Chem Biol Drug Des 2015; 86: 239–245 Research Article
Synthesis and Antimicrobial Activity of the Hybrid Molecules between Sulfonamides and Active Antimicrobial Pleuromutilin Derivative Liangzhu Chen1,2, Dexue Yang2, Zhikun Pan1, Lihong Lai2, Jianhua Liu2, Binghu Fang1,2,* and Shuning Shi2 1
Guangdong Dahuanong Animal Health Products Co. Ltd., Yunfu 527400, China 2 Department of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China *Corresponding author: Binghu Fang, [email protected] A series of novel hybrid molecules between sulfonamides and active antimicrobial 14-o-(3-carboxy-phenylsulfide)-mutilin were synthesized, and their in vitro antibacterial activities were evaluated by the broth microdilution. Results indicated that these compounds displayed potent antimicrobial activities in vitro against various drug-susceptible and drug-resistant Gram-positive bacteria such as Staphylococci and streptococci, including methicillin-resistant Staphylococcus aureus, and mycoplasma. In particular, sulfapyridine analog (6c) exhibited more potent inhibitory activity against Gram-positive bacteria and mycoplasma, including Staphylococcus aureus (MIC = 0.016–0.063 lg/mL), methicillin-resistant Staphylococcus aureus (MIC = 0.016 lg/mL), Streptococcus pneumoniae (MIC = 0.032–0.063 lg/mL), Mycoplasma gallisepticum (MIC = 0.004 lg/mL), with respect to other synthesized compounds and reference drugs sulfonamide (MIC = 8–128 lg/mL) and valnemulin (MIC = 0.004–0.5 lg/mL). Furthermore, comparison between MIC values of pleuromutilin-sulfonamide hybrids 6a–f with pleuromutilin parent compound 3 revealed that these modifications at 14 position side chain of the pleuromutilin with benzene sulfonamide could greatly improve the antibacterial activity especially against Gram-positives. Key words: antibacterial activity, hybrid molecules, pleuromutilin derivative, sulfonamides Received 23 September 2014, revised 18 November 2014 and accepted for publication 19 November 2014
The emergence and spread of multidrug-resistant strains, especially Gram-positive bacteria including methicillinresistant Staphylococcus aureus (MRSA), penicillin-resisª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12486
tant Streptococcus pneumoniae (PRSP), and vancomycinresistant enterococci (VRE) (1,2), stimulated our interest in the search for novel and more efficient antibacterial agents, particularly semi-synthetic pleuromutilin derivatives with unique mechanism of action and consequently no cross-resistance to the marketed antibacterial agents. Pleuromutilin (1) (Figure 1) was a naturally occurring crystalline compound with a rare fused 5-6-8 tricyclic diterpenoid structure, first isolated from basidiomycetes Pleurotus and P. passeckerianus in 1951, and exhibited modest antibacterial activity against Gram-positive pathogens and mycoplasmas in vitro (3,4). Further studies have revealed that this class of antibiotics selectively interfere with bacterial protein synthesis through a specific complexation with the 23 S rRNA of the 50 S prokaryotic ribosome subunit (5). To find a novel class of antibiotics with a new mechanism of action, many studies had been carried out to exploit new pleuromutilin derivatives with potent antibacterial activity (6,7). As a result, the valnemulin (2) was developed as excellent veterinary medicine. And through chemical modifications of 1, a new pleuromutilin derivative retapamulin (3) was first provided for human use as topical antimicrobial agent in 2007. Furthermore, researchers found that modification of the side chain at C-14 of 1 with polar heterocyclic basic groups and thioether bond could be the most promising method to obtain outstanding antibacterial activity (8). Sulfonamides were well known as broad-spectrum antimicrobial antifolic agents. Many hybrid drugs, incorporating moieties both of a sulfonamide and a different antibacterial agent, such as fluoroquinolone, were reported in the literatures as being very active (9–12). Moreover, several pleuromutilin analogs with aromatic or pyridine ring have been described excellent antibacterial activity, especially good inhibition of the growth of penicillin-resistant staphylococci, recently, such as BC 7013 (4) (13–16). According to the report, the method of rational drug design based on the synthesis of dual- or multiple-ligands, can produce drugs of superior clinical effect, compared with single targeted ones (17). Within antibacterial durgs, ‘dual targeting’ compounds may be a prime objective, also because they would reduce the odds of developing
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recorded with Thermo Scientific Orbitrap Elite MS (Thermo Fisher Scientific Inc., Waltham, MA, USA) using the electrospray ionization (ESI) method. Elemental analysis was performed on a Elementar (Germany) Vario EL Elemental Analyzer, for C, H, N and S. The found values for C, H, N, and S were always 0.4% of the theoretical ones.
Figure 1: Pleuromutilin and pleuromutilin derivatives.
bacterial resistance. To prove the impact of the sulfonamide group on the antibacterial activity and antibacterial spectrum of pleuromutilin derivatives, we synthesized and evaluated for antibacterial properties a series of novel pleuromutilin-benzenesulfonamide derivatives via modification of the side chain at C-14 position. The synthetic route was as showed in Figure 2. The antibacterial activity of the hybrids was investigated with drug-susceptible and drug-resistant pathogens by standard procedure in vitro. The results indicated that these molecules inhibited excellent in vitro antimicrobial properties against Gram-positive bacteria such as Staphylococci and streptococci, including methicillin-resistant Staphylococcus aureus (MRSA), and mycoplasma.
Materials and Methods Reagents and analysis Synthetic starting materials, reagents, and all solvents used in this study were purchased from J & K Chemical LTD (Beijing, China) or Sigma–Aldrich. Melting points were determined with XRC-1 melting point apparatus (Sichuan University Instrument Inc., Chengdu, China) without corrected. The IR spectrum was obtained on a Shimadzu IR Affinity-1 spectrometer (potassium bromide disks). 1H NMR spectrum were recorded using a Bruker AVANCE AV400 spectrometer (Bruker BioSpin Corp., Faellanden, Switzerland) in DMSO, and chemical shifts are given in d (ppm) with TMS as internal standard. Mass spectrum were
Chemistry The synthetic process for the preparation of the target compounds is outlined in Figure 2. The structures of the synthesized compounds were supported by the spectral IR, 1H NMR, and 13C NMR data, the results of HR-MS (ESI) and EA which were in agreement with the proposed structures.
14-o-(p-toluene sulfonyloxyacetyl) mutilin (2) Potassium carbonate solution (6.5 mL, 2 M) was slowly dropped to a mixture of pleuromutilin (3.78 g, 10 mmol) and p-toluene sulfonyl chloride (1.91 g, 10 mmol) in the mixed solution of ethyl acetate and water (40 mL, ethyl acetate: water = 1:3, volume). The mixture was stirred at 35 °C for 5 h, then evaporated in vacuum, and the residue was diluted in the water. The precipitates were filtrated and washed with water, dried under vacuum, dissolved in acetone (20 mL), followed by crystallizing for 2 days. Filtration afforded the pure product (2) 4.72 g. White solid; yield: 88.7%; 1H NMR (400 MHz, DMSO-d6) d: 7.79 (2 H, d, J = 4.0 Hz), 7.34 (2 H, d, J = 5.2 Hz), 6.41 (1 H, dd, J1 = 15.1 Hz, J2 = 9.8 Hz), 5.67 (1 H, d, J = 4.4 Hz), 5.25 (1H, d, J = 6.2 Hz), 5.01 (1H, d, J = 9.2 Hz), 4.48 (s, 1H), 3.33–3.37 (1 H, m), 2.41 (3 H, s), 2.02–2.33 (4 H, m), 1.73–1.76 (1 H, m), 1.61–1.68 (2 H, m), 1.60 (3 H, s), 1.43–1.47 (5 H, m), 1.38–1.41 (2 H, m), 1.27 (3 H, s), 0.89 (3 H, d, J = 6.4 Hz), 0.63 (3 H, d, J = 6.4 Hz); 13C NMR (100 MHz, DMSO-d6)d:216.9, 164.7, 145.2, 140.6, 132.2, 130.1, 127.7, 115.3, 72.5, 70.3, 65.7, 57.1, 44.9, 44.1, 43.1, 41.5, 36.4, 36.1, 33.9, 30.0, 29.6, 26.5, 24.4, 21.1, 15.8, 14.4, 11.5.
Figure 2: Scheme of synthesis and structure of compounds 5a–5f and 6a–6f. Reagent and conditions: (i) TsCl, KCO3 (2 M)/ethyl acetate and water, 35 °C, 5 h. (ii) KOH/CH3OH, m-mercaptobenzoic acid, 45 °C, 6 h. (iii) (A) Oxalyl chloride/CH2Cl2, m-mercaptobenzoic acid, C5H5N, N2, rt, 12 h; (B) CH2Cl2, Et3N, THF, reflux, 12 h. (iv) EtONa/ethanol, rt, over 6 h, N2.
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14-O-[(4- carboxy – benzenethiol)acetyl-] mutilin (3) Small portions of potassium hydroxide (0.32 g, 5.6 mmol) were added to a stirred mixture of 2 (5 g, 9.4 mmol) and m-mercaptobenzoic acid (1.5 g, 9.4 mmol) in methanol (40 mL) at room temperature for 30 min and then heated to 45 °C for 6 h. The concentrated reaction mixture was poured into water and crushed ice (80 mL, 20 g), acidified with conc. HCl and stirred at rt for additional 1 h. The mixture was then filtered and washed thoroughly with water (3 9 15 mL) and dried in vacuum. The gained filter residue was purified by silica gel chromatography, to give 3 (2.38 g, 49.2%); mp: 199–201 °C; IR (KBr, cm-1): mmax: 3482.8, 3166.5, 3075.9, 2985.3, 2937.1, 2877.3, 1722.1, 1581.3, 1457.9, 1417.4, 1380.8, 1288.2, 1126.2, 1004.7, 935.3; 1H NMR (400 MHz, DMSO-d6)d: 12.64(1 H, s), 7.28(1 H, s), 7.22–7.26 (1 H, dd, J1 = 7.2 Hz, J2 = 2.6 Hz), 7.21 (1 H, d, J = 7.2 Hz), 7.15 (1 H, d, J = 7.4 Hz), 6.02–6.07 (1 H, dd, J1 = 16.5 Hz, J2 = 9.8 Hz), 5.50 (1 H, d, J = 4.7 Hz), 4.97–4.99 (1 H, d, J = 7.2 Hz),4.91 (1 H, d, J = 8.8 Hz), 4.46 (1 H, s), 3.75– 3.84 (1 H, d, J = 5.3 Hz), 2.37 (1 H, s), 2.15–2.20 (1 H, m), 1.97–2.09 (4 H, m), 1.57–1.66 (2 H, m), 1.44–1.48 (1 H, m), 1.35–1.42 (1 H, m), 1.32 (3 H, s), 1.13–1.28 (3 H, m), 0.99 (3 H, s), 0.81 (3 H, d, J = 5.8 Hz), 0.57 (3 H, d, J = 6.4 Hz). 13C NMR (100 MHz, DMSO-d6) d:217.1, 167.4, 166.7, 140.7, 135.8, 132.4, 131.6, 129.1, 128.6, 127.0, 115.1, 72.5, 69.9, 57.2, 44.9, 44.0, 43.6, 41.4, 36.4, 36.3, 35.0, 34.0, 30.1, 28.6, 26.6, 24.5, 16.0, 14.5, 11.5; HR-MS (ESI) Calcd for C29H38O6S (M+H+): 515.2474; Found: 515.2462.
d:11.14 (1 H, s), 10.33(2 H, s), 7.95 (2 H, d, J = 7.6 Hz), 7.89 (2 H, d, J = 8.1 Hz), 7.48 (1 H, d, J = 7.7 Hz),7.27 (1 H, t), 7.14 (1 H, d, J = 6.6 Hz), 6.98 (1 H, s).
4-(3-mercapto-benzamido)-N-(4, 6-dimethyl-2pyrimidinyl)benzenesulfonamide (5b) Prepared according to the method A. White solid; yield: 53.6%; mp: 198–200 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.84 (1 H, s), 10.66 (1 H, s), 8.11 (1 H, s), 7.98 (2 H, d, J = 5.6 Hz), 7.91 (2 H, d, J = 10.5 Hz), 7.88 (1 H, d, J = 11.2 Hz), 7.78 (1 H, d, J = 6.8 Hz), 7.58 (1 H, t), 6.76 (1 H, s), 2.26 (6 H, s).
3-mercapto-N-[4-[(2-pyridinylamino)sulfonyl] phenyl]-benzamide (5c) Prepared according to the method A. White solid; yield: 62.5%; mp: 186–188 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.54 (1 H, s), 10.66 (1 H, s), 8.15 (1 H, s), 7.75 (2 H, d, J = 6.7 Hz), 7.48 (2 H, d, J = 8.2 Hz), 7.26 (3 H, t), 7.14 (2 H, d, J = 10.9 Hz), 7.07 (1 H, d, J = 4.7 Hz), 6.56 (1 H, d, J = 13.8 Hz),5.80 (1 H, s).
3-mercapto-N-[4-[(2-thizaylamino)sulfonyl]phenyl]benzamide (5d) Prepared according to the method A. White solid; yield: 45.8%; mp: 169–171 °C; 1
Method A: general procedure for the synthesis of sulfonamide-thiophenol 5a–f Oxalyl chloride (1.65 g, 13 mmol) in dichloride methane (20 mL) was dropped slowly to an ice-cooled solution of m-mercaptobenzoic acid (2.1 g, 13 mmol) and pyridine (0.47 mL, 5.8 mmol) in dichloride methane (30 mL). The cooling bath was then removed and the resultant solution stirred for 12 h at room temperature under an atmosphere of nitrogen before being concentrated to dryness in vacuum. The obtained residue was dissolved in dichloride methane (50 mL) and added slowly to a mixture of sulfanilamide 4a–f (13 mmol) and triethylamine (1.79 mL, 13 mmol) in THF (30 mL). The reaction mixture was then heated to reflux for 12 h, and the volatiles were removed at reduced pressure. The residue was then dissolved in ethyl acetate (30 mL), and the mixture was washed with 1 M hydrochloric acid and brine. The organic phase was then dried (magnesium sulfate), concentrated, and the residue purified by silica gel chromatography to give 5a–f.
3-mercapto-N-[4-(amino-sulfonyl)phenyl]benzamide (5a) Prepared according to the method A. White solid; yield: 50.9%; mp: 179–181 °C; 1H NMR (400 MHz, DMSO-d6) Chem Biol Drug Des 2015; 86: 239–245
H NMR (400 MHz, DMSO-d6) d:11.25 (1 H, s), 10.54 (1 H, s), 7.98 (2 H, d, J = 5.8 Hz), 7.86 (3 H, d, J = 7.9 Hz), 7.66 (1 H, d, J = 16.4 Hz), 7.51 (1 H, d, J = 12.1 Hz), 7.43 (1 H, t), 6.89 (1 H, s), 6.27 (1 H, s).
3-mercapto-N-[4-[(6-methoxy-2-pyrimidinylamino) sulfonyl]phenyl]-benzamide (5e) Prepared according to the method A. White solid; yield: 68.2%; mp: 190–192 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.73 (1 H, s), 10.54 (1 H, s), 8.31 (1 H, s), 7.88–7.95 (5 H, m), 7.72 (1 H, d, J = 15.6 Hz), 7.58 (1 H, s), 7.44 (1 H, t), 6.31 (1 H, s), 3.78 (3 H, d, J = 6.8 Hz).
3-mercapto-N-[4-[(5-methoxy-3- isoxazolylamino) sulfonyl]phenyl]-benzamide (5f) Prepared according to the method A. White solid; yield: 72.9%; mp: 175–177 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.37 (1 H, s), 10.64 (1 H, s), 7.97 (2 H, d, J = 6.4 Hz),7.87 (3 H, d, J = 3.4 Hz), 7.67 (1 H, d, J = 5.7 Hz), 7.53 (1 H, d, J = 10.5 Hz), 7.41 (1 H, t), 6.14 (1 H, s), 5.78 (1 H, s), 2.29 (3 H, s). 241
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Method B: general procedure for the synthesis of sulfoether A solution of 5a–f (10 mmol) in ethanol was added to an ice-cooled mixture of sodium ethoxide (1.72 g, 25 mmol) in ethanol (50 mL) under a nitrogen atmosphere. After 1 h, the resultant solution was treated with 2 (5.41 g, 10 mmol) in acetone (80 mL) and stirred over 6 h at rt. The reaction mixture was then concentrated at reduced pressure, and partitioned between ethyl acetate and water and the aqueous phase re-extracted with ethyl acetate. The combined organic extracts were washed with water and brine, dried (magnesium sulfate) and concentrated. The residue was purified by silica gel chromatography to give 6a–f.
14-O-[[3-[[[4-(aminosulfonyl) phenyl] amino] carbonyl] phenylthio] acetyl-] mutilin (6a) Prepared according to the method B. White solid; yield: 57.5%; mp: 195–197 °C; IR (KBr, cm 1) mmax: 3340.1, 3263, 3097.1, 2981.4, 2935.1, 2875.3, 1722.1, 1672, 1592.9, 1525.4, 1461.8, 1394.3, 1322.9, 1261.2, 1157.1, 1114.7, 1018.2; 1H NMR (400 MHz, DMSO-d6) d:11.32 (1 H, s), 10.56 (1 H, s), 7.90 (2 H, d, J = 6.5 Hz), 7.75 (1 H, d, J = 8.4 Hz), 7.59 (1 H, d, J = 7.3 Hz), 7.46 (1 H, t), 7.36 (2 H, s), 7.25–7.29 (1 H, dd, J1 = 8.3 Hz, J2 = 6.7 Hz), 6.75 (1 H, s), 6.01–6.05 (1 H, dd, J1 = 14.3 Hz, J2 = 6.1 Hz), 5.51 (1 H, d, J = 5.5 Hz), 4.92–4.94 (1 H, d, J = 6.2 Hz), 4.90 (1 H, d, J = 7.4 Hz), 4.49 (1 H, s), 3.75–3.88 (2 H, dd, J1 = 10.6 Hz, J2 = 4.3 Hz), 2.35 (1 H, s), 2.27 (6 H, s), 2.15–2.20 (1 H, m), 1.96–2.09 (3 H, m), 1.54–1.64 (2 H, m), 1.32–1.49 (2 H, m), 1.31 (3 H, s), 1.20–1.25 (1 H, t), 1.12–1.19 (2 H, m), 0.98 (3 H, s), 0.76 (3 H, d, J = 4.6 Hz), 0.56 (3 H, d, J = 6.3 Hz); 13C NMR (100 MHz, DMSO-d6) d:217.1, 167.5, 165.1, 142.0, 140.7, 138.8, 135.8, 135.0, 131.3, 129.1, 127.0, 126.5, 125.5, 119.9, 115.1, 72.5, 69.9, 57.2, 44.9, 44.0, 43.9, 41.4, 36.4, 36.3, 34.0, 30.1, 28.5, 26.6, 24.4, 16.1, 14.5, 11.7; HR-MS (ESI) Calcd for C35H45N2O7S2 (M+H+): 669.2663; Found: 669.2658; Anal. Calcd for C35H44N2O7S2 (668.26): C, 62.85; H, 6.63; N, 4.19; S, 9.58. Found: C, 62.73; H, 6.69; N, 4.15; S, 9.40.
14-O-[[3-[[[4-N-[(4,6-dimethyl-2-pyrimidinylamino) sulfonyl]phenyl]amino]carbonyl]phenylthio] acetyl-] mutilin (6b) Prepared according to the method B. White solid; yield: 48.0%; mp: 218–220 °C; IR (KBr, cm 1): 3444.2, 3382.5, 3104.8, 2935.1, 2873.4, 1726, 1677.8, 1596.8, 1525.4, 1434.8, 1313.3, 1265.1, 1155.2, 1118.5, 1083.8, 1020.2; 1 H NMR (400 MHz, DMSO-d6) d:11.5 6 (1 H, s), 10.55 (1 H, s), 7.93–7.99 (5 H, m), 7.75 (1 H, d, J = 6.9 Hz), 7.59 (1 H, d, J = 2.8 Hz), 7.47 (1 H, t), 6.76 (1 H, s), 5.97–6.04 (1 H, dd, J1 = 5.3 Hz, J2 = 17.4 Hz), 5.51 (1 H, d, J = 7.2 Hz), 4.89–4.92 (2 H, dd, J1 = 13.2 Hz, J2 = 4.5 Hz), 4.54 (1 H, s), 3.96–3.89 (2 H, dd, J1 = 8.9 Hz, J2 = 6.8 Hz), 2.34 (1 H, s), 2.26 (6 H, s), 2.14–2.19 (1 H, m), 1.94–2.08 (3 H, m), 1.56–1.63 (2 H, 242
m), 1.31–1.46 (2 H, m), 1.29 (3 H, s), 1.23–1.25 (1 H, t), 1.12–1.20 (2 H, m), 0.94 (3 H, s), 0.78 (3 H, d, J = 6.0 Hz), 0.57 (3 H, d, J = 7.8 Hz). 13C NMR (100 MHz, DMSO-d6) d:217.6, 167.9, 165.6, 156.2, 141.1, 140.7, 136.3, 135.4, 131.6, 129.5, 127.4, 125.9, 119.8, 115.5, 113.5, 73.0, 70.4, 57.7, 45.4, 44.4, 44.1, 41.9, 36.8, 36.7, 35.4, 34.4, 30.5, 28.9, 26.9, 24.8, 16.5, 14.9, 11.9; HR-MS (ESI) Calcd for C41H51N4O7S2 (M + H+): 775.3193; Found: 775.3186; Anal. Calcd for C41H50N4O7S2 (774.31): C, 63.54; H, 6.51; N, 7.23; S, 8.27. Found: C, 63.41; H, 6.68; N, 7.25; S, 8.11.
14-O-[[3-[[[4-N-[(2-pyridinylamino)sulfonyl]phenyl] amino]carbonyl]phenylthio] acetyl-] mutilin (6c) Prepared according to the method B. White solid; yield: 72.1%; mp: 212–214 °C; IR (KBr, cm 1): 3494.4, 3357.5, 2935.1, 2875.3, 2763.5, 1726, 1677.8, 1629.6, 1594.8, 1523.5, 1461.8, 1390.4, 1261.2, 1141.7, 1093.4, 1010.5; 1 H NMR (400 MHz, DMSO-d6) d:11.7 2 (1 H, s), 10.50 (1 H, s), 8.03 (1 H, s), 7.99–7.93 (5 H, m), 7.77 (1 H, d, J = 6.7 Hz), 7.69 (1 H, d, J = 7.2 Hz), 7.58 (1 H, d, J = 7.5 Hz), 7.46 (1 H, t), 7.15 (1 H, d, J = 9.3 Hz), 6.88 (1 H, t), 6.01–6.06 (1 H, dd, J1 = 6.1 Hz, J2 = 8.5 Hz), 5.50 (1 H, d, J = 16.5 Hz), 4.94–4.97 (2 H, m), 4.51 (1 H, s), 3.94–4.02 (2 H, dd, J1 = 10.9 Hz, J2 = 5.4 Hz), 2.37 (1 H, s), 2.14–2.18 (1 H, m), 1.97–2.03 (3 H, m), 1.55–1.64 (2 H, m), 1.43–1.46 (1 H, m), 1.37–1.41 (1 H, m), 1.34 (3 H, s), 1.15–1.24 (3 H, m), 0.98.00 (3 H, s), 0.76 (3 H, d, J = 6.4 Hz), 0.57 (3 H, d, J = 8.3 Hz); 13C NMR (100 MHz, DMSO-d6) d:217.6, 167.8, 165.5, 143.0, 141.2, 141. 1, 131.6, 128.7, 128.1, 127.1, 120.3, 115.6, 73.0, 70.5, 57.6, 45.4, 44.5, 44.0, 36.8, 34.7, 34.4, 30.6, 29.0, 27.0, 24.9, 16.6, 15.0, 12.0; HR-MS (ESI) Calcd for C40H48N3O7S2 (M + H+): 746.2928; Found: 746.2923; Anal. Calcd for C40H47N3O7S2 (745.29): C, 64.45; H, 6.35; N, 5.63; S, 8.60. Found: C, 64.60; H, 6.52; N, 5.43; S, 8.41.
14-O-[[3-[[[4-N-[(2-thizaylamino)sulfonyl]phenyl] amino]carbonyl]phenylthio] acetyl-] mutilin (6d) Prepared according to the method B. White solid; yield: 63.4%; mp: 198.8–201 °C; IR (KBr, cm 1): 3542.6, 3305.4, 3102.9, 2929.3, 2875.3, 2813.6, 2684.4, 1726, 1662.3, 1591, 1527.3, 1405.9, 1315.2, 1261.2, 1143.6, 1089.6, 1079.9, 1016.3; 1H NMR(400 MHz, DMS O-d6) d:11.72 (1 H, s), 10.60 (1 H, s), 7.99 (2 H, d, J = 6.8 Hz), 7.93 (1 H, d, J = 9.3 Hz), 7.85 (2 H, d, J = 7.5 Hz), 7.76 (1 H, d, J = 6.4 Hz), 7.61 (1 H, d, J = 3.4 Hz), 7.50 (1 H, d, J = 6.0 Hz), 6.60 (1 H, d, J = 4.2 Hz) 6.33 (1 H, d, J = 5.7 Hz), 5.98–6.05 (1 H, dd, J1 = 5.7 Hz, J2 = 19.1 Hz), 5.52 (1 H, d, J = 4.9 Hz), 4.91–4.98 (2 H, m), 4.48 (1 H, s), 3.92–3.98 (2 H, dd, J1 = 16.1 Hz, J2 = 5.2 Hz), 2.37 (1 H, s), 2.17–2.21 (1 H, m), 1.96–2.05 (3 H, m), 1.44–1.64 (3 H, m), 1.36–1.40 (1 H, m), 1.32 (3 H, s), 1.15–1.25 (3 H, m), 0.97 (3 H, s), 0.79 (3 H, d, J = 3.5 Hz), 0.55 (3 H, d, J = 9.6 Hz). 13C NMR (100 MHz, DMSO-d6) d:217.1, 170.3, 168.7, 165.2, 152.3, Chem Biol Drug Des 2015; 86: 239–245
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142.4, 141.3, 136.7, 135.7, 131.1, 129.2, 128.3, 127.9, 126.8, 126.1, 124.5, 119.7, 108.1, 73.1, 70.5, 65.1, 59.8, 57.1, 49.8, 44.9, 44.0, 43.8, 41.5, 36.7, 36.3, 34.0, 30.4, 28.7, 26.7, 24.5, 16.4, 14.5, 11.5; HR-MS (ESI) Calcd for C38H46N3O7S3 (M + H+): 752.2492; Found: 752.2487; Anal. Calcd for C38H45N3O7S3 (751.25): C, 60.61; H, 6.16; N, 5.58; S, 12.78. Found: C, 60.52; H, 6.29; N, 5.66; S, 12.53.
135.9, 134.9, 133.6, 131.3, 129.1, 127.9, 127.0, 125.6, 120.1, 115.1, 95.4, 72.6, 70.0, 57.2, 44.9, 44.0, 43.9, 41.5, 36.4, 36.3, 35.0, 34.0, 30.1, 28.5, 26.5, 24.4, 16.1, 14.5, 12.1, 11.5; HR-MS (ESI) Calcd for C39H48N3O8S2 (M+H+): 750.2877; Found: 750.2872; Anal. Calcd for C39H47N3O8S2 (749.29): C, 62.46; H, 6.32; N, 5.61; S, 8.55. Found: C, 62.57; H, 6.46; N, 5.75; S, 8.47.
14-O-[[3-[[[4-N-[(6-methoxy-2-pyrimidinylamino) sulfonyl] phenyl] amino]carbonyl]phenylthio] acetyl-] mutilin (6e) Prepared according to the method B. White solid; yield: 65.4%;mp: 220–222.5 °C; IR (KBr, cm 1): 3486.7, 3093.3, 2939, 2875.3, 1726, 1681.6, 1625.7, 1591, 1523.5, 1481.1, 1392.4, 1317.1, 1282.4, 1253.5, 1151.3, 1093.4; 1H NMR (400 MHz, DMSO-d6) d:11.8 5 (1 H, s), 10.60 (1 H, s), 8.42 (1 H, s), 7.89–7.97 (5 H, m), 7.75 (1 H, d, J = 7.0 Hz), 7.60 (1 H, s), 7.47 (1 H, t), 6.34 (1 H, s), 5.96–6.03 (1 H, dd, J1 = 12.7 Hz, J2 = 8.9 Hz), 5.50 (1 H, d, J = 4.8 Hz), 4.88–4.92 (2 H, t), 4.50 (1 H, s), 3.92– 4.01 (2 H, dd, J1 = 8.6 Hz, J2 = 11.1 Hz), 3.93 (3 H, s), 2.35 (1 H, s), 2.143–2.18 (1 H, m), 1.97–2.07 (3 H, m), 1.55–1.65 (2 H, m), 1.41–1.48 (1 H, m), 1.34–1.38 (1 H, m), 1.29 (3 H, s), 1.15–1.23 (3 H, m), 0.95 (3 H, s), 0.79 (3 H, d, J = 4.4 Hz), 0.55 (3 H, d, J = 5.2 Hz). 13C NMR (100 MHz, DMSO-d6) d: 217.7, 170.4, 167.8, 165.7, 158.9, 143.6, 141.3, 141.1, 131.5, 128.7, 128.4, 127.0, 120.4, 115.7, 91.3, 73.0, 70.5, 57.7, 54.7, 45.4, 44.5, 44.1, 36.8, 34.4, 30.5, 29.0, 27.0, 24.9, 16.6, 15.0, 11.9; HR-MS (ESI) Calcd for C40H49N4O8S2 (M + H+): 777.2986; Found: 777.2971; Anal. Calcd for C40H48N4O8S2 (776.29): C, 61.83; H, 6.23; N, 7.21; S, 8.23. Found: C, 61.85; H, 6.36; N, 7.11; S, 8.15.
Bacterial strains Staphylococcus aureus ATCC 29213, S. aureus CMCC 26003, Staphylococcus equi CICC 10373, S. equi CVCC 3307, E. coli ATCC 25922, and M. gallisepticum S6 were purchased from China Institute of Veterinary Drugs Control. MRSA (11) was separated from diseased pig.
14-O-[[3-[[[4-N-[(5-methyl-3- isoxazolylamino) sulfonyl] phenyl]amino]carbonyl]phenylthio] acetyl-] mutilin (6f) Prepared according to the method B. White solid; yield: 45.8%; mp: 215–217 °C; IR (KBr, cm 1): 3492.5, 3372.9, 3097.1, 2937.1, 2875.3, 1724, 1679.7, 1614.1, 1592.9, 1519.6, 1461.8, 1396.2, 1319.1, 1265.1, 1160.9, 1118.5, 1095.4, 1018.2; 1H NMR (400 MHz, D MSO-d6) d: 11.40 (1 H, s), 10.62 (1 H, s), 7.98 (2 H, d, J = 9.7 Hz), 7.90 (1 H, s), 7.86 (2 H, d, J = 3.9), 7.75 (1 H, d, J = 6.5 Hz), 7.60 (1H, d, J = 5.9 Hz), 7.48 (1 H, t), 6.15 (1 H, s), 5.97–6.05 (1 H, dd, J1 = 5.8 Hz, J2 = 16.7 Hz), 5.49 (1 H, d, J = 6.3 Hz), 4.88–4.93 (2 H, dd, J1 = 7.5 Hz, J2 = 4.2 Hz), 4.50 (1 H, d, J = 5.2 Hz), 4.49 (1 H, s), 3.93–3.96 (2 H, dd, J1 = 19.4 Hz, J2 = 6.6 Hz), 2.36 (1 H, s), 2.30 (3 H, s), 2.12–2.19 (1 H, m), 1.95–2.08 (3 H, m), 1.53–1.63 (2 H, m), 1.31– 1.45 (2 H, m), 1.29 (3 H, s), 1.20–1.24 (1 H, t), 1.11– 1.18 (2 H, m), 0.94 (3 H, s), 0.77 (3 H, d, J = 11.3 Hz), 0.56 (3 H, d, J = 4.7 Hz); 13C NMR (100 MHz, D MSOd6) d: 217.1, 170.3, 167.4, 165.3, 157.6, 143.4, 140.7, Chem Biol Drug Des 2015; 86: 239–245
Antimicrobial activity By the broth microdilution method, the new compounds and standard antibacterial agents (valnemulin and sulfanilamide) were detected in vitro for their antimicrobial activities against several typical pathogenic micro-organisms (Table 1) on the base of CLSI guideline. The compounds under test were dissolved in DMSO and diluted in the Muller–Hinton broth to give the concentrations ranging from 0.016 to 128 lg/mL. To ensure that the solvent had no impact on the growth of bacteria, a control test with the same medium as used in the experiment was carried out. An inocula of 1 9 106 CFU/mL which was prepared by diluting a bacteria solution (about 0.5 McFarland standard) was added. After cultured for 18–20 h at 37 °C (bacterial) and for 36 h at 37 °C in 5% CO2 atmosphere (mycoplasma), the minimum inhibitory concentrations (MIC, lg/mL) of compounds were determined, which resulted in no visible growth on the plate. Compound should be further diluted and tested according to the same process as above until the MICs were determined; in case, it could inhibit the growing of the bacteria at the concentration of 0.016 lg/mL.
Results and Discussion Chemistry The synthetic process of the new compounds is shown in Figure 2. The reaction of pleuromutilin (1) with equal chemical equivalent of p-toluenesulfonyl chloride in the presence of KCO3 at 35°C gave the intermediate 2. Treatment of mmercaptobenzoic acid with potassium hydroxide in absolute methanol followed by reaction with compound 2 at 45 °C provided the compound 3 in good yield. The m-mercaptobenzoic acid was treated with oxalyl chloride under the action of pyridine, in a cooling bath, followed by evaporating excess oxalyl chloride in vacuum, and then, 243
Chen et al. Table 1: In vitro antimicrobial activities of compounds 6a–f (MICs in lg/mL) MIC (lg/mL)
Compound
S. aureus ATCC 29213
S. aureus CMCC 26003
MRSA 11
S. equi CICC 10373
S. equi CVCC 3307
E. coli ATCC 25922
MG S6
6a 6b 6c 6d 6e 6f 3 Sulfanilamide Valnemulin
0.016 0.125 0.016 0.008 0.25 0.063 0.125 >128 0.016
0.125 4 0.063 0.5 4 4 32 8 0.5
0.032 8 0.016 0.25 16 8 0.5 >128 0.5
0.125 0.5 0.063 0.5 4 8 0.5 16 0.063
0.063 4 0.032 0.125 16 1 8 32 0.25
64 >128 16 64 >128 >128 >128 128 16
0.004 0.032 0.004 0.008 0.032 0.016 0.032 2 0.004
MG, M. gallisepticum (S6); MRSA, methicillin-resistant Staphylococcus aureus; S. equi, Staphylococcus equi.
the residue reacted with sulfanilamides (4a–f) at reflux in THF with triethylamine, to afford the crucial intermediates sulfonamide-thiophenol 5a–f. Compounds 6a–f were prepared by reacting sulfonamide-thiophenol 5a–f with sodium ethoxide in ethanol at 0–5 °C for 1 h, followed by reaction with compound 2 at room temperature for over 6 h, and purified on silica gel column chromatography.
Biological activity The in vitro antimicrobial activity of hybrids (6a–6f) was evaluated, and their minimum inhibitory concentration values (MICs) against various bacteria were determined by comparison to the parent benzoxy pleuromutilin derivative (3) and sulfanilamide, commercial antibiotic valnemulin as reference drug (Table 1). The MIC values of 6 novel synthetic pleuromutilin-sulfanilamide hybrids against Gram-positive pathogens indicated that some compounds possess a comparable or better activity compared with valnemulin. These new hybrids also exhibited potent antibacterial activity against MRSA and M. gallisepticum. Compound 6d exhibited the most potent inhibitory activity against S. aures (MIC = 0.008 lg/mL), and compound 6c showed the most inhibitory activity against S. epidemidis (MIC = 0.032 lg/mL), which were twofold and eightfold more potent than reference drug valnemulin, respectively. The MIC values of compound 6a–f against methicillin-resistant Staphylococcus aureus (MRSA 11) indicated that sulfanilamide analogs 6a and 6c showed excellent antibacterial activity (MIC = 0.032, 0.016 lg/mL). Their activities were 16- and 32-fold more than that of valnemulin (MIC = 0.5 lg/mL). Additionally, compound 6d exhibited potent activity (MIC = 0.25 lg/mL) comparable to valnemulin. Furthermore, the data obtained exhibited that compounds 6a, 6c, and 6d have more or equal inhibitory activity against M. gallisepticum (MIC = 0.004– 0.008 lg/mL), in contrast with reference drug valnemulin (MIC = 0.004 lg/mL). On the other hand, most tested compounds had less in vitro activity against Gram-negative bacterium E. coli, but were more active than reference 244
drugs sulfanilamide, with the exception of 6c (MIC = 16 lg/mL), which was eightfold more effective than sulfanilamide (MIC = 128 lg/mL). All the new hybrids showed more potent antibacterial activity than that of sulfanilamide. Comparison between MIC values of pleuromutilin-sulfonamide hybrids 6a–f with pleuromutilin parent compound 3 revealed that these modifications at 14 position side chain of the pleuromutilin with benzene sulfonamide could greatly improve the antibacterial activity especially against Gram-positives. Moreover, Compound 6a, having a terminal sulfa group at the 14 position side chain of the hybrid, is more potent than their corresponding hybrids 6b, 6e, and 6f, particularly against Gram-positives. Similarly, the antibacterial activity seems to be enhanced by the azine group, because the pyridine substituted hybrid 6c was more active than the unsubstituted analogs 6a.
Conclusion In conclusion, a series of novel pleuromutilin-sulfonamide hybrids were synthesized and evaluated. The results of antibacterial activities indicated that the hybrids gained appreciable antibacterial activity against Gram-positives when the sulfonamide was introduced into the C-14 side chain of pleuromutilin, especially the hybrids 6a and 6c which antibacterial activity has been improved clearly. More importantly, our discovery had provided a new approach to researching and developing novel potent antibiotic.
Acknowledgments We appreciate the financial support from the National Science Foundation of China (No. 31172359) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13063), NMR and Elemental analysis provided by SUN YAT-SEN University Instrumental Analysis & Research Center. Chem Biol Drug Des 2015; 86: 239–245
Hybrid Molecules, Antibacterial Activity
References 1. Wakatake H., Fujitani S., Kodama T. (2012) Positive clinical risk factors predict a high rate of methicillinresistant Staphylococcus aureus colonization in emergency department patients. Am J Infect Control;40:988–991. 2. Guducuoglu H., Aktas E., Comert F.B. (2009) First isolation and detection of multiple clones of vancomycinresistant enterococci in the pediatric unit of Van Yuzuncu Yil university, Turkey. Mikrobiyol Bul;43:535–543. 3. Kavanagh F., Herxey A., Robbins W.J. (1951) Antibiotic substance from basidiomycetes: VIII. pleurotus mutilus (Fr.) Sacc. and Pleurotus passeckerianus Pilat. Proc Natl Acad Sci USA;37:570–574. 4. Anchel M. (1952) Chemical studies with pleuromutilin. J Biol Chem;199:133–139. 5. Davidovich C., Bashan A., Yaggie R., Auerbach-Nevo T., Gontarek R.R., Yonath A. (2007) Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity. Proc Natl Acad Sci USA;104:4291–4294. 6. Egger H., Reinshagen H. (1976) New pleuromutilin derivatives with enhanced antimicrobial activity. I. Synthesis. J Antibiot;29:915–919. 7. Brooks G., Burgess W., Colthrust D., Hinks J.D., Hunt E., Pearson M.J., Shea B., Takle A.K., Wilson J.M., Woodnutt G. (2001) Pleuromutilins. Part 1: The identification of novel mutilin 14-carbamates. Bioorg Med Chem;9:1221–1231. 8. Egger H., Reinshagen H. (1976) New pleuromutilin derivatives with enhanced antimicrobial activity. J Antibiot;29:923–927. 9. Nieto M.J., Alovero F.L., Manzo R.H., Mazzieri M.R. (2005) Benzenesulfonamide analogs of fluoroquinol-
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Synthesis and Antimicrobial Activity of the Hybrid Molecules between Sulfonamides and Active Antimicrobial Pleuromutilin Derivative Liangzhu Chen1,2, Dexue Yang2, Zhikun Pan1, Lihong Lai2, Jianhua Liu2, Binghu Fang1,2,* and Shuning Shi2 1
Guangdong Dahuanong Animal Health Products Co. Ltd., Yunfu 527400, China 2 Department of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China *Corresponding author: Binghu Fang, [email protected] A series of novel hybrid molecules between sulfonamides and active antimicrobial 14-o-(3-carboxy-phenylsulfide)-mutilin were synthesized, and their in vitro antibacterial activities were evaluated by the broth microdilution. Results indicated that these compounds displayed potent antimicrobial activities in vitro against various drug-susceptible and drug-resistant Gram-positive bacteria such as Staphylococci and streptococci, including methicillin-resistant Staphylococcus aureus, and mycoplasma. In particular, sulfapyridine analog (6c) exhibited more potent inhibitory activity against Gram-positive bacteria and mycoplasma, including Staphylococcus aureus (MIC = 0.016–0.063 lg/mL), methicillin-resistant Staphylococcus aureus (MIC = 0.016 lg/mL), Streptococcus pneumoniae (MIC = 0.032–0.063 lg/mL), Mycoplasma gallisepticum (MIC = 0.004 lg/mL), with respect to other synthesized compounds and reference drugs sulfonamide (MIC = 8–128 lg/mL) and valnemulin (MIC = 0.004–0.5 lg/mL). Furthermore, comparison between MIC values of pleuromutilin-sulfonamide hybrids 6a–f with pleuromutilin parent compound 3 revealed that these modifications at 14 position side chain of the pleuromutilin with benzene sulfonamide could greatly improve the antibacterial activity especially against Gram-positives. Key words: antibacterial activity, hybrid molecules, pleuromutilin derivative, sulfonamides Received 23 September 2014, revised 18 November 2014 and accepted for publication 19 November 2014
The emergence and spread of multidrug-resistant strains, especially Gram-positive bacteria including methicillinresistant Staphylococcus aureus (MRSA), penicillin-resisª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12486
tant Streptococcus pneumoniae (PRSP), and vancomycinresistant enterococci (VRE) (1,2), stimulated our interest in the search for novel and more efficient antibacterial agents, particularly semi-synthetic pleuromutilin derivatives with unique mechanism of action and consequently no cross-resistance to the marketed antibacterial agents. Pleuromutilin (1) (Figure 1) was a naturally occurring crystalline compound with a rare fused 5-6-8 tricyclic diterpenoid structure, first isolated from basidiomycetes Pleurotus and P. passeckerianus in 1951, and exhibited modest antibacterial activity against Gram-positive pathogens and mycoplasmas in vitro (3,4). Further studies have revealed that this class of antibiotics selectively interfere with bacterial protein synthesis through a specific complexation with the 23 S rRNA of the 50 S prokaryotic ribosome subunit (5). To find a novel class of antibiotics with a new mechanism of action, many studies had been carried out to exploit new pleuromutilin derivatives with potent antibacterial activity (6,7). As a result, the valnemulin (2) was developed as excellent veterinary medicine. And through chemical modifications of 1, a new pleuromutilin derivative retapamulin (3) was first provided for human use as topical antimicrobial agent in 2007. Furthermore, researchers found that modification of the side chain at C-14 of 1 with polar heterocyclic basic groups and thioether bond could be the most promising method to obtain outstanding antibacterial activity (8). Sulfonamides were well known as broad-spectrum antimicrobial antifolic agents. Many hybrid drugs, incorporating moieties both of a sulfonamide and a different antibacterial agent, such as fluoroquinolone, were reported in the literatures as being very active (9–12). Moreover, several pleuromutilin analogs with aromatic or pyridine ring have been described excellent antibacterial activity, especially good inhibition of the growth of penicillin-resistant staphylococci, recently, such as BC 7013 (4) (13–16). According to the report, the method of rational drug design based on the synthesis of dual- or multiple-ligands, can produce drugs of superior clinical effect, compared with single targeted ones (17). Within antibacterial durgs, ‘dual targeting’ compounds may be a prime objective, also because they would reduce the odds of developing
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recorded with Thermo Scientific Orbitrap Elite MS (Thermo Fisher Scientific Inc., Waltham, MA, USA) using the electrospray ionization (ESI) method. Elemental analysis was performed on a Elementar (Germany) Vario EL Elemental Analyzer, for C, H, N and S. The found values for C, H, N, and S were always 0.4% of the theoretical ones.
Figure 1: Pleuromutilin and pleuromutilin derivatives.
bacterial resistance. To prove the impact of the sulfonamide group on the antibacterial activity and antibacterial spectrum of pleuromutilin derivatives, we synthesized and evaluated for antibacterial properties a series of novel pleuromutilin-benzenesulfonamide derivatives via modification of the side chain at C-14 position. The synthetic route was as showed in Figure 2. The antibacterial activity of the hybrids was investigated with drug-susceptible and drug-resistant pathogens by standard procedure in vitro. The results indicated that these molecules inhibited excellent in vitro antimicrobial properties against Gram-positive bacteria such as Staphylococci and streptococci, including methicillin-resistant Staphylococcus aureus (MRSA), and mycoplasma.
Materials and Methods Reagents and analysis Synthetic starting materials, reagents, and all solvents used in this study were purchased from J & K Chemical LTD (Beijing, China) or Sigma–Aldrich. Melting points were determined with XRC-1 melting point apparatus (Sichuan University Instrument Inc., Chengdu, China) without corrected. The IR spectrum was obtained on a Shimadzu IR Affinity-1 spectrometer (potassium bromide disks). 1H NMR spectrum were recorded using a Bruker AVANCE AV400 spectrometer (Bruker BioSpin Corp., Faellanden, Switzerland) in DMSO, and chemical shifts are given in d (ppm) with TMS as internal standard. Mass spectrum were
Chemistry The synthetic process for the preparation of the target compounds is outlined in Figure 2. The structures of the synthesized compounds were supported by the spectral IR, 1H NMR, and 13C NMR data, the results of HR-MS (ESI) and EA which were in agreement with the proposed structures.
14-o-(p-toluene sulfonyloxyacetyl) mutilin (2) Potassium carbonate solution (6.5 mL, 2 M) was slowly dropped to a mixture of pleuromutilin (3.78 g, 10 mmol) and p-toluene sulfonyl chloride (1.91 g, 10 mmol) in the mixed solution of ethyl acetate and water (40 mL, ethyl acetate: water = 1:3, volume). The mixture was stirred at 35 °C for 5 h, then evaporated in vacuum, and the residue was diluted in the water. The precipitates were filtrated and washed with water, dried under vacuum, dissolved in acetone (20 mL), followed by crystallizing for 2 days. Filtration afforded the pure product (2) 4.72 g. White solid; yield: 88.7%; 1H NMR (400 MHz, DMSO-d6) d: 7.79 (2 H, d, J = 4.0 Hz), 7.34 (2 H, d, J = 5.2 Hz), 6.41 (1 H, dd, J1 = 15.1 Hz, J2 = 9.8 Hz), 5.67 (1 H, d, J = 4.4 Hz), 5.25 (1H, d, J = 6.2 Hz), 5.01 (1H, d, J = 9.2 Hz), 4.48 (s, 1H), 3.33–3.37 (1 H, m), 2.41 (3 H, s), 2.02–2.33 (4 H, m), 1.73–1.76 (1 H, m), 1.61–1.68 (2 H, m), 1.60 (3 H, s), 1.43–1.47 (5 H, m), 1.38–1.41 (2 H, m), 1.27 (3 H, s), 0.89 (3 H, d, J = 6.4 Hz), 0.63 (3 H, d, J = 6.4 Hz); 13C NMR (100 MHz, DMSO-d6)d:216.9, 164.7, 145.2, 140.6, 132.2, 130.1, 127.7, 115.3, 72.5, 70.3, 65.7, 57.1, 44.9, 44.1, 43.1, 41.5, 36.4, 36.1, 33.9, 30.0, 29.6, 26.5, 24.4, 21.1, 15.8, 14.4, 11.5.
Figure 2: Scheme of synthesis and structure of compounds 5a–5f and 6a–6f. Reagent and conditions: (i) TsCl, KCO3 (2 M)/ethyl acetate and water, 35 °C, 5 h. (ii) KOH/CH3OH, m-mercaptobenzoic acid, 45 °C, 6 h. (iii) (A) Oxalyl chloride/CH2Cl2, m-mercaptobenzoic acid, C5H5N, N2, rt, 12 h; (B) CH2Cl2, Et3N, THF, reflux, 12 h. (iv) EtONa/ethanol, rt, over 6 h, N2.
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14-O-[(4- carboxy – benzenethiol)acetyl-] mutilin (3) Small portions of potassium hydroxide (0.32 g, 5.6 mmol) were added to a stirred mixture of 2 (5 g, 9.4 mmol) and m-mercaptobenzoic acid (1.5 g, 9.4 mmol) in methanol (40 mL) at room temperature for 30 min and then heated to 45 °C for 6 h. The concentrated reaction mixture was poured into water and crushed ice (80 mL, 20 g), acidified with conc. HCl and stirred at rt for additional 1 h. The mixture was then filtered and washed thoroughly with water (3 9 15 mL) and dried in vacuum. The gained filter residue was purified by silica gel chromatography, to give 3 (2.38 g, 49.2%); mp: 199–201 °C; IR (KBr, cm-1): mmax: 3482.8, 3166.5, 3075.9, 2985.3, 2937.1, 2877.3, 1722.1, 1581.3, 1457.9, 1417.4, 1380.8, 1288.2, 1126.2, 1004.7, 935.3; 1H NMR (400 MHz, DMSO-d6)d: 12.64(1 H, s), 7.28(1 H, s), 7.22–7.26 (1 H, dd, J1 = 7.2 Hz, J2 = 2.6 Hz), 7.21 (1 H, d, J = 7.2 Hz), 7.15 (1 H, d, J = 7.4 Hz), 6.02–6.07 (1 H, dd, J1 = 16.5 Hz, J2 = 9.8 Hz), 5.50 (1 H, d, J = 4.7 Hz), 4.97–4.99 (1 H, d, J = 7.2 Hz),4.91 (1 H, d, J = 8.8 Hz), 4.46 (1 H, s), 3.75– 3.84 (1 H, d, J = 5.3 Hz), 2.37 (1 H, s), 2.15–2.20 (1 H, m), 1.97–2.09 (4 H, m), 1.57–1.66 (2 H, m), 1.44–1.48 (1 H, m), 1.35–1.42 (1 H, m), 1.32 (3 H, s), 1.13–1.28 (3 H, m), 0.99 (3 H, s), 0.81 (3 H, d, J = 5.8 Hz), 0.57 (3 H, d, J = 6.4 Hz). 13C NMR (100 MHz, DMSO-d6) d:217.1, 167.4, 166.7, 140.7, 135.8, 132.4, 131.6, 129.1, 128.6, 127.0, 115.1, 72.5, 69.9, 57.2, 44.9, 44.0, 43.6, 41.4, 36.4, 36.3, 35.0, 34.0, 30.1, 28.6, 26.6, 24.5, 16.0, 14.5, 11.5; HR-MS (ESI) Calcd for C29H38O6S (M+H+): 515.2474; Found: 515.2462.
d:11.14 (1 H, s), 10.33(2 H, s), 7.95 (2 H, d, J = 7.6 Hz), 7.89 (2 H, d, J = 8.1 Hz), 7.48 (1 H, d, J = 7.7 Hz),7.27 (1 H, t), 7.14 (1 H, d, J = 6.6 Hz), 6.98 (1 H, s).
4-(3-mercapto-benzamido)-N-(4, 6-dimethyl-2pyrimidinyl)benzenesulfonamide (5b) Prepared according to the method A. White solid; yield: 53.6%; mp: 198–200 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.84 (1 H, s), 10.66 (1 H, s), 8.11 (1 H, s), 7.98 (2 H, d, J = 5.6 Hz), 7.91 (2 H, d, J = 10.5 Hz), 7.88 (1 H, d, J = 11.2 Hz), 7.78 (1 H, d, J = 6.8 Hz), 7.58 (1 H, t), 6.76 (1 H, s), 2.26 (6 H, s).
3-mercapto-N-[4-[(2-pyridinylamino)sulfonyl] phenyl]-benzamide (5c) Prepared according to the method A. White solid; yield: 62.5%; mp: 186–188 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.54 (1 H, s), 10.66 (1 H, s), 8.15 (1 H, s), 7.75 (2 H, d, J = 6.7 Hz), 7.48 (2 H, d, J = 8.2 Hz), 7.26 (3 H, t), 7.14 (2 H, d, J = 10.9 Hz), 7.07 (1 H, d, J = 4.7 Hz), 6.56 (1 H, d, J = 13.8 Hz),5.80 (1 H, s).
3-mercapto-N-[4-[(2-thizaylamino)sulfonyl]phenyl]benzamide (5d) Prepared according to the method A. White solid; yield: 45.8%; mp: 169–171 °C; 1
Method A: general procedure for the synthesis of sulfonamide-thiophenol 5a–f Oxalyl chloride (1.65 g, 13 mmol) in dichloride methane (20 mL) was dropped slowly to an ice-cooled solution of m-mercaptobenzoic acid (2.1 g, 13 mmol) and pyridine (0.47 mL, 5.8 mmol) in dichloride methane (30 mL). The cooling bath was then removed and the resultant solution stirred for 12 h at room temperature under an atmosphere of nitrogen before being concentrated to dryness in vacuum. The obtained residue was dissolved in dichloride methane (50 mL) and added slowly to a mixture of sulfanilamide 4a–f (13 mmol) and triethylamine (1.79 mL, 13 mmol) in THF (30 mL). The reaction mixture was then heated to reflux for 12 h, and the volatiles were removed at reduced pressure. The residue was then dissolved in ethyl acetate (30 mL), and the mixture was washed with 1 M hydrochloric acid and brine. The organic phase was then dried (magnesium sulfate), concentrated, and the residue purified by silica gel chromatography to give 5a–f.
3-mercapto-N-[4-(amino-sulfonyl)phenyl]benzamide (5a) Prepared according to the method A. White solid; yield: 50.9%; mp: 179–181 °C; 1H NMR (400 MHz, DMSO-d6) Chem Biol Drug Des 2015; 86: 239–245
H NMR (400 MHz, DMSO-d6) d:11.25 (1 H, s), 10.54 (1 H, s), 7.98 (2 H, d, J = 5.8 Hz), 7.86 (3 H, d, J = 7.9 Hz), 7.66 (1 H, d, J = 16.4 Hz), 7.51 (1 H, d, J = 12.1 Hz), 7.43 (1 H, t), 6.89 (1 H, s), 6.27 (1 H, s).
3-mercapto-N-[4-[(6-methoxy-2-pyrimidinylamino) sulfonyl]phenyl]-benzamide (5e) Prepared according to the method A. White solid; yield: 68.2%; mp: 190–192 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.73 (1 H, s), 10.54 (1 H, s), 8.31 (1 H, s), 7.88–7.95 (5 H, m), 7.72 (1 H, d, J = 15.6 Hz), 7.58 (1 H, s), 7.44 (1 H, t), 6.31 (1 H, s), 3.78 (3 H, d, J = 6.8 Hz).
3-mercapto-N-[4-[(5-methoxy-3- isoxazolylamino) sulfonyl]phenyl]-benzamide (5f) Prepared according to the method A. White solid; yield: 72.9%; mp: 175–177 °C; 1
H NMR (400 MHz, DMSO-d6) d:11.37 (1 H, s), 10.64 (1 H, s), 7.97 (2 H, d, J = 6.4 Hz),7.87 (3 H, d, J = 3.4 Hz), 7.67 (1 H, d, J = 5.7 Hz), 7.53 (1 H, d, J = 10.5 Hz), 7.41 (1 H, t), 6.14 (1 H, s), 5.78 (1 H, s), 2.29 (3 H, s). 241
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Method B: general procedure for the synthesis of sulfoether A solution of 5a–f (10 mmol) in ethanol was added to an ice-cooled mixture of sodium ethoxide (1.72 g, 25 mmol) in ethanol (50 mL) under a nitrogen atmosphere. After 1 h, the resultant solution was treated with 2 (5.41 g, 10 mmol) in acetone (80 mL) and stirred over 6 h at rt. The reaction mixture was then concentrated at reduced pressure, and partitioned between ethyl acetate and water and the aqueous phase re-extracted with ethyl acetate. The combined organic extracts were washed with water and brine, dried (magnesium sulfate) and concentrated. The residue was purified by silica gel chromatography to give 6a–f.
14-O-[[3-[[[4-(aminosulfonyl) phenyl] amino] carbonyl] phenylthio] acetyl-] mutilin (6a) Prepared according to the method B. White solid; yield: 57.5%; mp: 195–197 °C; IR (KBr, cm 1) mmax: 3340.1, 3263, 3097.1, 2981.4, 2935.1, 2875.3, 1722.1, 1672, 1592.9, 1525.4, 1461.8, 1394.3, 1322.9, 1261.2, 1157.1, 1114.7, 1018.2; 1H NMR (400 MHz, DMSO-d6) d:11.32 (1 H, s), 10.56 (1 H, s), 7.90 (2 H, d, J = 6.5 Hz), 7.75 (1 H, d, J = 8.4 Hz), 7.59 (1 H, d, J = 7.3 Hz), 7.46 (1 H, t), 7.36 (2 H, s), 7.25–7.29 (1 H, dd, J1 = 8.3 Hz, J2 = 6.7 Hz), 6.75 (1 H, s), 6.01–6.05 (1 H, dd, J1 = 14.3 Hz, J2 = 6.1 Hz), 5.51 (1 H, d, J = 5.5 Hz), 4.92–4.94 (1 H, d, J = 6.2 Hz), 4.90 (1 H, d, J = 7.4 Hz), 4.49 (1 H, s), 3.75–3.88 (2 H, dd, J1 = 10.6 Hz, J2 = 4.3 Hz), 2.35 (1 H, s), 2.27 (6 H, s), 2.15–2.20 (1 H, m), 1.96–2.09 (3 H, m), 1.54–1.64 (2 H, m), 1.32–1.49 (2 H, m), 1.31 (3 H, s), 1.20–1.25 (1 H, t), 1.12–1.19 (2 H, m), 0.98 (3 H, s), 0.76 (3 H, d, J = 4.6 Hz), 0.56 (3 H, d, J = 6.3 Hz); 13C NMR (100 MHz, DMSO-d6) d:217.1, 167.5, 165.1, 142.0, 140.7, 138.8, 135.8, 135.0, 131.3, 129.1, 127.0, 126.5, 125.5, 119.9, 115.1, 72.5, 69.9, 57.2, 44.9, 44.0, 43.9, 41.4, 36.4, 36.3, 34.0, 30.1, 28.5, 26.6, 24.4, 16.1, 14.5, 11.7; HR-MS (ESI) Calcd for C35H45N2O7S2 (M+H+): 669.2663; Found: 669.2658; Anal. Calcd for C35H44N2O7S2 (668.26): C, 62.85; H, 6.63; N, 4.19; S, 9.58. Found: C, 62.73; H, 6.69; N, 4.15; S, 9.40.
14-O-[[3-[[[4-N-[(4,6-dimethyl-2-pyrimidinylamino) sulfonyl]phenyl]amino]carbonyl]phenylthio] acetyl-] mutilin (6b) Prepared according to the method B. White solid; yield: 48.0%; mp: 218–220 °C; IR (KBr, cm 1): 3444.2, 3382.5, 3104.8, 2935.1, 2873.4, 1726, 1677.8, 1596.8, 1525.4, 1434.8, 1313.3, 1265.1, 1155.2, 1118.5, 1083.8, 1020.2; 1 H NMR (400 MHz, DMSO-d6) d:11.5 6 (1 H, s), 10.55 (1 H, s), 7.93–7.99 (5 H, m), 7.75 (1 H, d, J = 6.9 Hz), 7.59 (1 H, d, J = 2.8 Hz), 7.47 (1 H, t), 6.76 (1 H, s), 5.97–6.04 (1 H, dd, J1 = 5.3 Hz, J2 = 17.4 Hz), 5.51 (1 H, d, J = 7.2 Hz), 4.89–4.92 (2 H, dd, J1 = 13.2 Hz, J2 = 4.5 Hz), 4.54 (1 H, s), 3.96–3.89 (2 H, dd, J1 = 8.9 Hz, J2 = 6.8 Hz), 2.34 (1 H, s), 2.26 (6 H, s), 2.14–2.19 (1 H, m), 1.94–2.08 (3 H, m), 1.56–1.63 (2 H, 242
m), 1.31–1.46 (2 H, m), 1.29 (3 H, s), 1.23–1.25 (1 H, t), 1.12–1.20 (2 H, m), 0.94 (3 H, s), 0.78 (3 H, d, J = 6.0 Hz), 0.57 (3 H, d, J = 7.8 Hz). 13C NMR (100 MHz, DMSO-d6) d:217.6, 167.9, 165.6, 156.2, 141.1, 140.7, 136.3, 135.4, 131.6, 129.5, 127.4, 125.9, 119.8, 115.5, 113.5, 73.0, 70.4, 57.7, 45.4, 44.4, 44.1, 41.9, 36.8, 36.7, 35.4, 34.4, 30.5, 28.9, 26.9, 24.8, 16.5, 14.9, 11.9; HR-MS (ESI) Calcd for C41H51N4O7S2 (M + H+): 775.3193; Found: 775.3186; Anal. Calcd for C41H50N4O7S2 (774.31): C, 63.54; H, 6.51; N, 7.23; S, 8.27. Found: C, 63.41; H, 6.68; N, 7.25; S, 8.11.
14-O-[[3-[[[4-N-[(2-pyridinylamino)sulfonyl]phenyl] amino]carbonyl]phenylthio] acetyl-] mutilin (6c) Prepared according to the method B. White solid; yield: 72.1%; mp: 212–214 °C; IR (KBr, cm 1): 3494.4, 3357.5, 2935.1, 2875.3, 2763.5, 1726, 1677.8, 1629.6, 1594.8, 1523.5, 1461.8, 1390.4, 1261.2, 1141.7, 1093.4, 1010.5; 1 H NMR (400 MHz, DMSO-d6) d:11.7 2 (1 H, s), 10.50 (1 H, s), 8.03 (1 H, s), 7.99–7.93 (5 H, m), 7.77 (1 H, d, J = 6.7 Hz), 7.69 (1 H, d, J = 7.2 Hz), 7.58 (1 H, d, J = 7.5 Hz), 7.46 (1 H, t), 7.15 (1 H, d, J = 9.3 Hz), 6.88 (1 H, t), 6.01–6.06 (1 H, dd, J1 = 6.1 Hz, J2 = 8.5 Hz), 5.50 (1 H, d, J = 16.5 Hz), 4.94–4.97 (2 H, m), 4.51 (1 H, s), 3.94–4.02 (2 H, dd, J1 = 10.9 Hz, J2 = 5.4 Hz), 2.37 (1 H, s), 2.14–2.18 (1 H, m), 1.97–2.03 (3 H, m), 1.55–1.64 (2 H, m), 1.43–1.46 (1 H, m), 1.37–1.41 (1 H, m), 1.34 (3 H, s), 1.15–1.24 (3 H, m), 0.98.00 (3 H, s), 0.76 (3 H, d, J = 6.4 Hz), 0.57 (3 H, d, J = 8.3 Hz); 13C NMR (100 MHz, DMSO-d6) d:217.6, 167.8, 165.5, 143.0, 141.2, 141. 1, 131.6, 128.7, 128.1, 127.1, 120.3, 115.6, 73.0, 70.5, 57.6, 45.4, 44.5, 44.0, 36.8, 34.7, 34.4, 30.6, 29.0, 27.0, 24.9, 16.6, 15.0, 12.0; HR-MS (ESI) Calcd for C40H48N3O7S2 (M + H+): 746.2928; Found: 746.2923; Anal. Calcd for C40H47N3O7S2 (745.29): C, 64.45; H, 6.35; N, 5.63; S, 8.60. Found: C, 64.60; H, 6.52; N, 5.43; S, 8.41.
14-O-[[3-[[[4-N-[(2-thizaylamino)sulfonyl]phenyl] amino]carbonyl]phenylthio] acetyl-] mutilin (6d) Prepared according to the method B. White solid; yield: 63.4%; mp: 198.8–201 °C; IR (KBr, cm 1): 3542.6, 3305.4, 3102.9, 2929.3, 2875.3, 2813.6, 2684.4, 1726, 1662.3, 1591, 1527.3, 1405.9, 1315.2, 1261.2, 1143.6, 1089.6, 1079.9, 1016.3; 1H NMR(400 MHz, DMS O-d6) d:11.72 (1 H, s), 10.60 (1 H, s), 7.99 (2 H, d, J = 6.8 Hz), 7.93 (1 H, d, J = 9.3 Hz), 7.85 (2 H, d, J = 7.5 Hz), 7.76 (1 H, d, J = 6.4 Hz), 7.61 (1 H, d, J = 3.4 Hz), 7.50 (1 H, d, J = 6.0 Hz), 6.60 (1 H, d, J = 4.2 Hz) 6.33 (1 H, d, J = 5.7 Hz), 5.98–6.05 (1 H, dd, J1 = 5.7 Hz, J2 = 19.1 Hz), 5.52 (1 H, d, J = 4.9 Hz), 4.91–4.98 (2 H, m), 4.48 (1 H, s), 3.92–3.98 (2 H, dd, J1 = 16.1 Hz, J2 = 5.2 Hz), 2.37 (1 H, s), 2.17–2.21 (1 H, m), 1.96–2.05 (3 H, m), 1.44–1.64 (3 H, m), 1.36–1.40 (1 H, m), 1.32 (3 H, s), 1.15–1.25 (3 H, m), 0.97 (3 H, s), 0.79 (3 H, d, J = 3.5 Hz), 0.55 (3 H, d, J = 9.6 Hz). 13C NMR (100 MHz, DMSO-d6) d:217.1, 170.3, 168.7, 165.2, 152.3, Chem Biol Drug Des 2015; 86: 239–245
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142.4, 141.3, 136.7, 135.7, 131.1, 129.2, 128.3, 127.9, 126.8, 126.1, 124.5, 119.7, 108.1, 73.1, 70.5, 65.1, 59.8, 57.1, 49.8, 44.9, 44.0, 43.8, 41.5, 36.7, 36.3, 34.0, 30.4, 28.7, 26.7, 24.5, 16.4, 14.5, 11.5; HR-MS (ESI) Calcd for C38H46N3O7S3 (M + H+): 752.2492; Found: 752.2487; Anal. Calcd for C38H45N3O7S3 (751.25): C, 60.61; H, 6.16; N, 5.58; S, 12.78. Found: C, 60.52; H, 6.29; N, 5.66; S, 12.53.
135.9, 134.9, 133.6, 131.3, 129.1, 127.9, 127.0, 125.6, 120.1, 115.1, 95.4, 72.6, 70.0, 57.2, 44.9, 44.0, 43.9, 41.5, 36.4, 36.3, 35.0, 34.0, 30.1, 28.5, 26.5, 24.4, 16.1, 14.5, 12.1, 11.5; HR-MS (ESI) Calcd for C39H48N3O8S2 (M+H+): 750.2877; Found: 750.2872; Anal. Calcd for C39H47N3O8S2 (749.29): C, 62.46; H, 6.32; N, 5.61; S, 8.55. Found: C, 62.57; H, 6.46; N, 5.75; S, 8.47.
14-O-[[3-[[[4-N-[(6-methoxy-2-pyrimidinylamino) sulfonyl] phenyl] amino]carbonyl]phenylthio] acetyl-] mutilin (6e) Prepared according to the method B. White solid; yield: 65.4%;mp: 220–222.5 °C; IR (KBr, cm 1): 3486.7, 3093.3, 2939, 2875.3, 1726, 1681.6, 1625.7, 1591, 1523.5, 1481.1, 1392.4, 1317.1, 1282.4, 1253.5, 1151.3, 1093.4; 1H NMR (400 MHz, DMSO-d6) d:11.8 5 (1 H, s), 10.60 (1 H, s), 8.42 (1 H, s), 7.89–7.97 (5 H, m), 7.75 (1 H, d, J = 7.0 Hz), 7.60 (1 H, s), 7.47 (1 H, t), 6.34 (1 H, s), 5.96–6.03 (1 H, dd, J1 = 12.7 Hz, J2 = 8.9 Hz), 5.50 (1 H, d, J = 4.8 Hz), 4.88–4.92 (2 H, t), 4.50 (1 H, s), 3.92– 4.01 (2 H, dd, J1 = 8.6 Hz, J2 = 11.1 Hz), 3.93 (3 H, s), 2.35 (1 H, s), 2.143–2.18 (1 H, m), 1.97–2.07 (3 H, m), 1.55–1.65 (2 H, m), 1.41–1.48 (1 H, m), 1.34–1.38 (1 H, m), 1.29 (3 H, s), 1.15–1.23 (3 H, m), 0.95 (3 H, s), 0.79 (3 H, d, J = 4.4 Hz), 0.55 (3 H, d, J = 5.2 Hz). 13C NMR (100 MHz, DMSO-d6) d: 217.7, 170.4, 167.8, 165.7, 158.9, 143.6, 141.3, 141.1, 131.5, 128.7, 128.4, 127.0, 120.4, 115.7, 91.3, 73.0, 70.5, 57.7, 54.7, 45.4, 44.5, 44.1, 36.8, 34.4, 30.5, 29.0, 27.0, 24.9, 16.6, 15.0, 11.9; HR-MS (ESI) Calcd for C40H49N4O8S2 (M + H+): 777.2986; Found: 777.2971; Anal. Calcd for C40H48N4O8S2 (776.29): C, 61.83; H, 6.23; N, 7.21; S, 8.23. Found: C, 61.85; H, 6.36; N, 7.11; S, 8.15.
Bacterial strains Staphylococcus aureus ATCC 29213, S. aureus CMCC 26003, Staphylococcus equi CICC 10373, S. equi CVCC 3307, E. coli ATCC 25922, and M. gallisepticum S6 were purchased from China Institute of Veterinary Drugs Control. MRSA (11) was separated from diseased pig.
14-O-[[3-[[[4-N-[(5-methyl-3- isoxazolylamino) sulfonyl] phenyl]amino]carbonyl]phenylthio] acetyl-] mutilin (6f) Prepared according to the method B. White solid; yield: 45.8%; mp: 215–217 °C; IR (KBr, cm 1): 3492.5, 3372.9, 3097.1, 2937.1, 2875.3, 1724, 1679.7, 1614.1, 1592.9, 1519.6, 1461.8, 1396.2, 1319.1, 1265.1, 1160.9, 1118.5, 1095.4, 1018.2; 1H NMR (400 MHz, D MSO-d6) d: 11.40 (1 H, s), 10.62 (1 H, s), 7.98 (2 H, d, J = 9.7 Hz), 7.90 (1 H, s), 7.86 (2 H, d, J = 3.9), 7.75 (1 H, d, J = 6.5 Hz), 7.60 (1H, d, J = 5.9 Hz), 7.48 (1 H, t), 6.15 (1 H, s), 5.97–6.05 (1 H, dd, J1 = 5.8 Hz, J2 = 16.7 Hz), 5.49 (1 H, d, J = 6.3 Hz), 4.88–4.93 (2 H, dd, J1 = 7.5 Hz, J2 = 4.2 Hz), 4.50 (1 H, d, J = 5.2 Hz), 4.49 (1 H, s), 3.93–3.96 (2 H, dd, J1 = 19.4 Hz, J2 = 6.6 Hz), 2.36 (1 H, s), 2.30 (3 H, s), 2.12–2.19 (1 H, m), 1.95–2.08 (3 H, m), 1.53–1.63 (2 H, m), 1.31– 1.45 (2 H, m), 1.29 (3 H, s), 1.20–1.24 (1 H, t), 1.11– 1.18 (2 H, m), 0.94 (3 H, s), 0.77 (3 H, d, J = 11.3 Hz), 0.56 (3 H, d, J = 4.7 Hz); 13C NMR (100 MHz, D MSOd6) d: 217.1, 170.3, 167.4, 165.3, 157.6, 143.4, 140.7, Chem Biol Drug Des 2015; 86: 239–245
Antimicrobial activity By the broth microdilution method, the new compounds and standard antibacterial agents (valnemulin and sulfanilamide) were detected in vitro for their antimicrobial activities against several typical pathogenic micro-organisms (Table 1) on the base of CLSI guideline. The compounds under test were dissolved in DMSO and diluted in the Muller–Hinton broth to give the concentrations ranging from 0.016 to 128 lg/mL. To ensure that the solvent had no impact on the growth of bacteria, a control test with the same medium as used in the experiment was carried out. An inocula of 1 9 106 CFU/mL which was prepared by diluting a bacteria solution (about 0.5 McFarland standard) was added. After cultured for 18–20 h at 37 °C (bacterial) and for 36 h at 37 °C in 5% CO2 atmosphere (mycoplasma), the minimum inhibitory concentrations (MIC, lg/mL) of compounds were determined, which resulted in no visible growth on the plate. Compound should be further diluted and tested according to the same process as above until the MICs were determined; in case, it could inhibit the growing of the bacteria at the concentration of 0.016 lg/mL.
Results and Discussion Chemistry The synthetic process of the new compounds is shown in Figure 2. The reaction of pleuromutilin (1) with equal chemical equivalent of p-toluenesulfonyl chloride in the presence of KCO3 at 35°C gave the intermediate 2. Treatment of mmercaptobenzoic acid with potassium hydroxide in absolute methanol followed by reaction with compound 2 at 45 °C provided the compound 3 in good yield. The m-mercaptobenzoic acid was treated with oxalyl chloride under the action of pyridine, in a cooling bath, followed by evaporating excess oxalyl chloride in vacuum, and then, 243
Chen et al. Table 1: In vitro antimicrobial activities of compounds 6a–f (MICs in lg/mL) MIC (lg/mL)
Compound
S. aureus ATCC 29213
S. aureus CMCC 26003
MRSA 11
S. equi CICC 10373
S. equi CVCC 3307
E. coli ATCC 25922
MG S6
6a 6b 6c 6d 6e 6f 3 Sulfanilamide Valnemulin
0.016 0.125 0.016 0.008 0.25 0.063 0.125 >128 0.016
0.125 4 0.063 0.5 4 4 32 8 0.5
0.032 8 0.016 0.25 16 8 0.5 >128 0.5
0.125 0.5 0.063 0.5 4 8 0.5 16 0.063
0.063 4 0.032 0.125 16 1 8 32 0.25
64 >128 16 64 >128 >128 >128 128 16
0.004 0.032 0.004 0.008 0.032 0.016 0.032 2 0.004
MG, M. gallisepticum (S6); MRSA, methicillin-resistant Staphylococcus aureus; S. equi, Staphylococcus equi.
the residue reacted with sulfanilamides (4a–f) at reflux in THF with triethylamine, to afford the crucial intermediates sulfonamide-thiophenol 5a–f. Compounds 6a–f were prepared by reacting sulfonamide-thiophenol 5a–f with sodium ethoxide in ethanol at 0–5 °C for 1 h, followed by reaction with compound 2 at room temperature for over 6 h, and purified on silica gel column chromatography.
Biological activity The in vitro antimicrobial activity of hybrids (6a–6f) was evaluated, and their minimum inhibitory concentration values (MICs) against various bacteria were determined by comparison to the parent benzoxy pleuromutilin derivative (3) and sulfanilamide, commercial antibiotic valnemulin as reference drug (Table 1). The MIC values of 6 novel synthetic pleuromutilin-sulfanilamide hybrids against Gram-positive pathogens indicated that some compounds possess a comparable or better activity compared with valnemulin. These new hybrids also exhibited potent antibacterial activity against MRSA and M. gallisepticum. Compound 6d exhibited the most potent inhibitory activity against S. aures (MIC = 0.008 lg/mL), and compound 6c showed the most inhibitory activity against S. epidemidis (MIC = 0.032 lg/mL), which were twofold and eightfold more potent than reference drug valnemulin, respectively. The MIC values of compound 6a–f against methicillin-resistant Staphylococcus aureus (MRSA 11) indicated that sulfanilamide analogs 6a and 6c showed excellent antibacterial activity (MIC = 0.032, 0.016 lg/mL). Their activities were 16- and 32-fold more than that of valnemulin (MIC = 0.5 lg/mL). Additionally, compound 6d exhibited potent activity (MIC = 0.25 lg/mL) comparable to valnemulin. Furthermore, the data obtained exhibited that compounds 6a, 6c, and 6d have more or equal inhibitory activity against M. gallisepticum (MIC = 0.004– 0.008 lg/mL), in contrast with reference drug valnemulin (MIC = 0.004 lg/mL). On the other hand, most tested compounds had less in vitro activity against Gram-negative bacterium E. coli, but were more active than reference 244
drugs sulfanilamide, with the exception of 6c (MIC = 16 lg/mL), which was eightfold more effective than sulfanilamide (MIC = 128 lg/mL). All the new hybrids showed more potent antibacterial activity than that of sulfanilamide. Comparison between MIC values of pleuromutilin-sulfonamide hybrids 6a–f with pleuromutilin parent compound 3 revealed that these modifications at 14 position side chain of the pleuromutilin with benzene sulfonamide could greatly improve the antibacterial activity especially against Gram-positives. Moreover, Compound 6a, having a terminal sulfa group at the 14 position side chain of the hybrid, is more potent than their corresponding hybrids 6b, 6e, and 6f, particularly against Gram-positives. Similarly, the antibacterial activity seems to be enhanced by the azine group, because the pyridine substituted hybrid 6c was more active than the unsubstituted analogs 6a.
Conclusion In conclusion, a series of novel pleuromutilin-sulfonamide hybrids were synthesized and evaluated. The results of antibacterial activities indicated that the hybrids gained appreciable antibacterial activity against Gram-positives when the sulfonamide was introduced into the C-14 side chain of pleuromutilin, especially the hybrids 6a and 6c which antibacterial activity has been improved clearly. More importantly, our discovery had provided a new approach to researching and developing novel potent antibiotic.
Acknowledgments We appreciate the financial support from the National Science Foundation of China (No. 31172359) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13063), NMR and Elemental analysis provided by SUN YAT-SEN University Instrumental Analysis & Research Center. Chem Biol Drug Des 2015; 86: 239–245
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