Pleuromutilin derivative with natural amino acid amide phenyl derivative side chain as well as preparation method and application of pleuromutilin derivative

Abstract

The invention belongs to the field of medicinal chemistry, and discloses a pleuromutilin derivative with a natural amino acid amide phenyl derivative side chain as well as a preparation method and application of the pleuromutilin derivative. The derivative is a compound with a structure as shown in a formula 2 or a pharmaceutically acceptable salt thereof, or a solvent compound of the compound with the structure as shown in the formula 2 or the pharmaceutically acceptable salt thereof. The pleuromutilin derivative has good antibacterial and anti-mycoplasma activity and low toxicity, and is especially suitable for being used as a novel antibacterial agent for systemic system infection of animals or human beings. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain, and a preparation method and application thereof. Background Art

[0002] Mycoplasma is a prokaryotic microorganism similar to bacteria but without cell walls discovered by Nocard et al. in 1898. It can grow and reproduce on inanimate artificial culture medium, with a diameter of 50-300nm and can pass through bacterial filters. It is the smallest and simplest prokaryotic organism discovered so far. The number of genes is 480. The only visible organelle in mycoplasma cells is the ribosome. Among human pathogenic mycoplasmas, Mycoplasma pneumoniae causes pneumonia, Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium mainly cause urogenital tract infections; animal pathogenic mycoplasmas often cause respiratory infections in livestock and poultry in veterinary clinics. Because it has no cell wall, antibiotics such as penicillin and cephalosporin that act on the bacterial cell wall are ineffective against it. In human clinical medicine, macrolide antibiotics such as erythromycin, roxithromycin and azithromycin are the first choice for the treatment of mycoplasma infections. Fluoroquinolones and tetracyclines are also used to treat Mycoplasma pneumonia. In veterinary clinics, in addition to the above-mentioned drugs, pleuromutilin drugs such as tylosin and valnemulin are also commonly used to treat mycoplasma infections such as respiratory diseases in livestock and poultry caused by mycoplasma infections.

[0003] As one of the major pathogens with high morbidity and mortality, multidrug-resistant Staphylococcus aureus (MRSA) often causes healthcare-associated infections, posing a serious threat to human health worldwide. Therefore, there is an urgent need to develop new antibiotics with new modes of action for the treatment of infections caused by multidrug-resistant Staphylococcus aureus (MRSA).

[0004] Pleuromutilin (Formula 1) is a natural tricyclic diterpenoid compound produced by culturing Pleurotus mutilus and Pleurotus Passeckerianus. It has a structure including a 5-6-8 tricyclic carbon skeleton with eight stereocenters and a glycolic acid chain at C(14).

[0005]

[0006] Studies have shown that pleuromutilin and its derivatives exhibit effective antibacterial activity against Gram-positive bacteria. Unlike other antibacterial drugs widely used in clinical practice, pleuromutilin and its derivatives can inhibit bacterial protein synthesis by binding to the V region of the peptidyl transferase center (PTC) of the 23s RNA of the bacterial 50s ribosomal subunit. Due to the unique mechanism of action, pleuromutilin and its derivatives have a low incidence of cross-resistance with other antibacterial agents and a low tendency for bacteria to develop resistance to bacteria. At the same time, they basically do not interact with mammalian cell ribosomes and do not interfere with protein synthesis in eukaryotic cells. What caught the attention of researchers is that the side chain on C14 in the pleuromutilin structural molecule can penetrate deep into the hydrophobic group of the ribosomal subunit and improve its antibacterial activity. Therefore, chemical modification of the C14 side chain has always been an important means to improve the drugability of pleuromutilin derivatives.

[0007] So far, by modifying its C14 side chain, four antibacterial drugs have been successfully launched on the market, including the veterinary antibacterial drugs Tiamulin and Valnemulin, the human skin topical drug Retapamulin, and the human drug Lefamulin, which was approved by the US FDA in 2019 for the treatment of community-acquired bacterial pneumonia (CABP).

[0008] Compared with the dozens of drugs successfully developed based on the same parent core, such as penicillin, cephalosporin, and xacin, only four antibacterial drugs have been successfully developed for pleuromutilin, and drug-resistant bacteria against pleuromutilin antibacterial drugs are still rare. Therefore, it is necessary to develop more pleuromutilin antibacterial drugs. Summary of the invention

[0009] In order to overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide a pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain, which has good antibacterial and antimycoplasma activity and is particularly suitable as a new type of antibacterial drug for systemic infections in animals or humans.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain.

[0011] Another object of the present invention is to provide the application of the above-mentioned pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] A pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain, wherein the derivative is a compound having a structure shown in Formula 2 or a pharmaceutically acceptable salt thereof, or a solvent compound of a compound having a structure shown in Formula 2 or a pharmaceutically acceptable salt thereof:

[0014]

[0015] Wherein R1 is hydrogen or C 1-4 Alkyl, preferably hydrogen;

[0016] X is O, S or NR5, R5 is hydrogen or C 1-4 Alkyl, preferably O;

[0017] R2 is hydrogen, C 1-8 Alkyl or its derivatives, preferably the side chain group of amino acid, including hydrogen (i.e. the side chain group of glycine), methyl (i.e. the side chain group of alanine), isopropyl (i.e. the side chain group of valine), sec-butyl (i.e. the side chain group of isoleucine); C directly connected to R2 has chirality, and its optical isomer configuration is D-type or L-type, preferably L-type;

[0018] R3 is hydrogen, alkyl or amino acid;

[0019] The R2 and R3 can be connected and closed to form a ring to form a tetrahydropyrrol-2-yl group (i.e., the side chain of proline);

[0020] The specific groups of the compounds of the above preferred structures are summarized in Table 1:

[0021] Table 1 Compound numbers and specific groups

[0022]

[0023] The pharmaceutically acceptable salt is a salt formed by the compound of formula 2 and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid;

[0024] The pharmaceutically acceptable salt preferably has the following structural formula:

[0025]

[0026] The preparation method of the pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain comprises the following steps:

[0027] (1) Add pleuromutilin and p-toluenesulfonyl chloride to solvent 1 and mix, add triethylamine dropwise while stirring, react at room temperature for 3 to 4 hours, add 2-aminothiophenol dropwise, react at room temperature for 3 to 4 hours, add water dropwise, filter, wash, and dry, and then add the obtained crude product to solvent 2 for pulping purification, filter, wash, and dry to obtain an intermediate I with a structure shown in formula 3;

[0028]

[0029] (2) adding the intermediate I obtained in step (1), the R4 group donor and the catalyst to the solvent 3, stirring at room temperature for 4 h to 12 h to carry out the amidation reaction, and extracting, drying and purifying the obtained reaction product to obtain the intermediate II with the structure shown in formula 4;

[0030]

[0031] The R4 group donor is any one of Boc-Gly-OH, Boc-Ala-OH, Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH.

[0032] (3) The synthesis of pleuromutilin derivatives includes the following four approaches:

[0033] Way 1:

[0034] Add intermediate II to solvent 4, stir at room temperature for 2 to 5 hours, then add solvent 5 to precipitate solid, stir at room temperature for 1 to 2 hours, filter, wash and dry to obtain a pleuromutilin derivative with a structure shown in formula 2;

[0035] Way 2:

[0036] The intermediate II is added to the solvent 6, stirred at room temperature for 12 hours, a 50% by mass concentration of NaOH aqueous solution is added to adjust the pH value, extracted, dried, and concentrated to obtain a pleuromutilin derivative with a structure shown in Formula 2;

[0037] Path 3:

[0038] Add intermediate II and p-toluenesulfonic acid to solvent 7, stir at room temperature for 1 to 3 hours, add alkaline solution to adjust the pH value, extract, dry and concentrate to obtain a pleuromutilin derivative with a structure shown in Formula 2;

[0039] Path 4:

[0040] The intermediate II is added to the solvent 8, and stirred at room temperature for 0.5 to 3 hours. The resulting reaction product is concentrated under vacuum to obtain a pleuromutilin derivative having a structure as shown in Formula 2;

[0041] The molar volume ratio of pleuromutilin, p-toluenesulfonyl chloride, 2-aminothiophenol, solvent 1, water and triethylamine in step (1) is 1mmol: 1-10mmol: 1.2-10mmol: 5-20ml: 50-80ml: 1.5-10mmol; the mass volume ratio of the crude product to solvent 2 is 1g: 5-10ml;

[0042] The solvent 1 is any one of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and 1,4-dioxane; the most preferred solvent is acetonitrile;

[0043] The solvent 2 is any one of methyl tert-butyl ether, tetrahydrofuran, isopropyl ether and diethyl ether; the most preferred is methyl tert-butyl ether;

[0044] The step of adding water dropwise, filtering, washing and drying is as follows: adding water dropwise to the reaction solution, precipitating solid, filtering, washing with water for three times and drying under vacuum.

[0045] The pulping purification, filtration, washing and drying are specifically as follows: the crude product is added to methyl tert-butyl ether and stirred at room temperature for 1 to 2 hours, filtered, washed with methyl tert-butyl ether for 3 times, and dried under vacuum.

[0046] The molar volume ratio of the intermediate I, R4 group donor, catalyst and solvent 3 in step (2) is 1-3 mmol: 1-6 mmol: 1-10 mmol: 3-10 ml;

[0047] The solvent 3 is any one of dichloromethane, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and N,N-dimethylacetamide; the most preferred solvent is dichloromethane;

[0048] The catalyst is any one of a mixture of DIPEA and HATU / HBTU / HCTU, a mixture of DIPEA / triethylamine / N-methylmorpholine, EDCI / DCC / DIC and HOBT / HOAT, a mixture of DIPEA and PyAop / PyBop, a mixture of NMI and TCFH, and a mixture of phosphorus oxychloride and pyridine; the most preferred is a mixture of DIPEA and HATU in a molar ratio of 2-12 mmol:1.5-9 mmol;

[0049] The extraction is performed using dichloromethane, the drying is performed using anhydrous sodium sulfate, and the purification is performed using column chromatography.

[0050] Step (3) The mass volume ratio of the intermediate I, solvent 4 and solvent 5 described in route 1 is 1 g: 2-5 ml: 20-50 ml;

[0051] The solvent 4 is a mixture of trifluoroacetic acid, thioanisole, phenol, ethanedithiol and water in a volume ratio of 35:2:1:1:1, a mixture of dichloromethane and trifluoroacetic acid in a volume ratio of 3:1, or a mixture of trifluoroacetic acid, triethylsilane and water in a volume ratio of 38:1:1;

[0052] The solvent 5 is any one of petroleum ether, isopropyl ether, ethyl ether, and methyl tert-butyl ether;

[0053] The washing is performed using solvent 5, and the drying is performed under vacuum;

[0054] Step (3) The mass volume ratio of the intermediate II and the solvent 6 described in the second route is 1 g: 5-10 ml;

[0055] The solvent 6 is a mixture of 85% by mass percent phosphoric acid aqueous solution and acetonitrile, wherein the volume ratio of the phosphoric acid aqueous solution to the acetonitrile is 1-3 ml:1 ml;

[0056] The pH value is adjusted to 8-10; the extraction is performed by using ethyl acetate and deionized water; the drying is performed by using anhydrous sodium sulfate; and the drying is concentrated and dried under vacuum conditions.

[0057] Step (3) The molar volume ratio of the intermediate II, p-toluenesulfonic acid, and solvent 7 described in route 3 is 1 mmol: 2-20 mmol: 3-10 ml;

[0058] The solvent 7 is any one of dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, and toluene; the most preferred solvent is ethyl acetate;

[0059] The alkaline solution is any one of KOH, NaOH, K2CO3, Na2CO3, CsOH, and Cs2CO3 aqueous solutions;

[0060] The pH value is adjusted to 8-10; the extraction is performed by using ethyl acetate and water; the drying is performed by using anhydrous sodium sulfate; and the drying is concentrated and dried under vacuum conditions.

[0061] Step (3) The mass volume ratio of the intermediate II and the solvent 8 in the fourth pathway is 1 g: 2-10 ml;

[0062] The solvent 8 is any one of hydrochloric acid isopropanol organic solvent, hydrochloric acid methanol organic solvent, hydrochloric acid ethanol organic solvent, hydrochloric acid ethyl acetate organic solvent, hydrochloric acid 1,4-dioxane organic solvent; the most preferred is hydrochloric acid isopropanol organic solvent;

[0063] Application of the above-mentioned pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain in the preparation of products for treating drug-resistant Staphylococcus aureus, multidrug-resistant bacteria and mycoplasma infectious diseases.

[0064] The medicament may contain one or more pharmaceutically acceptable carriers, excipients or diluents;

[0065] The drug preparations include a variety of clinical drug dosage forms, such as tablets, injections, liposome nanoparticles, controlled release agents, etc.

[0066] An antibiotic drug, comprising a therapeutically effective amount of a pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain, and the remainder being pharmaceutical excipients or other compatible drugs;

[0067] The pharmaceutical excipients refer to conventional pharmaceutical excipients, such as solvents, disintegrants, flavoring agents, preservatives, colorants and adhesives;

[0068] The other compatible drugs refer to the effective dose of pleuromutilin derivatives having natural amino acid amide phenyl derivative side chains as drug raw materials, and then combined with other natural drugs or chemical drugs;

[0069] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0070] (1) The pleuromutilin derivatives provided by the present invention are new types of compounds that have never been reported.

[0071] (2) After extensive and in-depth research, the present invention has synthesized a large number of pleuromutilin derivatives with new structures and natural amino acid amide phenyl derivative side chains, and carried out extensive in vitro antibacterial and antimycoplasma activity screening. It is the first time to find that this type of compound not only has good in vitro antibacterial activity, but also has antimycoplasma activity, and also has the advantage of lower preparation cost than valnemulin and retapamulin. Therefore, it is particularly suitable as a new type of antibacterial drug for the prevention and treatment of bacterial or mycoplasma infectious diseases in humans or animals.

[0072] (3) Compared with some compounds reported in patent CN115850137A and most compounds reported in patent CN104803911B (most compounds of the invention have a solubility in pure water between 20 and 80 mg / ml), the truncated pleuromutilin derivatives having a natural amino acid amide phenyl derivative side chain prepared by the present invention have higher solubility in pure water, which is conducive to better absorption of the compounds in animals and has the potential to be developed into anti-infective drugs.

[0073] (4) The truncated pleuromutilin derivatives having a natural amino acid amide phenyl derivative side chain prepared in the present invention have a better inhibitory effect on the growth of human Mycoplasma pneumoniae ATCC15531 than some compounds reported in patent CN115850137A, and have the potential to be developed into drugs against Mycoplasma pneumoniae infection.

[0074] (5) Compared with some compounds reported in patent CN115850137A, the truncated pleuromutilin derivatives having natural amino acid amide phenyl derivative side chains prepared by the present invention have lower cytotoxicity to 16-HBE cells, lower toxicity to animal cells, better safety, and are more conducive to drug development.

[0075] (6) The predicted clogp value of the pleuromutilin derivatives having a natural amino acid amide phenyl derivative side chain prepared by the present invention is closer to 5 than that of some compounds reported in patent CN115850137A, so the compounds of the present invention are more druggable. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 This is the NMR spectrum of compound 1.

[0077] Figure 2 This is the H NMR spectrum of compound 2.

[0078] Figure 3 This is the H NMR spectrum of compound 3.

[0079] Figure 4 This is the H NMR spectrum of compound 4.

[0080] Figure 5 This is the H NMR spectrum of compound 5.

[0081] Figure 6 This is the H NMR spectrum of compound 6.

[0082] Figure 7 This is the H NMR spectrum of compound 7.

[0083] Figure 8 This is the H NMR spectrum of compound 8.

[0084] Fig. 9 This is the H NMR spectrum of compound 9.

[0085] Fig.10 This is the H NMR spectrum of compound 10.

[0086] Fig.11 This is the H NMR spectrum of compound 11.

[0087] Fig.12 This is the H NMR spectrum of compound 12.

[0088] Fig.13 This is the H NMR spectrum of compound 13.

[0089] Fig.14 It is the dissolved state of compound 1-13 in pure water.

[0090] Fig.15 This is the result of the effect of compounds 1-13 on the viability of 16-HBE cells. DETAILED DESCRIPTION

[0091] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0092] Example 1

[0093] (1) Preparation of intermediate I: 10 g of pleuromutilin and 5.27 g (27.73 mmol) of p-toluenesulfonyl chloride were dissolved in 50 ml of acetonitrile, and 6.69 g (66.10 mmol) of triethylamine was added dropwise under stirring in an ice bath. After the addition was completed, the mixture was stirred at room temperature for 3 h. Then, 3.97 g (31.75 mmol) of 2-aminobenzenethiol was added dropwise under stirring at room temperature. After the addition was completed, the mixture was stirred at room temperature for 12 h. Then, 250 ml of deionized water was added dropwise to precipitate a solid. The solid was stirred for 30 min, filtered, and washed with 250 ml of deionized water. The crude product was dried under vacuum. The crude product was added to 50 ml of methyl tert-butyl ether, stirred at room temperature for 2 h, filtered, washed with 50 ml of methyl tert-butyl ether, and dried to obtain the following:

[0094] Intermediate I of the structure shown in formula 3, yield: 95%;

[0095]

[0096] (2) Preparation of intermediate II: 4.94 mmol of R4 donor was dissolved in 10 ml of dichloromethane, and 1.28 g (9.88 mmol) of DIPEA, 2.81 g (7.41 mmol) of HATU and 1.2 g (2.47 mmol) of

[0097] The intermediate I prepared in Example 1 was reacted at room temperature for 12 hours under stirring, and 50 ml of dichloromethane and 100 ml of deionized water were added, and then transferred to a separatory funnel for shaking, and allowed to stand for stratification; the organic phase was taken and washed again with 100 ml of deionized water; dried over anhydrous sodium sulfate, purified by column chromatography (petroleum ether: ethyl acetate = 2:1 ~ dichloromethane: methanol = 10:1), and concentrated to obtain intermediates II-1 to II-5 having structures shown in formula (4);

[0098] Wherein, in the above reaction process, the R4 group donor is Boc-Gly-OH, Boc-Ala-OH, Boc-Pro-OH, Boc-Ile-OH, Boc-Val-OH;

[0099]

[0100] In the obtained intermediate II-1, R4 is N-tert-butoxycarbonyl-aminomethyl, in the intermediate II-2, R4 is (R)-N-tert-butoxycarbonyl-1-aminoethyl, in the intermediate II-3, R4 is (R)-N-tert-butoxycarbonyl-tetrahydropyrrol-2-yl, in the intermediate II-4, R4 is (R)-N-tert-butoxycarbonyl-1-amino-2-methylpropyl, and in the intermediate II-5, R4 is (1R, 2S)-N-tert-butoxycarbonyl-1-amino-2-methylbutyl.

[0101] Example 2

[0102] 1. Preparation of pleuromutilin derivatives 1 to 5

[0103] Path 1

[0104] 10 mmol of intermediates II-1 to II-5 were added to 1 mL of solvent (trifluoroacetic acid: thioanisole: phenol: ethanedithiol: water = 35:2:1:1:1), respectively, stirred at room temperature for 3 h, and 5 mL of isopropyl ether was added; a solid was precipitated, stirred at room temperature for 1 h, filtered, and washed with 10 mL of isopropyl ether. The product was dried under vacuum to obtain pleuromutilin derivatives 1 to 5 (i.e., compounds 1 to 5 shown in Table 1);

[0105] Path 2

[0106] 1 mmol of intermediate II-1 to 5 and 4.9 g (50 mmol) of 85% phosphoric acid aqueous solution were added to 2 mL of acetonitrile, stirred at room temperature for 12 h, 50% sodium hydroxide aqueous solution was added until the solution pH = 8 to 9, 50 mL of ethyl acetate and 100 mL of deionized water were added, and then transferred to a separatory funnel for shaking, and allowed to stand for stratification; the organic phase was taken and washed again with 100 mL of deionized water; dried over anhydrous sodium sulfate, and the product was concentrated under vacuum to obtain pleuromutilin derivatives 1 to 5 (i.e., compounds 1 to 5 shown in Table 1);

[0107] Path 3

[0108] 1 mmol of intermediate II-1 to 5 and 5 mmol of p-toluenesulfonic acid were added to 2 mL of ethyl acetate, stirred at room temperature for 2 h, saturated sodium carbonate aqueous solution was added until the solution pH = 8 to 9, 50 mL of ethyl acetate and 100 mL of deionized water were added, and then transferred to a separatory funnel and shaken, and allowed to stand for stratification; dried over anhydrous sodium sulfate, and the product was concentrated under vacuum to obtain pleuromutilin derivatives 1 to 5 (i.e., compounds 1 to 5 shown in Table 1).

[0109] Path 4

[0110] 1 g of intermediate II-1-5 was added to 5 mL of hydrochloric acid isopropanol organic solvent, stirred at room temperature for 1 h, and the product was concentrated under vacuum to obtain pleuromutilin derivatives 1-5 (i.e., compounds 1-5 shown in Table 1). The NMR spectrum of compound 1-5 is shown in Figure 1-5 As shown;

[0111] The above-mentioned preparation synthetic route is shown in the following formula:

[0112]

[0113] The yields of the above compounds are summarized in Table 2.

[0114] Table 2 Compound numbers and yields

[0115]

[0116]

[0117] Comparative Example 1

[0118] According to the method disclosed in the Chinese patent No. 202211415953.5, some of the compounds disclosed therein were prepared, including compounds 6 to 13 of the following structural formulas, and the structural characterization data are as follows Figure 6-13 As shown; the prepared compound was used as a control compound in subsequent experiments:

[0119]

[0120] Effect Example

[0121] (1) In vitro antibacterial test

[0122] The experiment used the broth dilution method. The experimental control drugs were tylosin, valnemulin, retapamulin, and vancomycin.

[0123] The strains used in the experiment were methicillin-resistant Staphylococcus aureus ATCC43300 and Staphylococcus aureus ATCC29213, clinical Staphylococcus aureus AD3 and clinical Staphylococcus aureus 144 (clinical bacteria AD3 and 144 were isolated and identified in the Pharmacology Laboratory of the School of Veterinary Medicine of South China Agricultural University, and have been published in the literature Zhe ZA, Kang LA, Gyz A, et al. Design, synthesis and biological activities of novel pleuromutilin derivatives with a substituted triazole moiety as potent antibacterial agents-Science Direct [J]. European Journal of Medicinal Chemistry, 2020.).

[0124] Preparation of target compound stock solution: 6.4 mg of target compound (compounds 1 to 5 prepared in Example 2) were accurately weighed and placed in a 10 mL volumetric flask, dissolved with 0.25 mL DMSO, added with 9.5 mL distilled water, 0.25 mL Tween 80 to volume, and shaken well to obtain a stock solution (6.4 mg / mL), sterilized with a 0.22 μm filter membrane, divided into small tubes, and stored at -20°C. The control drug tylosin was also prepared according to the above method.

[0125] Preparation of bacterial solution: Take out the strains that are well preserved at -20℃ and inoculate them on new MH plates. After culturing at 37℃ for 24 hours, pick out a single colony and inoculate it in MH medium for another 24 hours. Select a single colony and transfer it to sterile physiological saline and adjust its turbidity to 0.6McF. At this time, the concentration of bacterial solution is 10 6 CFU / mL.

[0126] MIC plate preparation: dilute the target compound stock solution (6.4 mg / mL) 10 times to obtain a target compound solution with a concentration of 640 μg / mL; take a sterile 96-well plate, add 180 μL MH broth medium to the first well, add 100 μL MH broth medium to the second to tenth wells, add 20 μL of antibacterial drugs with a concentration of 640 μg / mL to the first well, mix well, take 100 μL and add it to the second well, mix well, and then draw 100 μL to the third well, and so on, draw 100 μL from the 12th well and discard it. At this time, the drug concentrations of each well are: 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03 μg / mL, and three groups of parallel drugs are made for each concentration.

[0127] Inoculation: Add 100 μL of bacterial solution to each well from 1 to 12, so that the final bacterial concentration in each well is about 5×10 5 CFU / mL, the drug concentrations in wells 1 to 12 were 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, and 0.015 μg / mL, respectively. The inoculated 96-well plate was placed in a 37°C incubator for culture, and the growth of the bacterial solution was observed for 24 hours. The control drug tylosin was determined in the same way, and the lowest drug concentration that completely inhibited bacterial growth in the small well was MIC. The bacteria in the positive control well (i.e., without drug) needed to grow significantly. When a single jump hole appeared in the microbroth dilution method, the highest drug concentration that inhibited the bacteria was recorded. If multiple jump holes appeared, the test needed to be repeated.

[0128] Table 3 shows the MIC results, which shows that the target compound has good antibacterial activity against the selected strains and has good inhibitory activity against drug-resistant Staphylococcus aureus. It is particularly suitable as a new antibacterial drug for the prevention and treatment of infectious diseases caused by human or animal or drug-resistant Staphylococcus aureus or multidrug-resistant bacteria.

[0129] Table 3 In vitro antibacterial data

[0130]

[0131]

[0132] (2) In vitro mycoplasma activity inhibition test

[0133] The experiment used the agar double dilution method to determine the minimum inhibitory concentration (MIC) of some of the series of compounds obtained in Example 2 of the present invention.

[0134] The mycoplasma strains selected in the experiment were Mycoplasma pneumoniae ATCC15531, Mycoplasma gallinarum MS, Mycoplasma gallinarum CH13, Mycoplasma gallinarum CH18, Mycoplasma gallinarum CH19, and Mycoplasma gallinarum CH20.

[0135] Preparation of test compound stock solution: All test compounds (compounds 1-5 obtained in Example 2, control drug tylosin, control compounds 6-13 obtained in Example 3) were dissolved in 1 mL of DMF, and then diluted to an initial concentration of 5120 μg / mL in a 250 mL volumetric flask as a stock solution. The stock solution was diluted in a culture dish by a doubling dilution method, each culture dish containing 2 mL of the drug solution, and diluted to 20 mL with melted MH medium, so that the final concentrations of the test compounds in the series of culture dishes were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.0152 μg / mL, respectively.

[0136] Arrange 15 sterile 2mL EP tubes per row, add 0.9mL of liquid culture medium to the first tube and 0.5mL to the remaining tubes, take 0.1mL of the prepared liquid and add it to the first tube and mix thoroughly, then take 0.5mL and add it to the second tube, and so on. Discard the last 0.5mL, then add 0.5mL of diluted bacterial solution to each tube, cover the lid, make a blank control, and culture at 37℃ to observe the growth of mycoplasma.

[0137] Tables 4 and 5 show the MIC results of anti-mycoplasma activity (unit: μg / mL).

[0138] Table 4 In vitro antibacterial data

[0139]

[0140] Table 5 In vitro antibacterial data

[0141]

[0142] The data in Table 4 show that the compounds of the present invention have better inhibitory effects on the growth of human Mycoplasma pneumoniae ATCC15531 than some compounds disclosed in Chinese Patent No. 202211415953.5, and have the potential to be developed into drugs against Mycoplasma pneumoniae infection.

[0143] (3) Determination of the solubility of the preferred salt of pleuromutilin derivatives in pure water

[0144] Weigh 1 mg of sample (compounds 1-5 obtained in Example 2, control compounds 6-13 obtained in Example 3) into a 2 ml EP tube, add 1 ml of pure water, and sonicate for 30 min. If the solution is clear, repeat the above steps until the solution becomes turbid. The experimental results are shown in Table 6. The dissolution state of the compound is as follows Fig.14 shown.

[0145] Table 6 Solubility of pleuromutilin derivatives in pure water

[0146]

[0147]

[0148] The data in the above table show that the salts of the preferred compounds of the present invention have higher solubility in pure water than some compounds disclosed in Chinese Patent No. 202211415953.5 and most of the compounds reported in the patent application disclosed in Chinese Patent No. 201410032730.X (most of the compounds of the invention have a solubility in pure water between 20 and 80 mg / ml), and have greater potential for development as injectable anti-infective drugs.

[0149] (4) Cytotoxicity assay

[0150] The experiment used the MTT method.

[0151] The cell line used in the experiment was human bronchial epithelial cell line (16-HBE).

[0152] Preparation of target compound stock solution: 8 mg of target compound (compounds 1-5 obtained in Example 2) was accurately weighed, dissolved in 1 mL of DMSO, and shaken thoroughly to obtain a stock solution (8 mg / mL), sterilized with a 0.22 μm filter membrane, and stored at -20° C. Control compounds 6-13 were also prepared according to the above method.

[0153] MTT incubation and OD value determination: Collect cells in good growth state and add 100 μL of each well of a 96-well plate, about 5×10 5 Cells with a density of 100 / mL were plated. The 96-well plate was placed in a constant temperature incubator with 5% CO2 overnight. The complete culture medium was discarded, and the test compound was added with a final concentration of 8μg / mL (dissolved in DMSO to prepare 5mg / mL stock solution), and it was placed in a constant temperature incubator with 5% CO2 for incubation for 24h. The supernatant was discarded, and freshly prepared MTT with a concentration of 0.5mg / mL (dissolved in DMEM to prepare 5mg / mL stock solution, stored in aliquots at -20℃) was added and incubated in the dark for 2h. The supernatant was discarded, 150μL DMSO was added, and it was placed in an oscillator at room temperature and oscillated at 70r / min for 10min. The absorbance value was measured at 490nm with an enzyme reader, and the cell survival rate was calculated. The experiment was repeated three times independently. The formula is as follows:

[0154] Cell survival rate (%) = OD drug group / OD control group × 100%

[0155] The toxicity test results of the compounds are as follows Fig.15 As shown, compounds 1 to 5 have lower toxicity to the selected cell lines at 8 μg / mL than some compounds disclosed in Chinese patent No. 202211415953.5, and have a certain growth-promoting effect, and are suitable as new antibacterial drugs for the prevention and treatment of infectious diseases caused by humans or animals or drug-resistant Staphylococcus aureus or multidrug-resistant bacteria.

[0156] (5) Prediction of lipid-water partition coefficient of compounds

[0157] Chem3D was used to predict the clogP value of each compound. The experimental results are shown in Table 7

[0158] Table 7 clogP values ​​of pleuromutilin derivatives

[0159]

[0160] The data in the above table show that the predicted clogP values ​​of most compounds of the present invention are closer to 5 than those of some compounds disclosed in Chinese patent No. 202211415953.5, so the compounds of the present invention are more drugable.

[0161] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain, characterized in that: The derivative is a compound of the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof, or a solvent compound of the compound of the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof: Wherein R1 is hydrogen or C 1-4 alkyl; X is O, S or NR5, R5 is hydrogen or C 1-4 alkyl; R2 is hydrogen, C 1-8 Alkyl or its derivatives; C directly connected to R2 is chiral, and its optical isomer configuration is D-type or L-type; R3 is hydrogen, alkyl or amino acid.

2. The pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain according to claim 1, characterized in that: Said R1 is hydrogen; The X is O; R2 is a side chain group of an amino acid, including hydrogen, methyl, isopropyl or sec-butyl; The optical isomerization configuration of C directly connected to R2 is L-type; The R2 and R3 are connected and closed to form a ring to form a tetrahydropyrrol-2-yl group.

3. The pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound represented by formula (2) and hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, glutamic acid, aspartic acid, succinic acid, citric acid or malic acid.

4. The method for preparing a pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain according to any one of claims 1 to 3, characterized in that The following steps are included: (1) Add pleuromutilin and p-toluenesulfonyl chloride to solvent 1 and mix, add triethylamine dropwise while stirring, react at room temperature for 3 to 4 hours, add 2-aminothiophenol dropwise, react at room temperature for 3 to 4 hours, add water dropwise, filter, wash, and dry, and then add the obtained crude product to solvent 2 for pulping purification, filter, wash, and dry to obtain an intermediate I with a structure shown in formula 3; (2) adding the intermediate I obtained in step (1), the R4 group donor and the catalyst to the solvent 3, stirring at room temperature for 4 h to 12 h to carry out the amidation reaction, and extracting, drying and purifying the obtained reaction product to obtain the intermediate II with the structure shown in formula 4; The R4 group donor is any one of Boc-Gly-OH, Boc-Ala-OH, Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH; (3) The synthesis of pleuromutilin derivatives includes the following four approaches: Way 1: Add intermediate II to solvent 4, stir at room temperature for 2 to 5 hours, then add solvent 5 to precipitate solid, stir at room temperature for 1 to 2 hours, filter, wash and dry to obtain a pleuromutilin derivative with a structure shown in formula 2; Way 2: The intermediate II is added to the solvent 6, stirred at room temperature for 12 hours, a 50% by mass concentration of NaOH aqueous solution is added to adjust the pH value, extracted, dried, and concentrated to obtain a pleuromutilin derivative with a structure shown in Formula 2; Path 3: Add intermediate II and p-toluenesulfonic acid to solvent 7, stir at room temperature for 1 to 3 hours, add alkaline solution to adjust the pH value, extract, dry and concentrate to obtain a pleuromutilin derivative with a structure shown in Formula 2; Path 4: The intermediate II is added to the solvent 8, and the mixture is stirred at room temperature for 0.5 to 3 hours. The obtained reaction product is concentrated under vacuum to obtain a pleuromutilin derivative having a structure as shown in Formula 2.

5. The preparation method according to claim 4, characterized in that: The molar volume ratio of pleuromutilin, p-toluenesulfonyl chloride, 2-aminothiophenol, solvent 1, water and triethylamine in step (1) is 1mmol: 1-10mmol: 1.2-10mmol: 5-20ml: 50-80ml: 1.5-10mmol; the mass volume ratio of the crude product to solvent 2 is 1g: 5-10ml; The solvent 1 is any one of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and 1,4-dioxane; The solvent 2 is any one of methyl tert-butyl ether, tetrahydrofuran, isopropyl ether and diethyl ether; The step of adding water dropwise, filtering, washing and drying is as follows: adding water dropwise to the reaction solution, precipitating solid, filtering, washing with water for three times and drying under vacuum. The pulping purification, filtration, washing and drying are specifically as follows: the crude product is added to methyl tert-butyl ether and stirred at room temperature for 1 to 2 hours, filtered, washed with methyl tert-butyl ether for 3 times, and dried under vacuum.

6. The preparation method according to claim 4, characterized in that: The molar volume ratio of the intermediate I, R4 group donor, catalyst and solvent 3 in step (2) is 1-3 mmol: 1-6 mmol: 1-10 mmol: 3-10 ml; The solvent 3 is any one of dichloromethane, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and N,N-dimethylacetamide; The catalyst is any one of a mixture of DIPEA and HATU / HBTU / HCTU, a mixture of DIPEA / triethylamine / N-methylmorpholine, EDCI / DCC / DIC and HOBT / HOAT, a mixture of DIPEA and PyAop / PyBop, a mixture of NMI and TCFH, and a mixture of phosphorus oxychloride and pyridine; The extraction is performed using dichloromethane, the drying is performed using anhydrous sodium sulfate, and the purification is performed using column chromatography.

7. The preparation method according to claim 4, characterized in that: Step (3) The mass volume ratio of the intermediate II, solvent 4 and solvent 5 described in route 1 is 1 g: 2-5 ml: 20-50 ml; The solvent 4 is a mixture of trifluoroacetic acid, thioanisole, phenol, ethanedithiol and water in a volume ratio of 35:2:1:1:1, a mixture of dichloromethane and trifluoroacetic acid in a volume ratio of 3:1, or a mixture of trifluoroacetic acid, triethylsilane and water in a volume ratio of 38:1:1; The solvent 5 is any one of petroleum ether, isopropyl ether, ethyl ether, and methyl tert-butyl ether; The washing is performed using solvent 5, and the drying is performed under vacuum; Step (3) The mass volume ratio of the intermediate II and the solvent 6 described in the second route is 1 g: 5-10 ml; The solvent 6 is a mixture of 85% by mass percent phosphoric acid aqueous solution and acetonitrile, wherein the volume ratio of the phosphoric acid aqueous solution to the acetonitrile is 1-3 ml:1 ml; The pH value is adjusted to 8-10; the extraction is performed by using ethyl acetate and deionized water; the drying is performed by using anhydrous sodium sulfate; and the drying is concentrated and dried under vacuum conditions.

8. The preparation method according to claim 4, characterized in that: Step (3) The molar volume ratio of the intermediate II, p-toluenesulfonic acid, and solvent 7 described in route 3 is 1 mmol: 2-20 mmol: 3-10 ml; The solvent 7 is any one of dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, and toluene; The alkaline solution is any one of KOH, NaOH, K2CO3, Na2CO3, CsOH, and Cs2CO3 aqueous solutions; The pH value is adjusted to 8-10; the extraction is performed by using dichloromethane and water; the drying is performed by using anhydrous sodium sulfate; and the drying is concentrated and dried under vacuum conditions. Step (3) The mass volume ratio of the intermediate II and the solvent 8 in the fourth pathway is 1 g: 2-10 ml; The solvent 8 is any one of an organic solvent of isopropanol hydrochloride, an organic solvent of ethanol hydrochloride, an organic solvent of methanol hydrochloride, an organic solvent of ethyl acetate hydrochloride, and an organic solvent of 1,4-dioxane hydrochloride.

9. Use of the pleuromutilin derivative having a natural amino acid amide phenyl derivative side chain according to claim 1 in the preparation of a product for treating drug-resistant Staphylococcus aureus, multidrug-resistant bacteria and mycoplasma infectious diseases.

Images