concentrated sulfuric acid was added to precipitate a large amount of solids, and the mixture was refluxed for 30 minutes. The solution was allowed to cool and crystallize, then stirred overnight at room temperature, suction filtered, washed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. This was air-dried at 60°C to a constant mass. This was then oven-dried to a constant mass to obtain 4.4 g of white crystals with a yield of 81.8% and a melting point of 236.4-237.8°C.

[0086] Example 12: Preparation of Irdenafil D-tartrate

[0087] [ka]

[0088] 1.0 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 31 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. A clear solution containing 0.31 g of D-tartaric acid and 5 ml of absolute ethanol was added to precipitate the solid, and the mixture was refluxed for 30 minutes. The solution was allowed to cool and crystallize, stirred overnight at room temperature, suction filtered, rinsed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. This was blown dry at 60°C to a constant mass. This was then oven-dried to a constant mass to obtain 1.0 g of white crystals with a melting point of 207.0-208.0°C.

[0089] Example 13: Preparation of Irdenafil DL-tartrate

[0090] [ka]

[0091] 1.0 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 31 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify the powder. A clear solution containing 0.31 g of DL-tartaric acid and 5 ml of absolute ethanol was added to precipitate the solid, and the mixture was refluxed for 30 minutes. The solution was allowed to cool and crystallize, stirred overnight at room temperature, suction filtered, rinsed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. The solid was blown dry at 60°C until a constant mass was obtained. The solid was oven-dried until a constant mass was obtained, yielding 1.0 g of white crystals with a melting point of 197.0-198.0°C.

[0092] Example 14: Preparation of irdenafil meso-tartrate

[0093] [ka]

[0094] 1.0g of irdenafil white crystalline powder was added to a 250ml bottle, 31ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. The clear solution containing 0.31g of mesotartaric acid and 5ml of absolute ethanol was added to precipitate the solid, and the mixture was refluxed for 30 minutes after addition. The solution was allowed to cool and crystallize, and the solution was stirred overnight at room temperature, suction filtered, rinsed twice with 5ml of absolute ethanol, and then dried by suction to obtain a white solid. The solid was blown dry at 60°C until it reached a constant weight. The solid was oven-dried until it reached a constant weight, obtaining 1.1g of white crystals.

[0095] Example 15: Preparation of irdenafil hemi-L-tartrate

[0096] [ka]

[0097] 2.45 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 60 ml of absolute ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. A clear solution containing 0.38 g of L-tartaric acid and 5 ml of absolute ethanol was added to precipitate the solid, resulting in a cloudy solution, and refluxed for 30 minutes. After natural cooling, the solution was stirred overnight at room temperature, suction filtered, rinsed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. This was blown dry at 60°C to a constant mass. This was then oven-dried to a constant mass to obtain 2.4 g of white crystals with a yield of 85% and a melting point of 210.0-211.5°C.

[0098] Example 16: Preparation of Irdenafil L-tartrate

[0099] [ka]

[0100] 1.0 g of irdenafil white crystalline powder was placed in a 250 ml bottle, 19 ml of 95% ethanol was added, and the mixture was heated and refluxed to dissolve and clarify. A clear solution containing 0.32 g of L-tartaric acid and 5 ml of 95% ethanol was added, resulting in a cloudy solution and refluxing for 30 minutes. After cooling to allow crystallization, the solution was stirred overnight at room temperature, suction filtered, rinsed twice with 5 ml of absolute ethanol, and then dried by suction to obtain a white solid. This was blown dry at 60°C to a constant mass. This was then oven-dried to a constant mass to obtain 1.2 g of white crystals with a yield of 91.6% and a melting point of 206.0-207.0°C.

[0101] Example 17: Room temperature water solubility of different irdenafil salts The saturation solubility of different irdenafil salts in water was measured at room temperature, and the results are as follows:

[0102] [Table 1]

[0103] Experimental Results: After salification, all irdenafil salts were white crystals. However, the saturated solubilities of different salts in water varied greatly, with the difference between maximum and minimum solubility exceeding 200-fold. Among these, L-tartrate was readily soluble in water at room temperature, reaching a saturated concentration of approximately 50 mg / ml after dissolution. The saturated concentration of phosphate dissolved in water at room temperature was approximately 50 mg / ml. In contrast, the maximum concentration of other salts after saturation reached only approximately 6 mg / ml (6 mg / ml for hydrochloride). Gluconate, which has a similar structure to tartrate, was virtually insoluble in water at room temperature, with its concentration after saturation being less than 0.2 mg / ml. The solubility of irdenafil L-tartrate and phosphate in water was quite surprising. Additionally, irdenafil L-tartrate and phosphate dissolved rapidly in water, becoming clear within 15 seconds after manual shaking, which is highly beneficial for the reconstitution of lyophilized powder for injection. Furthermore, in terms of the yield of salt formation, the yield of irdenafil L-tartrate and phosphate exceeded 95%, which basically met the requirements of atomic economy.

[0104] Example 18: Sterilization stability test for aqueous solutions of airdenafil citrate, airdenafil phosphate, and airdenafil L-tartrate 10 mg each of airdenafil L-tartrate, airdenafil phosphate, and airdenafil citrate was added to a 20 ml volumetric flask, and purified water was added to the volume. They were sterilized in a sterilizer at 121°C for 30 minutes, and samples were taken before and after sterilization. Related substances were measured according to the following method.

[0105] Related substances: A sample was taken and dissolved in the mobile phase to prepare a solution containing 0.5 mg per ml, which was used as the test solution; the detection wavelength was 290 nm, and the filler was octadecylsilane-bonded silica gel. The mobile phase was phosphate buffer (800 ml water, 7 ml triethylamine added, pH adjusted to 3.0 with phosphoric acid, diluted to 1000 ml with water)-methanolacetonitrile (58:25:17).

[0106] Experimental Results: Aqueous solutions of 0.5 mg / ml airdenafil L-tartrate, airdenafil phosphate, and airdenafil citrate were sterilized at 121°C for 30 minutes, and no impurities were found. There was no difference in the HPLC images of the solutions before and after sterilization. The HPLC images before and after sterilization are shown in Figures 2 to 10. The results are shown in the table below.

[0107] Comparison of related substances of different salts at 0.5mg / ml sterilized at 121°C for 30 minutes

[0108] [Table 2]

[0109] Example 19: pH measurement of different representative irdenafil salts dissolved in water The most water-soluble salts of airdenafil were determined: airdenafil tartrate and airdenafil phosphate, and compared with airdenafil citrate. The results are as follows:

[0110] [Table 3]

[0111] Unexpectedly, it was discovered that the pH of phosphate salts is close to physiological pH and is suitable for injection administration.

[0112] Example 20: Preparation of Irdenafil Citrate Formulation Irdenafil citrate can be made into various preparations including tablets, granules, capsules, sublingual patches, suppositories, topical preparations, oral solutions, injections, etc. A typical oral immediate-release tablet can be prepared as follows:

[0113] The formulation for irdenafil citrate tablets is as follows: The formula (calculated by alkali) for 60 mg of irdenafil tablets is as follows (5000 tablets):

[0114] [Table 4]

[0115] The tablet preparation process was as follows:

[0116] Microcrystalline cellulose, irdenafil raw material, anhydrous calcium hydrogen phosphate, and cross-linked sodium carboxymethylcellulose were weighed according to the prescribed amount (5,000 tablets), milled through an 80-mesh sieve, and mixed. The mixed, milled, and sieved materials were added with purified water and wet-granulated for 15 minutes to prepare a soft material, which was then passed through a 16-mesh sieve for granulation. The amount of water was adjusted according to the granulation condition. The prepared granules were dried in an oven at 60°C ± 5°C for 4 hours by blowing air, and the mass loss during drying was measured. After drying, the mass loss was quantified, and the dried granules were sieved and sized through a 10-mesh sieve to obtain granules. After granulation, the mixture was placed in a 3D motion mixer, and a specified amount of magnesium stearate was added and mixed for 100 revolutions. Samples were taken and tested. All the mixed materials were fed into the feeding port of a tablet press, and tablets were pressed under controlled feeding height. The tablet weight was adjusted to 360 mg with a hardness of 30-60 N and a friability of less than 0.3%. The difference between tablet weights was controlled within ±4%.

[0117] Example 21: Comparative study on the repair effect of oral administration of irdenafil citrate and sildenafil citrate on nerve injury in MCAO rats 1. Experimental Drugs Irdenafil Citrate: White powder, solid, with a purity of over 99%. Homemade, batch number 20191127-1. Sildenafil Citrate (USP): White powder, solid, with a purity of over 99%. Manufacturer: AZICO BIOPHORE, India; Batch number: 4002 / 4 / 005 / 20.

[0118] 2. Experimental Animals SD rat, 210-230g.

[0119] 3. Drug form Irdenafil citrate: Dissolve in purified water to prepare a 2 mg / ml drug solution, and store in a refrigerator at 4°C before use. Sildenafil citrate: Dissolve in purified water to prepare a 2 mg / ml drug solution, and store refrigerated at 4°C before use.

[0120] 4. Route of administration, dosage, and frequency Route of administration: oral gavage; Dosage: 4 mg / kg body weight, twice daily; Dosage frequency: Two oral gavage doses per day, morning and evening (administered immediately after modeling).

[0121] 5. Generation of MCAO Model A rat model of middle cerebral artery ischemia / reperfusion (MCAO / R) was created using the suture method.

[0122] 6. Experimental Grouping The experimental rats were randomly divided into four groups, each with 30 rats, and labeled as MCAO model group (referred to as model group), sham group, treatment group 1 (ildenafil group), and treatment group 2 (sildenafil group).

[0123] (1) MCAO model group: cerebral ischemia-reperfusion group; (2) Sham group: after surgical exposure of the carotid artery, suture was performed without ischemia-reperfusion injury; (3) Treatment group 1: MCAO model + irdenafil citrate drug treatment group; (4) Treatment group 2: MCAO model + sildenafil citrate drug treatment group;

[0124] 7. Experimental Metrics Weight monitoring The physical recovery of rats after cerebral infarction treated with different therapeutic agents was indirectly evaluated by monitoring the weight gain of rats in different groups at different times during the experimental process.

[0125] Neuropathy (mNSS) score: Scores were assessed using the mNSS rat neuropathy scoring method. Neuropathy in experimental rats was assessed 14 days after modeling.

[0126] Behavioral experiments: Morris water maze experiment: Testing the learning and memory ability of rats.

[0127] The Morris water maze experiments were performed for 6 consecutive days at each time, with training being carried out on the first 5 days and testing on the sixth day.

[0128] For each test, rats were trained four times a day for a fixed period of time during the first five days (once in each of the first, second, third, and fourth quadrants). At the beginning of training, the platform was placed in the first quadrant, and rats were placed in the pool facing the pool wall from any starting point in the four quadrants on the pool wall. A camera system recorded the time when the rat found the platform and the rat's swimming path. Four training sessions required placing rats in the water from four different starting points. If the rat found the platform or was unable to find it within 60 seconds, the experimenter guided the rat to the platform, allowed it to rest on the platform for 10 seconds, and then proceeded to the next trial.

[0129] The rats were removed from the platform, dried with a soft cloth and returned to their cages, after which the next trial for the rat was performed until the numbered animal finished swimming, and then the next set of trials was performed.

[0130] The experiment was carried out on day 14 after administration. The experiment was carried out for 6 consecutive days, with the first 5 days being swim training and the 6th day being testing.

[0131] 8. Experimental Results Weight monitoring

[0132] [Table 5]

[0133] The weight results showed that the sham-operated group (sham group) showed normal weight gain, with only a slight decrease on the fourth day after surgery. On the fourth day after surgery, the weight loss in the model and treatment groups was approximately 1 / 6, indicating a significant decrease and indicating that the surgery caused serious damage to the rats. The treatment group recovered weight faster than the model group. Statistics showed that the distribution of weight data at different time points in the model group, sham group, and two treatment groups had homogeneity of variance. The probability value of T for the weight of the model group and the sham-operated group after a T-test was 0.005, indicating a significant difference between the two groups. This indicates that the model surgery had a very significant effect on the rats. As shown in the data graph, the weight recovery in the irdenafil group (treatment group 1) was faster than that in the sildenafil group (treatment group 2), indicating that irdenafil is more effective than the sildenafil group in promoting recovery from ischemic brain injury. The data are shown in Figure 11.

[0134] Neuropathy score The neurological impairment and damage scores of rats after 14 days of administration using the nMSS neurobehavioral score are shown in the table below.

[0135] [Table 6]

[0136] After 14 days of administration, the recovery of nerve function is better in the group treated with irdenafil than in the group treated with sildenafil, with a highly significant difference (p=0.01).The irdenafil group also shows a highly significant recovery compared with the model group (p=0.002).The experimental results are very surprising, showing that irdenafil can promote the recovery of nerve function in ischemic injury rats more effectively than sildenafil, and there is a significant difference.

[0137] Morris water maze experiment results The results of the water maze test on day 14 showed that the average latency of the sham group was 4.9 seconds, the model group was 32.51 seconds, the airdenafil group (treatment group 1) had an average latency of 8.93 seconds, and the sildenafil group (treatment group 2) had an average latency of 16.88 seconds. The experiment also unexpectedly found that the airdenafil group had a shorter latency than the model group, with a significant difference (P=0.03), but there was no significant difference in latency between the sildenafil group and the model group (P=0.148). This indicates that airdenafil has a better promoting effect on ischemic stroke recovery than sildenafil, and that airdenafil can significantly promote the recovery of brain memory.

[0138] Example 22: Comparison of the therapeutic effects of intravenously administered irdenafil citrate and sildenafil citrate in ischemic stroke model rats 1. Test Reagents and Drugs

[0139] [Table 7]

[0140] 2. Test equipment

[0141] [Table 8]

[0142] 3. Laboratory Animal Ordering Information

[0143] [Table 9]

[0144] Feeding and Care All rats were housed at temperatures between 20.5 and 24.5°C, humidity between 40 and 75%, and a 12-hour light and 12-hour dark light cycle. Up to five rats were housed in each cage, measuring 48 cm x 35 cm x 20 cm. Sterilized shaving padding was used under the cage and was replaced twice a week. During the experiment, all experimental rats were allowed to eat ad libitum, and padding and drinking water were sterilized under high pressure and replaced twice a week. Each cage had a clear and detailed corresponding abel.

[0145] 4. Test Method Model preparation On the day of the experiment, rats were anesthetized with isoflurane gas and placed in a supine position for immobilization. The skin was incised along the midline of the neck to expose the right common carotid artery. Perivascular nerves and fascia from the common carotid artery bifurcation to the base of the skull were carefully removed, and the external and internal carotid arteries were sequentially isolated. A MCAO suture was inserted from the free end of the external carotid artery, and a nylon suture was introduced from the distal end of the external carotid artery to the internal carotid artery. This was inserted into the circle of Willis of the middle cerebral artery, effectively blocking the middle cerebral artery. The length of the inserted suture was 18–20 mm from the bifurcation of the common carotid artery. The free end of the external carotid artery was then ligated along the intravascular suture to prevent bleeding. The subcutaneous fascia and skin were sutured in layers, and penicillin was applied topically to prevent infection. For the sham group, only the internal carotid artery was isolated. Two hours after the initiation of MCAO, the suture was carefully removed from the lumen of the internal carotid artery to allow reperfusion of the internal carotid artery. The animals were kept in their cages.

[0146] Two hours after embolization and subsequent reperfusion, the degree of neurological damage in the animals was scored in a blinded manner according to the scoring criteria in the attached table. Those with significant neurological impairment (≥8 points) were considered successful modeling.

[0147] Grouping and administration Based on their scores, the successfully modeled animals were randomly divided into three groups, each with 10 animals: model control group, 10 mg / kg Compound 1 AD group (sildenafil citrate group), and 10 mg / kg Compound 2 XD group (sildenafil citrate group). Immediately after 2 hours of cerebral ischemia and reperfusion, the test drug was administered intravenously once a day for 7 consecutive days. The experimental endpoint was 14 days after administration, and the dosage is detailed in the table below.

[0148] [Table 10]

[0149] Mode of administration In this experiment, the drug was administered intravenously at a dose of 10 mg / kg in a volume of 5 mL / kg once daily for seven consecutive days.

[0150] Compound AD: formulated as follows: dosage 10 mg / kg, administration volume 5 ml / kg, i.e., 10 mg / 5 ml = 2 mg / ml; use immediately and discard any remaining material; Compound XD: formulated as a dosage of 10 mg / kg, administration volume of 5 ml / kg, i.e., 10 mg / 5 ml = 2 mg / ml; prepare and use as needed and discard any remaining material.

[0151] Detection indicators Neurological function score Scoring is done before treatment, 3 days after treatment, 7 days after treatment, and 14 days after treatment.

[0152] Determining the extent of cerebral infarction At the end of the experiment, rat brain tissue was collected by bloodletting, frozen in a -20°C freezer, and then sliced ​​at a thickness of 2 mm per slice. The brain tissue slices were placed in a 2% red tetrazolium (TTC) solution and incubated at 37°C for 5 minutes. Infarcted tissue appeared white, while non-infarcted tissue appeared red. The area of ​​cerebral infarction was measured using Image J software, and the percentage of the infarcted area relative to the total brain area was calculated.

[0153] Data analysis methods The measurement data,

[0154]

number

[0155] 5. Test Results Effects on behavioral scores in rats Compared with the model control group, the AD 10 mg / kg group showed significantly improved behavioral scores (P<0.05) on days 7 and 14 after administration. The XD 10 mg / kg group showed a tendency for improvement, but no statistical difference was observed. The results are shown in Table 4.

[0156] [Table 11]

[0157] Effect on the extent of cerebral infarction in rats Compared with the model control group, the improvement rate of cerebral infarction area in the AD 10 mg / kg group was 29.6% (P<0.05) 14 days after administration. The results are shown in Table 5.

[0158] [Table 12]

[0159] The experimental results were unexpected: sildenafil citrate had a statistically significant therapeutic effect in stroke model rats, significantly improved their neurological function, and reduced the extent of cerebral infarction.However, sildenafil citrate did not have a statistically significant therapeutic effect in stroke model rats, and was unable to significantly improve neurological function or significantly reduce the extent of cerebral infarction in stroke model rats.

[0160] Example 23: Comparative study on repair of nerve damage in MCAO rats by oral administration of Irdenafil Citrate, Yukinafil Citrate, and Tadalafil 1. Purpose of the experiment The restorative effects of three different compounds (airdenafil citrate, yukinafil citrate, and tadalafil) on nerve injury in MCAO rats were evaluated.

[0161] 2. Experimental Materials a) Experimental drugs Irdenafil citrate was over 99% pure and homemade. Physical and chemical properties: White powder, solid, slightly soluble in water, soluble in DMF or DMSO. Storage method: Closed at room temperature. Shelf life: 2 years.

[0162] Yukinafil citrate was homemade with a purity of over 99%. Physical and chemical properties: White powder, solid, soluble in water, soluble in DMF or DMSO. Storage method: Closed at room temperature. Shelf life: 2 years.

[0163] Tadalafil was homemade with a purity of over 99%. Physical and chemical properties: White powder, solid, insoluble in water, soluble in DMF or DMSO. Storage method: Closed at room temperature. Shelf life: 2 years.

[0164] b) Laboratory animals SD rats (210-230 g, male, purchased from Sibeifu (Beijing) Biotechnology) were kept properly in the laboratory for 1-2 weeks before the subsequent experiments.

[0165] 3. Experimental methods and processes a) Drug Preparation Preparation of airdenafil citrate: 1600 mg of airdenafil citrate drug was accurately weighed and dissolved in 800 ml of purified water to prepare a 2 mg / ml drug solution, which was refrigerated and stored at 4°C for later use.

[0166] Preparation of yukinafil citrate: 1600 mg of yukinafil citrate drug was accurately weighed and dissolved in 800 ml of purified water to prepare a 2 mg / ml drug suspension solution, which was refrigerated and stored at 4°C for later use.

[0167] Tadalafil Preparation: 1600 mg of tadalafil drug is accurately weighed and dissolved in 800 ml of 5% CMC Na to prepare a 2 mg / ml drug suspension solution, which is prepared extemporaneously as needed.

[0168] b) Route of administration, dosage, and frequency of administration Route of administration: oral gavage; Dosage: 8 mg / kg body weight, twice daily; Dosage frequency: Oral gavage twice daily in the morning and evening (administered immediately after modeling).

[0169] c) Generation of the MCAO model A rat model of middle cerebral artery ischemia / reperfusion (MCAO / R) was created using the suture-occlusion technique.

[0170] Two hours after embolization and subsequent reperfusion, the degree of neurological damage in the animals was scored in a blinded manner according to the scoring criteria in the attached table. Those with significant neurological impairment (≥8 points) were considered successful modeling.

[0171] d) Experimental grouping Based on their scores, the successfully modeled rats were randomly divided into four groups, each with an average of 20 rats, and labeled as the MCAO model group (referred to as the model group), irdenafil-treated group 1, yukinafil-treated group 2, and tadalafil-treated group 3, respectively.

[0172] 1) MCAO model group: cerebral ischemia-reperfusion group; 2) Treatment group 1: MCAO model + irdenafil citrate drug treatment group; 3) Treatment group 2: MCAO model + yukinafil citrate drug treatment group; 4) Treatment group 3: MCAO model + tadalafil drug treatment group;

[0173] e) Experimental Measurement Indicators Neurological function scores: Assessments were performed before treatment, and 3, 7, and 14 days after treatment.

[0174] Determining the extent of cerebral infarction At the end of the experiment, rat brain tissue was collected by bloodletting, frozen in a -20°C freezer, and then sliced ​​at a thickness of 2 mm per slice. The brain tissue slices were placed in a 2% red tetrazolium (TTC) solution and incubated at 37°C for 5 minutes. Infarcted tissue appeared white, while non-infarcted tissue appeared red. The area of ​​cerebral infarction was measured using Image J software, and the percentage of the infarcted area relative to the total brain area was calculated.

[0175] f) Data analysis method The measurement data,

[0176]

number

[0177] 4. Test Results ·Effects on behavioral scores of rats Compared with the model control group, the irdenafil group showed significant improvements in behavioral scores after 3, 7, and 14 days of administration (P<0.05). There was a trend toward improvement between the yukinafil and tadalafil groups, but no statistically significant difference was observed. After 14 days of administration, the irdenafil-treated group was more effective than the yukinafil and tadalafil-treated groups, with significant differences (P=0.037 between the irdenafil and sildenafil groups, and P=0.026 between the irdenafil and tadalafil groups).

[0178] Effect on the extent of cerebral infarction in rats Compared with the model control group, the improvement rate of cerebral infarction area in the irdenafil group after 14 days was 31.5% (P<0.05). The results are shown in Table 6.

[0179] [Table 13]

[0180] Notes: 1. Compared with the model control group, the P value of the airdenafil group was 0.0002, *P<0.05, indicating that the airdenafil group could significantly improve the extent of cerebral infarction. 2. Compared with the model group, the yukinafil group and tadalafil group showed a tendency to improve the extent of cerebral infarction between the yukinafil group and the tadalafil group, with P>0.05, but no significant statistical difference was observed. 3. Compared with the group treated with yukinafil, the P value of the group treated with airdenafil was 0.035 with P<0.05, indicating that airdenafil was also significantly more effective than yukinafil. 4. Compared with the tadalafil group, the P value of the airdenafil group was 0.048 and P<0.05, indicating that airdenafil was also significantly more effective than tadalafil.

[0181] Example 24: Study on the efficacy of injection of different doses of irdenafil phosphate in MCAO rats 1. Experimental Design Animal selection: Healthy SD rats were used as experimental animals. Ten rats were used in each group to estimate the influence of individual animal variation on the results. The experimental process strictly followed the requirements of the protocol to ensure animal welfare. Dosage and administration route: The low dose of 2.5 mg / kg, the medium dose of 5 mg / kg, and the high dose of 10 mg / kg of the test drug were administered using irdenafil phosphate (code ADLS1026). Two hours after cerebral ischemia-reperfusion, the drug was immediately injected into the tail vein for seven consecutive days.

[0182] 2. Test Materials Test substance: Irdenafil phosphate; Batch number: 20220908.

[0183] [Table 14]

[0184] [Table 15]

[0185] [Table 16]

[0186] All rats were housed at temperatures between 20.5 and 24.5°C, humidity between 40 and 75%, and a 12-hour light and 12-hour dark light cycle. Up to five rats could be housed in each cage, measuring 48 cm x 35 cm x 20 cm. Sterilized shaving padding was used under the cage and was replaced twice a week. During the experiment, all experimental rats were allowed to eat ad libitum, and padding and drinking water were sterilized under high pressure and replaced twice a week.

[0187] 3. Test Method Model preparation On the day of the experiment, rats were anesthetized with isoflurane gas and fixed in a supine position. The skin was incised along the midline of the neck to expose the right common carotid artery. Perivascular nerves and fascia were carefully removed from the common carotid artery bifurcation at the base of the skull, and the external and internal carotid arteries were sequentially isolated. A MCAO suture (250–280 g) was inserted from the free end of the external carotid artery, and a nylon suture was introduced from the distal end of the external carotid artery to the internal carotid artery. This was inserted into the middle cerebral artery at the circle of Willis, effectively blocking the middle cerebral artery. The length of the inserted suture was 18–20 mm from the bifurcation of the common carotid artery. The free end of the external carotid artery was then ligated along the intraluminal suture to prevent bleeding. The subcutaneous fascia and skin were sutured in layers, and penicillin was applied topically to prevent infection. In the sham group, only the internal carotid artery was isolated. Two hours after the start of MCAO, the suture was carefully removed from the lumen of the internal carotid artery to allow reperfusion of the internal carotid artery. The animals were kept in cages.

[0188] Grouping and administration Successfully modeled rats with neurological function scores of 8 to 12 were randomly divided into four groups, each with seven rats, based on their scores: a model control group, a low-dose test drug group (2.5 mg / kg), a medium-dose test drug group (5 mg / kg), and a high-dose test drug group (10 mg / kg). The test drug groups were administered irdenafil phosphate (code ADLS1026), while the model control group was administered an equal volume of vehicle. Immediately after 2 hours of cerebral ischemia-reperfusion, the drug was administered via tail vein injection for seven consecutive days. The experimental endpoint was 14 days after the first administration. See Table 7 for details.

[0189] [Table 17]

[0190] Detection indicators (1) Balance beam and forelimb placement detection The balance beam and forelimb placing tests were performed 7 and 14 days after treatment, respectively.

[0191] (2) Extent of cerebral infarction (TTC) After 14 days of drug treatment, rats were anesthetized with isoflurane gas, blood collected, and euthanized by cervical dislocation. Brain tissue was removed and placed in a -20°C freezer for freezing. It was then sliced ​​anteriorly and posteriorly, each 2 mm thick. Brain tissue slices were incubated in a 2% red tetrazolium (TTC) solution at 37°C for 5 minutes. Infarcted tissue appears white, while non-infarcted tissue appears red. The area of ​​cerebral infarction was measured using Image J software, and the percentage of the infarcted area relative to the total brain area was calculated.

[0192] (3) Data analysis method For analysis, the quantitative data were processed using the DPS data processing system of Zhejiang University.

[0193]

number

[0194] 4. Test Results Balance beam and forelimb placing tests In the rat balance beam test, the ischemic stroke model group showed significant neurological symptoms in the paralyzed limbs, such as sideways gait, falling during walking, etc. Compared with the model control group, the ADLS1026 medium-dose group and high-dose group (5 mg / kg, 10 mg / kg) significantly improved the walking ability of rats on the balance beam 14 days after treatment, as shown in Table 8 (P<0.05).

[0195] In the rat forelimb placement test, the ischemic stroke model group showed significant impairment in placing the paralyzed limb, such as not lifting or lifting less frequently.Compared with the model control group, the ADLS1026 medium and high dose groups (5 mg / kg, 10 mg / kg) improved the rats' forelimb placement rate after 14 days of treatment (P<0.05).The results are shown in Table 9.

[0196] [Table 18]

[0197] [Table 19]

[0198] Effect on the extent of cerebral infarction in rats The rat ischemic stroke model showed significant cerebral infarction. Compared with the model control group, different doses of ADLS1026 were able to improve the extent of cerebral infarction in the model rats to various degrees. After 14 days of administration, the improvement rates in the low (2.5 mg / kg), medium (5 mg / kg), and high (10 mg / kg) dose groups were 22.6% (P > 0.05), 46.5% (P < 0.05), and 43.4% (P < 0.05), respectively. The results are shown in Table 10 and Figure 12.

[0199] [Table 20]

Claims

1. Use of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same in the preparation of a medicament for the treatment or prevention of a human disease caused by cerebral ischemia. 【Chemistry 1】

2. The use according to claim 1, characterized by the use of irdenafil or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing same, in the preparation of a drug for the treatment or prevention of human focal ischemic cerebral infarction, human ischemic stroke, human focal cerebral ischemic memory impairment, and senile dementia, human energy metabolic depletion due to global cerebral ischemia, or human cerebral ischemic neurological symptoms caused by cerebral trauma.

3. 2. The use according to claim 1, characterized in that the pharmaceutically acceptable salt of irdenafil is citrate, sulfate, hydrochloride, phosphate, tartrate or hemi-tartrate, nicotinate, lactate, gluconate, maleate or aspartate.

4. The use according to claim 3, characterized in that the pharmaceutically acceptable salt of irdenafil is citrate, sulfate, hydrochloride, phosphate, tartrate or hemi-tartrate.

5. 4. The use according to claim 3, wherein the tartrate or hemi-tartrate of irdenafil is D-tartrate or hemi-D-tartrate, L-tartrate or hemi-L-tartrate, DL-tartrate or hemi-DL-tartrate, or meso-tartrate or hemi-meso-tartrate.

6. The use according to claim 5, characterized in that the tartrate or hemi-tartrate of airdenafil is airdenafil D-tartrate or hemi-D-tartrate, or airdenafil L-tartrate or hemi-L-tartrate.

7. 2. The use according to claim 1, characterized in that the pharmaceutical composition comprising irdenafil or a pharmaceutically acceptable salt thereof is an oral or injectable preparation.

8. The use according to claim 7, characterized in that the pharmaceutical composition containing irdenafil or a pharmaceutically acceptable salt thereof is a tablet, an oral liquid, or an injectable liquid.

9. 8. The use according to claim 7, characterized in that the effective dosage of the compound used in humans is 0.1 mg / kg / day to 20 mg / kg / day, administered in one or more doses per day.

10. The use according to claim 9, characterized in that the oral dosage used in humans is 0.2 mg / kg / day to 10 mg / kg / day and the injectable dosage used in humans is 0.1 mg / kg / day to 5 mg / kg / day.

11. The use according to any one of claims 1 to 10, wherein airdenafil is obtained by cyclization of 4-[2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)benzamido]-1-methyl-3-n-propylpyrazole-5-formamide, and the base catalyst is sodium tert-butoxide.

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