A sulfate of a pleuromutilin compound, and a preparation method and application thereof

Abstract

The present application relates to the technical field of drug salt type, and specifically discloses a sulfate of a truncated pleuromutilin compound, specifically, a sulfate of 14-O-[(4-(pyrrolidinyl-1-yl)-6-methylpyridine-2-yl) mercaptoacetyl] pleuromutilin PMT, and a preparation method and application thereof are provided.Compared with the original drug PMT, the water solubility and oral bioavailability of the PMT sulfate are obviously improved, and the PMT sulfate has the possibility of being developed into an oral drug for treating human or animal infections caused by bacteria or mycoplasma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical salt types, and particularly relates to a sulfate of a pleuromutilin compound and a preparation method and application thereof. BACKGROUND

[0002] In the process of chemical drug research and development, the physicochemical properties of drugs are of great significance to drug absorption, distribution, metabolism and excretion (ADME), dosage form research and development or production, etc. in the body. Most chemical active pharmaceutical ingredients (APIs) belong to class II compounds in the BCS classification, which are water-insoluble compounds, thus leading to low oral bioavailability, which is not conducive to the clinical use of drugs. In order to improve water solubility, chemical drugs are generally prepared into salt types or co-crystals, so as to change their physicochemical properties, improve water solubility and increase their oral bioavailability.

[0003] Pleuromutilin is a diterpenoid compound with antibacterial activity, which was first isolated from higher fungi Pleurotus multilus (Fr.) Sacc. and Pleurotus Passeckrianus Pilat in the 1950s. The antibacterial activity of this compound can be significantly improved by structural modification of the C-4 side chain. Through the structural modification of pleuromutilin, four drugs, i.e. Tiamulin, Valnemulin, Retapamulin and Lefamulin, have been developed. The first two are used as veterinary drugs to treat or prevent diseases caused by Brachyspira hyodysenteriae and Mycoplasma spp. The last two are used as human drugs to treat skin infections caused by Staphylococcus and Streptococcus and community-acquired bacterial pneumonia, respectively.

[0004] 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl) mercaptoacetyl] mutilin (PMT) is a pleuromutilin compound containing a pyrimidine ring side chain in the side chain. Studies have shown that the compound has good activity against gram-positive bacteria and mycoplasma. In addition, the compound has the advantages of low toxicity and low production cost. However, due to the poor water solubility of PMT, the oral bioavailability of the compound is low. SUMMARY

[0005] The first object of the present application is to solve the problem of poor water solubility and low oral bioavailability of PMT, and to provide a sulfate of a pleuromutilin compound with high oral bioavailability.

[0006] The second object of the present application is to provide a preparation method of the above-mentioned sulfate of a pleuromutilin compound.

[0007] The third object of the present application is to provide the use of the sulfate salt of the above-mentioned pleuromutilin compound.

[0008] The object of the present application is achieved by the following technical solutions.

[0009] A sulfate salt of a pleuromutilin compound, the sulfate salt of the pleuromutilin compound being a sulfate salt of 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)mercaptoacetyl]mutilin having the following structure,

[0010]

[0011] A preparation method of the above-mentioned sulfate salt of the pleuromutilin compound, the 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)mercaptoacetyl]mutilin is dissolved in an organic solvent with sulfuric acid, heated and stirred to react, cooled to room temperature, and the obtained solid is the sulfate salt of 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)mercaptoacetyl]mutilin after standing.

[0012] As preferred, the amount ratio of the 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)mercaptoacetyl]mutilin, sulfuric acid, and organic solvent is: 5.6g of 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)mercaptoacetyl]mutilin corresponds to 40-50mL of 29.4mg / mL aqueous sulfuric acid solution and 80-150mL of organic solvent.

[0013] As preferred, the organic solvent includes one or a mixture of several of alcohol, ester, ketone, and acetonitrile solvent.

[0014] As preferred, the heating temperature is 50-70℃, the reaction time is 0.5-3h, and the standing time is 1-3 days.

[0015] The above-mentioned sulfate salt of the pleuromutilin compound is used in the preparation of an oral drug for treating bacterial or mycoplasma-induced human or animal infection.

[0016] As preferred, the bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus, and Streptococcus agalactiae; and the mycoplasma includes Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma flocculare, Mycoplasma hyopneumoniae, and Mycoplasma bovigenitalium.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] The sulfate salt of the compound 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)thioacetyl] merazole provided by the present invention has good stability and water solubility, and significantly improves the oral bioavailability of the compound PMT. It has the potential to be developed into an oral drug for treating human or animal infections caused by bacteria or mycoplasmas. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the PXRD pattern of the pleuromutilin compound PMT sulfate prepared in Example 1;

[0020] Figure 2 is the FT-IR image of the pleuromutilin compound PMT sulfate prepared in Example 1;

[0021] Figure 3 1 is the DSC and TG graph of the pleuromutilin compound PMT sulfate prepared in Example 1;

[0022] Figure 4 The dissolution curves of PMT and its sulfate in different pH buffers: (A) is the dissolution curve of PMT and its sulfate in pH 1.2 buffer; (B) is the dissolution curve of PMT sulfate in pH 4.5 and 6.8 buffers;

[0023] Figure 5 is the positive ion spectrum of PMT;

[0024] Figure 6 This is the positive ion spectrum of IS (Tiamulin);

[0025] Figure 7 The graph shows the average blood concentration-time curve of PMT in rats: (A) is the intravenous administration of PMT original drug; (B) is the oral administration of PMT original drug; (C) is the oral administration of PMT sulfate. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0027] Unless otherwise specified, the raw materials used in the examples are all commercially available products. Among them, PMT was prepared in this laboratory. The preparation method can be found in reference (Eur. J. Med. Chem. 2020, 207, 12735.).

[0028] The product characterization methods obtained in the following examples are as follows:

[0029] Powder X-ray diffraction (PXRD) detection method:

[0030] Before use, the instrument is calibrated with the standard sample provided. The sample is placed on an organic glass slide and tested at room temperature. CuKa radiation is used Voltage 40kv, current 40mA. Detection conditions: 2θ angle 5-40°, step 0.02°, speed 5s / step.

[0031] Fourier infrared spectroscopy (FT-IR) detection method:

[0032] Nicolet FT-IR Magna 550 instrument is used for detection. About 5mg of sample is pressed into a transparent film by KBr, and placed on the sample holder for detection. The resolution is 4cm -1 , and the spectral range is 4000-400cm -1 .

[0033] Differential scanning calorimetry (DSC) method:

[0034] A differential scanning calorimeter (TAQ2000) is used for analysis. Before analysis, the instrument is calibrated with metallic indium. 3-5mg of sample is sealed in an aluminum crucible under high-purity nitrogen protection (nitrogen flow rate 50mL / min), and heated to 600℃ at a rate of 10℃ / min.

[0035] Thermogravimetric (TG) analysis method:

[0036] A thermogravimetric analyzer (TA Q500) is used for analysis. 5-10mg of sample is placed in a platinum pan under high-purity nitrogen protection (nitrogen flow rate 40mL / min), and heated to 600℃ at a rate of 20℃ / min.

[0037] Example 1

[0038] According to the molar ratio of PMT to sulfuric acid of 1:1.5, 0.56g (1mmol) of PMT is dispersed in 10mL of acetone, stirred at 50±1℃ until completely dissolved, then 5mL of aqueous solution containing 1.5mmol of sulfuric acid (29.4mg / mL) is added, stirred at this temperature for 1h, then 5mL of distilled water is added again (distilled water can also not be added), stirred for 1h, then cooled to room temperature and placed for 2d. The obtained solid is suction filtered, and washed with 10mL of distilled water twice. The filter cake is dried at 50℃ for 5h to obtain 0.59g of light yellow solid, which is PMT sulfate.

[0039] Example 2

[0040] According to the PMT and sulfuric acid in a molar ratio of 1:1.5, 0.56 g (1 mmol) of PMT was dispersed in 15 mL of ethanol, stirred at 55±1°C until completely dissolved, then 5 mL of aqueous solution containing 1.5 mmol of sulfuric acid (29.4 mg / mL) was added, stirred at this temperature for 1 h, then 5 mL of distilled water was added again (also can not add distilled water), after stirring for 1 h, cooled to room temperature and placed for 3 d, the obtained solid was suction filtered, washed with 10 mL of distilled water twice, the filter cake was dried at 50°C for 7 h, 0.57 g of light yellow solid was obtained, which was PMT sulfate.

[0041] Example 3

[0042] According to the PMT and sulfuric acid in a molar ratio of 1:1.5, 0.56 g (1 mmol) of PMT was dispersed in 15 mL of acetonitrile, stirred at 60±1°C until completely dissolved, then 5 mL of aqueous solution containing 1.5 mmol of sulfuric acid (29.4 mg / mL) was added, stirred at this temperature for 1 h, then 5 mL of distilled water was added again (also can not add distilled water), after stirring for 1 h, cooled to room temperature and placed for 3 d, the obtained solid was suction filtered, washed with 10 mL of distilled water twice, the filter cake was dried at 50°C for 7 h, 0.60 g of light yellow solid was obtained, which was PMT sulfate.

[0043] The PMT sulfate prepared in the examples was characterized by PXRD, FT-IR, DSC, TG and other solid state chemical methods, and the results were basically consistent. The spectrum of the product of Example 1 is shown in the accompanying drawings Figures 1-3 .

[0044] Example 4

[0045] Dissolution kinetics study

[0046] The PMT and the PMT sulfate obtained in Example 1 were respectively subjected to dissolution test in ammonium acetate buffer at pH values of 1.2, 4.5 and 6.8.

[0047] Test method: The test sample was ground and sieved (100 mesh) to eliminate the difference caused by different particle sizes. 20 mg of sieved sample was added into 10 mL of ammonium acetate buffer with different pH values, and dissolved in a vortex at a speed of 300 rpm in a 37°C environment, then 1 mL of solution was taken at 2.5, 5, 10, 20, 40, 60, 120 and 240 min, respectively, filtered through a 0.45 μm filter membrane, and the content of PMT and its sulfate was determined by HPLC.

[0048] Ultra-high performance liquid chromatograph: Agilent Infinity 1290 equipped with binary pump, diode array detector (DAD); Agilent, USA.

[0049] Chromatographic conditions: The chromatographic column was ZORBAX Eclipse Plus C18 (4.6 μm × 250 mm); the flow rate was 1 mL / min; the detection wavelength was set at 265 nm; the column temperature was 22°C; and the mobile phase was water and acetonitrile (80:20).

[0050] Experimental results: The dissolution curves of PMT and its sulfate at different sampling times in different pH buffers are shown in the appendix of the manual. Figure 4 At pH 1.2 ( Figure 4 In Figure A), PMT sulfate showed much faster solubility than PMT, with the maximum solubility concentration (C max ), 1972±74μg / mL, and then the solubility gradually decreased and was lower than the PMT of the original drug at about 57min, but the area under the curve (AUC) of PMT sulfate was 1.12 times higher than that of PMT. At pH 4.5 and 6.8 ( Figure 4 In Figure B), the original PMT was not detected in the buffer. However, PMT sulfate exhibited a similar dissolution curve, reaching maximum solubility at 2.5 min, with concentrations of 87.66 ± 18.48 and 0.55 ± 0.14 μg / mL, respectively, demonstrating the high water solubility of the PMT sulfate provided by the present invention.

[0051] Example 5

[0052] Preclinical pharmacokinetic studies

[0053] Pharmacokinetic studies were conducted by intravenously injecting PMT original drug, orally administering PMT original drug and its sulfate to SD rats, and measuring the content of PMT original drug in rat plasma at different times.

[0054] Detection method: LC-MS / MS analysis was performed using an AB SCIEX QTRAP 5500 ultra-high performance liquid chromatography-mass spectrometer.

[0055] Liquid phase conditions: The chromatographic column was a Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm ID, 1.8 μm), and gradient elution was performed according to the mobile phase ratio in Table 1. The flow rate was 0.2 mL / min, the column temperature was 40°C, the run time was 7 min, and the injection volume was 2.5 μL.

[0056] Table 1 Gradient elution time and mobile phase ratio

[0057] Time (min) Flow rate (mL / min) A (0.1% formic acid water) B (acetonitrile) 0 0.2 90 10 1 0.2 90 10 1.5 0.2 20 80 3.8 0.2 20 80 4.8 0.2 90 10 7 0.2 90 10

[0058] Mass spectrometry conditions: positive ion mode, ion source is electrospray ion source (ESI), scanning in multi-reactions monitoring (MRM) mode. The parameter settings are as follows: electrospray voltage is +5500V, ion source temperature is 550°C, entrance potential (EP) is 10V, collision cell exit potential (CXP) is 13V, atomizer and auxiliary pressure are both 55psi, gas curtain pressure is 20psi, and collision gas pressure is set to moderate.

[0059] All data are obtained by German Bruker 3.4MS Workstation V8.2 software integration. The specific PMT and internal standard IS (tylosin) quantitative and qualitative ion pair parameters are shown in Table 2.

[0060] Table 2 PMT and internal standard (tylosin) quantitative and qualitative ion pair parameters

[0061]

[0062] Preparation of sample test solution: chromatographically pure acetonitrile is used to prepare PMT stock solution and working solution, and the working solution concentrations are 1, 5, 10, 50, 100, 500, 1000, 1500, and 3000 ng / mL, respectively. The internal standard stock solution and working solution (100 ng / mL) are also prepared using chromatographically pure acetonitrile.

[0063] Plasma sample processing method: 100 μL and 10 μL of internal standard (internal standard concentration is 100 ng / mL) are added to a 4 mL centrifuge tube, vortexed for 30 s, then 2 mL of acetonitrile is added, vortexed and shaken for 5 min, centrifuged at 12000 rpm at 4°C for 10 min, the supernatant is taken into a 4 mL centrifuge tube, the solvent is evaporated at 40°C, 100 μL of mobile phase is used for reconstitution, vortexed for 10 min, centrifuged at 12000 rpm at 4°C for 10 min, transferred to a sample injection vial, 2.5 μL is injected, and the obtained PMT ion spectrum is shown in Figure 5 , and the ion spectrum of the internal standard tylosin is shown in Figure 6 .

[0064] The above PMT extraction and detection method in plasma is verified for specificity, precision and accuracy, extraction recovery, matrix effect and stability, etc. The established method is stable and reliable, and can be applied to the subsequent pharmacokinetic study of PMT and its salt type.

[0065] Drug metabolism and kinetics studies: Drug metabolism and kinetics studies were conducted using 18 Sprague-Dawley rats (half male and half female, weighing 180-200 g). The 18 rats were randomly divided into three groups: an intravenous injection group (PMT stock), an oral administration group (PMT stock), and an oral administration group (PMT sulfate), with 6 rats in each group. All rats were fasted for 12 hours before the experiment. The intravenous injection group received a 5 mg / kg intravenous injection of PMT stock. Prior to administration, the PMT stock was dissolved in DMSO, Tween-80, and then saline at a ratio of 0.5:0.5:9. The oral administration groups (PMT stock and sulfate) received a 60 mg / kg oral dose. Prior to administration, the PMT stock or its sulfate was dissolved in DMSO, Tween-80, and then saline at a ratio of 0.5:1:8.5.

[0066] From the intravenous injection group, 0.5 mL of blood was collected from the orbital cavity before administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration. The blood was centrifuged at 5000 rpm for 10 minutes to obtain plasma samples, which were then stored at -20°C until use. From the oral drug group and the sulfate oral group, 0.5 mL of blood was collected from the orbital cavity before administration and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours after administration. The blood was centrifuged at 5000 rpm for 10 minutes to obtain plasma samples, which were then stored at -20°C until use.

[0067] The plasma concentrations of the above plasma samples were detected using the established LC-MS / MS method. The measured plasma concentration-time curves were fitted using PK Solver software. The pharmacokinetic parameters of each animal were analyzed using a non-compartmental model, and the oral absolute and relative bioavailability of PMT was calculated according to the following formula.

[0068] F abs. =(AUC extravascular / (AUC i.v )×(dose i.v. / dose extravascular )×100%;

[0069] F rel =(AUC salt / (AUC extravascular )×(dose extravascular. / dose salt )×100%.

[0070] According to the established LC-MS / MS method, data collection of plasma samples at different time points after administration to rats was completed, and the blood drug concentrations of different administration routes were calculated. The average blood drug concentration-time curve is shown in the attached Figure 7 .

[0071] The blood concentration time data is analyzed by using a non-compartment model with PK Solver software to calculate the main pharmacokinetic parameters of PMT and its sulfate, and the results are shown in Table 3. According to the formula Fabs=(AUCT·Div) / (AUCiv·DT)×100%, the absolute bioavailability of PMT and its sulfate is calculated to be 0.57% and 5.92%, respectively. The above studies show that the PMT sulfate provided by the present application is superior to the original drug PMT in terms of drug metabolism parameters and oral bioavailability, and the bioavailability is increased by 10.39 times.

[0072] Table 3 Average drug metabolism parameters of PMT in rat plasma (n=6, mean ± SD).

[0073]

[0074] F=(AUCT·Div) / (AUCiv·DT)×100%

[0075] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a sulfate of a pleuromutilin compound, characterized in that: 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)thioacetyl] tert-butyl ester having the following structure Dissolve the compound with sulfuric acid in an organic solvent, heat and stir to react, cool to room temperature, and allow to stand to obtain a solid, which is the sulfate salt of 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)thioacetyl] methoxazole; The ratio of the amount of 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)mercaptoacetyl] emtholin, sulfuric acid, and organic solvent is as follows: for every 5.6 g of 14-O-[(4-(pyrrolidin-1-yl)-6-methylpyridin-2-yl)mercaptoacetyl] emtholin, add 40-50 mL of a 29.4 mg / mL sulfuric acid aqueous solution and 80-150 mL of the organic solvent; The organic solvent is one of acetone, ethanol and acetonitrile.

2. The method for preparing the sulfate of pleuromutilin compounds according to claim 1, characterized in that: The heating temperature is 50-70° C.; the reaction time is 0.5-3 hours; and the standing time is 1-3 days.

3. The sulfate of pleuromutilin compounds obtained according to the preparation method of claim 1 or 2.

4. Use of the sulfate salt of the pleuromutilin compound according to claim 3 in the preparation of an oral medicament for treating bacterial or mycoplasma infections in humans or animals.

5. The use according to claim 4, characterized in that The bacteria are Staphylococcus aureus, methicillin-resistant Epidermidis, and Streptococcus agalactiae; the mycoplasmas are Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma hyorhinis, Mycoplasma hyopneumoniae, and Mycoplasma bovis.

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