Pleuromutilin derivative containing quinoxaline 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 containing a quinoxaline 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. The pleuromutilin derivative has good antibacterial 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 present invention belongs to the field of medicinal chemistry, and particularly relates to a pleuromutilin derivative with a quinoxaline side chain, a preparation method thereof, and an application thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) is a major human pathogen in community and healthcare settings and can cause life-threatening infections. Although the emergence of methicillin was a powerful treatment strategy against S. aureus infections, methicillin-resistant S. aureus (MRSA) has been discovered since 1961. With the sharp increase in its numbers, MRSA has become a global health crisis. To address this challenge, researchers have been working on developing new and highly effective antibiotics.

[0003] Pleuromutilin (Formula 1) is a natural tricyclic diterpenoid compound, initially isolated from Pleurotus mutilus and Pleurotus passeckerianus. Structurally, it consists of an eight-membered ring, a five-membered ring, a six-membered ring, and a glycolate side chain at the C14 position. Pleuromutilin derivatives generally have good activities against Gram-positive bacteria and mycoplasmas, and also show excellent activities in terms of anti-MRSA activity. Therefore, the development of pleuromutilin derivatives is an effective strategy to address the problem of the sharp increase in MRSA drug resistance.

[0004]

[0005] Research shows that, different from most other antibacterial drugs widely used clinically, pleuromutilin has a unique antibacterial mechanism. It can bind to the P and A sites in the peptidyl transferase center (PTC) V region of the 23S RNA of the bacterial 50S ribosomal subunit, thereby inhibiting bacterial protein synthesis. This unique antibacterial mechanism makes pleuromutilin less likely to develop drug resistance to MRSA and less likely to show cross-drug resistance with other antibiotics compared with other antibiotics. Moreover, pleuromutilin generally does not interact with mammalian cell ribosomes and does not interfere with protein synthesis in eukaryotic cells, having good bacterial targeting. The side chain at C14 in the pleuromutilin structural molecule is the site that affects the antibacterial activity of pleuromutilin. It can penetrate deep into the hydrophobic group inside the ribosomal subunit and form a tighter binding with the PTC, thereby enhancing its antibacterial activity. Therefore, modifying the C14 side chain is an important means to improve the activity of pleuromutilin derivatives. At the same time, compared with other sites, it has a higher feasibility of modification and more development prospects.

[0006] So far, among the antibacterial drugs successfully launched by modifying their C14 side chains, there are only four: the veterinary antibacterial drug tiamulin, valnemulin, the human topical skin 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).

[0007] Compared with dozens of drugs successfully developed based on the same parent nucleus, such as penicillins, cephalosporins, and quinolone antibacterial drugs, only four antibacterial drugs have been successfully developed for pleuromutilins, and there are not many drug-resistant bacteria against pleuromutilin antibacterial drugs. Therefore, it is very necessary to develop more pleuromutilin antibacterial drugs. Summary of the Invention

[0008] The primary object of the present invention is to provide a pleuromutilin derivative containing a quinoxaline side chain. Such pleuromutilin derivatives have good antibacterial activity and low toxicity and are expected to be used as novel antibacterial drugs for systemic infections in animals or humans.

[0009] Another object of the present invention is to provide a preparation method for the above-mentioned pleuromutilin derivative containing a quinoxaline side chain.

[0010] Another object of the present invention is to provide the application of the above-mentioned pleuromutilin derivative containing a quinoxaline side chain.

[0011] The object of the present invention is achieved by the following technical solutions:

[0012] A pleuromutilin derivative containing a quinoxaline side chain, wherein the derivative is a compound of the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof:

[0013]

[0014] Wherein, R is a hydrogen atom, a phenyl group, a 4-nitrophenyl group, a 3-methylphenyl group, a 3-hydroxyphenyl group, a 4-cyanophenyl group, a 4-formylphenyl group, a 4-pyridyl group, or a 5-pyrimidinyl group;

[0015] The specific groups of the compounds with the above preferred structures are summarized in Table 1:

[0016] Table 1 Compound numbers and specific groups

[0017]

[0018] The pharmaceutically acceptable salts are salts formed by the compound of the structure shown in 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;

[0019] The pharmaceutically acceptable salts preferably have the following structural formula:

[0020]

[0021] The preparation method of the pleuromutilin derivative containing a quinoxaline side chain comprises the following steps:

[0022] (1) React pleuromutilin with p-toluenesulfonyl chloride to obtain Intermediate I of the structure shown in Formula 3;

[0023] (2) Use o-phenylenediamine as a raw material and react it with ethyl glyoxylate to obtain Intermediate II of the structure shown in Formula 4;

[0024] (3) Dissolve Intermediate II prepared in step (2) in acetic acid and react it with Br2 to obtain Intermediate III of the structure shown in Formula 5;

[0025] (4) Dissolve Intermediate III prepared in step (3) in pyridine and react it with P2S5 to obtain Intermediate IV of the structure shown in Formula 6;

[0026] (5) Use Intermediate I prepared in step (1) as a raw material, further activate it with sodium iodide, and then react it with Intermediate IV prepared in step (4) to obtain Intermediate V of the structure shown in Formula 7;

[0027] (6) Dissolve Intermediate V prepared in step (5) in 1,4-dioxane containing water and react it with aromatic boric acid to obtain a pleuromutilin derivative containing a quinoxaline side chain of the structure shown in Formula 2, where R is phenyl, 4-nitrophenyl, 3-methylphenyl, 3-hydroxyphenyl, 4-cyanophenyl, 4-formylphenyl, 4-pyridyl or 5-pyrimidinyl;

[0028] The Intermediate I, II, III, IV, and V respectively have the structural formulas of Formulas 3 to 7:

[0029]

[0030] In step (1), the molar ratio of p-toluenesulfonyl chloride to pleuromutilin is 1.1:1;

[0031] In step (2), the molar ratio of o-phenylenediamine to ethyl glyoxylate is 1:1;

[0032] In step (3), the molar ratio of intermediate II to Br2 is 1:1.05;

[0033] In step (4), the molar ratio of intermediate III to P2S5 is 1:1;

[0034] The specific operation steps of step (5) are as follows:

[0035] Using acetonitrile as a solvent, first dissolve intermediate I, then add sodium iodide and a base, heat under reflux at 78 °C for 1 - 2 h, and then add intermediate IV and continue to react for 3 h to obtain intermediate V;

[0036] The amount of acetonitrile used is 30 - 40 times the mass of intermediate I, the molar ratio of the base to intermediate I is 2:1, and the molar amount of sodium iodide is 10% of the molar amount of the base;

[0037] The base is sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate or cesium carbonate.

[0038] The specific operation steps of step (6) are as follows:

[0039] Under nitrogen protection, dissolve intermediate V, aromatic boric acid, tris(dibenzylideneacetone)dipalladium (Pd2(dba)3), 2 - dicyclohexylphosphino - 2',4',6' - triisopropylbiphenyl (X - Phos), and a base in a solvent of 1,4 - dioxane containing water, and heat under reflux at 100 °C for 2 - 3 h;

[0040] The amount of the solvent used is preferably 30 - 40 times the mass of intermediate V, the molar ratio of the base to intermediate V is preferably 2:1, the molar ratio of tris(dibenzylideneacetone)dipalladium, 2 - dicyclohexylphosphino - 2',4',6' - triisopropylbiphenyl to intermediate V is preferably 1:1:10, and the molar ratio of the aromatic boric acid to intermediate V is preferably 3:2;

[0041] The base is sodium carbonate, potassium acetate, potassium carbonate or cesium carbonate.

[0042] The preparation method of the above - mentioned pleuromutilin derivative containing a quinoxaline side chain further includes the following operation steps:

[0043] (1) React pleuromutilin with p - toluenesulfonyl chloride to obtain intermediate I with the structure shown in formula 3;

[0044] (2) Use o - phenylenediamine as a raw material and react it with ethyl glyoxylate to obtain intermediate II with the structure shown in formula 4;

[0045] (3) Dissolve the intermediate II prepared in step (2) in pyridine and react it with P2S5 to obtain intermediate VI with the structure shown in formula 8;

[0046] (4) Using the intermediate I prepared in step (1) as a raw material, further activating it with sodium iodide, and then reacting it with the intermediate VI prepared in step (3) to obtain a pleuromutilin derivative containing a quinoxaline side chain with the structure shown in Formula 2, where R is H;

[0047] The said intermediate VI has the structural formula of Formula 8:

[0048]

[0049] Use of the above-mentioned pleuromutilin derivative containing a quinoxaline side chain in the preparation of an antibacterial product, where the antibacterial product is a drug for treating infectious diseases.

[0050] The said infectious diseases are infectious diseases caused by human or animal infection with drug-resistant Staphylococcus aureus or multi-drug resistant bacteria.

[0051] The said drug may contain one or more pharmaceutically acceptable carriers, excipients or diluents;

[0052] The preparations of the said drug include various clinical pharmaceutical dosage forms, such as tablets, injections, liposome nanoparticles, controlled-release agents, etc.;

[0053] An antibiotic drug contains an effective amount of a pleuromutilin derivative with a quinoxaline side chain, and the balance is a pharmaceutical excipient or other compatible drugs;

[0054] The said pharmaceutical excipient refers to conventional pharmaceutical excipients, such as solvents, disintegrants, flavoring agents, preservatives, coloring agents and binders, etc.;

[0055] The said other compatible drugs refer to using an effective dose of a pleuromutilin derivative with a quinoxaline side chain as a drug raw material and then formulating it with other natural drugs or chemical drugs;

[0056] The synthetic route of the above preparation method is shown in the following formula:

[0057]

[0058] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0059] (1) The pleuromutilin derivative provided by the present invention is a new type of compound that has not been reported before.

[0060] (2) Through extensive and in-depth research, the present invention synthesized a large number of pleuromutilin derivatives with a quinoxaline side chain and a completely new structure, and conducted extensive antibacterial activity screening. It was found that this type of compound not only has good in vitro antibacterial activity, but also has good ability to treat intracellular MRSA infections, and has the advantage of lower preparation cost compared with valnemulin and retapamulin. Therefore, it is particularly suitable as a new antibacterial drug for preventing and treating bacterial infectious diseases in humans or animals, especially infectious diseases caused by drug-resistant Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is the NMR spectrum diagram of Compound 1.

[0062] Figure 2 It is the NMR spectrum diagram of Compound 2.

[0063] Figure 3 It is the NMR spectrum diagram of Compound 3.

[0064] Figure 4 It is the NMR spectrum diagram of Compound 4.

[0065] Figure 5 It is the NMR spectrum diagram of Compound 5.

[0066] Figure 6 It is the NMR spectrum diagram of Compound 6.

[0067] Figure 7 It is the NMR spectrum diagram of Compound 7.

[0068] Figure 8 It is the NMR spectrum diagram of Compound 8.

[0069] Figure 9 It is the NMR spectrum diagram of Compound 9.

[0070] Figure 10 It is the cytotoxicity diagram of Compounds 1 - 9 against 16HBE cells at a concentration of 8 μg / ml.

[0071] Figure 11 It is the cytotoxicity diagram of Compounds 1 - 9 against A549 cells at a concentration of 8 μg / ml.

[0072] Figure 12 It is the cytotoxicity diagram of Compounds 1 - 9 against RAW 264.7 cells at a concentration of 8 μg / ml. DETAILED DESCRIPTION OF THE INVENTION

[0073] The present invention will be further described in detail below in conjunction with the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0074] In the embodiment, the R group is specifically shown in Table 1, and the raw materials and reagents are all commercially available.

[0075] Example 1

[0076] (1) Preparation of Intermediate I: 18.9 g (50 mmol) of pleuromutilin was dissolved in 20 ml of acetonitrile and placed in an ice bath; 11.5 g (60 mmol) of p-toluenesulfonyl chloride was dissolved in 10 ml of acetonitrile, and then slowly added to the above-mentioned pleuromutilin acetonitrile solution. Sodium hydroxide (4.0 g, 100.0 mmol) was dissolved in 15 ml of water and slowly added dropwise to this system. Next, the mixture was stirred at room temperature until the reaction was complete, and the acetonitrile was removed. The residue was suspended in dichloromethane and then washed with water. The organic layer was collected, dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude product. The crude product was recrystallized in 30 ml of ethanol, a large amount of white powder was precipitated, filtered by suction, and the filter residue was washed with isopropanol and dried to obtain Intermediate I with the structure shown in Formula 3, with a yield of 86%;

[0077]

[0078] (2) Preparation of Intermediate II: Ethyl glyoxylate (12.3 g, 120 mmol) was added dropwise to an ethanol solution containing o-phenylenediamine (10.8 g, 100 mmol) in 200 ml. The mixture was heated and maintained at 45 °C for 8 hours. The resulting precipitate was filtered, thoroughly washed with water, and dried under vacuum to obtain Intermediate II with the structure shown in Formula 4 with a yield of 80% (11.8 g, 81 mmol).

[0079]

[0080] (3) Preparation of Intermediate III: Intermediate II (10 g, 68 mmol) was added to 500 ml of acetic acid. After stirring for 5 minutes, liquid bromine (3.4 ml, 68 mmol) was added dropwise. The mixture was stirred overnight at room temperature in the dark. Subsequently, the reaction mixture was poured into ice water. The resulting precipitate was collected by filtration, thoroughly washed with water, and dried under vacuum to obtain Intermediate III with the structure shown in Formula 5 with a yield of 64% (9.8 g, 44 mmol).

[0081]

[0082] (4) Preparation of Intermediate Ⅳ: Under ice bath conditions, P2S5 (3 g, 14 mmol) was dissolved in 100 ml of pyridine, and then Intermediate III (2 g, 14 mmol) was added. The mixture was stirred for 2 minutes. Then the ice bath was removed, and the mixture was heated to 80 °C. After 6 hours, the reaction mixture was poured into ice water. The resulting precipitate was collected by filtration, washed thoroughly with water, and dried under vacuum to obtain Intermediate Ⅳ with the structure shown in Formula 6 in a yield of 79% (1.8 g, 11.1 mmol).

[0083]

[0084]

[0085] (5) Preparation of Intermediate Ⅴ: Take Intermediate Ⅰ (1.07 g, 2 mmol) and dissolve it in 40 ml of acetonitrile. Add anhydrous sodium iodide (0.6 g, 0.4 mmol) and anhydrous potassium carbonate (0.55 g, 4 mmol), and heat the reaction mixture under reflux at 78 °C for 2 h. Then add Intermediate Ⅳ (0.58 g, 2.4 mmol) to the above system and continue the reaction at 78 °C for 3 h. Pour the reaction solution into a separatory funnel, add 50 ml of distilled water and 50 ml of dichloromethane in sequence, shake well, and let it stand for layering. Take the organic phase and wash it twice with an aqueous sodium chloride solution (15% w / v) and dry it with anhydrous sodium sulfate. Take the organic phase; the obtained organic phase was evaporated to dryness by rotary evaporation, and the resulting mixture was redissolved in dichloromethane. Add 2 g of silica gel with a mesh size of 100 - 200 and mix well. After the solvent has evaporated completely, purify the above-mentioned crude product - silica gel powder mixture by column chromatography (using silica gel powder with a mesh size of 100 - 200 as the stationary phase and ethyl acetate: petroleum ether = 1:6 (V:V) as the mobile phase) to obtain Intermediate Ⅴ with the structure shown in Formula 7.

[0086]

[0087] Example 2 Preparation of 22 - ((7 - phenylquinoxalin - 2 - yl)thio)deoxypleconaril (Compound 2)

[0088] Under nitrogen protection, the intermediate V obtained in Example 1 (0.2 g, 0.33 mmol) was dissolved in a mixed solution of 5 ml of 1,4-dioxane and 1 ml of water, and phenylboronic acid (0.39 mmol) was added. Subsequently, the resulting reaction mixture was degassed with nitrogen for 10 minutes. Then potassium carbonate (0.66 mmol), tris(dibenzylideneacetone)dipalladium (0.033 mmol), and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (0.033 mmol) were added, and the reaction mixture was heated to 100 °C. After 2 hours, the resulting solution was collected by filtration and then extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate, evaporated under vacuum, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1). Compound 2 was obtained, and its NMR spectrum is as shown in Figure 2 below. The 1H NMR data and the compound structure are shown below. Structure characterization confirmed it to be the pure product of 22-((7-phenylquinoxalin-2-yl)thio)pleuromutilin.

[0089] 22-((7-Phenylquinoxalin-2-yl)thio)pleuromutilin (Compound 2)

[0090] 1 H NMR (600 MHz, Chloroform-d) δ 8.64 (1H, s), 8.08 (1H, d, J = 8.6 Hz), 8.04 (1H, d, J = 2.0 Hz), 7.90 (1H, dd, J = 8.6, 2.1 Hz), 7.71 (2H, dd, J = 8.2, 1.3 Hz), 7.50 (2H, t, J = 7.7 Hz), 7.46–7.40 (1H, m), 6.42 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.76 (1H, d, J = 8.5 Hz, H14), 5.23–4.89 (2H, m,

[0091]

[0092] H20), 4.18–3.90 (2H, m, H22), 3.27 (1H, d, J = 6.5 Hz, H11), 2.30–2.13 (3H, m, H2, H10), 2.04 (1H, d, J = 2.7 Hz, 11-OH), 1.91 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.77–1.48 (5H, m, H1, H6, H7), 1.42 (4H, m, H15, H8), 1.37–1.33 (1H, m, H8), 1.24 (1H, d, J = 16.0 Hz, H13), 1.11 (1H, td, J = 14.2, 4.7 Hz, H4), 0.91 (3H, s, H18), 0.81 (6H, dd, J = 11.4, 7.1 Hz, H17, H16).

[0093] Preparation of Compounds 3 - 9 in Example 3

[0094] Other steps were the same as in Example 2, except that phenylboronic acid was replaced with the corresponding equimolar arylboronic acid to obtain the corresponding products shown in Formula 2, numbered 3 - 9 in sequence. Their NMR spectra are as shown in Figures 3 - 9 shown below. The compound names, 1H NMR data, and compound structures are as follows:

[0095] 22 - ((7-(4-Nitrophenyl)-quinoxalin-2-yl)thio)pleuromutilin (Compound 3)

[0096] 11H NMR (600 MHz, Chloroform-d) δ 8.69 (1H, s), 8.37 (2H, d, J = 8.7 Hz), 8.15 (1H, d, J = 8.7 Hz), 8.09 (1H, d, J = 2.0 Hz), 7.90 (1H, dd, J = 8.6, 2.1 Hz), 7.88–7.85 (2H, m), 6.44 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.76 (1H, d, J = 8.5 Hz, H14), 5.23–4.93 (2H, m, H20), 4.18–3.95 (2H, m, H22), 3.28 (1H, d, J = 6.4 Hz, H11), 2.31–2.13 (3H, m, H2, H10), 2.04 (1H, d, J = 2.6 Hz, 11-OH), 1.93 (1H, dd, J = 16.0, 8.7 Hz, H13), 1.76–1.49 (5H, m, H1, H6, H7), 1.42 (4H, m, H15, H8), 1.38–1.33 (1H, m, H8), 1.24 (1H, d, J = 16.0 Hz, H13), 1.11 (1H, td, J = 14.2, 4.4 Hz, H4), 0.93 (3H, s, H18), 0.83 (3H, d, J = 7.1 Hz, H17), 0.80 (3H, d, J = 7.1 Hz, H16).

[0097]

[0098] 22-((7-(3-Methylphenyl)-quinoxalin-2-yl)thio)pleuromutilin (Compound 4)

[0099] 11H NMR (600 MHz, Chloroform-d) δ 8.63 (1H, s), 8.07 (1H, d, J = 8.6 Hz), 8.03 (1H, d, J = 2.0 Hz), 7.90 (1H, dd, J = 8.6, 2.0 Hz), 7.52 (1H, d, J = 1.8 Hz), 7.52–7.49 (1H, m), 7.39 (1H, t, J = 7.6 Hz), 7.24 (1H, d, J = 7.5 Hz), 6.43 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.76 (1H, d, J = 8.5 Hz, H14), 5.23–4.92 (2H, m, H20), 4.19–3.93 (2H, m, H22), 3.27 (1H, d, J = 6.4 Hz, H11), 2.46 (3H, s), 2.31–2.13 (3H, m, H2, H10), 2.06–2.01 (1H, m, 11-OH), 1.91 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.77–1.48 (5H, m, H1, H6, H7), 1.42 (4H, m, H15, H8), 1.36 (1H, dq, J = 15.4, 4.4, 4.0 Hz, H8), 1.24 (1H, d, J = 16.0 Hz, H13), 1.11 (1H, td, J = 14.1, 4.4 Hz, H4), 0.92 (3H, s, H18), 0.83 (3H, d, J = 7.1 Hz, H17), 0.80 (3H, d, J = 7.1 Hz, H16).

[0100]

[0101] 22-((7-(3-Hydroxyphenyl)-quinoxalin-2-yl)thio)deoxypleuromutilin (Compound 5)

[0102] 11H NMR (600 MHz, Chloroform-d) δ 8.63 (1H, s), 8.05 (1H, d, J = 8.6 Hz), 8.01 (1H, d, J = 2.0 Hz), 7.84 (1H, dd, J = 8.6, 2.1 Hz), 7.34 (1H, t, J = 7.9 Hz), 7.24 (1H, dt, J = 7.7, 1.2 Hz), 7.18 (1H, t, J = 2.1 Hz), 6.90 (1H, ddd, J = 8.0, 2.5, 0.9 Hz), 6.42 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.76 (1H, d, J = 8.5 Hz, H14), 5.22–4.92 (2H, m, H20), 4.16–3.94 (2H, m, H22), 3.28 (1H, d, J = 6.5 Hz, H11), 2.30–2.13 (3H, m, H2, H10), 2.06–2.03 (1H, m, 11-OH), 1.91 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.77–1.47 (5H, m, H1, H6, H7), 1.43 (4H, m, H15, H8), 1.35 (1H, dd, J = 14.4, 3.7 Hz, H8), 1.24 (1H, d, J = 16.2 Hz, H13), 1.10 (1H, td, J = 14.2, 4.5 Hz, H4), 0.92 (3H, s, H18), 0.83 (3H, d, J = 7.0 Hz, H17), 0.79 (3H, d, J = 7.1 Hz, H16).

[0103]

[0104] 22-((7-(4-Cyanophenyl)-quinoxalin-2-yl)thio)deoxypleuromutilin (Compound 6)

[0105] 11H NMR (600 MHz, Chloroform-d) δ 8.68 (1H, s), 8.13 (1H, d, J = 8.6 Hz), 8.05 (1H, d, J = 2.0 Hz), 7.87 (1H, dd, J = 8.6, 2.1 Hz), 7.81 (4H, s), 6.43 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.76 (1H, d, J = 8.5 Hz, H14), 5.25–4.87 (2H, m, H20), 4.23–3.92 (2H, m, H22), 3.28 (1H, d, J = 6.4 Hz, H11), 2.31–1.88 (5H, m, H2, H10, 11-OH, H13), 1.76–1.49 (5H, m, H1, H6, H7), 1.42 (4H, m, H15, H8), 1.35 (1H, dq, J = 14.5, 3.7 Hz, H8), 1.23 (1H, d, J = 16.0 Hz, H13), 1.11 (1H, td, J = 14.2, 4.4 Hz, H4), 0.92 (3H, s, H18), 0.83 (3H, d, J = 7.0 Hz, H17), 0.79 (3H, d, J = 7.1 Hz, H16).

[0106]

[0107] 22 - ((7-(4-Formylphenyl)-quinoxalin-2-yl)thio)pleuromutilin (Compound 7)

[0108] 11H NMR (600 MHz, Chloroform-d) δ 10.11 (1H, s), 8.68 (1H, s), 8.12 (1H, d, J = 8.6 Hz), 8.09 (1H, d, J = 2.1 Hz), 8.05–8.00 (2H, m), 7.92 (1H, dd, J = 8.6, 2.1 Hz), 7.89–7.86 (2H, m), 6.43 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.76 (1H, d, J = 8.5 Hz, H14), 5.20–4.92 (2H, m, H20), 4.23–3.90 (2H, m, H22), 3.28 (1H, d, J = 6.4 Hz, H11), 2.31–2.13 (3H, m, H2, H10), 2.06–2.03 (1H, m, 11-OH), 1.92 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.76–1.50 (5H, m, H1, H6, H7), 1.42 (4H, m, H15, H8), 1.38–1.33 (1H, m, H8), 1.24 (1H, d, J = 16.0 Hz, H13), 1.11 (1H, td, J = 14.2, 4.5 Hz, H4), 0.91 (3H, s, H18), 0.83 (3H, d, J = 7.0 Hz, H17), 0.80 (3H, d, J = 7.1 Hz, H16).

[0109]

[0110] 22 - ((7-(Pyridin-4-yl)-quinoxalin-2-yl)thio)pleuromutilin (Compound 8)

[0111] 11H NMR (600 MHz, Chloroform-d) δ 8.75 (2H, d, J = 5.2 Hz), 8.69 (1H, s), 8.14 (1H, d, J = 8.6 Hz), 8.11 (1H, d, J = 2.0 Hz), 7.91 (1H, dd, J = 8.6, 2.0 Hz), 7.64 (2H, d, J = 5.3 Hz), 6.43 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.77 (1H, d, J = 8.5 Hz, H14), 5.24–4.92 (2H, m, H20), 4.20–3.94 (2H, m, H22), 3.29 (1H, d, J = 6.4 Hz, H11), 2.31–2.12 (3H, m, H2, H10), 2.05 (1H, d, J = 2.7 Hz, 11-OH), 1.93 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.79–1.47 (5H, m, H1, H6, H7), 1.43 (4H, m, H15, H8), 1.36 (1H, dq, J = 14.4, 3.6 Hz, H8), 1.24 (1H, d, J = 16.2 Hz, H13), 1.11 (1H, td, J = 14.2, 4.3 Hz, H4), 0.93 (3H, s, H18), 0.83 (3H, d, J = 7.0 Hz, H17), 0.80 (3H, d, J = 7.1 Hz, H16).

[0112]

[0113] 22 - ((7-(Pyrimidin-5-yl)-quinoxalin-2-yl)thio)pleuromutilin (Compound 9)

[0114] 11H NMR (600 MHz, Chloroform-d) δ 9.30 (1H, s), 9.09 (2H, s), 8.18 (1H, d, J = 8.5 Hz), 8.07 (1H, d, J = 1.9 Hz), 7.86 (1H, dd, J = 8.6, 2.0 Hz), 6.45 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.77 (1H, d, J = 8.5 Hz, H14), 5.23–4.95 (2H, m, H20), 4.17–3.98 (2H, m, H22), 3.29 (1H, d, J = 6.4 Hz, H11), 2.30–2.13 (3H, m, H2, H10), 2.07–2.03 (1H, m, 11-OH), 1.95 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.83–1.47 (5H, m, H1, H6, H7), 1.42 (4H, m, H15, H8), 1.36 (1H, dd, J = 14.5, 3.7 Hz, H8), 1.25 (1H, d, J = 16.0 Hz, H13), 1.11 (1H, td, J = 14.1, 4.4 Hz, H4), 0.95 (3H, s, H18), 0.84 (3H, d, J = 7.0 Hz, H17), 0.80 (3H, d, J = 7.1 Hz, H16).

[0115]

[0116] Preparation of Compound 1 in Example 4

[0117] Other steps are the same as in Example 1, except that step (3) is not required, and the corresponding product shown in Formula 2 is obtained, numbered 1, and its NMR spectrum is as shown in Figure 1 shown below. The compound name, 1H NMR data, and compound structure are as follows:

[0118] 22 - ((Quinoxalin-2-yl)thio)deoxypleconaril (Compound 1)

[0119] 11H NMR (600 MHz, Chloroform-d) δ 8.66 (1H, s), 8.03 (1H, dd, J = 8.1, 1.5 Hz), 7.85 (1H, dd, J = 8.3, 1.4 Hz), 7.66 (2H, dddd, J = 25.3, 8.3, 6.9, 1.5 Hz), 6.43 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.76 (1H, d, J = 8.5 Hz, H14), 5.29–5.02 (2H, m, H20), 4.11–3.96 (2H, m, H22), 3.31 (1H, d, J = 6.5 Hz, H11), 2.34–2.13 (3H, m, H2, H10), 2.06 (1H, d, J = 2.6 Hz, 11-OH), 1.96 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.76–1.50 (5H, m, H1, H6, H7), 1.42 (4H, m, H15, H8), 1.36 (1H, dq, J = 14.5, 3.6 Hz, H8), 1.29 (1H, d, J = 16.0 Hz, H13), 1.12 (1H, td, J = 14.2, 4.5 Hz, H4), 1.05 (3H, s, H18), 0.85 (3H, d, J = 7.1 Hz, H17), 0.79 (3H, d, J = 7.1 Hz, H16).

[0120]

[0121] The preparation yields of the above Compounds 1-9 are summarized in Table 2 as follows.

[0122] Table 2 Compound Numbers and Yields

[0123]

[0124] Effect Examples

[0125] (1) In vitro antibacterial experiment

[0126] The broth dilution method was used in the experiment. Tiamulin was selected as the experimental control drug. Tiamulin is a pleuromutilin antibiotic and is one of the world's top ten veterinary antibiotics.

[0127] 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 strains AD3 and 144 were isolated and identified in the Pharmacology Laboratory of the College of Veterinary Medicine, South China Agricultural University and have been published in the literature Zhe Z A, Kang L A, Gyz A, et al. Design, synthesis and biological activities of novel pleuromutilin derivatives with a substituted triazole moiety as potent antibacterial agents - ScienceDirect[J]. European Journal of Medicinal Chemistry, 2020.).

[0128] Preparation of the stock solution of the target compound: Weigh accurately 6.4 mg of the compounds 1 - 9 obtained in Examples 1 - 4 and place them in a 10 mL volumetric flask. Dissolve them with 0.25 mL of DMSO, add 9.5 mL of distilled water, and make up the volume to the mark with 0.25 mL of Tween 80. Shake well to obtain the stock solution of the target compound (6.4 mg / mL). Filter it through a 0.22 μm filter membrane to sterilize, dispense it into small tubes, and store it at -20 °C. The control drug tiamulin was prepared in the same manner as above.

[0129] Preparation of the bacterial suspension: Take out the strains stored intact at -20 °C and inoculate them on a new MH plate. After culturing at 37 °C for 24 h, pick a single colony and inoculate it in MH medium and culture for another 24 h; Select a single colony and transfer it to sterile physiological saline and adjust its turbidity to 0.6 McF. At this time, the bacterial concentration is 10 6 CFU / mL.

[0130] Preparation of the MIC plate: Dilute the stock solution of the target compound (6.4 mg / mL) by 10 times respectively to obtain a target compound solution with a concentration of 640 μg / mL; Take a sterile 96-well plate. Add 180 μL of MH broth medium to the first well, add 100 μL of MH broth medium to each of the second to tenth wells. Add 20 μL of the antibacterial drug with a concentration of 640 μg / mL to the first well, mix well, then take 100 μL and add it to the second well, mix well, and then pipette 100 μL to the third well, and so on. Discard 100 μL from the twelfth well. At this time, the drug concentrations in each well are: 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03 μg / mL. Each drug concentration is made in three parallels.

[0131] Inoculation of bacterial solution: Add 100 μL of bacterial solution to each well from well 1 to well 12, so that the final bacterial solution concentration in each well is approximately 5×10 5 CFU / mL, and the drug concentrations in wells 1 to 12 are 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 is placed in an incubator at 37 °C for cultivation, and the growth of the bacterial solution is observed after 24 h. The control drug tiamulin is determined in the same way, and the lowest drug concentration that completely inhibits bacterial growth in the small wells is the MIC. Bacteria in the positive control wells (i.e., wells without drugs) should grow significantly. When a single skipped well appears in the microbroth dilution method, record the highest drug concentration that inhibits bacteria. If multiple skipped wells appear, the experiment needs to be repeated.

[0132] Table 3 shows the MIC results. It can be seen that the target compound has good antibacterial activity against the selected strains and good activity against resistant Staphylococcus aureus, and is particularly suitable as a new antibacterial drug for preventing and treating infectious diseases caused by humans or animals or resistant Staphylococcus aureus or multidrug-resistant bacteria.

[0133] Table 3 In vitro antibacterial data

[0134]

[0135]

[0136] (2) Cytotoxicity experiment

[0137] The MTT method is used in the experiment. The control drug tiamulin is selected for the experiment.

[0138] The cell lines used in the experiment are human bronchial epithelial cells (16HBE), human lung cancer cells (A549), and mouse macrophages (RAW 264.7).

[0139] Preparation of the target compound stock solution: Weigh accurately 4 mg of compounds 1-9 obtained in Examples 1-4 respectively, dissolve them with 1 mL of DMSO, shake well, and obtain the target compound stock solution (4 mg / mL). Filter and sterilize it with a 0.22 μm filter membrane and store it at -20 °C. The control drug tiamulin is prepared in the same way as above.

[0140] Cell resuscitation: Take out the cells from liquid nitrogen and quickly put them into a 37°C constant temperature water bath. Quickly shake the cryopreservation tube to make the cells melt rapidly. Then add the cryopreserved cells to 4 mL of 10% Dulbecco's Modified Eagle's medium (DMEM, Gbico, C11995500BT) complete medium prepared in advance and containing 10% (v / v) fetal bovine serum (FBS, Gibco, 10091148) and 1% (v / v) penicillin-streptomycin (Gibco, 15140122). Mix well and centrifuge at 1000 r / min for 3 min. Pour off the supernatant, resuspend the cells with freshly prepared 10% DMEM complete medium, and place the cell suspension in a dedicated T25 cell culture flask (Gibco). Place the culture dish in an incubator at 37°C with 5% CO2 for subculture.

[0141] Cell culture: Subculture the cells with a growth confluence rate of 90%. First, discard the supernatant and rinse the cells three times with sterile PBS (0.01 M, pH 7.2 - 7.4). Digest with 1 mL of trypsin containing EDTA for 1 min 20 s until some cells fall off. Aspirate and discard the trypsin, add 3 mL of 10% DMEM complete medium, and pipette the cells. Collect the cell suspension. After cell counting, transfer the cell suspension to a culture dish at a ratio of 1:3, shake it to make it evenly distributed, and then place it in an incubator with 5% CO2 for continuous static culture.

[0142] Cell cryopreservation: Cryopreserve and store the cells with good growth status. Collect the cells according to the above method, add cell cryopreservation solution (serum: DMSO = 9:1), and transfer them to a cell cryopreservation tube (Gibco). Then place the cryopreservation tube in a gradient cooling box, place it at -80°C overnight, and store it in liquid nitrogen for a long time.

[0143] MTT incubation and OD value measurement: Collect the cells with good growth status. Add 100 μL of cells at a density of about 5×10 5 cells / mL to each well of a 96-well plate for seeding. Place the 96-well plate in an incubator with 5% CO2 and let it stand overnight. Discard the complete culture medium, add the test compound with a final concentration of 4 μg / mL (dissolved in DMSO to prepare a 4 mg / mL stock solution), and incubate it in an incubator with 5% CO2 for 24 h. Discard the supernatant, add freshly prepared MTT with a concentration of 0.5 mg / mL (dissolved in DMEM to prepare a 5 mg / mL stock solution, stored in aliquots at -20°C) and incubate it in the dark for 4 h. Discard the supernatant, add 150 μL of DMSO, and place it on a shaker at room temperature and shake at 70 r / min for 10 min. Measure the absorbance value at 490 nm with an enzyme-linked immunosorbent assay reader and calculate the cell survival rate. The experiment is repeated independently three times. The formula is as follows:

[0144] Cell survival rate (%) = OD of drug group / OD of control group × 100%

[0145] Figure 10 , Figure 11 , Figure 12 As the results of the toxicity experiment, it can be seen that the target compound has no toxicity to the selected cell lines at 8 μg / mL, and has a certain growth-promoting effect, and is suitable as a new antibacterial drug for preventing and treating infectious diseases caused by humans or animals or drug-resistant Staphylococcus aureus or multi-drug resistant bacteria.

[0146] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A pleuromutilin derivative containing a quinoxaline side chain, characterized in that: The derivative is a compound of the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof: wherein R is a hydrogen atom, phenyl, 4-nitrophenyl, 3-methylphenyl, 3-hydroxyphenyl, 4-cyanophenyl, 4-formylphenyl, 4-pyridyl or 5-pyrimidyl.

2. The pleuromutilin derivative with a quinoxaline side chain according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound of the structure shown in 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.

3. The preparation method of the pleuromutilin derivative containing a quinoxaline side chain according to claim 1 or 2, characterized in that It includes the following operation steps: (1) React pleuromutilin with p-toluenesulfonyl chloride to obtain Intermediate I of the structure shown in Formula 3; (2) Use o-phenylenediamine as a raw material and react it with ethyl glyoxylate to obtain Intermediate II of the structure shown in Formula 4; (3) Dissolve Intermediate II prepared in step (2) in acetic acid and react it with Br2 to obtain Intermediate III of the structure shown in Formula 5; (4) Dissolve Intermediate III prepared in step (3) in pyridine and react it with P2S5 to obtain Intermediate IV of the structure shown in Formula 6; (5) Use Intermediate I prepared in step (1) as a raw material, further activate it with sodium iodide, and then react it with Intermediate IV prepared in step (4) to obtain Intermediate V of the structure shown in Formula 7; (6) Dissolve Intermediate V prepared in step (5) in 1,4-dioxane containing water and react it with aromatic boronic acid to obtain a pleuromutilin derivative with a quinoxaline side chain of the structure shown in Formula 2, wherein R is phenyl, 4-nitrophenyl, 3-methylphenyl, 3-hydroxyphenyl, 4-cyanophenyl, 4-formylphenyl, 4-pyridyl or 5-pyrimidyl; The said Intermediates I, II, III, IV, V have the structural formulas of Formulas 3 - 7 respectively:

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of p-toluenesulfonyl chloride to pleuromutilin is 1.1:1; In step (2), the molar ratio of o-phenylenediamine to ethyl glyoxylate is 1:1; In step (3), the molar ratio of Intermediate II to Br2 is 1:1.05; In step (4), the molar ratio of Intermediate III to P2S5 is 1:

1.

5. The preparation method according to claim 3, characterized in that: The said step (5) is specifically carried out according to the following operation steps: Use acetonitrile as a solvent to first dissolve Intermediate I, then add sodium iodide and a base, heat and reflux at 78 °C for 1 - 2 h, and then add Intermediate IV and continue to react for 3 h to obtain Intermediate V; The dosage of acetonitrile is 30 - 40 times the mass of Intermediate I, the molar ratio of the base to Intermediate I is 2:1, and the molar number of sodium iodide is 10% of the molar number of the base; The said base is sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate or cesium carbonate.

6. The preparation method according to claim 3, characterized in that: The said step (6) is specifically carried out according to the following operation steps: Under nitrogen protection, dissolve intermediate V, aromatic boronic acid, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, and base in a solvent of 1,4-dioxane containing water, and heat under reflux at 100 °C for 2 - 3 h; The amount of the solvent used is 30 - 40 times the mass of intermediate V, the molar ratio of the base to intermediate V is 2:1, the molar ratio of tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl to intermediate V is 1:1:10, and the molar ratio of the aromatic boronic acid to intermediate V is 3:2; The base is sodium carbonate, potassium acetate, potassium carbonate, or cesium carbonate.

7. A method for preparing a pleuromutilin derivative with a quinoxaline side chain according to claim 1 or 2, characterized in that It also includes the following operating steps: (1) React pleuromutilin with p-toluenesulfonyl chloride to obtain intermediate I with the structure shown in Formula 3; (2) Use o-phenylenediamine as a raw material and react it with ethyl glyoxylate to obtain intermediate II with the structure shown in Formula 4; (3) Dissolve the intermediate II prepared in step (2) in pyridine and react it with P2S5 to obtain intermediate VI with the structure shown in Formula 8; (4) Use the intermediate I prepared in step (1) as a raw material, further activate it with sodium iodide, and then react it with the intermediate VI prepared in step (3) to obtain a pleuromutilin derivative containing a quinoxaline side chain with the structure shown in Formula 2, where R is H; 8. Use of the pleuromutilin derivative containing a quinoxaline side chain according to claim 1 or 2 in the preparation of an antibacterial product, characterized in that: The antibacterial product is a drug for treating infectious diseases.

9. The application according to claim 8, wherein: The infectious disease is an infectious disease caused by human or animal infection with drug-resistant Staphylococcus aureus or multidrug-resistant bacteria.

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