WO2023109513A1 - 爱地那非或其盐在制备预防或治疗缺血性脑损伤的药物中的应用 - Google Patents

爱地那非或其盐在制备预防或治疗缺血性脑损伤的药物中的应用 Download PDF

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WO2023109513A1
WO2023109513A1 PCT/CN2022/135218 CN2022135218W WO2023109513A1 WO 2023109513 A1 WO2023109513 A1 WO 2023109513A1 CN 2022135218 W CN2022135218 W CN 2022135218W WO 2023109513 A1 WO2023109513 A1 WO 2023109513A1
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tartrate
group
edenafil
aldenafil
day
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French (fr)
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林善良
田昕
林贺锡
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真奥金银花药业有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention relates to the field of medicine, in particular to the compound 1-[4-ethoxy-3-[5-(6,7-dihydro-1-methyl-7-oxo-3-n-propyl-1H-pyridine
  • azolo[4,3-d]pyrimidine)]benzenesulfonyl]-3,5-dimethylpiperazine especially in the preparation of diseases caused by cerebral ischemia, especially for ischemic brain Use in medicines for neuronal injury and necrosis.
  • t-PA is the first and so far the only thrombolytic drug for the treatment of acute cerebral ischemia approved by the US FDA. It was approved for marketing in 1987, but the drug is only effective within 3 hours of onset, beyond this time window, whose role is unclear and can lead to a higher incidence of bleeding.
  • Edaravone approved for marketing in Japan, protects brain cells by scavenging oxygen free radicals and inhibiting lipid peroxidation. This product has serious adverse reactions, and serious adverse reactions of death have been reported. Butadipine peptide approved by China in 2004 can only be used for acute cerebral ischemia.
  • the above-mentioned drugs are only used for acute cerebral ischemia, and their therapeutic effect is limited.
  • the compounds involved in the present invention are disclosed in the patent CN1127506C and their use in medicines for the treatment of impotence, but so far there is no relevant report on the prevention and treatment of cerebral ischemia-related diseases.
  • Aldenafil citrate products currently on the market are tablets.
  • oral tablet administration has a process of disintegration and dissolution, a long time to reach the peak blood concentration, and a slow onset speed. ) patients are difficult to administer and other defects. Therefore it is very necessary to develop a kind of edenafil salt injection and oral solution to solve the deficiency in clinical use.
  • the solubility of aldenafil citrate in water at room temperature is only about 2mg/ml. If it is made into an injection made of aldenafil citrate dissolved in water, the concentration of administration is low, and the total volume of administration is large, which is difficult to obtain. Reconstitute. This limits the speed at which the blood concentration of edenafil increases in the body and the administration method of ardenafil injection, for example, it is limited to intravenous drip or large volume injection.
  • High-concentration Aldenafil Injection can directly and quickly complete the administration, quickly reach the peak blood concentration in the body, and take effect quickly, avoiding large-volume and long-term administration.
  • Aldenafil injection and oral solution have important medicinal value for patients with acute or severe swallowing dysfunction (such as stroke, acute thrombolysis, confusion, inability to eat, etc.).
  • the purpose of the present invention is to provide compounds with formula (I) or pharmaceutically acceptable salts thereof or pharmaceutical compositions containing them in the preparation of medicines for treating or preventing diseases caused by human cerebral ischemia:
  • the 3,5-dimethyl group on the piperazine ring of the compound of formula (I) has cis-trans isomers, and the cis-trans isomer structures are as follows:
  • the compound of formula (I) having the cis structure is Edenafil or claiming Edenafil, and its chemical name is 1-[3-(6,7-dihydro-1-methyl-7-oxo-3 -n-Propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxybenzenesulfonyl]-3,5-dimethylpiperazine.
  • the present invention provides a use of edenafil or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing them in the preparation of a medicament for treating or preventing diseases caused by human cerebral ischemia.
  • the present invention provides a kind of edenafil or its pharmaceutically acceptable salt or the pharmaceutical composition containing them in the preparation for the treatment or prevention of human ischemic cerebral infarction, human ischemia It can be used in medicines for acute cerebral apoplexy, human partial cerebral ischemic memory impairment and senile dementia, energy metabolism exhaustion of human global cerebral ischemia, or human brain trauma leading to cerebral ischemic neurological symptoms.
  • the pharmaceutically acceptable salt of edenafil described in the present invention is citrate, sulfate, hydrochloride, phosphate, tartrate or hemitartrate, nicotinate, lactate , gluconate, maleate or aspartate; preferably citrate, sulfate, hydrochloride, phosphate, tartrate or hemitartrate.
  • the idenafil tartrate or hemi-tartrate described in the present invention is D-tartrate or hemi-D-tartrate, L-tartrate or hemi-L-tartrate, DL-tartrate or hemi-tartrate DL-tartrate, meso tartrate or hemi-meso tartrate; preferably edenafil D-tartrate or hemi-D-tartrate, or edenafil L-tartrate or hemi-D-tartrate.
  • the pharmaceutical composition containing edenafil or a pharmaceutically acceptable salt thereof in the present invention can be various pharmaceutical dosage forms in the art, for example including but not limited to oral agents, suppositories, external preparations, injections or compound preparations. It may include edenafil or its pharmaceutically acceptable salt or a pharmaceutical composition containing any of them and a pharmaceutically acceptable carrier, and the preferred human effective dosage range is 0.1 to 20 mg/kg/day. day, single or multiple doses per day; the preferred oral dose for humans is 0.2 mg/kg/day to 10 mg/kg/day, and the dose for human injections is 0.1 mg/kg/day to 5 mg/kg /sky.
  • the dosage when administered orally, can be 0.25mg/kg/day, 1.0mg/kg/day, 2.0mg/kg/day; when administered by injection, the dosage can be 0.2mg/kg/day , 0.5mg/kg/day, 1.0mg/kg/day.
  • the oral preparations include tablets, granules, capsules or oral liquids
  • the injections include injection liquids and freeze-dried powders.
  • the pharmaceutical composition containing edenafil or a pharmaceutically acceptable salt thereof of the present invention is an oral agent or an injection; preferably a tablet, an oral solution or an injection.
  • the compound edenafil and its pharmaceutically acceptable salt is edenafil citrate.
  • the drug combination when it is in tablet form, it may include edenafil citrate, microcrystalline cellulose, calcium hydrogen phosphate anhydrous, cross-linked carboxymethyl cellulose
  • the sodium and magnesium stearate preferably comprise 420 g of edenafil citrate, an appropriate amount of microcrystalline cellulose, an appropriate amount of anhydrous calcium hydrogen phosphate, an appropriate amount of croscarmellose sodium and an appropriate amount of magnesium stearate.
  • the present invention also provides a pharmaceutically acceptable salt of idenafil, which is idenafil phosphate or idenafil tartrate.
  • the idenafil tartrate described in the present invention is D-tartrate, L-tartrate, DL-tartrate, or meso tartrate; preferably idenafil D-tartrate or idenafil Not L-tartrate.
  • Aldenafil In order to solve the solubility problem of Aldenafil, the inventor accidentally found that Aldenafil can be formed into phosphate or tartrate in the experiment of making Aldenafil into various salts, which can greatly increase the solubility: Aldenafil
  • the solubility of non-phosphate salt in room temperature water reaches nearly 50mg/ml, and the pH of the aqueous solution is between 5-6, which is close to neutral; while the solubility of aidenafil L-tartrate in room temperature water also reaches about 50mg/ml, while aidenafil
  • the solubility of gluconate in water at room temperature is actually less than 0.2 mg/ml, that is, the solubility of aidenafil phosphate and L-tartrate in room temperature water is more than 200 times greater than that of aidenafil gluconate.
  • the present invention provides edenafil salts with high solubility and high stability in water - edenafil phosphate and tartrate.
  • Aldenafil and tartaric acid can form a salt with a molar ratio of 1:1 or 2:1, and the tartaric acid can be D-tartaric acid or L-tartaric acid or meso-tartaric acid or DL-tartaric acid.
  • the present invention also provides the application of ardenafil phosphate and tartrate in preparing ardenafil injection and solution.
  • the solubility of Aldenafil Phosphate is nearly 50mg/ml, and the pH of the aqueous solution is close to neutral, and the solution is stable, which is very suitable for injection; Preparation of Nafil High Concentration Injection and Solution. After preparation of lyophilized powder for injection with phosphate and tartrate, the high concentration reconstitution time is very short, and the solution is clear, which cannot be achieved by other salts or other solubilization methods. best choice.
  • edenafil phosphate and tartrate of the present invention have the following beneficial effects:
  • Aidenafil phosphate and tartrate of the present invention are easy to dissolve in water at room temperature, dissolve rapidly, and the solubility is surprisingly increased by more than 200 times compared with other salts such as gluconate, so that Aidenafil injection can be realized
  • Small-volume rapid intravenous administration greatly increases the rate of increase in blood drug concentration and shortens the onset time of the drug. It is especially suitable for patients who need rapid drug administration (such as stroke, acute embolism, etc.) and patients with swallowing disorders.
  • Aldenafil phosphate and tartrate of the present invention have high solubility in water, which provides the possibility for the preparation of high-concentration injections, and can realize rapid intravenous administration in small volumes, avoiding long-term injections of large volumes or multiple injections Injection, reduce side effects caused by large volume injection or multiple injections, such as body burden caused by high volume, toxicity caused by excessive sodium chloride, glucose and other tonicity infusions, etc.
  • Aldenafil phosphate and tartrate of the present invention have good stability, less degradation impurities, fast reconstitution speed when made into freeze-dried powder, and no drug precipitation at high concentration.
  • the synthesis process of aldenafil phosphate and tartrate of the present invention is simple, the yield of salt formation is as high as nearly 100%, the process is stable, the reproducibility is good, the operation is easy, the controllability is strong, and it is suitable for industrial production.
  • the present invention also provides a preparation method of edenafil or a pharmaceutically acceptable salt thereof, the method comprising 4-[2-ethoxyl-5-(cis-3,5-dimethylpiperazine- 1-sulfonyl)benzamido]-1-methyl-3-n-propylpyrazole-5-carboxamide is cyclized to synthesize aidenafil, wherein the base catalyst is sodium tert-butoxide.
  • Figure 1 L-aldenafil tartrate in Example 3-HNMR.
  • Fig. 2 Blank water in embodiment 18.
  • FIG. 4 Aldenafil citrate in Example 18.
  • Fig. 5 Samples sterilized with ardenafil citrate for half an hour in Example 18.
  • Fig. 7 A half-hour sample sterilized with ardenafil phosphate in Example 18.
  • Figure 8 L tartaric acid in Example 18.
  • Figure 10 L tartrate ardenafil sterilized half an hour sample in embodiment 18
  • FIG. 11 Results of rat body weight monitoring in Example 21.
  • Figure 12 Typical illustration of ADLS1026 in Example 24 for the treatment of cerebral infarction range in the acute phase of ischemic stroke in rats, A: model control group, B: low-dose group, C: middle-dose group, D: high-dose group.
  • Embodiment 1 Preparation of Aldenafil and Aldenafil Citrate
  • the second step the preparation of 2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)benzoic acid (III)
  • the third step is the preparation of 2-ethoxy-5-(cis-3,5-dimethylpiperazine-1-sulfonyl)benzoyl chloride
  • Detection method use octadecylsilane bonded silica gel as filler; 0.05mol/L triethylamine phosphate (take 7ml of triethylamine, dilute it to 1000ml with water, adjust the pH value to 3.0 with phosphoric acid)-methanol-acetonitrile ( 58:25:17) is the mobile phase; the detection wavelength is 290nm.
  • Embodiment 2 Preparation of Aldenafil Citrate
  • Embodiment 4 Preparation of Edenafil Phosphate
  • Embodiment 5 Preparation of Aldenafil Maleate
  • Embodiment 7 Preparation of lactic acid aldenafil
  • Embodiment 8 Preparation of Aldenafil Aspartate
  • Embodiment 9 Preparation of Niacin Aldenafil
  • Embodiment 10 Preparation of Aldenafil Hydrochloride
  • Embodiment 11 Preparation of Aldenafil Sulfate
  • Example 19 Typical pH determination of different aidenafil salts dissolved in water
  • Aldenafil citrate can be made into various preparations, such as tablets, granules, capsules, sublingual patches, suppositories, external preparations, oral liquids, injections, etc.
  • Common oral immediate release tablets can be prepared as follows:
  • Aldenafil citrate tablets are prescribed as follows:
  • the prescription of 60mg Aldenafil Tablets is as follows (5000 tablets):
  • the preparation process of tablet can be as follows:
  • Aldenafil citrate white powder, solid, with a purity greater than 99%. Self-made, batch number 20191127-1.
  • Sildenafil citrate (USP): white powder, solid, with a purity greater than 99%.
  • Aldenafil citrate Dissolve in purified water to make a 2mg/ml drug solution, refrigerate at 4°C for later use.
  • Sildenafil citrate Dissolve in purified water to make a 2mg/ml drug solution, refrigerate at 4°C until use.
  • Administration method intragastric administration
  • Dosage 4mg/kg body weight, 2 times/day;
  • Administration time gavage twice a day in the morning and evening (immediately after the model is made).
  • MAO/R middle cerebral artery ischemia/reperfusion
  • the experimental rats were randomly divided into 4 groups, labeled as MCAO model group (abbreviated as model group), Sham group, treatment group 1 (Aidi group), and treatment group 2 (Xidi group), 30 rats in each group.
  • 1MCAO model group cerebral ischemia-reperfusion group
  • 2Sham group the carotid artery was sutured after surgical exposure, and no ischemia-reperfusion injury operation was performed;
  • 3Treatment group 1 MCAO model + edenafil citrate drug treatment group
  • 4Treatment group 2 MCAO model + sildenafil citrate treatment group
  • mNSS Neurological deficit
  • the neurological deficit scoring method of mNSS rats was used. 14 days after modeling, the experimental rats were evaluated for neurological deficits.
  • the Morris water maze experiment was carried out for 6 consecutive days each time, with training on the first 5 days and testing on the 6th day.
  • the rats were trained 4 times at a fixed time period every day for the first 5 days (training once in the first, second, third and fourth quadrants respectively).
  • the platform was placed in the first quadrant, and the mice were put into the pool facing the pool wall from any starting point of the four quadrants of the pool wall.
  • a camera system records the time the rat finds the platform and the swimming path.
  • the 4 training sessions were to put the rats into the water from four different starting points respectively. If the rat finds the platform or cannot find the platform within 60 seconds, the experimenter will guide it to the platform, rest on the platform for 10 seconds, and then proceed to the next experiment.
  • test was carried out 14 days after administration.
  • the experiment was tested for 6 consecutive days, of which the first 5 days were training swimming, and the 6th day was testing.
  • Body weight results showed that the body weight of the sham operation group (Sham group) basically increased normally, and only slightly decreased on the 4th day after the operation; while in the model group level administration group, the body weight decreased by about 1/6 on the 4th day after the operation. It is very large, indicating that the injury of the operation to the rat is very large.
  • the weight recovery of the drug treatment group was faster than that of the model group.
  • Statistics show that the distribution of body weight data at different time points in the model group, the sham operation group, and the two administration groups has homogeneous variances.
  • the weight of the model group and the sham operation group is tested by T.
  • the probability value of T is 0.005.
  • the results of the neurological deficit damage score in rats are as follows after 14 days of administration:
  • the experimental results are very surprising. The results show that Aldenafil can promote the recovery of nerve function in rats with ischemic injury more than Sildenafil, and there is a very significant difference.
  • Reagent name source Isoflurane Shenzhen Ruiwode Life Technology Co., Ltd. Penicillin G sodium salt Phygene Corporation Aldenafil Citrate Injection self made Cinafil Citrate Injection self made
  • All rats were reared at a temperature of 20.5-24.5° C., a humidity of 40-75%, and a photoperiod of 12 hours of light and 12 hours of darkness. Up to 5 rats were raised in each raising cage, and the size of the raising cage was 48cm ⁇ 35cm ⁇ 20cm.
  • the bedding material used under the cage was sterilized wood shavings bedding material, which was replaced twice a week. During the experiment, all experimental rats were allowed to eat and drink freely, and the litter and drinking water were sterilized by autoclaving and replaced twice a week. Each breeding cage has a corresponding clear and detailed label.
  • model control group compound 1 AD group (aldenafil citrate group) 10 mg/kg, compound 2 XD group (cinafil citrate group) ) 10mg/kg, 10 rats in each group.
  • test drug was administered intravenously, once a day, for 7 consecutive days, and the end point of the test was 14 days after drug administration. The dosage is shown in the table below.
  • the rats were bled and their brains were taken out, and the brain tissue was frozen in a -20°C refrigerator and then sliced, each with a thickness of 2mm.
  • the brain tissue slices were placed in 2% red tetrazolium (TTC) solution, incubated at 37°C for 5 min, the infarcted tissue was white, and the non-infarcted tissue was red.
  • TTC red tetrazolium
  • the AD10mg/kg group could significantly improve the behavioral scores 7d and 14d after treatment (P ⁇ 0.05).
  • XD10mg/kg group had a tendency to improve, but no statistical difference was found. The results are shown in Table 4.
  • Aldenafil citrate has a statistically significant therapeutic effect on stroke model rats, can significantly improve the neurological function of stroke model rats, and significantly reduce the size of cerebral infarction; while sildenafil citrate There is no statistically significant therapeutic effect on stroke model rats, neither can significantly improve the neurological function of stroke model rats, nor can it significantly reduce the size of cerebral infarction.
  • Example 23 Comparative study on the effect of oral administration of edenafil citrate, eucafil citrate, and tadalafil on nerve injury repair in MCAO rats
  • Aldenafil citrate self-made, purity>99%. Physical and chemical properties: white powder, solid, slightly soluble in water, soluble in DMF or DMSO, storage method: sealed at room temperature, storage period: 2 years.
  • Eucafil citrate self-made, purity>99%. Physical and chemical properties: white powder, solid, soluble in water, soluble in DMF or DMSO, storage method: sealed at room temperature, storage period: 2 years.
  • Tadalafil homemade, >99% pure. Physical and chemical properties: white powder, solid, insoluble in water, soluble in DMF or DMSO, storage method: sealed at room temperature, storage period: 2 years.
  • Aldenafil citrate configuration Accurately weigh 1600 mg of edenafil citrate, dissolve it in 800 ml of pure water to make a 2 mg/ml drug solution, and refrigerate at 4°C for later use.
  • Configuration of ukenafil citrate Accurately weigh 1600mg of ukenafil citrate, dissolve in 800ml of pure water to make a 2mg/ml drug solution, and refrigerate at 4°C for use.
  • tadalafil Accurately weigh 1600mg of tadalafil medicine, dissolve in 5% CMC-Na 800ml, make the medicine suspension solution of 2mg/ml, use now and prepare now.
  • Administration method intragastric administration
  • Dosage 8mg/kg body weight, 2 times/day;
  • Administration time gavage twice a day in the morning and evening (immediately after the model is made).
  • MAO/R middle cerebral artery ischemia/reperfusion
  • the rats with successful modeling were randomly divided into 4 groups according to their scores, with an average of 20 rats in each group, which were respectively marked as MCAO model group (referred to as model group), edenafil treatment group 1, euknafil treatment group 2, other groups, etc. Dalafil treatment group three.
  • MCAO model group cerebral ischemia-reperfusion group
  • Treatment group 1 MCAO model + edenafil citrate treatment group
  • Treatment group 2 MCAO model + eukenafil citrate drug treatment group
  • Treatment group 3 MCAO model + tadalafil drug treatment group
  • Neurological function score Evaluation was performed before treatment, 3 days, 7 days, and 14 days after treatment.
  • the rats were bled and their brains were taken out, and the brain tissue was frozen in a -20°C refrigerator and then sliced, each with a thickness of 2mm.
  • the brain tissue slices were placed in 2% red tetrazolium (TTC) solution, incubated at 37°C for 5 min, the infarcted tissue was white, and the non-infarcted tissue was red.
  • TTC red tetrazolium
  • the P of edenafil group is 0.048, P ⁇ 0.05, indicating that edenafil is also significantly more effective than tadalafil.
  • test drug group was given aidenafil phosphate (code ADLS1026), cerebral ischemia Immediately after 2 hours of reperfusion, the drug was injected into the tail vein for 7 consecutive days.
  • Test article Aldenafil phosphate; batch number: 20220908.
  • All rats were reared at a temperature of 20.5-24.5° C., a humidity of 40-75%, and a photoperiod of 12 hours of light and 12 hours of darkness. Up to 5 rats were raised in each raising cage, and the size of the raising cage was 48cm ⁇ 35cm ⁇ 20cm.
  • the bedding material used under the cage was sterilized wood shavings bedding material, which was replaced twice a week. During the experiment, all experimental rats were allowed to eat and drink freely, and the litter and drinking water were sterilized by autoclaving and replaced twice a week.
  • the patient On the day of the test, the patient was anesthetized with isoflurane gas and fixed in the supine position. The skin was incised along the midline of the neck to expose the right common carotid artery. The nerves and fascia around the blood vessels from the bifurcation of the common carotid artery to the base of the skull were carefully removed, and the outer neck was separated in turn.
  • the test drug group was given edenafil phosphate (code ADLS1026), and the model control group was given an equal volume of vehicle; 2 hours after cerebral ischemia-reperfusion, the drug was injected into the tail vein immediately for 7 consecutive days. The end point of the test was 14 days after the first drug. See Table 7 below for details.
  • the balance beam and forelimb placement were tested 7 and 14 days after administration, respectively.
  • TTC cerebral infarction
  • the rats were anesthetized with isoflurane gas to collect blood, and then sacrificed by cervical dislocation.
  • the brain was taken out, and the brain tissue was frozen in a -20°C refrigerator, and sliced from front to back, with a thickness of 2 mm each.
  • the brain tissue slices were placed in 2% red tetrazolium (TTC) solution, incubated at 37°C for 5 min, the infarcted tissue was white, and the non-infarcted tissue was red.
  • TTC red tetrazolium
  • the size of the cerebral infarction was measured using Image J software, and the percentage of the infarct size to the total brain area was calculated.
  • the ischemic stroke model rats had obvious cerebral infarction.
  • different dose groups of ADLS1026 can improve the range of cerebral infarction in model rats to varying degrees, and the low (2.5mg/kg), medium (5mg/kg), and high (10mg/kg) dose groups improved
  • the rates were 22.6% (P>0.05), 46.5% (P ⁇ 0.05), and 43.4% (P ⁇ 0.05).
  • the results are shown in Table 10 and Figure 12.

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Abstract

本发明涉及化合物1-[4-乙氧基-3-[5-(6,7-二氢-1-甲基-7-氧代-3-正丙基-1H-吡唑并[4，3-d]嘧啶)]苯磺酰]-3，5-二甲基哌嗪的用途，特别是在制备预防和治疗脑缺血引起的疾病，尤其是对缺血性脑神经元损伤和坏死的药物中的用途。

Description

爱地那非或其盐在制备预防或治疗缺血性脑损伤的药物中的应用 技术领域
本发明涉及医药领域，具体涉及化合物1-[4-乙氧基-3-[5-(6,7-二氢-1-甲基-7-氧代-3-正丙基-1H-吡唑并[4，3-d]嘧啶)]苯磺酰]-3，5-二甲基哌嗪的用途，特别是在制备预防和治疗脑缺血引起的疾病，尤其是对缺血性脑神经元损伤和坏死的药物中的用途。
背景技术
至今为止，临床正式批准用于治疗脑缺血引起疾病的药物非常有限。t-PA是美国FDA批准上市的第一个也是迄今为止唯一一个治疗急性脑缺血的溶栓药物，于1987年获批上市，但该药仅在发病的3小时内治疗有效，超过此时间窗，其作用不明确，且可导致较高的出血发生率。日本批准上市的依达拉奉通过清除氧自由基，抑制脂质过氧化，从而保护脑细胞，该品不良反应大，曾有死亡严重不良反应报道。中国在2004年批准上市的丁苯肽，仅可用于急性脑缺血。上述这些药物仅用于急性脑缺血，且治疗作用有限。本发明中涉及的化合物，在专利CN1127506C中公了所述化合物及其在治疗阳痿药物中的用途，至今未见有预防和治疗脑缺血相关疾病的相关报道。
目前上市枸橼酸爱地那非产品为片剂，然而，口服片给药存在崩解溶出过程及血药浓度达峰时间长、起效速度慢、对于重病(如中风、昏迷或无法进食等)患者难以施用等缺陷。因此开发一种爱地那非盐注射剂及口服溶液剂对解决在临床使用上不足是非常必要的。但是，枸橼酸爱地那非室温下在水中溶解度约仅为2mg/ml，如果采用枸橼酸爱地那非溶于水做成注射剂，给药浓度较低，给药总体积大，难以复溶。这限制了爱地那非体内血药浓度提升的速度和爱地那非注射液的施用方式，例如仅限于静脉滴注或大体积注射。
现有技术中尚未有爱地那非注射剂或口服溶液剂的开发，尚未实现高浓度的注射剂或口服溶液剂药物组合物的开发。高浓度爱地那非注射液可直接快速完成给药，迅速在体内达到血药浓度峰值，快速起效，避免大容量长时间给药。爱地那非注射剂及口服溶液剂对于急重症或吞咽功能障碍(如中风、急性溶栓、错迷、无法进食等)患者具有重要的药用价值。
发明内容
本发明旨在提供具有式(I)的化合物或其药学上可接受的盐或含有它们的药物组合物在制备用于治疗或预防人类脑缺血引起的疾病的药物中的用途：
Figure PCTCN2022135218-appb-000001
式(I)化合物的哌嗪环上3，5-二甲基有顺反异构体存在，顺反异构体结构分别如下：
Figure PCTCN2022135218-appb-000002
具有顺式结构的式(I)化合物为爱地那非或称艾地那非，其化学名称为1-[3-(6,7-二氢-1-甲基-7-氧代-3-正丙基-1H-吡唑并[4，3-d]嘧啶-5-基)-4-乙氧基苯磺酰基]-3，5-二甲基哌嗪。
作为实施方案之一，本发明提供一种爱地那非或其药学上可接受的盐或含有它们的药物组合物在制备用于治疗或预防人类脑缺血引起的疾病的药物中的用途。
作为实施方案之一，本发明提供一种爱地那非或其药学上可接受的盐或含有它们的药物组合物在制备用于治疗或预防人类局部脑缺血性的脑梗塞、人类缺血性脑卒中、人类局部脑缺血性记忆障碍及老年痴呆、人类全脑缺血的能量代谢耗竭或人类脑外伤导致脑缺血性神经症状的药物中的用途。
作为实施方案之一，本发明所述爱地那非药学上可接受的盐为枸橼酸盐、硫酸盐、盐酸盐、磷酸盐、酒石酸盐或半酒石酸盐、烟酸盐、乳酸盐、葡萄糖酸盐、马来酸盐或天冬氨酸盐；优选枸橼酸盐、硫酸盐、盐酸盐、磷酸盐、酒石酸盐或半酒石酸盐。
作为实施方案之一，本发明所述爱地那非酒石酸盐或半酒石酸盐为D-酒石酸盐或半D-酒石酸盐、L-酒石酸盐或半L-酒石酸盐、DL-酒石酸酸盐或半DL-酒石酸酸盐、meso酒石酸酸盐或半meso酒石酸酸；优选爱地那非D-酒石酸盐或半D-酒石酸盐，或者爱地那非L-酒石酸盐或半D-酒石酸盐。
本发明中作为示例性的说明，1-[4-乙氧基-3-[5-(6,7-二氢-1-甲基-7-氧代-3-正丙基-1H-吡唑并[4，3-d]嘧啶)]苯磺酰]-顺-3，5-二甲基哌嗪(爱地那非)的半L-酒石酸盐(2：1)，结构如下所示：
Figure PCTCN2022135218-appb-000003
1-[4-乙氧基-3-[5-(6,7-二氢-1-甲基-7-氧代-3-正丙基-1H-吡唑并[4，3-d]嘧啶)]苯磺酰]-顺-3，5-二甲基哌嗪(爱地那非)的L-酒石酸盐(1：1)，结构如下所示：
Figure PCTCN2022135218-appb-000004
本发明中含有爱地那非或其药学上可接受的盐的药物组合物可以为本领域的各种药物剂型，例如包括但不限于口服剂、栓剂、外用剂、注射剂或复方制剂，药物中可包括爱地那非或其药学上可接受的盐或含有它们中任何一种的药物组合物和药用可接受的载体，优选的人用有效使用剂量范围为每日0.1～20mg/kg/天，每日单次或多次给药；优选人用口服给药剂量为0.2mg/kg/天～10mg/kg/天，人用注射给药剂量为0.1mg/kg/天～5mg/kg/天。作为示例性的说明，若口服给药时，剂量可以为0.25mg/kg/天，1.0mg/kg/天，2.0mg/kg/天；注射给药时，剂量可以为0.2mg/kg/天，0.5mg/kg/天，1.0mg/kg/天。
本发明，作为实施方案之一，所述口服剂包括片剂、颗粒剂、胶囊剂或口服液，所述注射剂包括注射液、冻干粉。作为实施方案之一，本 发明所述含有爱地那非或其药学上可接受的盐的药物组合物为口服剂或注射剂；优选片剂、口服溶液或注射液。
本发明的用途中，作为实施方案之一，所述化合物爱地那非其药学上可接受的盐为枸橼酸爱地那非。
本发明的用途中，作为实施方案之一，所述药物组合为片剂形式时，其可以包括橼酸爱地那非、微晶纤维素、无水磷酸氢钙、交联羧甲基纤维素钠和硬脂酸镁，优选包括枸橼酸爱地那非420g、微晶纤维素适量、无水磷酸氢钙适量、交联羧甲基纤维素钠适量和硬脂酸镁适量组成。
本发明还提供一种爱地那非药学上可接受的盐，所述爱地那非药学上可接受的盐为爱地那非磷酸盐或爱地那非酒石酸盐。
作为实施方案之一，本发明所述爱地那非酒石酸盐为D-酒石酸盐、L-酒石酸盐、DL-酒石酸盐、或meso酒石酸盐；优选爱地那非D-酒石酸盐或爱地那非L-酒石酸盐。
为了解决爱地那非溶解性问题，发明人在将爱地那非做成各种盐的试验中，意外发现爱地那非成磷酸盐或酒石酸盐，能极大的增加溶解度：爱地那非磷酸盐室温水中溶解度达近50mg/ml，且水溶液pH在5-6之间，接近中性；而爱地那非L-酒石酸盐室温水中溶解度也达约50mg/ml，而爱地那非葡萄糖酸盐室温水中溶解度竟然小于0.2mg/ml，即爱地那非磷酸盐和L-酒石酸盐室温水中溶解度较爱地那非葡萄糖酸盐大200倍以上。不仅如此，爱地那非磷酸盐和L-酒石酸盐水中溶解非常迅速，且水溶液非常稳定，经121℃高温高压灭菌30分钟，没有杂质产生。因此，意外解决了爱地那非高浓度注射液和口服溶液剂的高溶解性问题。
本发明一方面提供了水中高溶解度高稳定性的爱地那非盐——爱地那非磷酸盐和酒石酸盐。爱地那非与酒石酸可以以摩尔比1：1或2：1成盐，酒石酸可以是D-酒石酸或L-酒石酸或meso-酒石酸或DL-酒石酸。
另一方面，本发明还提供了爱地那非磷酸盐、酒石酸盐在制备爱地那非注射剂和溶液剂中的应用。爱地那非磷酸盐溶解度近50mg/ml，且水溶液pH接近中性，溶液稳定性好，非常适合注射使用；爱地那非酒石酸盐室温在水中溶解度大于50mg/ml，可方便用于爱地那非高浓度注射剂和 溶液剂的制备。爱地那非磷酸盐和酒石酸盐制备冻干粉针后，高浓度复溶时间非常短，且溶液澄清，是其它盐或其它增溶方法无法达到的，是制备爱地那非冻干粉针的最佳选择。
与现有技术相比，本发明的爱地那非磷酸盐和酒石酸盐具有以下有益效果：
1.本发明的爱地那非磷酸盐和酒石酸盐室温在水中易于溶解，溶解迅速，溶解度较其它盐如葡萄糖酸盐令人惊奇地增加达200倍以上，从而能实现爱地那非注射液小体积快速静脉给药，大大提升了血药浓度上升速度，缩短了药物起效时间，尤其适用于需要快速给药的患者(如卒中、急性栓塞等)和吞咽有障碍的患者。
2.本发明的爱地那非磷酸盐和酒石酸盐在水中具有高溶解性，为制备高浓度注射液提供了可能，可实现小体积快速完成静脉给药，避免大体积长时间注射或多次注射，减少大体积注射或多次注射引发的副作用，如高容量所致的机体负担、过量的氯化钠、葡萄糖等等渗剂输液所致的毒性等。
3.本发明的爱地那非磷酸盐和酒石酸盐稳定性好，降解杂质少，做成冻干粉针复溶速度快，高浓度无药物析出。
4.本发明的爱地那非磷酸盐和酒石酸盐合成工艺简单，成盐收率高达近100％，工艺稳定，重现性好，易于操作，可控性强，适用于工业化生产。
本发明还提供一种爱地那非或其药学接受的盐的制备方法，所述方法包括由4-[2-乙氧基-5-(顺式-3，5-二甲基哌嗪-1-磺酰基)苯甲酰胺基]-1-甲基-3-正丙基吡唑-5-甲酰胺环合合成爱地那非，其中碱催化剂为叔丁醇钠。
附图说明
图1：实施例3中L-酒石酸爱地那非-HNMR。
图2：实施例18中空白水。
图3：实施例18中枸橼酸。
图4：实施例18中枸橼酸爱地那非。
图5：实施例18中枸橼酸爱地那非灭菌半小时样品。
图6：实施例18中磷酸盐爱地那非。
图7：实施例18中磷酸爱地那非灭菌半小时样品。
图8：实施例18中L酒石酸。
图9：实施例18中L酒石酸爱地那非。
图10：实施例18中L酒石酸爱地那非灭菌半小时样品
图11：实施例21中的大鼠体重监测结果。
图12：实施例24中ADLS1026对大鼠缺血性脑卒中急性期治疗脑梗死范围典型图例，A：模型对照组、B：低剂量组、C：中剂量组、D：高剂量组。
具体实施方式
下面通过实施例对本发明作进一步说明。应该理解的是，本发明实施例不是对本发明的限制，在本发明的构思前提下对本发明的简单改进都属于本发明要求保护的范围。
实施例1 爱地那非及枸橼酸爱地那非的制备
第一步.5-氯磺酰基-2-乙氧基苯甲酸的制备
在250ml四口瓶中，氮气保护下加入23ml氯化亚砜，冷冻机浴液温度为-5度，滴加81ml氯磺酰氯；将2-乙氧基苯甲酸用热水浴溶解成溶液，称量滴加2-乙氧基苯甲酸52.5g，滴加放热。加完2-乙氧基苯甲酸，搅拌反应过夜。反应液棕色粘稠。将反应液取出，置室温放置。
在冷冻液中支上1000ml三口瓶，加入750ml水，冷冻至3度。滴加反应液，控制温度小于10度。滴加反应液即析出白色固体，刚开始固体有点粘壁，后来能搅拌分散。加完再室温搅拌1小时以上。过滤,室温50ml*3水洗，抽干，得类白色结晶状固体，湿重68.8g。室温晾过夜干燥。 测熔点为113-116℃。
第二步：2-乙氧基-5-(顺式-3，5-二甲基哌嗪-1-磺酰基)苯甲酸(III)的制备
Figure PCTCN2022135218-appb-000005
在1000ml三口瓶中，加入170ml水，冷却至5℃，将湿重60g第一步产品5-氯磺酰基-2-乙氧基苯甲酸加入，分散。搅拌下分批加入顺-2，6-二甲基哌嗪，反应放热，控制反应温度小于10℃，加完，关闭冷冻，搅拌过夜，测pH10，搅拌析晶2小时，过滤，冰水洗三次，抽干，得湿白色产品95.0g，50℃鼓风干燥，取样检测，滤液加85％磷酸调pH至6-7，析出结晶，调完后搅拌1小时，过滤，水洗，又得到白色结晶湿重30克左右，50℃鼓风干燥，总共得67.0g。研碎，加丙酮200ml，回流1小时，基本不溶，冷却至室温，静置1小时，过滤，丙酮50ml*3淋洗，抽干，50℃鼓风干燥2小时，得类白色结晶67.2g。熔点为258.1～258.7℃。
第三步 2-乙氧基-5-(顺式-3，5-二甲基哌嗪-1-磺酰基)苯甲酰氯的制备
Figure PCTCN2022135218-appb-000006
将67.2g第二步产品加入500ml单口瓶中，加入氯化亚砜150ml，溶解冒泡，加热至50度，溶清，加热回流3小时，放出的氯化氢气体用碱 水吸收，旋干，加入乙酸乙酯300ml，摇晃，成均匀溶液，很快析出黄色结晶，静置析晶过夜，过滤，乙酸乙酯淋洗，抽干，室温风干1小时，再50℃鼓风干燥2小时，得黄色结晶产品65.0g。
第四步 4-[2-乙氧基-5-(顺式-3，5-二甲基哌嗪-1-磺酰基)苯甲酰胺基]-1-甲基-3-正丙基吡唑-5-甲酰胺的制备
Figure PCTCN2022135218-appb-000007
在1000ml三口瓶中加入DMPA 1.0g，加入吡唑甲酰胺30.0g，加入二氯甲烷320ml，溶解澄清，加入三乙胺34.0g，搅拌冷却至0℃。反应液温度为-3℃，分批加入第三步产品共63.5g，加入反应放热，控制反应液温度小于5℃。加完后，关闭冷冻，升温至室温，搅拌反应2小时以上。减压蒸馏至干，加入300ml水，搅拌析晶，过滤，滤液为红棕色，产品黄色，水洗100ml*3次涤，乙酸乙酯100ml*3洗涤，乙酸乙酯洗出红色，至滤液基本无色，抽干，得类白色松散结晶湿得88.6g，先室温风干，再50℃鼓风干燥3小时，得69.0g，测熔点为194.3-195.1℃。
将69克上述产品粗品加入1000ml单口瓶中，加入水560ml，加入甲醇280ml，加热回流。发现不能溶清，补加甲醇100ml，回流溶清，趁热过滤，滤液很快析出白色结晶。静置5小时，过滤，150ml甲醇：水＝1：2淋洗，抽干，50℃鼓风干燥至恒重，得60.0g。测熔点199.4-199.9℃。
检测方法：用十八烷基硅烷键合硅胶为填充剂；0.05mol/L磷酸三乙胺(取三乙胺7ml，用水稀释至1000ml，用磷酸调节pH值至3.0)-甲醇一乙睛(58:25:17)为流动相；检测波长为290nm。
第五步 环合5-【2-乙氧基-5-(顺式-3，5-二甲基哌嗪-1-磺酰基)苯基】-1-甲基-3-正丙基-7，6-二氢-1H-吡唑并【4，3-d】嘧啶-7-酮的制 备
Figure PCTCN2022135218-appb-000008
在干燥的1000ml单口瓶中，加入300ml叔丁醇，室温磁力搅拌下分批剪入金属钠3.0g加完金属钠，加热到70℃，磁力搅拌反应至金属钠消失(原位生成叔丁醇钠，也可以直接加市售叔丁钠产品)。搅拌下分批加入57g第四步产品，加完搅拌回流反应8小时以上。冷却至室温，室温下往反应液中滴加水500ml，搅拌均匀。减压蒸馏蒸去叔丁醇，室温搅拌2小时以上，过滤，水洗至中性，50℃鼓风干燥，得粗品40g。加乙醇1000ml，回流至溶清，冷却析晶。过滤，50度鼓风干燥，得35.1g爱地那非产品。
第六步 枸橼酸爱地那非的制备
Figure PCTCN2022135218-appb-000009
在1000ml单口瓶中，加入无水乙醇870ml，加入爱地那非34.8g，加入枸橼酸一水合物17.4g，加热回流1.5小时，关闭加热，搅拌自然降温 至室温，析晶5小时以上，过滤，乙醇淋洗，抽干，50℃鼓风干燥至恒重，得枸橼酸爱地那非产品47.4克，为白色结晶，收率97.7％。熔点为：210.2～211.9℃(熔融分解)，纯度>99.9％。粉碎过80目筛，得白色粉末状产品。质谱[M+H] +为489.50。
实施例2 枸橼酸爱地那非的制备
Figure PCTCN2022135218-appb-000010
在250ml单口瓶中，加入无水乙醇60ml和爱地那非2.45g，加热回流溶清。加入枸橼酸一水合物1.1g与5ml无水乙醇配成的溶液，关闭加热，搅拌自然降温至室温，析晶5小时以上。过滤，乙醇淋洗，抽干，60℃鼓风干燥至恒重，得枸橼酸爱地那非产品3.33克，为白色结晶，收率97.7％。熔点为：216.5～217.0℃(熔融分解)，纯度100％。
实施例3 L-酒石酸爱地那非的制备
Figure PCTCN2022135218-appb-000011
取爱地那非白色结晶粉末2.45g置250ml单口瓶中，加无水乙醇60ml，加热回流溶清。加入L-酒石酸0.77g和5ml无水乙醇配成的澄清 溶液，析出固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶3.2g，收率为100％，熔点为205.8-207.4℃，HNMR图参见图1。
实施例4 磷酸爱地那非的制备
Figure PCTCN2022135218-appb-000012
取爱地那非白色结晶粉末4.89g置250ml单口瓶中，加无水乙醇120ml，加热回流溶清。加入85％磷酸1.15g，反应液变混浊，析出固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽干，得到白色固体。80℃鼓风干燥至恒重。烘至恒重得白色结晶5.8g，收率为98.8％，熔点为225.3-227.0℃。
实施例5 马来酸爱地那非的制备
Figure PCTCN2022135218-appb-000013
取爱地那非白色结晶粉末2.45g置250ml单口瓶中，加无水乙醇60ml，加热回流溶清。加入马来酸0.59g，析出固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽 干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶2.8g，收率为92.4％，熔点为221.0-222.3℃。
实施例6 葡萄糖酸爱地那非的制备
Figure PCTCN2022135218-appb-000014
取爱地那非白色结晶粉末1.00g置250ml单口瓶中，加无水乙醇26ml，加热回流溶清。滴加葡萄糖酸溶液(49-53％)0.81g，加完回流30分钟。自然冷却析晶，在约45℃时析出结晶变混浊，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶0.6g，收率为42.8％，熔点为195.0-195.8℃。
实施例7 乳酸爱地那非的制备
Figure PCTCN2022135218-appb-000015
取爱地那非白色结晶粉末2.45g置250ml单口瓶中，加无水乙醇60ml，加热回流溶清。加入乳酸0.51g，加完后很快溶清，加完回流30分钟。自然冷却析晶，40℃开始析晶，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至 恒重得白色结晶2.2g，收率为75.9％，熔点为180.6-181.8℃。
实施例8 天冬氨酸爱地那非的制备
Figure PCTCN2022135218-appb-000016
取爱地那非白色结晶粉末1.00g置250ml单口瓶中，加无水乙醇26ml，加热回流溶清。加入天冬氨酸0.31g，加完后很快溶清，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶1.0g，收率为78.9％，熔点为193.1-194.4℃。
实施例9 烟酸爱地那非的制备
Figure PCTCN2022135218-appb-000017
取爱地那非白色结晶粉末2.45g置250ml单口瓶中，加无水乙醇60ml，加热回流溶清。加入烟酸0.62g，加完后很快溶清，加完回流30分钟。自然冷却析晶，约45℃开始析晶，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶2.8g，收率为91.5％，熔点为204.0℃-205.0℃。
实施例10 盐酸爱地那非的制备
Figure PCTCN2022135218-appb-000018
取爱地那非白色结晶粉末4.89g置250ml单口瓶中，加无水乙醇120ml，加热回流溶清。加入36.5％盐酸1.01g，析出固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶5.2g，收率为99.0％，熔点为>250℃。
实施例11 硫酸爱地那非的制备
Figure PCTCN2022135218-appb-000019
取爱地那非白色结晶粉末4.89g置250ml单口瓶中，加无水乙醇120ml，加热回流溶清。加入浓硫酸0.52g，析出大量固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml的无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶4.4g，收率为81.8％，熔点为236.4-237.8℃。
实施例12 D-酒石酸爱地那非的制备
Figure PCTCN2022135218-appb-000020
取爱地那非白色结晶粉末1.0g置250ml单口瓶中，加无水乙醇31ml，加热回流溶清。加入D-酒石酸0.31g和5ml无水乙醇配成的澄清溶液，析出固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶1.0g，熔点为207.0-208.0℃。
实施例13 DL-酒石酸爱地那非的制备
Figure PCTCN2022135218-appb-000021
取爱地那非白色结晶粉末1.0g置250ml单口瓶中，加无水乙醇31ml，加热回流溶清。加入DL-酒石酸0.31g和5ml无水乙醇配成的澄清溶液，析出固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶1.0g，熔点为197.0-198.0℃。
实施例14 meso-酒石酸爱地那非的制备
Figure PCTCN2022135218-appb-000022
取爱地那非白色结晶粉末1.0g置250ml单口瓶中，加无水乙醇31ml，加热回流溶清。加入meso-酒石酸0.31g和5ml无水乙醇配成的澄清溶液，析出固体，加完回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶1.1g。
实施例15 L-半酒石酸爱地那非的制备
Figure PCTCN2022135218-appb-000023
取爱地那非白色结晶粉末2.45g置250ml单口瓶中，加无水乙醇60ml，加热回流溶清。加入L-酒石酸0.38g和5ml无水乙醇配成的澄清溶液，析出固体变混，回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶2.4g，收率85％，熔点：210.0～211.5℃。
实施例16 L-酒石酸爱地那非的制备
Figure PCTCN2022135218-appb-000024
取爱地那非白色结晶粉末1.0g置250ml单口瓶中，加95％乙醇19ml，加热回流溶清。加入L-酒石酸0.32g和5ml95％乙醇配成的澄清溶液，大约5分钟析出固体变混，回流30分钟。自然冷却析晶，室温搅拌过夜。抽滤，加5ml无水乙醇淋洗2次，抽干，得到白色固体。60℃鼓风干燥至恒重。烘至恒重得白色结晶1.2g，收率91.6％，熔点：206.0～207.0℃。
实施例17 不同爱地那非盐的室温水溶解性
取不同爱地那非盐室温测定水中的饱和溶解度，结果如下：
Figure PCTCN2022135218-appb-000025
Figure PCTCN2022135218-appb-000026
试验结果：爱地那非成盐后均为白色结晶，但不同盐在水中饱和溶解度相差非常大，溶解度最大跟最小竟相差>200倍。其中，L-酒石酸盐室温在水中易溶，溶解后饱和浓度约50mg/m；其中磷酸盐室温水中溶解后饱和浓度约50mg/ml；而其它盐溶解饱和后最大浓度只能达到约6mg/ml(盐酸盐6mg/ml)；结构与酒石酸相似的葡萄糖酸盐室温在水中竟然基本不溶，溶解饱和后浓度竟然小于0.2mg/ml。爱地那非成L-酒石盐和磷酸盐后的水中溶解度非常令人意外；另外，爱地那非L-酒石酸盐和磷酸盐在水中溶解非常快，手摇在15秒之内就溶清了，这对于冻干粉针剂复溶非常有利；再者，从成盐的收率来看，爱地那非成L-酒石酸盐和磷酸盐，收率达95％以上，基本达到原子经济要求。
实施例18 爱地那非枸橼酸盐、爱地那非磷酸盐、爱地那非L-酒石酸盐水溶液灭菌稳定性试验
取L-酒石酸爱地那非、磷酸爱地那非、枸橼酸爱地那非各10mg置20ml量瓶中，加纯化水溶解定容。121℃置灭锅中灭菌30分钟，灭菌前和灭菌后取样，按下面方法测有关物质：
有关物质取本品，加流动相溶解制成每1ml中含0.5mg的溶液，作为供试品溶液；以十八烷基硅烷键合硅胶为填充剂，磷酸盐缓冲液(取水800ml，加入7ml三乙胺，用磷酸调pH至3.0，加水稀释至1000ml)-甲醇-乙腈(58:25:17)为流动相，检测波长为290nm。
试验结果：爱地那非L-酒石酸盐、磷酸盐、枸橼酸盐0.5mg/ml水溶 液在121℃灭菌30分钟，均未见杂质产生。灭菌前和灭菌后溶液HPLC图未见差别，灭菌前后HPLC图见附图2～图10。结果如下表：
不同盐0.5mg/ml于121℃灭菌30分钟有关物质比较
Figure PCTCN2022135218-appb-000027
实施例19 典型不同爱地那非盐溶于水中的pH测定
测定水中溶解性最好的爱地那非盐：酒石酸爱地那非和磷酸爱地那非，并与枸橼酸爱地那非进行了比较，结果如下：
Figure PCTCN2022135218-appb-000028
意外发现，磷酸盐pH更接近生理pH，适用于注射给药。
实施例20 枸橼酸爱地那非制剂的制备
枸橼酸爱地那非可以制成各种制剂，可制成片剂、颗粒剂、胶囊剂、舌下帖剂、栓剂、外用制剂、口服液、注射液等。普通的口服速释片剂可如下制备：
枸橼酸爱地那非片处方如下：
60mg爱地那非片(按碱计)处方如下(5000片)：
名称 用量 质量百分比(％)
枸橼酸爱地那非 420g 23.33％
微晶纤维素 940g 52.2％
无水磷酸氢钙 314g 17.4％
交联羧甲基纤维素钠 90g 5.00％
硬脂酸镁 36g 2.0％
总计 1800  
纯化水 900g  
片剂的制备工艺可如下：
将按照上面处方量(5000片)称好微晶纤维素、爱地那非原料、无水磷酸氢钙、交联羧甲基纤维素钠。原辅料粉碎过80目筛，混合。取上述混合粉碎过筛的物料，加纯化水，湿法制粒混合15分钟，制软材，再过16目筛制粒。水量可以根据制粒情况调整。将制好的颗粒置烘箱中鼓风干燥，温度为60℃±5，鼓风干燥4小时，测干燥失重。干燥失重合格后，将干燥的颗粒过10目筛，整粒，得颗粒。整粒后将混料倒入三维运动混合机中，并加入处方量的硬脂酸镁，混合100转。取样检测。将总混后的物料加入压片机装料口，控制装料高度，进行压片。调节片重至360mg，控制片硬度为30-60N，脆碎度小于0.3％；控制片重差异(±4％)。
实验例21 枸橼酸爱地那非、枸橼酸西地那非口服给药对MCAO大鼠神经损伤修复作用的比较研究
1.实验药物
枸橼酸爱地那非：白色粉末，固体，纯度大于99％。自制，批号20191127-1。
枸橼酸西地那非(USP)：白色粉末，固体，纯度大于99％。厂家印度AZICO BIOPHORE；批号：4002/4/005/20。
2.实验动物
SD大鼠，210-230g。
3.药物配置
枸橼酸爱地那非：溶于纯化水中，制成2mg/ml的药物溶液，4℃冷藏待用。
枸橼酸西地那非：溶于纯化水中，制成2mg/ml的药物溶液，4℃冷藏待用。
4.给药方式、给药剂量、给药时间
给药方式：灌胃给药；
给药剂量：4mg/kg体重，2次/天；
给药时间：每日早、晚灌胃2次(造完模后立即给药)。
5.MCAO模型的制作
利用线栓法制作大鼠大脑中动脉缺血/再灌注(MCAO/R)模型。
6.实验分组
将实验大鼠随机分为4组，分别标记为MCAO模型组(简称模型组)、Sham组、治疗组一(爱地组)、治疗组二(西地组)，每组30只。
①MCAO模型组：脑缺血再灌注组；
②Sham组：手术暴露颈动脉后缝合，不进行缺血再灌注损伤操作；
③治疗组一：MCAO模型+枸橼酸爱地那非药物治疗组；
④治疗组二：MCAO模型+枸橼酸西地那非药物治疗组；
7.实验测定指标
Figure PCTCN2022135218-appb-000029
体重监测
通过监测不同时间不同组别大鼠在实验过程中的体重增长情况，侧面评价不同治疗药物对脑梗后大鼠的身体恢复情况。
Figure PCTCN2022135218-appb-000030
神经功能缺损(mNSS)评分：
采用mNSS大鼠神经功能缺损评分法。于造模后14天对实验大鼠进行一次神经功能缺损评价。
Figure PCTCN2022135218-appb-000031
行为学实验：
Morris水迷宫实验：测试大鼠学习记忆能力。
Morris水迷宫实验每次连续进行6天，前5天进行训练，第6天进行测试。
每一次测试，大鼠在前5天每天定于固定时间段训练4次(于第一、二、三、四4个象限分别训练一次)。训练开始时，将平台置于第一象限，从池壁四个象限的任一起始点，将鼠面向池壁放入水池。摄像系统记录大鼠找到平台的时间和游泳路径。4次训练是将鼠分别从四个不同的起始点放入水中。大鼠找到平台或者60秒内找不到平台，则由实验者将其引导到平台，在平台上休息10秒后，再进行下一实验。
将动物从平台拿下，用软布将大鼠擦干，放回笼内。之后再进行下一只大鼠的实验，直到编号的动物游完，再进行下一组实验。
本实验于给药后14天进行测试。实验连续测试6天，其中前5天训练游泳，第6天测试。
8.实验结果
Figure PCTCN2022135218-appb-000032
体重监测
表1
天数 模型组(g) Sham组(g) 爱地组(g) 西地组(g)
D0 298.4 295.9 303.9 297.7
D4 248.3 285.3 251.4 251.4
D7 250.8 300.3 253.8 249.0
D11 261.2 319.7 274.1 266.9
D14 273.7 334.9 287.2 282.4
D18 296.4 359.1 312.0 307.6
D21 305.2 364.4 321.3 318.3
D25 319.1 378.9 341.9 339.6
D28 316.6 380.7 345.0 345.9
体重结果表明，假手术组(Sham组)体重基本正常增加，只在手术后的第4天体重微微下降；而模型组级给药组，手术后的第4天体重下降约1/6，下降非常大，说明手术对大鼠的伤害是非常大的。给药组均比 模型组体重恢复得更快。统计表明，模型组、假手术组及两给药组不同时间点体重数据分布方差齐性，模型组与假手术组体重经T检验，T的概率值为0.005,两组间有显著差异，说明模型手术对大鼠造成了非常显著影响，由数据图可见，爱地组(治疗组一)较西地组(治疗组二)体重恢复更快，说明爱地那非较西地那非更能促进大脑缺血性损伤的恢复，数据如图11所示。
Figure PCTCN2022135218-appb-000033
神经功能缺损评分
采用nMSS神经行为评分，给药14天时大鼠神经功能缺损伤评分结果如下表：
表2.给药14天大鼠神经功能缺损评分表
Figure PCTCN2022135218-appb-000034
给药14天，爱地那非给药组较西地那非给药组神经功能恢复更好,两者有非常显著差异(p＝0.01)，爱地组也较模型组有非常显著性恢复(p＝0.002)。实验结果非常令人意外，结果表明爱地那非较西地那非更能促进缺血性损伤大鼠神经功能的恢复，并且具有非常显著的差异。
Figure PCTCN2022135218-appb-000035
Morris水迷宫实验结果
给药14天水迷宫试验结果表明，假手术组平均潜伏期为4.9秒，模型组平均潜伏期为32.51秒，爱地组(治疗一组)平均潜伏期为8.93秒，西地组(治疗组二)潜伏期为16.88秒。该实验也意外发现，爱地组与模型组相比，爱地组潜伏期较模型组短，具有显著差异(P＝0.03)，而西地组与模型组潜伏期无显著性差异(P＝0.148)，说明爱地那非较西那那非 对缺血性脑梗促进恢复作用更好，爱地那非能显著促进大脑记忆的恢复。
实施例22 枸橼酸爱地那非和枸橼酸西那非注射给药对缺血性卒中模型大鼠的治疗作用比较
1.试验试剂及药物
试剂名称 来源
异氟烷 深圳市瑞沃德生命科技有限公司
青霉素G钠盐 Phygene公司
枸橼酸爱地那非注射液 自制
枸橼酸西那非注射液 自制
2.试验仪器
仪器名称 型号/规格 厂家
电子天平 ECC2201 南京伯尼塔科学仪器有限公司
小动物麻醉机 R500IP 瑞沃德生命科技有限公司
电子天平 ME203E/02 梅特勒
3.实验动物订购信息
实验动物 大鼠
种属 SD
等级 SPF级
性别 雄性
数量 50只
体重 250-270g
来源 斯贝福(北京)生物技术有限公司
生产许可证号 SCXK(京)2019-0010
饲养及管理
所有的大鼠饲养温度为20.5～24.5℃，湿度为40～75％，光照周期12小时明12小时暗。每个饲养笼内最多饲养5只大鼠，饲养笼的规格为48cm×35cm×20cm，笼下使用垫料为灭菌后的刨花垫料，每周更换两次。试验过程中，所有试验大鼠均可以自由饮食，垫料和饮水均经过高压灭菌，每周更换2次。每只饲养笼均有对应的明确的详细标签。
4.试验方法
Figure PCTCN2022135218-appb-000036
模型制备
试验当天异氟烷气体麻醉，仰卧位固定，沿颈正中线切开皮肤，暴露右侧颈总动脉，小心去除颈总动脉分叉至颅底部的血管周围的神经及筋膜，依次分离颈外动脉和颈内动脉，从颈外动脉游离端插入MCAO栓线，从颈外动脉远心端将尼龙线导入到颈内动脉，插至Willis环大脑中动脉处，以有效阻断大脑中动脉，插入的栓线长度距颈总动脉分叉处18～20mm。然后将颈外动脉游离端连同腔内栓线一并结扎，以防出血。逐层缝合皮下筋膜和皮肤，局部滴加青霉素防止感染。假手术组动物仅分离出颈内动脉。MCAO开始后2h，小心抽出颈内动脉血管腔内的栓线，使颈内动脉再灌注。动物回笼饲养。
栓塞2h再灌注后，根据附表评分标准，采用盲法对动物进行神经功能损害程度评分，有明显神经功能缺陷者(≥8分)为造型成功。
Figure PCTCN2022135218-appb-000037
分组及给药
造模成功的动物根据分值随机分为3组，即模型对照组、化合物一AD组(枸橼酸爱地那非组)10mg/kg、化合物二XD组(枸橼酸西那那非组)10mg/kg，每组10只。脑缺血2h再灌注后立即给药，受试药静脉注射给予，每天一次，连续7d，试验终点为药后14d，给药剂量详见下表。
表3
Figure PCTCN2022135218-appb-000038
Figure PCTCN2022135218-appb-000039
给药方式
本试验采用静脉注射给药，给药剂量为10mg/kg，给药体积5mL/kg，每天1次，连续7d。
化合物AD：剂量10mg/kg，给药体积5mL/kg，即配制成10mg/5mL＝2mg/mL；现配现用，配制剩余废弃；
化合物XD：剂量10mg/kg，给药体积5mL/kg，即配制成10mg/5mL＝2mg/mL；现配现用，配制剩余废弃。
Figure PCTCN2022135218-appb-000040
检测指标
神经功能评分
分别于治疗前、治疗后3d、治疗后7d、治疗后14d进行评分。
脑梗死范围测定
试验终点，大鼠放血取脑，将脑组织置于-20℃冰箱内冷冻后进行切片，每片厚度为2mm。将脑组织切片置2％红四氮唑(TTC)溶液中，37℃孵育5min，梗死组织呈白色，非梗死组织为红色。采用Image J软件进行脑梗死面积测定，并计算梗死面积占全脑面积的百分比。
Figure PCTCN2022135218-appb-000041
数据分析方法
计量资料以
Figure PCTCN2022135218-appb-000042
表示，采用TTEST两两比较，P<0.05有统计学差异。
5.试验结果
对大鼠行为学评分的影响
与模型对照组相比，AD10mg/kg组药后7d、14d能明显改善行为学评分(P＜0.05)。XD10mg/kg组有改善趋势，但未见统计学差异。结果见表4。
表4.化合物对大鼠行为学评分的影响(
Figure PCTCN2022135218-appb-000043
n＝10)
Figure PCTCN2022135218-appb-000044
对大鼠脑梗死范围的影响
与模型对照组相比，AD10mg/kg组药后14d脑梗死范围改善率为29.6％(P<0.05)。结果见表5。
表5.化合物对大鼠脑梗死范围的影响(
Figure PCTCN2022135218-appb-000045
n＝10)
Figure PCTCN2022135218-appb-000046
实验结果令人意想不到：枸橼酸爱地那非对卒中模型大鼠有统计学意义的治疗作用，能显著改善卒中模型大鼠神经功能，显著缩小脑梗死范围；而枸橼酸西地那非对卒中模型大鼠没有统计意义的治疗作用，没有能显著改善卒中模型大鼠神经功能，也没能显著缩小脑梗死范围。
实施例23 枸橼酸爱地那非、枸橼酸尤克那非、他达拉非口服给药对MCAO大鼠神经损伤修复作用的比较研究
1.实验目的
评价三种不同化合物(枸橼酸爱地那非、枸橼酸尤克那非、他达拉非)对MCAO大鼠神经损伤的修复作用。
2.实验材料
a)实验药物
枸橼酸爱地那非，自制，纯度>99％。理化性征：白色粉末，固体，微溶于水，可溶于DMF或DMSO，保存方式：密封室温，保存期限：2年。
枸橼酸尤克那非，自制，纯度>99％。理化性征：白色粉末，固体，溶于水，可溶于DMF或DMSO，保存方式：密封室温，保存期限：2年。
他达拉非，自制，纯度>99％。理化性征：白色粉末，固体，不溶于 水，可溶于DMF或DMSO，保存方式：密封室温，保存期限：2年。
b)实验动物
SD大鼠(购于斯贝福(北京)生物技术有，210-230g，雄性。大鼠购买后在实验室适应性饲养1-2周，然后进行后续实验。
3.实验方法及过程
a)药物配置
枸橼酸爱地那非的配置：准确称量枸橼酸爱地那非药物1600mg，溶于800ml纯水中，制成2mg/ml的药物溶液，4℃冷藏待用。
枸橼酸尤克那非的配置：准确称量枸橼酸尤克那非药物1600mg，溶于800ml纯水中，制成2mg/ml的药物溶液，4℃冷藏待用。
他达拉非的配置：准确称量他达拉非药物1600mg，溶于5％CMC-Na 800ml中，制成2mg/ml的药物混悬溶液，现用现配。
b)给药方式、给药剂量、给药时间
给药方式：灌胃给药；
给药剂量：8mg/kg体重，2次/天；
给药时间：每日早、晚灌胃2次(造完模后立即给药)。
c)MCAO模型的制作
利用线栓法制作大鼠大脑中动脉缺血/再灌注(MCAO/R)模型。
栓塞2h再灌注后，根据附表评分标准，采用盲法对动物进行神经功能损害程度评分，有明显神经功能缺陷者(≥8分)为造型成功。
d)实验分组
造模成功的大鼠根据分值随机分为4组，平均每组20只，分别标记为MCAO模型组(简称模型组)、爱地那非治疗组一、尤克那非治疗组二、他达拉非治疗组三。
1)MCAO模型组：脑缺血再灌注组；
2)治疗组一：MCAO模型+枸橼酸爱地那非药物治疗组；
3)治疗组二：MCAO模型+枸橼酸尤克那非药物治疗组；
4)治疗组三：MCAO模型+他达拉非药物治疗组；
e)实验测定指标
神经功能评分：分别于治疗前、治疗后3天、7天、14天进行评价。
脑梗死范围测定
试验终点，大鼠放血取脑，将脑组织置于-20℃冰箱内冷冻后进行切片，每片厚度为2mm。将脑组织切片置2％红四氮唑(TTC)溶液中，37℃孵育5min，梗死组织呈白色，非梗死组织为红色。采用Image J软件进行脑梗死面积测定，并计算梗死面积占全脑面积的百分比。
f)数据分析方法
计量资料以
Figure PCTCN2022135218-appb-000047
表示，采用TTEST两两比较，P<0.05有统计学差异。
4.试验结果
Figure PCTCN2022135218-appb-000048
对大鼠行为学评分的影响
与模型对照组相比，爱地那非组药后3d、7d、14d能明显改善行为学评分(P＜0.05)；尤克那非组和他达拉非组有改善趋势，但未见统计学差异；给药后14d，爱地那非组较尤克那非和他达拉非组更有效，有显著性差异(爱地那非与西地那非组间P＝0.037；爱地那非与他达拉非组之间P＝0.026)。
Figure PCTCN2022135218-appb-000049
对大鼠脑梗死范围的影响
与模型对照组相比，爱地那非组药后14d脑梗死范围改善率为31.5％(P<0.05)。结果见表6。
表6.化合物对大鼠脑梗死范围的影响(
Figure PCTCN2022135218-appb-000050
n＝20)
组别 剂量 全脑梗死％ 改善率％
模型对照组 - 24.8±5.3  
爱地那非组 8mg/kg*2 17.0±6.7 * 31.5
尤克那非组 8mg/kg*2 21.2±6.3 13.1
他达拉非组 8mg/kg*2 21.3±6.5 14.2
注：
1.爱地那非组与模型对照组相比：P为0.0002，*P＜0.05，说明爱地那非能显著改善脑梗死范围；
2.尤克那非组与他达拉非组与模型组相比：P>0.05，说明尤克那非组和他达拉非组有改善脑梗死范围趋势，但未见明显统计学差异；
3.爱地那非组与尤克那非组相比，P为0.035，P＜0.05，说明爱地那非也较尤克那非显著有效；
4.爱地那非组与他达拉非组相比，P为0.048，P＜0.05，说明爱地那非也较他达拉非显著有效。
实施例24 不同剂量磷酸爱地那非注射给药对MCAO大鼠有效性研究
1.试验设计
Figure PCTCN2022135218-appb-000051
动物的选择：采用健康SD大鼠为实验动物。为了消除动物个体变异对结果产生影响，每组大鼠10只。试验过程严格按照方案要求进行，保证动物福利。
Figure PCTCN2022135218-appb-000052
剂量与给药途径：受试药低剂量2.5mg/kg组、中剂量5mg/kg组、高剂量10mg/kg组，受试药组给予爱地那非磷酸盐(代码ADLS1026)，脑缺血再灌注2h后立即尾静脉注射给药，连续7d。
2.试验材料
Figure PCTCN2022135218-appb-000053
供试品：磷酸爱地那非；批号：20220908。
Figure PCTCN2022135218-appb-000054
试验试剂：
试剂名称 批号 来源
青霉素G钠 C12854341 麦克林
红四氮唑(TTC) 20210220 Phygene
异氟烷 20220202 瑞沃德
Figure PCTCN2022135218-appb-000055
试验仪器
Figure PCTCN2022135218-appb-000056
Figure PCTCN2022135218-appb-000057
试验动物
实验动物 健康大鼠
种属 SD
等级 SPF
性别 雄性
引入数量 60只
体重 240-260g
来源 斯贝福(北京)生物技术有限公司
生产许可证号 SCXK(京)2019-0010
质量合格证号 110324220106384338
所有的大鼠饲养温度为20.5～24.5℃，湿度为40～75％，光照周期12小时明12小时暗。每个饲养笼内最多饲养5只大鼠，饲养笼的规格为48cm×35cm×20cm，笼下使用垫料为灭菌后的刨花垫料，每周更换两次。试验过程中，所有试验大鼠均可以自由饮食，垫料和饮水均经过高压灭菌，每周更换2次。
3.试验方法
Figure PCTCN2022135218-appb-000058
模型制备
试验当天异氟烷气体麻醉，仰卧位固定，沿颈正中线切开皮肤，暴露右侧颈总动脉，小心去除颈总动脉分叉至颅底部的血管周围的神经及筋膜，依次分离颈外动脉和颈内动脉，从颈外动脉游离端插入MCAO栓线(250～280g)，从颈外动脉远心端将尼龙线导入到颈内动脉，插至Willis环大脑中动脉处，以有效阻断大脑中动脉，插入的栓线长度距颈总动脉分叉处18～20mm。然后将颈外动脉游离端连同腔内栓线一并结扎，以防出血。逐层缝合皮下筋膜和皮肤，局部滴加青霉素防止感染。假手术组动物仅分离出颈内动脉。MCAO开始后2h，小心抽出颈内动脉血管腔内的栓线，使颈内动脉再灌注。动物回笼饲养。
Figure PCTCN2022135218-appb-000059
分组及给药
选取造模成功神功功能评分8-12分的大鼠，按评分随机分为4组，分别为模型对照组、受试药低剂量2.5mg/kg组、中剂量5mg/kg组、高剂量10mg/kg组，每组7只大鼠。受试药组给予爱地那非磷酸盐(代码ADLS1026)，模型对照组给予等体积溶媒；脑缺血再灌注2h后立即尾静脉注射给药，连续7d。试验终点为首次药后14d。详见下表7。
表7分组信息表
Figure PCTCN2022135218-appb-000060
Figure PCTCN2022135218-appb-000061
检测指标
(1)平衡木、前肢放置检测
分别于给药后7、14d后进行平衡木、前肢放置检测。
(2)脑梗死范围(TTC)
药后14d大鼠用异氟烷气体麻醉取血后脱颈椎处死，取脑，将脑组织置于-20℃冰箱内冷冻后，由前向后进行切片，每片厚度为2mm。将脑组织切片置2％红四氮唑(TTC)溶液中，37℃孵育5min，梗死组织呈白色，非梗死组织为红色。采用Image J软件进行脑梗死面积测定，并计算梗死面积占全脑面积的百分比。
(3)数据分析方法
应用浙大DPS数据处理系统分析，计量资料以
Figure PCTCN2022135218-appb-000062
表示，方差齐性数据比较采用LSD检验，方差不齐数据采用非参数Kruskal Wallis检验，P<0.05有统计学意义。
4.试验结果
Figure PCTCN2022135218-appb-000063
平衡木、前肢放置检测
在大鼠平衡木测试中，缺血性脑卒中模型组大鼠瘫痪肢体出现明显神经症状，如步态侧滑、走时掉落等等。与模型对照组相比，ADLS1026中、高剂量组(5mg/kg、10mg/kg)药后14d显著改善大鼠在平衡木上的行走能力(P＜0.05)，结果见表8。
大鼠前置放置测试中，缺血性脑卒中模型组大鼠瘫痪肢体出现明显前肢放置障碍，如无抬起、抬起次数少等等。与模型对照组相比，ADLS1026中、高剂量组(5mg/kg、10mg/kg)药后14d均改善大鼠前 肢放置率(P＜0.05)。结果见表9。
表8 ADLS1026对大鼠缺血性脑卒中急性期治疗平衡木的影响(
Figure PCTCN2022135218-appb-000064
n＝7)
Figure PCTCN2022135218-appb-000065
注：与模型对照组相比： *P＜0.05。
表9 ADLS1026对大鼠缺血性脑卒中急性期治疗前肢放置的影响(
Figure PCTCN2022135218-appb-000066
n＝7)
Figure PCTCN2022135218-appb-000067
注：与模型对照组相比： *P＜0.05。
Figure PCTCN2022135218-appb-000068
对大鼠脑梗死范围的影响
缺血性脑卒中模型大鼠出现明显脑梗死。与模型对照组相比，ADLS1026不同剂量组均可不同程度改善模型大鼠脑梗死范围，药后14d低(2.5mg/kg)、中(5mg/kg)、高(10mg/kg)剂量组改善率分别为22.6％(P>0.05)、46.5％(P<0.05)、43.4％(P<0.05)，结果见表10、图12。
表10 ADLS1026对大鼠缺血性脑卒中急性期治疗脑梗死范围的影响(
Figure PCTCN2022135218-appb-000069
n＝7)
Figure PCTCN2022135218-appb-000070
注：与模型对照组相比： *P＜0.05。

Claims (9)

  1. 具有式(I)的化合物或其药学上可接受的盐或含有它们的药物组合物在制备用于治疗或预防人类脑缺血引起的疾病的药物中的用途：
    Figure PCTCN2022135218-appb-100001
  2. 根据权利要求1所述的用途，其特征在于，爱地那非或其药学上可接受的盐或含有它们的药物组合物在制备用于治疗或预防人类局部脑缺血性的脑梗塞、人类缺血性脑卒中、人类局部脑缺血性记忆障碍及老年痴呆、人类全脑缺血的能量代谢耗竭或人类脑外伤导致脑缺血性神经症状的药物中的用途。
  3. 根据权利要求1所述的用途，其特征在于，爱地那非药学上可接受的盐为枸橼酸盐、硫酸盐、盐酸盐、磷酸盐、酒石酸盐或半酒石酸盐、烟酸盐、乳酸盐、葡萄糖酸盐、马来酸盐或天冬氨酸盐；优选枸橼酸盐、硫酸盐、盐酸盐、磷酸盐，酒石酸盐或半酒石酸盐。
  4. 根据权利要求3所述的用途，其特征在于，爱地那非酒石酸盐或半酒石酸盐为D-酒石酸盐或半D-酒石酸盐、L-酒石酸盐或半L-酒石酸盐、DL-酒石酸盐或半DL-酒石酸盐、或者meso酒石酸盐或半meso酒石酸盐；优选爱地那非D-酒石酸盐或半D-酒石酸盐，或者爱地那非L-酒石酸盐或半L-酒石酸盐。
  5. 根据权利要求1所述的用途，其特征在于，所述含有爱地那非或其药学上可接受的盐的药物组合物为口服剂或注射剂；优选片剂、口服溶液或注射液。
  6. 根据权利要求5所述的用途，其特征在于，所述化合物的人用有效使用剂量为0.1～20mg/kg/天，每日单次或多次给药；优选人用口服给药剂量为0.2mg/kg/天～10mg/kg/天，人用注射给药剂量为0.1mg/kg/天～5mg/kg/天。
  7. 一种权利要求1所述的爱地那非药学上可接受的盐，其特征在于，爱地那非药学上可接受的盐为爱地那非磷酸盐、爱地那非酒石酸盐。
  8. 根据权利要求7所述的爱地那非药学上可接受的盐，其特征在于，爱地那非酒石酸盐为D-酒石酸盐、L-酒石酸盐、DL酒石酸盐、或meso酒石酸盐；优选爱地那非D-酒石酸盐或爱地那非L-酒石酸盐。
  9. 一种制备权利要求1～8中任一所述化合物方法，其特征在于，所述方法包括由4-[2-乙氧基-5-(顺式-3，5-二甲基哌嗪-1-磺酰基)苯甲酰胺基]-1-甲基-3-正丙基吡唑-5-甲酰胺环合合成爱地那非，其中碱催化剂为叔丁醇钠。
PCT/CN2022/135218 2021-12-14 2022-11-30 爱地那非或其盐在制备预防或治疗缺血性脑损伤的药物中的应用 WO2023109513A1 (zh)

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CN1127506C (zh) 2001-06-29 2003-11-12 刘宝顺 一种治疗阳痿的新化合物
CN101671338A (zh) * 2009-09-27 2010-03-17 刘桂坤 枸橼酸爱地那非晶型d及其制备方法和用途
CN102180832A (zh) * 2011-03-18 2011-09-14 苏州沪云肿瘤研究中心有限公司 脑缺血保护用化合物及制备方法
CN103417543A (zh) * 2012-05-14 2013-12-04 陈凯 爱地那非及其盐的新用途
CN103613597A (zh) * 2013-11-22 2014-03-05 安徽悦康凯悦制药有限公司 一种枸橼酸爱地那非的制备工艺
CN110393723A (zh) * 2019-09-05 2019-11-01 北京中医药大学 松香烷衍生物在制备预防和治疗脑缺血性疾病药中的应用

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Publication number Priority date Publication date Assignee Title
CN1127506C (zh) 2001-06-29 2003-11-12 刘宝顺 一种治疗阳痿的新化合物
CN101671338A (zh) * 2009-09-27 2010-03-17 刘桂坤 枸橼酸爱地那非晶型d及其制备方法和用途
CN102180832A (zh) * 2011-03-18 2011-09-14 苏州沪云肿瘤研究中心有限公司 脑缺血保护用化合物及制备方法
CN103417543A (zh) * 2012-05-14 2013-12-04 陈凯 爱地那非及其盐的新用途
CN103613597A (zh) * 2013-11-22 2014-03-05 安徽悦康凯悦制药有限公司 一种枸橼酸爱地那非的制备工艺
CN110393723A (zh) * 2019-09-05 2019-11-01 北京中医药大学 松香烷衍生物在制备预防和治疗脑缺血性疾病药中的应用

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