A cilostazol derivative and its preparation method and use

Abstract

The present invention relates to lauroyl cilostazol, its preparation method, and use. The lauroyl cilostazol is sterile and preferably crystalline. The present invention also provides the use of lauroyl cilostazol in preparing a medicament for treating cerebral infarction, cerebrovascular disease, atherosclerosis, diabetic complications, peripheral vascular disease, Raynaud's disease, intermittent claudication, ischemic heart disease, and acute coronary syndrome, which exhibits significant long-lasting effects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a sterile lauroyl cilostazol crystal, a preparation method thereof, a pharmaceutical preparation thereof and medical uses thereof. Background Art

[0002] A recent report on treatment evidence published by the Scientific Research Council stated that antiplatelet drugs are the first-line treatment for patients in the acute phase of acute myocardial or cerebral infarction. However, a serious problem is that all antiplatelet drugs on the market are oral, making it difficult to administer these drugs to patients who have just experienced an acute myocardial or cerebral infarction, especially in the acute phase, and who also expect immediate results. All antiplatelet drug formulations on the market are oral because the active ingredients contained in these formulations are poorly soluble in water. Cilostazol, a commonly used antiplatelet drug, is no exception; it is virtually insoluble in water, with a solubility as low as approximately 3 μg / ml at room temperature.

[0003] Cilostazol is a new antiplatelet drug that significantly inhibits platelet aggregation and vasodilation, effectively preventing thrombosis and vascular obstruction. Cilostazol has been widely used in the treatment of intermittent claudication and other cardiovascular diseases both domestically and internationally, demonstrating its significant clinical value.

[0004] The chemical name of cilostazol is 6-[4-(1-cyclohexyl-1H-tetrazol-5-ylbutoxy]-3,4-dihydro-2(1H-quinolinone, and its structural formula is as follows:

[0005] Product Name Cilostazol is marketed for the treatment of chronic arterial occlusive disease, including diabetic complications of the peripheral vasculature. Cilostazol is also approved in Europe. The recommended daily dose is 100 mg twice daily, or 50 mg twice daily if co-administered with strong inhibitors of CYP3A4 and CYP2C19. Currently, cilostazol is in 15 ongoing clinical trials for cerebral infarction, cerebrovascular disease, atherosclerosis, diabetic complications, peripheral vascular disease, Raynaud's disease, intermittent claudication, ischemic heart disease, and acute coronary syndrome.

[0006] Other trials are investigating cilostazol in combination with other therapies. For example, a trial is investigating cilostazol in combination with aspirin in patients with ischemic stroke ("Overcoming Biochemical Aspirin Resistance with Cilostazol Combination Therapy (ARCC)"), and a trial is investigating the effect of aspirin combined with cilostazol versus aspirin alone on the progression of intracranial artery stenosis in 200 patients with chronic stroke with 50-99% stenosis. Cilostazol derivatives are described in the following publications: KR2012010800, WO2010063724, and WO2024002783.

[0007] Cilostazol is a white to off-white crystalline powder that is slightly soluble in methanol and ethanol but practically insoluble in water, 0.1N HCl, and 0.1N NaOH. Cilostazol is classified as a Class II drug by the Biopharmaceutical Classification System (BCS), indicating that it is a low-solubility, high-permeability drug. This means that the rate-limiting step in the oral bioavailability of cilostazol is the dissolution of the drug from its dosage form. These characteristics of cilostazol can present multiple challenges for formulation scientists in dosage form development. Furthermore, due to numerous factors, including frequent dosing, prolonged treatment duration, patient compliance, and low bioavailability, the use of oral dosage forms may not be a suitable approach for the treatment of cerebral infarction, cerebrovascular disease, atherosclerosis, diabetic complications, peripheral vascular disease, Raynaud's disease, intermittent claudication, ischemic heart disease, and acute coronary syndrome.

[0008] Despite the beneficial activity of cilostazol, there is still a need for new compounds for the treatment of the above-mentioned diseases and conditions.

[0009] Optimizing drug bioavailability has many potential benefits. For patient convenience and improved compliance, it is generally considered desirable to reduce dosing frequency. By extending the release time of the drug, the duration of action per dose is expected to be longer. This, in turn, will lead to overall improvements in dosing parameters, such as taking a drug once daily, where previously four times a day or once a week was required, or even less frequently where daily dosing was previously required. Many drugs are currently administered with a once-daily dosing frequency. However, not all of these drugs have pharmacokinetic properties suitable for a precise 24-hour dosing interval.

[0010] Extending the release period of these drugs would also be beneficial.

[0011] To date, no long-acting injectable drug has been approved in any market for the treatment of cerebral infarction, cerebrovascular disease, atherosclerosis, diabetic complications, peripheral vascular disease, Raynaud's disease, intermittent claudication, ischemic heart disease, and acute coronary syndrome. Furthermore, key parameters associated with drug development, such as the selection of an appropriate dose, dosage form, route of administration, dosing regimen, and effects in animals and humans, need to be successfully optimized. Therefore, there is an unmet need for an effective drug delivery system for cilostazol.

[0012] The bioavailability of cilostazol can be improved by the injection route of administration. More specifically, a long-acting (sustained-release or extended-release) injection formulation may provide better bioavailability after intramuscular or subcutaneous administration than the oral route. The expected increase in the bioavailability of the injection formulation can provide therapeutic plasma concentration levels, and its dosage can be administered intramuscularly or subcutaneously once a day, once every three days, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or once every six months. The total injection dose can be much lower than the oral daily dose required over the same time period, thereby reducing toxicity and improving patient compliance. Therefore, given the advantages associated with the long-acting formulation, there is an unmet need to develop a long-acting parenteral formulation of cilostazol, which would provide higher compliance rates while maintaining therapeutic levels of the drug in the patient's system for long periods of time (days, weeks, months, or even years) for the treatment of cerebral infarction, cerebrovascular disease, atherosclerosis, diabetic complications, peripheral vascular disease, Raynaud's disease, intermittent claudication, ischemic heart disease and acute coronary syndrome or other related diseases described herein.

[0013] Cilostazol and its various uses have been described in US Pat. No. 4,277,479, “Tetrazolylalkoxycassinyl derivatives and pharmaceutical compositions containing them”; US Pat. No. 6,187,790, “Use of Cilostazol for the treatment of sexual dysfunction”; US Pat. No. 6,515,128, “Process for the preparation of Cilostazol”; US Pat. Nos. 6,531,603, 6,573,382, 6,531,603, 6,657,061, and 6,660,864, “Polymorphs of 6-[4-1(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H-quinone)”; ”; US6525201, 6660773 and 6740758, “Processes for preparing 6-hydroxy-3,4-dihydroquinolinone, cilostazol and N-(4-methoxyphenyl)-3-chloropropionamide”, and US6825214, “Substantially pure cilostazol and method for making the same”. Formulations of cilostazol and its therapeutic uses are disclosed, for example, in WO2009 / 113741; WO2009 / 107864; and US published application US2009 / 0297596. All of these documents are incorporated herein by reference.

[0014] One of the fundamental considerations in drug therapy involves the relationship between blood levels and therapeutic activity. For most drugs, maintaining serum levels between the minimum effective concentration and the potentially toxic level is paramount. From a pharmacokinetic perspective, the peaks and troughs of drug blood levels ideally fit neatly within the therapeutic window of serum concentration. For some therapeutics, this window is so narrow that dose formulation becomes crucial.

[0015] The currently approved solid dosage oral form of cilostazol has several disadvantages. For example, its bioavailability is very low, so patients need a large daily pill burden (twice daily x 100 mg tablets). In addition, it causes high variability in patients' blood levels due to low bioavailability and a large food effect.

[0016] Non-oral routes of administration (e.g., parenteral routes) have been explored for other classes of drugs. However, to date, there is no sustained-release injectable prodrug formulation of cilostazol. Summary of the Invention

[0017] The present invention prepares a prodrug with long-lasting properties by modifying the structure of cilostazol. This type of drug is prepared by formulation means into a preparation that can be injected intramuscularly, subcutaneously, or intravenously. After intramuscular, subcutaneous, or intravenous injection, a drug reservoir is formed in the body. The drug is slowly, continuously, and stably released from the reservoir and converted into cilostazol, thereby exerting a long-lasting effect. The present invention adopts the following technical solutions to achieve this:

[0018] The purpose of the present invention is to remedy the deficiencies in the prior art and provide lauroyl cilostazol or a solvate thereof as shown in formula (I):

[0019]

[0020] The lauroyl cilostazol of the present invention is a sterile API. The purpose of the sterile API is that it can be directly fed into the injection dosage form. At the same time, the production process of the injection is very simple, for example, simple sterile filtration can be used to achieve the sterility of the injection.

[0021] The lauroyl cilostazol of the present invention is crystalline, and its X-ray powder diffraction pattern has characteristic peaks at diffraction angles 2θ of 5.30±0.2, 7.18±0.2, 9.54±0.2, 13.72±0.2, 14.60±0.2, 16.80±0.2, 16.90±0.2, 18.46±0.2, 19.86±0.2, 20.98±0.2, 21.08±0.2, 22.40±0.2, 23.74±0.2, 25.20±0.2, 26.36±0.2, 26.40±0.2, and 27.86±0.2°. The endothermic temperature of the crystals according to differential scanning calorimetry is 55-58°C.

[0022] Another object of the present invention is to provide a method for preparing lauroyl cilostazol, which comprises:

[0023] The method comprises the steps of reacting chloromethyl laurate and cilostazol in the presence of a solvent and a catalyst to prepare lauroyl cilostazol (Formula I).

[0024] Another object of the present invention is to provide a method for preparing lauroyl cilostazol, which comprises:

[0025] Step 1: preparing chloromethyl laurate by reacting dodecanoyl chloride with paraformaldehyde in the presence of a catalyst;

[0026] Step 2: The product of step 1 and cilostazol are reacted with a solvent and a catalyst to prepare lauroyl cilostazol (Formula I).

[0027] Another object of the present invention is to provide a method for preparing lauroyl cilostazol, which further comprises step 3:

[0028] Step 3: The crude product of formula (I) is used to prepare a pharmaceutical grade raw material of formula (I) in a crystallization solvent. The reaction formula is as follows:

[0029]

[0030] In the optimized process, the catalyst in step 1 is zinc chloride or aluminum chloride, preferably zinc chloride; the reaction solvent in step 2 is selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, dioxane, preferably N,N-dimethylformamide; the catalyst in step 2 is selected from sodium iodide; in step 3, a series of crystallization methods were screened when preparing lauroyl cilostazol crystals, and the results are as follows:

[0031] Table 1 Crystallization method

[0032]

[0033]

[0034] The solvent of the crystallization process provided by us is preferably one or a combination of ethyl acetate, n-hexane, anhydrous ethanol, and anhydrous methanol, preferably anhydrous methanol. Meanwhile, the temperature of the crystallization process is preferably 0-10°C.

[0035] Another object of the present invention is to provide a sterile lauroyl cilostazol, which is prepared by pre-filtration with a 0.45 μm microporous filter membrane before crystallization, and then undergoing two stages of 0.22 μm sterilization filtration and vacuum drying.

[0036] Yet another object of the present invention is the aforementioned lauroyl cilostazol, which is administered by injection, and the dosage forms include injection, freeze-dried powder, etc. Preferably, the lauroyl cilostazol injection preparation is used.

[0037] The injection preparation is prepared from the active sterile raw material lauroyl cilostazol and related pharmaceutical excipients according to a general method in pharmaceutical preparation. The excipients include those necessary in pharmaceutical preparation, such as suspending agents, antioxidants, stabilizers, wetting agents, preservatives, etc.

[0038] Another aspect of the present invention is to provide a use of lauroyl cilostazol in preparing a medicament, wherein the medicament is used to prepare a medicament for treating cerebral infarction, cerebrovascular disease, atherosclerosis, diabetic complications, peripheral vascular disease, Raynaud's disease, intermittent claudication, ischemic heart disease and acute coronary syndrome.

[0039] The main points of the present invention are:

[0040] The present invention unexpectedly discovered that compared with other prodrugs, the lauroyl cilostazol of the present application has the advantages of good physicochemical stability, not easy to decompose and produce impurities, easy to prepare into sterile raw materials, suitable for storage and transportation, high bioavailability, effectively prolonged drug action time, and low hepatotoxicity. Overall, it is more suitable for preparation into injections.

[0041] Disclosed herein are novel cilostazol prodrugs, lauroyl cilostazol, that cleave to release cilostazol and a safe and biodegradable lauric acid component. Compared to oral cilostazol formulations, the novel cilostazol prodrugs and formulations herein represent a breakthrough because they provide enhanced bioavailability, eliminate the food effect, reduce pill burden, reduce dosing frequency, and provide sustained, effective plasma levels of cilostazol, for example, for at least one week, typically at least two weeks, and up to ten weeks or longer, following administration of the cilostazol prodrug formulations. Furthermore, pharmacokinetic and pharmacodynamic studies of representative cilostazol prodrugs indicate that the novel lauroyl cilostazol and formulations are suitable for administration once a week, once a month, once every two months, once every three months, or even less frequently, a feature that alone represents a significant improvement over currently available cilostazol prodrugs. A significant improvement over tablets, which require patients to take a large number of tablets per day (twice daily x 100 mg tablets).

[0042] The present disclosure also demonstrates that administration of cilostazol lauroxil is generally well tolerated. For example, no hepatotoxicity was observed with the doses of cilostazol lauroxil administered. Furthermore, the present disclosure provides novel parenteral formulations of cilostazol lauroxil that may be more suitable for drug development. The compositions and methods described herein address a long-standing unmet need by providing an alternative to oral formulations that suffer from 1) low bioavailability; 2) interactions with ingested food; 3) delivery of highly variable parent drug blood levels, potentially reducing efficacy and increasing side effects; 4) daily dosing requirements and a high pill burden; 5) the need for castration; and 6) poor patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 This is the structure of lauroyl cilostazol.

[0045] Figure 2 This is the blood drug concentration-time curve of Example 14.

[0046] Figure 3 This is the HPLC analysis of lauroyl cilostazol in Example 11.

[0047] Figure 4 This is the hydrogen spectrum of lauroyl cilostazol in Example 11.

[0048] Figure 5 This is the carbon spectrum of lauroyl cilostazol in Example 11.

[0049] Figure 6 This is Example 11 Cilostazol Lauroyl LC-MS.

[0050] Figure 7 This is Example 11 Cilostazol Lauroyl DSC.

[0051] Figure 8 This is Example 11 lauroyl cilostazol TG.

[0052] Figure 9 This is Example 11 Cilostazol Lauroyl IR.

[0053] Figure 10 This is the XRD of lauroyl cilostazol in Example 11. DETAILED DESCRIPTION

[0054] The following examples are provided to further illustrate the present invention, however, these examples should not be construed as limiting the scope of the present invention. The following examples all relate to the preparation of lauroyl cilostazol (hereinafter referred to as YPK-008) and related experiments.

[0055] Example 1:

[0056] Step 1: Preparation of YPK-008 side chain

[0057] Reaction equation:

[0058]

[0059] Table 2 Material ratio

[0060]

[0061] Operation process:

[0062] To a 250 mL three-necked flask, 2.0 g of paraformaldehyde, 0.2 g of zinc chloride, and 10.9 g of lauroyl chloride were added in sequence. The mixture was stirred and heated to 105-115 ° C. for 16 hours (the system turned light black and the material was not dissolved). The temperature was lowered to room temperature (20-25 ° C). 100 mL of ethyl acetate was added and stirred for 5 minutes. The mixture was filtered under reduced pressure. The mother liquor was washed with 50 mL*2 of 10% sodium bicarbonate aqueous solution and the layers were separated. The organic phase was washed with 50 mL of water and dried over anhydrous sodium sulfate. The dried organic phase was concentrated under reduced pressure at 57 ° C to obtain 7.2 g of a gray oil. The mixture was purified by column chromatography (cyclohexane as eluent) to obtain 4.5 g of a colorless oil, which was the YPK-008 side chain.

[0063] Step 2: Preparation of YPK-008

[0064] Reaction equation:

[0065]

[0066] Table 3 Material ratio

[0067]

[0068] Operation process:

[0069] 2.0 g of cilostazol and 100 mL of dichloromethane were added to a 250 mL three-necked flask and stirred for several minutes. 2.5 g of triethylamine and 0.8 g of Dmap were added. After stirring for several minutes, 4.0 g of YPK-008 side chain (chloromethyl laurate) was added and allowed to react at room temperature (20-25°C). The reaction was monitored by TLC (λ = 254 nm, PE:EA = 1:1). The reaction was cooled to 0-10°C and quenched with water (20 mL). The layers were separated and the organic phase was washed with 50 mL of water. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated under reduced pressure to dryness to obtain 2.1 g of a yellow oil. The oil was purified by column chromatography (eluents: dichloromethane and PE:EA = 1:1, in sequence) to obtain 1.3 g of a light yellow oil. The TLC plate showed a pure spot. The product was submitted for H NMR analysis and the batch number was: CLTZ-LC-250320-01. The NMR data are as follows:

[0070] 1 H-NMR (400MHz, CDCl3): δ7.20~6.73(m,3H); 5.92~5.43(m,2H); 4.17~4.15(m,1H); 4.02(t,2H); 2.96~2 .88(m,4H); 2.72~2.67(m,2H); 2.39~2.29(m,2H); 2.10~1.38(m,16H); 1.32~1.25(m,16H); 0.90(t,3H).

[0071] Example 2:

[0072] Step 1: Preparation of YPK-008 side chain

[0073] Table 4 Material ratio

[0074]

[0075] Operation process:

[0076] 10 g of paraformaldehyde, 1.0 g of zinc chloride and 54.5 g of lauroyl chloride were added to a 500 mL three-necked flask in sequence, the temperature was raised to 90-95 ° C with stirring and the reaction was carried out for 16 hours (the system turned light black and the material was not dissolved), the temperature was lowered to room temperature (20-25 ° C), 500 mL of ethyl acetate was added and stirred for 5 minutes, and the mixture was filtered under reduced pressure. The mother liquor was washed with 250 mL*2 of 10% sodium bicarbonate aqueous solution and the layers were separated. The organic phase was washed with 250 mL of water and dried over anhydrous sodium sulfate. The dried organic phase was concentrated under reduced pressure at 57 ° C to obtain 34.2 g of a gray oil. It was purified by column chromatography (cyclohexane as eluent) to obtain 21.3 g of a colorless oil, which was the side chain of YPK-008. The H NMR spectrum was sent for inspection. The specific results are as follows: 1H-NMR (400MHz, CDCl3) δ: 5.739 (s, 2H); 2.414 (s, 2H); 1.688 (q, 2H); 1.44~1.23 (m, 16H); 0.917 (t, 3H).

[0077] Step 2: Preparation of YPK-008

[0078] Table 5 Material ratio

[0079]

[0080] Operation process:

[0081] 9.0 g of cilostazol and 180 mL of N, N-dimethylformamide were added to a 500 mL three-necked flask and stirred for several minutes. The material was completely dissolved and cooled to 0-5 ° C. 2.5 g of sodium hydride was added in batches and stirred for several minutes. 15 g of YPK-008 side chain (chloromethyl laurate) and 5.0 g of sodium iodide were added and stirred for several minutes. The mixture was transferred to room temperature (20-25 ° C for reaction). The reaction was monitored by TLC (λ = 254 nm, PE: EA = 1:1). The temperature was lowered to . After adding water (150 mL) at 0-10°C to quench the reaction, ethyl acetate (250 mL*2) was added and extracted twice. The organic phases were combined and washed twice with water (250 mL*2). The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated under reduced pressure to dryness to obtain 16 g of a yellow oil. The oil was purified by column chromatography (eluents were dichloromethane and PE:EA=1:1, in sequence) to obtain 13.5 g of a light yellow oil. The TLC spot plate showed a pure spot, which was the same spot as the product obtained in Case 1.

[0082] Example 3:

[0083] 18 g of cilostazol and 360 mL of N, N-dimethylformamide were added to a 500 mL three-necked flask and stirred for several minutes. The material was completely dissolved and cooled to 0-5 ° C. 5.0 g of sodium hydride was added in batches and stirred for several minutes. 30 g of YPK-008 side chain (chloromethyl laurate) and 10 g of sodium iodide were added. After stirring for several minutes, the mixture was transferred to room temperature (20-25 ° C for reaction). The reaction was monitored by TLC (λ = 254 nm, PE: EA = 1:1). The temperature was lowered to . After adding water (300 mL) at 0-10°C to quench the reaction, ethyl acetate (500 mL*2) was added and extracted twice. The organic phases were combined and washed twice with water (500 mL*2). The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to dryness under reduced pressure to obtain 33 g of a yellow oil. The oil was purified by column chromatography (eluents were dichloromethane and PE:EA=1:1, in sequence) to obtain 27.4 g of a light yellow oil. The TLC spot plate showed a pure spot, which was the same spot as the product in Case 1.

[0084] Example 4:

[0085] 18 g of cilostazol and 360 mL of N, N-dimethylformamide were added to a 500 mL three-necked flask and stirred for several minutes. The material was completely dissolved and cooled to 0-5 ° C. 5.0 g of sodium hydride was added in batches and stirred for several minutes. 15 g of YPK-008 side chain (chloromethyl laurate) and 5 g of sodium iodide were added. After stirring for several minutes, the mixture was transferred to room temperature (20-25 ° C for reaction). The reaction was monitored by TLC (λ = 254 nm, PE: EA = 1:1). The temperature was lowered to After adding water (300 mL) at 0-10°C to quench the reaction, ethyl acetate (500 mL*2) was added and extracted twice. The organic phases were combined and washed twice with water (500 mL*2). The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to dryness under reduced pressure to obtain 33 g of a yellow oil. The oil was purified by column chromatography (eluents were dichloromethane and PE:EA=1:1, in sequence) to obtain 27.4 g of a light yellow oil. The TLC spot plate showed a pure spot, which was the same spot as the product in Case 1.

[0086] Example 5:

[0087] 18 g of cilostazol and 180 mL of N, N-dimethylformamide were added to a 500 mL three-necked flask, stirred for several minutes, cooled to 0-5 ° C, 5.0 g of sodium hydride was added in batches, stirred for several minutes, 30 g of YPK-008 side chain (chloromethyl laurate) and 10 g of sodium iodide were added, stirred for several minutes, and then transferred to room temperature (20-25 ° C for reaction), TLC (λ = 254 nm, PE: EA = 1:1) was used to monitor the completion of the reaction, and the temperature was lowered to 0-1 After adding water (300 mL) at 0°C to quench the reaction, ethyl acetate (500 mL*2) was added and extracted twice. The organic phases were combined and washed twice with water (500 mL*2). The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated under reduced pressure to dryness to obtain 31 g of a yellow oil. The oil was purified by column chromatography (eluents were dichloromethane and PE:EA=1:1, respectively) to obtain 26.3 g of a light yellow oil. The TLC spot plate showed a pure spot, which was the same spot as the product in Case 1.

[0088] Example 6:

[0089] 20 g of cilostazol and 1000 mL of dichloromethane were added to a 2 L three-necked flask and stirred for several minutes. 20 g of triethylamine and 8 g of Dmap were added. After stirring for several minutes, 40 g of YPK-008 side chain was added and the mixture was reacted at room temperature (20-25 ° C). The reaction was monitored by TLC (λ = 254 nm, PE: EA = 1: 1). After cooling to 0-10 ° C, water (200 mL) was added to quench the reaction. The layers were separated and the organic phase was washed with 500 mL of water. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated under reduced pressure to dryness to obtain 21.4 g of a yellow oil. The oil was purified by column chromatography (eluents were dichloromethane and PE: EA = 1: 1, in sequence) to obtain 13.2 g of a light yellow oil. The TLC plate showed a pure spot, which was the same spot as the product in Case 1.

[0090] Example 7:

[0091] 20 g of cilostazol and 1000 mL of dichloromethane were added to a 2 L three-necked flask and stirred for several minutes. 20 g of triethylamine and 8 g of Dmap were added. After stirring for several minutes, 20 g of YPK-008 side chain was added and the mixture was reacted at room temperature (20-25 ° C). The reaction was monitored by TLC (λ = 254 nm, PE: EA = 1: 1). After cooling to 0-10 ° C, water (200 mL) was added to quench the reaction. The layers were separated and the organic phase was washed with 500 mL of water. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated under reduced pressure to dryness to obtain 20.3 g of a yellow oil. The oil was purified by column chromatography (eluents were dichloromethane and PE: EA = 1: 1, in sequence) to obtain 11.3 g of a light yellow oil. The TLC plate showed a pure spot, which was the same spot as the product in Case 1.

[0092] Example 8:

[0093] 20 g of cilostazol and 400 mL of dichloromethane were added to a 1 L three-necked flask and stirred for several minutes. 20 g of triethylamine and 8 g of Dmap were added. After stirring for several minutes, 40 g of YPK-008 side chain was added and the mixture was reacted at room temperature (20-25°C). The reaction was monitored by TLC (λ = 254 nm, PE:EA = 1:1). The temperature was lowered to 0-10°C and water (200 mL) was added to quench the reaction. The layers were separated and the organic phase was washed with 500 mL of water. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated under reduced pressure to dryness to obtain 21.3 g of a yellow oil. The oil was purified by column chromatography (eluents were dichloromethane and PE:EA = 1:1, in sequence) to obtain 13.0 g of a light yellow oil. The TLC plate showed a pure spot, which was the same spot as the product in Case 1.

[0094] Example 9:

[0095] Take 3.5 g of the light yellow oil obtained in step 2 of Example 2, add 7 mL of ethyl acetate and 35 mL of n-hexane, stir and heat until completely dissolved, slowly cool to 20-25 ° C without solid precipitation, continue cooling to -5-0 ° C, gradually solid precipitation, keep at -5-0 ° C for crystallization for 3 days, and filter the obtained solid under reduced pressure and dry in vacuo at 45 ° C to obtain 1.1 g of a white solid, sample batch number: YPK-008-250408-01, HPLC test result: 99.47%.

[0096] Example 10:

[0097] Take 3.5 g of the light yellow oil obtained in step 2 of Example 2, add 35 mL of anhydrous ethanol, stir and heat until completely dissolved, slowly cool to 20-25 ° C without solid precipitation, continue cooling to -5-0 ° C, gradually solid precipitation, keep at -5-0 ° C for 2 hours, and crystallize the obtained solid under reduced pressure and filter, and vacuum dry at 40 ° C to obtain 2.1 g of a white solid. The HPLC test result is: 99.33%.

[0098] Example 11:

[0099] Take 3.5 g of the light yellow oil obtained in step 2 of Example 1, add 35 mL of anhydrous methanol, stir and heat until completely dissolved, slowly cool to 20-25 ° C without solid precipitation, continue cooling to -5-0 ° C, gradually solid precipitation, keep at -5-0 ° C for 2 hours, and crystallize the obtained solid under reduced pressure and filter, and vacuum dry at 40 ° C to obtain 1.6 g of white solid. The HPLC test result is: 99.54%.

[0100]

[0101] Table 6 Nuclear magnetic hydrogen spectrum detection results

[0102]

[0103] Table 7 Mass spectrometry detection results

[0104] Mass-to-charge ratio (m / z) Remark 582.3 <![CDATA[[M+H] + ]]>

[0105] Table 8 Infrared detection results

[0106] <![CDATA[Absorption peak wave number (cm -1 )]]> Vibration type Group 3439 -C=O stretching vibration (overtone) -C=O 2920 Methylene / methyl stretching vibration Methylene, methyl 2852 Methylene-CH stretching vibration Methylene 1725 -C=O stretching vibration C=O of ester group 1697 -C=O stretching vibration C=O in amides 1589 R-COO-, CN stretching vibration Ester group, CN 1507 Skeletal vibration of benzene ring Benzene ring 1471 CH bending vibration Methylene 1458 -CH3 bending vibration -CH3 1446 -CH bending vibration -CH 1297 -CH bending vibration (in-plane) -CH 1236 -CC stretching vibration -CC 1163 -COC tensile vibration Ester--COC 938 -CH bending vibration (out-of-plane) -CH 868 -CH2 bending vibration (out-of-plane) -CH2 821 -CC skeleton vibration -CC 673 CH rocking vibration -CH 598 -CC skeleton vibration -CC

[0107] TGA test results

[0108] The TGA graph shows that this product does not contain crystalline water or solvent and begins to decompose at about 360°C.

[0109] DSC test results: From the DSC graph, we can see that this product does not contain crystal water or crystallization solvent, and the melting point is 57.39℃.

[0110] Table 9 XRD patterns

[0111] 2θ angle Diffraction intensity (I) 5.30 382 7.18 182 9.54 74 13.72 124 14.60 366 16.80 242 16.90 248 18.46 380 19.86 496 20.98 484 21.08 410 22.40 254 23.74 280 25.20 452 26.36 208 26.40 204 27.86 130

[0112] Table 10 NMR carbon spectrum test results

[0113]

[0114] Example 12: Stability Test

[0115] The compounds of this invention will be used as APIs for the preparation of suspension injections, making storage a critical issue. Impurities may be introduced or generated during storage, potentially impacting the API's performance. Therefore, the API was investigated for stability. Experiments were conducted using the influencing factors test, referring to the ICH guideline "Stability Testing of New APIs and Drug Products."

[0116] 1. Test conditions: The product is tested under high humidity conditions at 25℃±2℃, RH: 75%±5%, high temperature (60℃) with inner and outer packaging removed for 30 days, and light intensity (total illumination not less than 1.2×106Lux·hr) with inner and outer packaging removed.

[0117] 2 Sampling and testing: sampling is conducted on the 5th, 10th and 30th day under high temperature; sampling is conducted on the 5th and 10th day under high humidity; sampling is conducted on the 5th and 11th day under light conditions.

[0118] 3Investigation results: See the table below

[0119] Table 11 Stability test results

[0120]

[0121] Conclusion: The table shows that the API of the present invention is relatively stable under the relevant setting conditions and does not produce obvious impurities. Therefore, after the API is prepared, it can be stored at room temperature.

[0122] Example 13: Experimental study on cytotoxicity of normal human hepatocytes in vitro

[0123] 1 Experimental Materials

[0124] 1.1 Cells: LO2 cells, a human liver cell line

[0125] 1.2 Drug: Compound 11 of the present invention, HPLC purity 99.54%

[0126] 1.3 Reagents and instruments Modified RPMI-1640 culture medium, penicillin-streptomycin double antibody solution, 0.25% trypsin-EDTA, fetal bovine serum, MTT, dimethyl sulfoxide, CO-150 carbon monoxide incubator, SW-CJ-2F medical clean bench, CKX-41-32 inverted microscope, CU600 electric constant temperature water bath, RT-2100C enzyme label analyzer

[0127] 2 Experimental methods

[0128] 2.1 Reagent preparation

[0129] 2.1.1 Preparation of MTT: Weigh 0.25 g of MTT using a precision balance and place it in a 50 mL volumetric flask. Add an appropriate amount of PBS and place in a 50-60°C water bath. Shake well to dissolve thoroughly. Add PBS to the mark to prepare a 5 mg / kg solution. Sterilize by filtering through a 0.22 μm microporous filter membrane. Aliquot and store in a refrigerator at 4°C, protected from light.

[0130] 2.1.2 Preparation of cell freezing solution Cell freezing solution was prepared by mixing 20% ​​serum, 10% DMSO and 70% 1640 culture medium and stored at -20℃.

[0131] 2.1.3 Preparation of the compound of Example 11 of the present invention A stock solution of the drug was prepared with DMSO, and then diluted with culture medium to the concentration of the drug to be used. The final concentration of DMSO was controlled to be ≤0.1%.

[0132] 2.2 LO2 cell culture Normal human LO2 cells were placed in a 25 cm 2 Add approximately 4-5 mL of RPMI-1640 medium containing 10% FBS to the cell culture flask and incubate in a 37°C cell culture incubator with 5% CO2 saturated humidity. Change the medium every two days and observe cell growth daily. Passage or cryopreserve cells when they reach 80% confluency. Select cells from passages 5-7 for formal experiments.

[0133] 2.3 Grouping and Dosing The experiment was divided into a normal cell control group, and different concentrations of the drug-dosing groups of the present invention were set according to the results of the preliminary experiment, with concentrations of 6.0, 12.0, 24.0, 48.0, 96.0, 192, 384, 768, 1200, and 1500 μmol / L, respectively.

[0134] 2.4 Hepatocyte MTT Colorimetry: LO2 cells in the logarithmic growth phase were plated in a 96-well plate at a cell suspension of 5.0 × 10 cells / mL. A normal control group and groups treated with the compound of the present invention at various concentrations of 6.0, 12.0, 24.0, 48.0, 96.0, 192, 384, 768, 1200, and 1500 μmol / L were set up. After 24 hours of incubation, the culture medium was aspirated and the cells were washed two to three times with PBS. The compounds of the present invention were then added at various concentrations, with eight replicate wells per well. The plates were incubated at 37°C in a 5% CO2 incubator. After 24, 48, and 72 hours of incubation, MTT solution was added at each time point and incubated for 4 hours in the dark. After 4 hours, the supernatant was aspirated and 150 μL of DMSO solvent was added to each well to dissolve the thiazolyl blue crystals. The plates were gently shaken to ensure uniform dissolution. The absorbance of each well was measured at 490 nm using a microplate reader. The absorbance value of each well reflects the number of cells, and the two are directly proportional. The experiment was repeated 3 times. The cell viability rate was calculated as follows: Cell viability rate (%) = absorbance of each group (OD490) × 100 / absorbance of control group (OD490)

[0135] 2.5 Statistical Analysis All data are expressed as mean ± standard deviation and processed using SPSS 17.0 statistical software package. Statistical analysis was performed using the t-test. Paired t-tests were used for intragroup comparisons, and unpaired t-tests were used for intergroup comparisons. P < 0.05 was the standard.

[0136] 3 Experimental results

[0137] Table 12 Experimental results of in vitro cytotoxicity of normal human hepatocytes

[0138]

[0139] Conclusion: 72 hours after administration of different concentrations of the present invention, the OD values ​​of cells in each group showed an increasing trend, but the growth rate decreased, indicating that the cells were still in the growth stage and the growth rate was inhibited. The groups treated with different concentrations of the present invention had no inhibitory effect on the cells. Compared with the normal group, the cell survival rate was greater than 90%. Within the range of 6.0-1500 μmol / L, there was almost no inhibition on cell growth and no toxicity to the cells.

[0140] Example 14: Pharmacokinetic study in rats

[0141] 1 In vivo HPLC analysis method

[0142] Use octadecylsilane bonded silica gel as the filler (250 mm × 4.6 mm; 5 μm or equivalent performance chromatographic column; ghost peak trapping column: MORHCHEM, 4.6 mm × 50 mm); use water as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution according to the table below; the flow rate is 1.0 ml per minute, the column temperature is 40°C; the detection wavelength is 250 nm; the injection volume is 20 μl.

[0143] Table 13 Chromatographic mobile phase

[0144] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 65 35 5 65 35 10 0 100 50 0 100 51 65 35 60 65 35

[0145] 2 Preparation of plasma samples

[0146] Blank plasma sample: About 0.5 ml of blood was collected from the canthus of the rat and placed in a pre-prepared heparin-coated centrifuge tube. The tube was centrifuged at 10,000 rpm for 10 min, 200 μL of the supernatant was removed, 600 μL of anhydrous ethanol was added, the tube was vortexed for 10 min, and the tube was centrifuged at 10,000 rpm for 10 min. The supernatant was removed.

[0147] Plasma samples containing Example 11: 200 μL of blank rat plasma stored in the refrigerator was thawed at room temperature, and 20 μL of a series of prepared solutions of Example 11 (the compound of Example 11 was dissolved in a mixed solvent of sesame oil and benzyl alcohol) were added thereto to prepare plasma solutions of Example 11 at concentrations of 10, 5, 2, 1, 0.5, and 0.2 μg / ml. 600 μL of anhydrous ethanol was added to each solution to precipitate protein. The solution was vortexed for 10 minutes and centrifuged at 1000 rpm for 10 minutes. The supernatant was removed for injection.

[0148] 3. Dosage regimen and sample collection

[0149] Healthy rats were fasted for 12 hours before the experiment and had free access to water. The rats were divided into two groups. One group received a 120 mg / kg intramuscular injection of the solution of Example 11 (the compound of Example 11 was dissolved in a mixed solvent of sesame oil and benzyl alcohol) into the leg muscles of the rats. The other group received a 120 mg / kg intramuscular injection of the cilostazol solution (cilostazol was dissolved in a mixed solvent of sesame oil and benzyl alcohol) into the leg muscles of the rats.

[0150] 3. Dosage regimen and sample collection

[0151] Healthy rats were fasted for 12 hours before the experiment and had free access to water. The rats were divided into two groups. One group received a 120 mg / kg intramuscular injection of the solution of Example 11 (the compound of Example 11 was dissolved in a mixed solvent of sesame oil and benzyl alcohol) into the rat leg muscles, while the other group received a 120 mg / kg intramuscular injection of the cilostazol solution (cilostazol was dissolved in a mixed solvent of sesame oil and benzyl alcohol). Before and after administration, 0.5 mL of blood was collected from the rat ophthalmic vein at different time points. The blood was collected in a centrifuge tube pre-coated with heparin and centrifuged at 10,000 rpm for 10 minutes. 0.2 mL of the supernatant was collected in a centrifuge tube and stored in a -4°C refrigerator. When measuring plasma drug concentration, anhydrous ethanol was added to the plasma sample to precipitate protein, followed by vortexing for 10 minutes and centrifugation at 10,000 rpm for 10 minutes. The supernatant was taken for HPLC determination, and the blood drug concentration-time curve was drawn. The measured blood drug concentration-time data were fitted with a single-compartment model to obtain pharmacokinetic parameters for analysis.

[0152] Table 14 Pharmacokinetic experimental results

[0153]

[0154]

[0155] AUC of lauroyl cilostazol of Example 11 0-720h The AUC value of the cilostazol group was 390.28±19.56h·μg / mL, which was significantly higher than the AUC value of the same dose of cilostazol administered intramuscularly (P<0.05). At the same time, the plasma clearance rate (CL / F) was 0.30±0.01L / (h·kg), which was significantly lower than that of the cilostazol group (0.61±0.11L / (h·kg), P<0.05), indicating that the in vivo clearance rate of lauroyl cilostazol in Example 11 was reduced, the residence time was prolonged, and the AUC was significantly increased, showing a more obvious sustained-release effect than that of cilostazol.

Claims

1. Cilostazol lauroyl represented by formula (I): 。 2. The lauroyl cilostazol according to claim 1, characterized in that Cilostazol lauroyl is crystalline, and the X-ray powder diffraction pattern of the cilostazol lauroyl crystals has characteristic peaks at diffraction angles 2θ: 5.30±0.2, 7.18±0.2, 9.54±0.2, 13.72±0.2, 14.60±0.2, 16.80±0.2, 16.90±0.2, 18.46±0.2, 19.86±0.2, 20.98±0.2, 21.08±0.2, 22.40±0.2, 23.74±0.2, 25.20±0.2, 26.36±0.2, 26.40±0.2, and 27.86±0.2°.

3. The lauroyl cilostazol crystal according to claim 2, characterized in that The endothermic temperature of the lauroyl cilostazol crystals according to differential scanning calorimetry is 55-58°C.

4. The method for preparing lauroyl cilostazol according to claim 1, wherein: The following steps are involved: Step 1: preparing chloromethyl laurate by reacting dodecanoyl chloride with paraformaldehyde in the presence of a catalyst; Step 2: The product of step 1 and cilostazol are reacted with a solvent and a catalyst to prepare the compound of formula (I); The reaction formula is as follows: 。 5. The method for preparing lauroyl cilostazol according to claim 4, characterized in that: The catalyst in step 1 is zinc chloride or aluminum chloride; the reaction solvent in step 2 is selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, and dioxane; and the catalyst in step 2 is selected from sodium iodide.

6. The method for preparing lauroyl cilostazol according to claim 5, characterized in that: The catalyst in step 1 is zinc chloride.

7. The method for preparing lauroyl cilostazol according to claim 5, characterized in that: The reaction solvent in step 2 is N,N-dimethylformamide.

8. The method for preparing lauroyl cilostazol according to claim 4, wherein: The method further comprises step 3: preparing a pharmaceutical-grade raw material of formula (I) from the crude product of formula (I) in a crystallization solvent, wherein the crystallization solvent of step 3 is selected from one or a combination of ethyl acetate, n-hexane, anhydrous ethanol, and anhydrous methanol; and the temperature of the crystallization process is 0 to 10°C.

9. The method for preparing lauroyl cilostazol according to claim 8, characterized in that: The crystallization solvent in step 3 is selected from anhydrous methanol.

10. A pharmaceutical composition comprising the lauroyl cilostazol according to claim 1 and a pharmaceutically acceptable carrier.

11. Use of the lauroyl cilostazol according to claim 1 in preparing a medicament for treating cerebral infarction, atherosclerosis, diabetic complications, peripheral vascular disease, Raynaud's disease, intermittent claudication, ischemic heart disease and acute coronary syndrome.

Images