Synthesis and Biological Evaluation of Novel Thioether Pleuromutilin Derivatives

Abstract

To develop new pleuromutilin derivatives as veterinary antibiotic medicines, we designed and synthesized a series of new thioether pleuromutilin derivatives possessing acylthiazolyl moiety based on previously designed derivatives. The antibacterial properties of the prepared pleuromutilin derivatives were assessed in vitro by the broth dilution method against five kinds of bacteria and the mycoplasma Mycoplasma gallisepticum (MG). All of the tested compounds displayed moderate to good antibacterial activity to methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-sensitive Staphylococcus epidermidis (MSSE), methicillin-resistant S. aureus (MRSA), Streptococcus agalactiae (S. aga) and MG. However, the activity to Pyogeniccoccus (Pyogens) was generally poor. Compounds 13i and l showed potent antibacterial activity against MSSE and MRSA which are better than that of valnemulin. The structural modification for pleuromutilin affected the antibacterial activity. Amino substituents in the benzene ring can effectively improve activity. Compared with the analogue 13a that possesses unsubstitution benzoyl group, the nitro, methoxy, hydroxy and dichloro substituent contributed little to antibacterial activity. Increasing a methylene between benzene moiety and carbonyl group decreased the bioactivity of derivative. The analogues that obtained by the reaction of amino acids and intermediate 9 showed moderate activity.

In 1951, Kavanagh first isolated the fused 5-6-8 tricyclic diterpenoid from Clitopilus scyphoides and Pleurotus passckerianus and named it as pleuromutilin (1)^1,2) (Fig. 1). Compound 1 was characterized as antibiotic with modest in vitro activity against Gram-positive bacteria and mycoplasmas.^3,4) Further structural studies of complexes of ribosomal subunits with pleuromutilin derivatives suggested that this class of antibiotics selectively inhibited the synthesis of bacterial protein by interaction with the 23S ribosomal RNA (rRNA) of the 50S bacterial ribosome subunit. Thus, this class of antibiotics prevents the correct positioning of the tRNA for peptide transfer and inhibits peptide bond formation.^5,6) The specific interactions of pleuromutilin drugs with the ribosome lead to the difficulty of pleuromutilin to produce cross resistance with other antimicrobial agents. Subsequently, Davidovich et al. found that these compounds bind to the peptidyl transferase center (PTC) with their core at A-site and their C14 extensions pointing towards the P-site. The C11 hydroxyl group was located in a position suitable for hydrogen bonding to G2505 phosphate, and the C21 keto group of the C14 extension was involved in two to three H-bonds with G2061.⁷⁾

Fig. 1. Structure of Pleuromutilin Derivatives and NTZ

Since pleuromutilin was determined to have good antibacterial activity, much synthetic effort had been done by using it as the lead compound. In 1976, Egger and Reinshagen synthesized 66 derivatives of pleuromutilin and tried to observe the relationship between the structure and antibacterial activity. Their group concluded that the carbonyl group of the five-membered ring and the C11-OH were necessary activity functional groups, but there was no effect on the antibiotic activity whether the exocyclic vinyl group was hydrogenated or not. At the same time, the glycolate ester at 14 position was also necessary because the activity disappeared if pleuromutilin was transformed into mutilin with deacylation. Their group also suggested that the modifications of C14 side chain with a thioether group and a basic group offered the best bioactivities.^8,9) Subsequently, tiamulin (2) and valnemulin (3) were successfully developed as the veterinary antibiotic medicine.^3,10) As the first pleuromutilin class developed for human use, azamulin (4) is safe and has good antibacterial activity. Unfortunately, poor water solubility limits the bioavailability of azamulin and it has a strong irreversible inhibition effect on CYP P450, which leads to discontinued clinical research.¹¹⁾ Retapamulin (5), which was developed by GlaxoSmithKline in 2007, is the first pleuromutilin drug for human use as a topical antimicrobial agent to treat impetigo and secondarily infected traumatic lesions of skin infections.¹²⁾

More recently, BC-3205 (6), BC-7013 (7) and BC-3781 (8) are the new derivatives of pleuromutlin reported by Nabriva Therapeutics in the clinical research stage.¹³⁾ In 2011, 8 has successfully completed Phase II clinical trials and may be developed as the first systemic pleuromutilin antibiotic to treat acute bacterial skin and skin structure infections (ABSSSI).¹⁴⁾ In summary, all of these compounds either licensed or in clinical trials were attached with the different basic group through the thioether bond in C14 side chain. In 2012, Wang et al. reported a series of derivatives that were synthesized using 14-O-(2-aminothiazol)-4-methyl thioether mutilin (9) as the key intermediate.¹⁵⁾ These compounds had shown good antibacterial activity to methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-sensitive Staphylococcus epidermidis (MSSE), which were isolated from clinical bacteria. In 2014, Shang et al. reported a series of derivatives possessing substituted benzeneacyl group on the thiadiazole moiety of the 14-position side chain. Compound with meta amino substituted in the benzene ring showed the best antibacterial derivative among substituted benzeneamide derivatives.¹⁶⁾ Derivatives with an amino group on the terminal C14 side chain presented improved activity.¹⁷⁾ In addition, some efforts were focused on structural modification of valnemulin, one kinds of derivatives via modification the amino of dimethyl cysteine moiety of C14 side chain by general heterocyclic acid or chloromate or substituted benzene carboxylic acid.^18,19) Shang et al. found that the stronger resonance effect of substituents, like methoxy or amino, the more excellent antibacterial activity.¹⁸⁾ Xu et al. reported this kind of derivatives with furamide or pyrazolecarboxamide with excellent bioactivity.¹⁹⁾ Ling et al. systematically described the activity of pleuromutilin derivatives possessing different heterocyclic group on C14 side chain and the bioactivity data showed that derivatives containing pyridine ring exhibited the best activity. And a heterocyclic ring at the C14 position of pleuromutilin derivatives may increase hydrogen bonding and π–π stacking interactions and thereby derivatives have strong antibacterial properties.^15,20)

Nitazoxanide (NTZ) is a broad-spectrum anti-infective and antiviral agent,²¹⁾ has a 2-acetyloxybenzamide-thiazole structure. We concerned whether the anti-infective activity could be improved when NTZ was used as a substituent at the C14 position to modify pleuromutilin. Therefore, combined with the above mentioned ideas, we designed some novel analogues by using compound 9 as a lead compound. We expected that derivatives containing aromatic amide group and heterocyclic ring will improve their activity through their resonance effect. Derivative that possesses 2-hydroxybenzamide group attached on thiazole is similar to that of NTZ with extended antimicrobial spectrum and better activity. In addition, we designed and synthesized three derivatives, which were obtained by the reaction of amino acid and intermediate 9. They have an amino group on C-14 side chain may improve bioactivity through the similar effects as tiamulin and valnemulin. A total of 15 new derivatives were synthesized by the amino of thiazol acylation reaction¹⁸⁾ with a series of aromatic carboxylic acids, heterocyclic carboxylic acids and amino acids. The in vitro antibacterial activities of the prepared compounds were assessed by the broth dilution method against five bacteria, with erythomycin, tiamulin and valnemulin as positive controls. The control drug for Mycoplasma gallisepticum (MG) was tylosin instead of erythromycin.

MATERIALS AND METHODS

General

Melting point was measured on a WRR-Y drug melting point instrument (Shenguang, China); the thermometer reading was not corrected. IR spectra were obtained by a Nicolet IS10 spectrometer (Thermo Fisher Scientific, U.S.A.) with KBr thin films. ¹H-NMR spectra were obtained on an Avance 600/400 MHz spectrometer (Bruker, Switzerland). ¹³C-NMR data were collected on an Avance 150/100 MHz instrument (Bruker) with complete proton decoupling. The chemical shift values (δ) were expressed in parts per million (ppm). The internal standard was tetramethylsilane (TMS). High-resolution (HR)-MS data were obtained on Q-TOF (Waters & Bruker, U.S.A. and Switzerland). All reagents and solvents (AR) were purchased from the Sinopharm Chemical Reagent and Aladdin Reagent companies without further purification.

Chemistry

The synthetic route of the target compounds 13a–o is shown in Chart 1. First, pleuromutilin (1) reacts with 4-toluene sulphonyl chloride in the presence of triethylamine (TEA) to afford 14-O-(p-toluene sulphonyloxyacetyl) mutilin (11). Iodide 12 is obtained with good yield by the nucleophilic attack of potassium iodide on 11. Compound 12 reacts with S-(2-amino-4-thiazolylmethyl)-isothiourea dihydrochloride in the presence of 10% NaOH aqueous solution in tetrahydrofuran (THF) to afford 9.^22,23) Compound 9 is further treated with carboxylic acid or N-tert-butoxy-carbonyl (Boc) protected amino acid in presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT) to give the compounds 13a–g, 13j–l and 14h–i, 14m–o at room temperature in CH₂Cl₂. Compound 14h–i, 14m–o is further treated with trifluoroacetic acid (TFA) in CH₂Cl₂ and turns into the final compound 13h–i, 13m–o. Compound 13g was got from acetosalicylic acid instead of salicylic acid by using the general method without extra possessing. All of the target products were purified by column chromatography; their structures were characterized by IR, ¹H-NMR, ¹³C-NMR and HR-MS.

Chart 1. Reaction Conditions and Reagents

(a) TsCl, Et₃N, CH₂Cl₂, r.t., 22 h; (b) KI, acetone, reflux, 1.5 h; (c) S-(2-amino-4-thiazolylmethyl)-isothiourea dihydrochloride, 10% NaOH, THF, −5°C, 2 h; (d) carboxylic acid, EDCI and TEA or DCC, HOBT, CH₂Cl₂, 32–48 h; (e) TFA, CH₂Cl₂, r.t., 0.5 h.

Synthesis

14-O-(p-Toluene Sulphonyloxyacetyl) Mutilin (11)

Pleuromutilin (10.00 g) and TEA (2.95 g, 1.1 eq) were dissolved in 80 mL CH₂Cl₂. 4-Toluene sulphonyl chloride (5.53 g, 1.1 eq) was added to the above mentioned solution by batch. The mixture was stirred at room temperature for 22 h. The reaction mixture was washed with brine, dried with anhydrous sodium sulphate, filtered and evaporated in a vacuum. After recrystallization with ethyl acetate (EA) : petroleum ether (PE)=1 : 1 (v/v), compound 11 was produced (8.44 g, 60%). mp 144.6–146.3°C. ¹H-NMR (CDCl₃) δ: 7.74 (d, J=7.7 Hz, 2H), 7.28 (d, J=7.9 Hz, 2H), 6.34 (dd, J=17.4, 11.0 Hz, 1H), 5.70 (d, J=8.6 Hz, 1H), 5.27 (d, J=11.7 Hz, 1H), 5.12 (d, J=17.4 Hz, 1H), 4.48–4.34 (m, 2H), 3.27 (dd, J=10.5, 6.6 Hz, 1H), 2.38 (s, 3H), 2.24–2.08 (m, 3H), 2.02–1.94 (m, 2H), 1.69 (dd, J=14.5, 2.0 Hz, 1H), 1.61–1.54 (m, 2H), 1.52 (s, 3H), 1.46–1.35 (m, 3H), 1.34 (s, 3H), 1.28 (dd, J=14.4, 3.0 Hz, 1H), 1.16 (dd, J=22.2, 11.1 Hz, 1H), 1.10–1.00 (m, 4H), 0.80 (d, J=6.9 Hz, 3H), 0.55 (d, J=7.0 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.74, 164.86, 145.28, 138.64, 132.56, 129.89, 128.07, 117.40, 74.50, 70.24, 65.00, 57.99, 45.36, 44.46, 43.93, 41.80, 36.51, 35.98, 34.37, 30.30, 26.73, 26.33, 24.78, 21.67, 16.51, 14.73, 11.46. IR (KBr) cm⁻¹: 3447, 2941, 1732, 1453, 1361, 1307, 1117, 1035, 832, 664, 560. MS m/z: 577.2 [M−H]⁻.

14-O-1-Iodide Mutilin (12)

Compound 11 (3.00 g) was reacted with potassium iodide (1.07 g, 1.18 eq) in 100 mL acetone with refluxing for 1.5 h. After the reaction was completed, the reaction solution was concentrated. The residue was dissolved with CH₂Cl₂ and washed three times with a small amount of water. The organic phase was separated, dried, filtered and evaporated in a vacuum to obtain the white solid 12 (1.10 g) without purification, with a yield of 87%. mp 112.2–113.3°C. IR (KBr) cm⁻¹: 3585, 3301, 2989, 2933, 1735, 1711, 1454, 1278, 1084, 1023, 985, 976. MS m/z: 533.1 [M−H]⁻ (Calcd for C₂₉H₄₀NO₇S: 532.6981).

14-O-(2-Aminothiazol)-4-methyl Thioether Mutilin (9)

2-Amino-4-methyl thiazolyl isothiourea dihydrochloride (1.00 g) and 10 mL water were added to the flask with three necks. The mixture was cooled in an ice-water bath. A solution of 12 (2.00 g/50 mL THF) was dropped into the mixture, followed by adding 6 mL of a 10% NaOH aqueous solution. The reaction mixture was stirred for 2 h after the dropwise addition. TLC monitored the end of the reaction. The aqueous layer was identified, whereas the organic phase was removed under decompression. A white foam of crude 9 was obtained (yield of 93%), which used in the next step without further purification. mp 79.9–83.1°C. ¹H-NMR (CDCl₃) δ: 6.50 (dd, J=17.4, 11.0 Hz, 1H), 6.33 (s, 1H), 5.78 (d, J=8.4 Hz, 1H), 5.37 (dd, J=11.0, 1.3 Hz, 1H), 5.23 (dd, J=17.4, 1.4 Hz, 1H), 3.68 (s, 2H), 3.46–3.32 (m, 1H), 3.09 (s, 2H), 2.43–2.32 (m, 1H), 2.29–2.16 (m, 2H), 2.14–2.05 (m, 2H), 1.79 (dd, J=14.4, 2.7 Hz, 1H), 1.72–1.61 (m, 2H), 1.58 (dd, J=18.3, 7.4 Hz, 2H), 1.51–1.42 (m, 4H), 1.42–1.32 (m, 2H), 1.24–1.05 (m, 4H), 0.90 (d, J=7.0 Hz, 3H), 0.75 (d, J=6.9 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 217.07, 168.83, 168.20, 147.27, 139.21, 117.19, 105.78, 74.67, 69.26, 58.23, 45.48, 44.85, 43.96, 41.78, 36.81, 36.05, 34.48, 32.96, 31.84, 30.46, 26.87, 26.46, 24.87, 16.87, 14.97, 11.52. IR (KBr) cm⁻¹: 3431, 3336, 3199, 3119, 2933, 1724, 1617, 1526, 1281, 1115, 1017, 802, 691. FAB-MS m/z: 507.2342 [M−H]⁻ (Calcd for C₂₆H₃₉N₂O₄S₂: 507.2351).

General Procedure for the Synthesis of Compounds 13a–o

A mixture of carboxylic acid (2 eq), DCC (0.41 g, 2 eq), HOBT (0.27 g, 2 eq) and CH₂Cl₂ (15 mL) was stirred at room temperature for 1 h. Compound 9 (0.5 g, 1 eq) was added into the mixture, which was continuously stirred for 48 h. The mixture was filtered to remove insoluble solids and the filtrate was concentrated under vacuum. The crude residue was purified by silica gel column chromatography to afford the pure desired compounds 13a–g, 13j–l and 14h–i, 14m–o. Compounds 14h–i and 14m–o (0.50 g) were treated with a mixture of 4 mL TFA and 10 mL dichloromethane at room temperature for half an hour. The reaction was quenched with saturated aqueous NaHCO₃. The organic phase was separated, washed with water, dried with anhydrous Na₂SO₄ and removed under vacuum. The crude product was purified by silica gel column chromatography to obtain 13h–i, 13m–o.

14-O-(Benzamide-thiazolyl)-4-methyl Thioether Mutilin (13a)

White foam solid; yield: 44%; mp 104.2–105.8°C. ¹H-NMR (CDCl₃) δ: 10.24 (s, 1H), 7.92 (d, J=7.4 Hz, 2H), 7.58 (t, J=7.4 Hz, 1H), 7.49 (dd, J=7.8, 7.0 Hz, 2H), 6.79 (s, 1H), 6.47 (dd, J=17.4, 11.0 Hz, 1H), 5.74 (d, J=8.4 Hz, 1H), 5.33 (d, J=11.0 Hz, 1H), 5.20 (d, J=17.4 Hz, 1H), 3.70 (s, 2H), 3.43–3.31 (m, 1H), 3.00 (s, 2H), 2.39–2.29 (m, 1H), 2.26–2.13 (m, 2H), 2.13–2.02 (m, 2H), 1.75 (d, J=14.4 Hz, 1H), 1.62 (dd, J=21.1, 10.6 Hz, 4H), 1.57–1.47 (m, 1H), 1.47–1.38 (m, 4H), 1.38–1.28 (m, 2H), 1.21–1.04 (m, 4H), 0.86 (d, J=6.8 Hz, 3H), 0.70 (d, J=6.7 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.99, 168.59, 164.59, 158.83, 146.38, 139.15, 132.94, 131.90, 128.99, 127.43, 117.17, 111.39, 74.67, 69.29, 58.18, 45.44, 44.83, 43.91, 41.73, 36.75, 36.01, 34.43, 32.92, 31.58, 30.40, 26.83, 26.45, 24.84, 16.84, 14.92, 11.51. IR (KBr) cm⁻¹: 3262, 2933, 1732 1668, 1540, 1455, 1285, 1115, 705. FAB-MS m/z: 609.2443 [M−H]⁻ (Calcd for C₃₃H₄₁N₂O₅S₂: 609.2457).

14-O-(Benzacetamide-thiazolyl)-4-methyl Thioether Mutilin (13b)

White solid; yield: 65%; mp 191.9–193.7°C. ¹H-NMR (CDCl₃) δ: 9.00 (s, 1H), 7.38–7.29 (m, 3H), 7.26 (t, J=6.3 Hz, 2H), 6.72 (s, 1H), 6.44 (dd, J=17.4, 11.0 Hz, 1H), 5.72 (d, J=8.4 Hz, 1H), 5.31 (d, J=11.0 Hz, 1H), 5.17 (d, J=17.4 Hz, 1H), 3.78 (s, 2H), 3.72 (s, 2H), 3.35 (s, 1H), 3.03–2.92 (m, 2H), 2.37–2.25 (m, 1H), 2.25–2.10 (m, 2H), 2.05 (dd, J=15.8, 8.1 Hz, 2H), 1.74 (d, J=14.3 Hz, 1H), 1.68–1.48 (m, 4H), 1.49–1.37 (m, 4H), 1.36–1.20 (m, 2H), 1.16–1.04 (m, 4H), 0.86 (d, J=6.9 Hz, 3H), 0.68 (d, J=6.8 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 217.09, 168.74, 168.61, 158.01, 146.23, 139.21, 132.78, 129.61, 129.41, 128.10, 117.18, 111.40, 74.65, 69.34, 58.22, 45.48, 44.86, 43.95, 43.46, 41.77, 36.79, 36.04, 34.47, 32.88, 31.64, 30.44, 26.86, 26.48, 24.88, 16.85, 14.95, 11.53. IR (KBr) cm⁻¹: 3500, 3193, 2931, 1730, 1549, 1455, 1279, 1114, 1015, 730. FAB-MS m/z: 623.2599 [M−H]⁻ (Calcd for C₃₄H₄₃N₂O₅S₂: 623.2613).

14-O-(3-Methoxybenzamide-thiazolyl)-4-methyl Thioether Mutilin (13c)

White foam solid; yield: 32%; mp 87.7–89.4°C. ¹H-NMR (CDCl₃) δ: 9.81 (s, 1H), 7.89 (d, J=8.6 Hz, 2H), 6.98 (d, J=8.6 Hz, 2H), 6.78 (s, 1H), 6.48 (dd, J=17.4, 11.0 Hz, 1H), 5.76 (d, J=8.4 Hz, 1H), 5.35 (d, J=11.0 Hz, 1H), 5.21 (d, J=17.4 Hz, 1H), 3.87 (s, 3H), 3.78 (s, 2H), 3.36 (t, J=7.4 Hz, 1H), 3.03 (s, 2H), 2.39–2.28 (m, 1H), 2.27–2.13 (m, 2H), 2.12–2.03 (m, 2H), 1.76 (d, J=14.3 Hz, 1H), 1.69–1.59 (m, 2H), 1.59–1.48 (m, 2H), 1.48–1.40 (m, 4H), 1.40–1.29 (m, 2H), 1.20–1.07 (m, 4H), 0.87 (d, J=6.9 Hz, 3H), 0.72 (d, J=6.8 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.95, 168.62, 163.94, 163.37, 159.03, 146.24, 139.16, 129.40, 123.95, 117.16, 114.26, 111.18, 74.66, 69.31, 58.19, 55.51, 45.44, 44.85, 43.92, 41.75, 36.76, 36.02, 34.42, 32.94, 31.66, 30.41, 26.84, 26.43, 24.84, 16.83, 14.92, 11.48. IR (KBr) cm⁻¹: 3437, 3265, 2932, 1731, 1667, 1607, 1541, 1513, 1456, 1285, 1260, 1114, 1019, 980, 843. FAB-MS m/z: 641.2714 [M−H]⁻ (Calcd for C₃₄H₄₅N₂O₆S₂: 641.2719).

14-O-(2-Nitrobenzamide-thiazolyl)-4-methyl Thioether Mutilin (13d)

Light yellow foam solid; yield: 50%; mp 109.1–111.4°C. ¹H-NMR (CDCl₃) δ: 8.10 (d, J=7.9 Hz, 1H), 7.68 (ddd, J=19.9, 13.6, 7.4 Hz, 3H), 6.82 (s, 1H), 6.46 (dd, J=17.4, 11.1 Hz, 1H), 5.73 (d, J=8.4 Hz, 1H), 5.33 (d, J=10.9 Hz, 1H), 5.19 (d, J=17.4 Hz, 1H), 3.61 (s, 2H), 3.36 (dd, J=9.9, 6.6 Hz, 1H), 3.02 (s, 2H), 2.37–2.27 (m, 1H), 2.27–2.14 (m, 2H), 2.13–2.03 (m, 2H), 1.75 (d, J=14.4 Hz, 1H), 1.68–1.58 (m, 2H), 1.57–1.48 (m, 2H), 1.48–1.38 (m, 4H), 1.38–1.28 (m, 2H), 1.19–1.06 (m, 4H), 0.86 (d, J=6.9 Hz, 3H), 0.70 (d, J=6.8 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 217.06, 168.60, 163.56, 158.15, 146.73, 146.48, 139.18, 133.87, 131.66, 130.46, 128.70, 124.87, 117.15, 111.71, 74.62, 69.35, 58.16, 45.43, 44.79, 43.91, 41.73, 36.74, 35.99, 34.43, 32.99, 31.34, 30.40, 26.83, 26.39, 24.83, 16.85, 14.91, 11.47. IR (KBr) cm⁻¹: 3448, 3256, 3193, 2933, 1725, 1685, 1533, 1458, 1347, 1286, 1116, 1017, 980, 721. FAB-MS m/z: 654.2299 [M−H]⁻ (Calcd for C₃₃H₄₃N₃O₇S₂: 654.2308).

14-O-(4-Nitrobenzamide-thiazolyl)-4-methyl Thioether Mutilin (13e)

Saffron foam solid; yield: 54%; mp 118.6–120.1°C. ¹H-NMR (CDCl₃) δ: 10.16 (s, 1H), 8.44–8.26 (m, 2H), 8.12 (d, J=6.9 Hz, 2H), 6.86 (s, 1H), 6.48 (dd, J=17.4, 11.1 Hz, 1H), 5.76 (d, J=8.0 Hz, 1H), 5.35 (d, J=11.0 Hz, 1H), 5.21 (d, J=17.5 Hz, 1H), 3.79 (s, 2H), 3.42–3.30 (m, 1H), 3.05 (s, 2H), 2.39–2.29 (m, 1H), 2.28–2.15 (m, 2H), 2.15–2.03 (m, 2H), 1.77 (d, J=13.1 Hz, 3H), 1.64 (dd, J=21.2, 11.7 Hz, 2H), 1.58–1.49 (m, 2H), 1.49–1.41 (m, 4H), 1.41–1.21 (m, 4H), 1.21–1.05 (m, 4H), 0.88 (d, J=6.4 Hz, 3H), 0.72 (d, J=6.6 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.98, 168.59, 162.57, 158.14, 150.33, 146.66, 139.18, 137.32, 128.64, 124.20, 117.18, 111.88, 74.65, 69.41, 58.17, 45.44, 44.84, 43.93, 41.75, 36.74, 36.02, 34.43, 32.96, 31.55, 30.40, 26.85, 26.42, 24.84, 16.86, 14.92, 11.50. IR (KBr) cm⁻¹: 3450, 3253, 2932, 1727, 1676, 1548, 1528, 1346, 1288, 1115, 1015, 980, 850, 713. FAB-MS m/z: 654.2286 [M−H]⁻ (Calcd for C₃₃H₄₃N₃O₇S₂: 654.2308).

14-O-(2,4-Dichlorobenzamide-thiazolyl)-4-methyl Thioether Mutilin (13f)

White foam solid; yield: 46%; mp 102.5–104.1°C. ¹H-NMR (CDCl₃) δ: 7.82 (d, J=8.4 Hz, 1H), 7.50 (d, J=2.0 Hz, 1H), 7.39 (dd, J=8.4, 2.0 Hz, 1H), 6.85 (s, 1H), 6.48 (dd, J=17.4, 11.0 Hz, 1H), 5.76 (d, J=8.5 Hz, 1H), 5.35 (d, J=11.0 Hz, 1H), 5.21 (dd, J=17.4, 1.4 Hz, 1H), 3.78 (s, 2H), 3.36 (s, 1H), 3.06 (s, 2H), 2.40–2.30 (m, 1H), 2.30–2.15 (m, 2H), 2.14–2.03 (m, 2H), 1.77 (dd, J=14.3, 2.8 Hz, 1H), 1.69–1.61 (m, 2H), 1.61–1.48 (m, 2H), 1.48–1.41 (m, 4H), 1.40–1.29 (m, 2H), 1.20–1.06 (m, 4H), 0.88 (d, J=7.0 Hz, 3H), 0.73 (d, J=6.9 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.87, 168.58, 162.32, 157.65, 146.88, 139.17, 138.65, 132.13, 132.04, 130.65, 130.25, 127.94, 117.15, 111.70, 74.64, 69.34, 58.18, 45.44, 44.86, 43.94, 41.76, 36.76, 36.03, 34.42, 33.03, 31.54, 30.42, 26.85, 26.41, 24.84, 16.83, 14.92, 11.44. IR (KBr) cm⁻¹: 3449, 3256, 3193, 3080, 2960, 2927, 1728, 1676, 1541, 1454, 1284, 1262, 1103, 1018, 802, 697. FAB-MS m/z: 677.1660 [M−H]⁻ (Calcd for C₃₃H₃₉N₂O₅S₂Cl₂: 677.1677).

14-O-(2-Hydroxylbenzamide-thiazolyl)-4-methyl Thioether Mutilin (13g)

White solid; yield: 37.7%; mp 218.5–220.1°C. ¹H-NMR (CDCl₃) δ: 7.64 (d, J=8.0 Hz, 1H), 7.46 (t, J=7.7 Hz, 1H), 7.03 (d, J=8.4 Hz, 1H), 6.94 (t, J=7.6 Hz, 1H), 6.85 (s, 1H), 6.48 (dd, J=17.4, 11.0 Hz, 1H), 5.76 (d, J=8.4 Hz, 1H), 5.35 (d, J=11.0 Hz, 1H), 5.21 (d, J=17.3 Hz, 1H), 3.82 (s, 2H), 3.41–3.32 (m, 1H), 3.05 (s, 2H), 2.40–2.30 (m, 1H), 2.28–2.17 (m, 2H), 2.17–2.04 (m, 2H), 1.76 (d, J=14.2 Hz, 2H), 1.70–1.59 (m, 4H), 1.58–1.49 (m, 2H), 1.49–1.41 (m, 4H), 1.39–1.29 (m, 2H), 1.20–1.07 (m, 4H), 0.88 (d, J=7.0 Hz, 3H), 0.73 (d, J=6.9 Hz, 3H). δ: 11.9 (s, 1H), 11.65 (s, 1H), 7.96 (d, J=7.8 Hz, 1H), 7.44 (s, 1H), 6.97 (d, J=10.7 Hz, 3H), 6.13 (d, J=11.2 Hz, 1H), 5.54 (d, J=8.1 Hz, 1H), 5.05 (d, J=8.2 Hz, 2H), 4.46 (d, J=5.5 Hz, 1H), 3.79 (s, 2H), 3.4 (s, 1H), 2.37 (s, 1H), 2.33–1.94 (m, 4H), 1.7–1.53 (m, 2H), 1.52–1.32 (m, 5H), 1.31–1.18 (m, 5H), 1.07–0.92 (m, 4H), 0.8 (d, J=8 Hz, 3H), 0.61 (d, J=6.5 Hz, 3H). ¹H-NMR (DMSO-d₆): δ: 11.9 (s, 1H), 11.65 (s, 1H), 7.96 (d, J=7.8 Hz, 1H), 7.44 (s, 1H), 6.97 (d, J=10.7 Hz, 3H), 6.13 (d, J=11.2 Hz, 1H), 5.54 (d, J=8.1 Hz, 1H), 5.05 (d, J=8.2 Hz, 2H), 4.46 (d, J=5.5 Hz, 1H), 3.79 (s, 2H), 3.4 (s, 1H), 2.37 (s, 1H), 2.33–1.94 (m, 4H), 1.7–1.53 (m, 2H), 1.52–1.32 (m, 5H), 1.31–1.18 (m, 5H), 1.07–0.92 (m, 4H), 0.8 (d, J=8 Hz, 3H), 0.61 (d, J=6.5 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.98, 168.60, 139.14, 135.54, 119.67, 118.72, 117.21, 111.86, 74.65, 69.38, 58.18, 45.44, 44.84, 43.92, 41.74, 36.75, 36.01, 34.43, 32.96, 31.53, 30.41, 26.84, 26.39, 24.84, 16.87, 14.93, 11.50. ¹³C-NMR (DMSO-d₆) δ: 217.64, 168.66, 164.60, 157.82, 147.62, 141.39, 134.92, 130.71, 120.22, 117.64, 115.63, 111.47, 73.10, 69.99, 57.76, 45.46, 44.52, 44.22, 41.96, 36.87, 34.48, 33.81, 31.62, 30.61, 30.31, 29.02, 27.05, 24.96, 16.62, 15.07, 12.01. IR (KBr) cm⁻¹: 3446, 3247, 2952, 1731, 1646, 1540, 1463, 1262, 1114, 1017, 802, 756. δ: 217.64, 168.66, 164.60, 157.82, 147.62, 141.39, 134.92, 130.71, 120.22, 117.64, 115.63, 111.47, 73.10, 69.99, 57.76, 45.46, 44.52, 44.22, 41.96, 36.87, 34.48, 33.81, 31.62, 30.61, 30.31, 29.02, 27.05, 24.96, 16.62, 15.07, FAB-MS m/z: 625.2387 [M−H]⁻ (Calcd for C₃₃H₄₁N₂O₆S₂: 625.2406).

14-O-(3-Aminobenzamide-thiazolyl)-4-methyl Thioether Mutilin (13h)

White foam solid; yield: 40%; mp 108.5–110.4°C. ¹H-NMR (CDCl₃) δ: 7.26 (t, J=5.3 Hz, 2H), 7.23–7.19 (m, 1H), 6.87 (dd, J=7.1, 1.7 Hz, 1H), 6.80 (s, 1H), 6.49 (dd, J=17.4, 11.0 Hz, 1H), 5.76 (d, J=8.4 Hz, 1H), 5.35 (d, J=11.0 Hz, 1H), 5.21 (dd, J=17.4, 1.4 Hz, 1H), 3.80 (s, 2H), 3.36 (s, 1H), 3.05 (s, 2H), 2.41–2.30 (m, 1H), 2.28–2.14 (m, 2H), 2.14–2.04 (m, 2H), 1.76 (d, J=14.3 Hz, 1H), 1.69–1.59 (m, 3H), 1.59–1.50 (m, 1H), 1.48–1.40 (m, 4H), 1.39–1.30 (m, 2H), 1.19–1.06 (m, 4H), 0.88 (d, J=7.0 Hz, 3H), 0.73 (d, J=6.9 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.99, 168.63, 164.60, 158.67, 147.15, 146.39, 139.14, 132.87, 129.89, 119.28, 117.21, 116.59, 113.82, 111.33, 74.64, 69.29, 58.18, 45.44, 44.83, 43.92, 41.75, 36.77, 36.01, 34.44, 32.93, 31.66, 30.41, 26.84, 26.40, 24.84, 16.85, 14.94, 11.49. IR (KBr) cm⁻¹: 3455, 3370, 3235, 2929, 1727, 1667, 1541, 1456, 1280, 1115, 1017, 941, 844, 744. FAB-MS m/z: 626.2730 [M−H]⁻ (Calcd for C₃₃H₄₄N₃O₅S₂: 626.2722).

14-O-(4-Aminobenzamide-thiazolyl)-4-methyl Thioether Mutilin (13i)

White solid; yield: 16.4%; mp 134.8–137.4°C. ¹H-NMR (CDCl₃) δ: 7.78 (d, J=7.2 Hz, 2H), 6.76 (s, 1H), 6.69 (d, J=7.2 Hz, 2H), 6.48 (dd, J=17.0, 11.1 Hz, 1H), 5.76 (d, J=8.3 Hz, 1H), 5.36 (d, J=11.0 Hz, 1H), 5.21 (d, J=17.4 Hz, 1H), 3.80 (s, 2H), 3.36 (s, 1H), 3.04 (s, 2H), 2.39–2.29 (m, 1H), 2.29–2.15 (m, 2H), 2.14–2.04 (m, 2H), 1.76 (d, J=14.2 Hz, 1H), 1.64 (dd, J=21.4, 10.6 Hz, 2H), 1.56 (d, J=12.9 Hz, 1H), 1.52–1.40 (m, 5H), 1.40–1.29 (m, 2H), 1.19–1.06 (m, 4H), 0.87 (d, J=6.4 Hz, 3H), 0.73 (d, J=6.2 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.97, 168.61, 164.02, 159.63, 151.00, 145.47, 139.14, 129.55, 120.65, 117.22, 114.25, 111.00, 74.64, 69.29, 58.19, 45.44, 44.83, 43.92, 41.74, 36.77, 36.01, 34.44, 32.88, 31.43, 30.42, 26.84, 26.39, 24.84, 16.86, 14.93, 11.50. IR (KBr) cm⁻¹: 3466, 3362, 3231, 2926, 1724, 1624, 1540, 1282, 1182, 1016, 734. FAB-MS m/z: 624.2585 [M−H]⁻ (Calcd for C₃₃H₄₂N₃O₅S₂: 624.2566).

14-O-(2-Furoyl-thiazolyl)-4-methyl Thioether Mutilin (13j)

White foam solid; yield: 50%; mp 105.8–107.2°C. ¹H-NMR (CDCl₃) δ: 9.84 (s, 1H), 7.59–7.45 (m, 1H), 7.31 (d, J=3.6 Hz, 1H), 6.78 (s, 1H), 6.56 (dd, J=3.5, 1.7 Hz, 1H), 6.46 (dd, J=17.4, 11.0 Hz, 1H), 5.74 (d, J=8.4 Hz, 1H), 5.32 (d, J=11.0 Hz, 1H), 5.19 (dd, J=17.4, 1.4 Hz, 1H), 3.79 (s, 2H), 3.39–3.29 (m, 1H), 3.03 (s, 2H), 2.37–2.28 (m, 1H), 2.28–2.12 (m, 2H), 2.11–2.01 (m, 2H), 1.74 (dd, J=14.5, 2.4 Hz, 1H), 1.68–1.57 (m, 3H), 1.57–1.47 (m, 1H), 1.47–1.37 (m, 4H), 1.38–1.26 (m, 2H), 1.18–1.04 (m, 4H), 0.86 (d, J=7.0 Hz, 3H), 0.70 (d, J=6.8 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.99, 168.58, 157.58, 155.05, 146.64, 145.82, 145.52, 139.20, 117.10, 117.07, 112.88, 111.42, 74.60, 69.30, 58.18, 45.43, 44.83, 43.92, 41.73, 36.75, 36.02, 34.42, 32.91, 31.65, 30.40, 26.82, 26.49, 24.82, 16.81, 14.91, 11.47. IR (KBr) cm⁻¹: 3447, 3410, 2933, 1728, 1671, 1588, 1541, 1328, 1296, 1115, 1015, 856, 759. FAB-MS m/z: 599.2243 [M−H]⁻ (Calcd for C₃₁H₃₉N₂O₆S₂: 599.2250).

14-O-(Thiophenecarbonyl-thiazolyl)-4-methyl Thioether Mutilin (13k)

White solid; yield: 47%; mp 127.3–129.7°C. ¹H-NMR (CDCl₃) δ: 7.73 (d, J=3.7 Hz, 1H), 7.63 (d, J=5.0 Hz, 1H), 7.15 (t, J=4.4 Hz, 1H), 6.80 (s, 1H), 6.48 (dd, J=17.4, 11.1 Hz, 1H), 5.76 (d, J=8.4 Hz, 1H), 5.36 (d, J=11.0 Hz, 1H), 5.21 (d, J=17.4 Hz, 1H), 3.80 (s, 2H), 3.43–3.30 (m, 1H), 3.04 (s, 2H), 2.40–2.29 (m, 1H), 2.29–2.15 (m, 2H), 2.14–2.04 (m, 2H), 1.76 (d, J=14.5 Hz, 1H), 1.71–1.59 (m, 3H), 1.59–1.51 (m, 1H), 1.51–1.39 (m, 5H), 1.39–1.29 (m, 2H), 1.19–1.06 (m, 4H), 0.88 (d, J=7.0 Hz, 3H), 0.73 (d, J=6.8 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.96, 168.60, 158.95, 158.38, 146.28, 139.15, 136.31, 132.61, 129.95, 128.28, 117.20, 111.53, 74.64, 69.32, 58.18, 45.44, 44.84, 43.92, 41.75, 36.76, 36.02, 34.43, 32.89, 31.60, 30.41, 26.84, 26.39, 24.84, 16.85, 14.93, 11.49. IR (KBr) cm⁻¹: 3391, 3092, 2966, 1732, 1652, 1557, 1455, 1412, 1287, 1115, 1048, 849, 723. FAB-MS m/z: 615.2046 [M−H]⁻ (Calcd for C₃₁H₃₉N₂O₅S₂: 615.2021).

14-O-(4-Pyridinecarbonyl-thiazolyl)-4-methyl Thioether Mutilin (13l)

Light yellow foam solid; yield: 78%; mp 125.5–127.8°C. ¹H-NMR (MeOD) δ: 8.67 (d, J=4.7 Hz, 2H), 7.98 (d, J=4.7 Hz, 2H), 6.87 (s, 1H), 6.30 (dd, J=17.4, 11.3 Hz, 1H), 5.72 (d, J=8.3 Hz, 1H), 5.16 (dd, J=14.4, 7.7 Hz, 2H), 3.83 (s, 2H), 3.48 (d, J=6.0 Hz, 1H), 3.16 (s, 2H), 2.38–2.20 (m, 3H), 2.19–2.04 (m, 2H), 1.79 (d, J=14.0 Hz, 1H), 1.73–1.49 (m, 4H), 1.47–1.36 (m, 4H), 1.36–1.25 (m, 2H), 1.19–1.07 (m, 4H), 0.91 (d, J=6.9 Hz, 3H), 0.71 (d, J=6.0 Hz, 3H). ¹³C-NMR (MeOD) δ: 218.30, 169.14, 166.17, 149.30, 146.32, 143.62, 139.88, 122.16, 115.11, 110.37, 74.00, 69.83, 57.92, 45.39, 44.63, 43.87, 41.70, 36.76, 36.33, 33.90, 32.92, 31.47, 30.13, 26.88, 26.61, 24.43, 15.66, 14.04, 10.36. IR (KBr) cm⁻¹: 3446, 2932, 1730, 1670, 1550, 1456, 1284, 1115, 1018, 908, 801, 718. FAB-MS m/z: 612.2564 [M+H]⁺ (Calcd for C₃₂H₄₂N₃O₅S₂: 612.2566).

14-O-(2-Pyrrolidinecarbonyl-thiazolyl)-4-methyl Thioether Mutilin (13m)

White foam solid; yield: 70%; mp 137.8–140.2°C. ¹H-NMR (Pyr) δ: 7.06 (s, 1H), 6.91 (s, 1H), 6.75 (ddd, J=17.6, 14.0, 11.3 Hz, 2H), 6.17 (dd, J=15.8, 7.7 Hz, 1H), 6.04 (t, J=7.5 Hz, 2H), 5.85 (s, 1H), 5.57–5.48 (m, 2H), 5.43 (t, J=9.7 Hz, 2H), 4.97 (s, 3H), 4.30 (td, J=8.4, 4.3 Hz, 1H), 4.17–4.10 (m, 2H), 4.04 (dd, J=13.7, 7.8 Hz, 2H), 3.94 (d, J=13.5 Hz, 1H), 3.81 (d, J=5.9 Hz, 2H), 3.53 (s, 2H), 3.37 (d, J=14.5 Hz, 1H), 3.30 (d, J=14.5 Hz, 1H), 3.05 (dd, J=10.6, 4.0 Hz, 1H), 2.88 (dt, J=10.0, 6.8 Hz, 1H), 2.83 (dt, J=10.3, 6.5 Hz, 1H), 2.69 (td, J=11.9, 7.5 Hz, 1H), 2.57–2.42 (m, 5H), 2.34–2.25 (m, 3H), 2.20–2.14 (m, 4H), 2.06 (dt, J=20.4, 7.9 Hz, 1H), 1.98 (td, J=12.8, 6.4 Hz, 1H), 1.81–1.65 (m, 17H), 1.60–1.48 (m, 2H), 1.43 (s, 5H), 1.38–1.33 (m, 2H), 1.29 (t, J=13.3 Hz, 2H), 1.15 (t, J=6.6 Hz, 6H), 1.07 (dd, J=20.6, 8.1 Hz, 2H), 0.88 (d, J=6.6 Hz, 3H), 0.83 (d, J=6.4 Hz, 3H). ¹³C-NMR (Pyr) δ: 216.94, 177.19, 174.09, 169.98, 168.79, 168.77, 158.43, 147.80, 145.27, 141.37, 141.34, 115.84, 115.75, 110.52, 109.48, 73.93, 73.90, 70.18, 61.11, 60.71, 58.19, 54.84, 47.28, 47.17, 45.68, 45.00, 44.95, 44.82, 44.79, 42.24, 42.20, 37.16, 37.13, 37.03, 34.52, 33.81, 33.16, 32.72, 32.13, 30.82, 30.46, 29.80, 28.70, 27.07, 27.03, 26.29, 26.08, 25.10, 16.64, 16.62, 15.08, 15.06, 11.99. IR (KBr) cm⁻¹: 3445, 3257, 2935, 1728, 1575, 1457, 1280, 1115, 1017, 715. FAB-MS m/z: 602.2723 [M−H]⁻ (Calcd for C₃₁H₄N₃O₅S₂: 602.2722).

14-O-(2-Amino-3-methyl)butanoyl-thiazolyl)-4-methyl Thioether Mutilin (13n)

White foam solid; yield: 64%; mp 110.8–116.0°C. ¹H-NMR (Pyr) δ: 7.05 (s, 1H), 6.76 (dd, J=17.7, 11.1 Hz, 1H), 6.04 (d, J=8.2 Hz, 1H), 5.84 (s, 1H), 5.52 (d, J=17.7 Hz, 1H), 5.43 (d, J=11.2 Hz, 1H), 4.98 (s, 4H), 4.18–4.09 (m, 2H), 3.80 (d, J=4.4 Hz, 1H), 3.65 (d, J=5.0 Hz, 1H), 3.53 (s, 2H), 3.40 (s, 1H), 2.57–2.45 (m, 2H), 2.34–2.23 (m, 2H), 2.20–2.12 (m, 2H),1.81–1.65 (m, 9H), 1.43 (s, 3H), 1.40–1.31 (m, 1H), 1.27 (d, J=11.9 Hz, 1H), 1.19–1.11 (m, 4H), 1.10–1.03 (m, 1H), 1.02–0.93 (m, 6H), 0.87 (d, J=6.6 Hz, 4H). ¹³C-NMR (Pyr) δ: 216.94, 174.41, 168.79, 158.83, 147.84, 141.34, 115.82, 110.42, 73.92, 70.18, 60.92, 58.19, 45.67, 45.01, 44.81, 42.23, 37.13, 37.03, 34.51, 33.86, 32.26, 32.19, 30.45, 28.69, 27.06, 25.09, 19.59, 16.94, 16.64, 15.08, 11.99. ¹H-NMR (CDCl₃) δ: 6.79 (s, 1H), 6.49 (dd, J=17.4, 11.0 Hz, 1H), 5.77 (d, J=8.4 Hz, 1H), 5.37 (dd, J=11.0, 1.1 Hz, 1H), 5.22 (dd, J=17.4, 1.3 Hz, 1H), 3.80 (s, 1H), 3.49 (s, 1H), 3.37 (s, 1H), 3.12–3.00 (m, 2H), 2.45 (s, 1H), 2.41–2.29 (m, 1H), 2.29–2.15 (m, 2H), 2.15–2.05 (m, 2H), 1.78 (dd, J=14.4, 2.6 Hz, 1H), 1.72–1.60 (m, 2H), 1.54–1.42 (m, 5H), 1.41–1.30 (m, 2H), 1.21–1.08 (m, 4H), 1.04 (d, J=7.0 Hz, 3H), 0.88 (dd, J=11.7, 7.0 Hz, 6H), 0.74 (d, J=6.8 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 217.04, 168.70, 139.18, 117.23, 110.98, 74.67, 69.30, 59.80, 58.23, 45.48, 44.86, 43.96, 41.78, 36.81, 36.05, 34.47, 32.93, 31.77, 30.77, 30.45, 26.87, 26.44, 24.88, 19.60, 16.88, 16.11, 14.97, 11.52. IR (KBr) cm⁻¹: 3437, 2957, 1731, 1538, 1456, 1280, 1114, 1017, 730. FAB-MS m/z: 604.2875 [M−H]⁻ (Calcd for C₃₁H₄₇N₃O₅S₂: 604.2879).

14-O-(2-Amino-3-phenyl)propanoyl-thiazolyl)-4-methyl Thioether Mutilin (13o)

White foam solid; yield: 67%; mp 98.5–100.3°C. ¹H-NMR (Pyr) δ: 7.26 (dd, J=14.2, 6.9 Hz, 4H), 7.22–7.15 (m, 2H), 7.03 (s, 1H), 6.77 (dd, J=17.6, 11.1 Hz, 1H), 6.05 (d, J=8.3 Hz, 1H), 5.83 (s, 1H), 5.53 (d, J=17.8 Hz, 1H), 5.44 (d, J=11.4 Hz, 1H), 5.01 (s, 3H), 4.21–4.07 (m, 1H), 3.81 (d, J=5.4 Hz, 1H), 3.50 (s, 2H), 2.99 (dd, J=12.9, 9.3 Hz, 1H), 2.60–2.47 (m, 2H), 2.29 (dd, J=15.7, 8.3 Hz, 1H), 2.17 (dd, J=9.7, 6.3 Hz, 2H), 1.82–1.66 (m, 8H), 1.44 (s, 3H), 1.39–1.32 (m, 1H), 1.32–1.25 (m, 1H), 1.15 (d, J=7.0 Hz, 3H), 1.11–1.02 (m, 1H), 0.88 (t, J=10.9 Hz, 3H). ¹³C-NMR (Pyr) δ: 216.97, 168.81, 141.36, 138.39, 129.57, 128.71, 126.77, 115.84, 73.92, 70.18, 58.19, 57.07, 54.84, 45.68, 45.04, 44.81, 42.24, 37.13, 37.04, 34.53, 30.46, 28.70, 27.07, 25.10, 16.66, 15.10, 12.00. IR (KBr) cm⁻¹: 3458, 3294, 2929, 2860, 1730, 1538, 1455, 1279, 1114 1017, 701. FAB-MS m/z: 652.2873 [M−H]⁻ (Calcd for C₃₅H₄₇N₃O₅S₂: 652.2879).

14-O-(Boc-2-pyrrolidinecarbonyl-thiazolyl)-4-methyl Thioether Mutilin (14m)

White foam solid; yield: 58%; mp 79.8–81.3°C. ¹H-NMR (CDCl₃) δ: 10.31 (s, 1H), 9.47 (s, 1H), 6.70 (s, 1H), 6.42 (dd, J=17.4, 11.0 Hz, 1H), 5.70 (d, J=8.4 Hz, 1H), 5.29 (dd, J=11.0, 1.4 Hz, 1H), 5.15 (dd, J=17.4, 1.4 Hz, 1H), 4.46 (s, 1H), 4.32 (s, 1H), 3.73 (s, 2H), 3.40 (s, 1H), 3.30 (s, 1H), 2.99 (s, 2H), 2.36–2.08 (m, 4H), 2.08–1.96 (m, 3H), 1.91–1.80 (m, 2H), 1.71 (dd, J=14.4, 2.6 Hz, 1H), 1.65–1.48 (m, 3H), 1.48–1.34 (m, 12H), 1.33–1.14 (m, 5H), 1.14–0.97 (m, 4H), 0.82 (d, J=7.0 Hz, 3H), 0.66 (d, J=6.8 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.05, 167.69, 156.87, 145.55, 138.17, 116.21, 110.11, 80.29, 73.65, 68.26, 59.15, 57.21, 52.44, 46.35, 44.46, 43.85, 42.93, 40.76, 35.79, 35.04, 33.46, 31.94, 30.62, 29.44, 27.34, 25.85, 25.44, 23.86, 15.85, 13.95, 10.51. IR (KBr) cm⁻¹: 3491, 3214, 2978, 1698, 1549, 1409, 1279, 1160, 1116, 1017, 912, 731. FAB-MS m/z: 702.3234 [M−H]⁻ (Calcd for C₃₆H₅₂N₃O₇S₂: 702.3247).

14-O-(Boc-2-amino-3-methyl)butanoyl-thiazolyl)-4-methyl Thioether Mutilin (14n)

White foam solid; yield: 52%; mp 103.8–106.9°C. ¹H-NMR (CDCl₃) δ: 6.70 (s, 1H), 6.42 (dd, J=17.4, 11.0 Hz, 1H), 5.70 (d, J=8.3 Hz, 1H), 5.29 (dd, J=11.0, 1.2 Hz, 1H), 5.15 (dt, J=10.7, 5.4 Hz, 2H), 4.20 (s, 1H), 3.78–3.66 (m, 2H), 3.34 (dd, J=10.2, 6.5 Hz, 1H), 2.97 (s, 2H), 2.34–2.24 (m, 1H), 2.24–2.08 (m, 3H), 2.08–1.96 (m, 2H), 1.71 (dd, J=14.4, 2.4 Hz, 1H), 1.65–1.44 (m, 4H), 1.43–1.34 (m, 13H), 1.26 (ddd, J=30.1, 17.1, 6.2 Hz, 3H), 1.13–0.99 (m, 4H), 0.96–0.73 (m, 9H), 0.66 (d, J=6.9 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.12, 169.40, 167.72, 154.93, 145.32, 138.16, 116.23, 110.31, 79.60, 73.66, 68.34, 58.97, 57.19, 52.44, 44.48, 43.84, 42.94, 40.76, 35.78, 35.03, 33.47, 31.85, 30.66, 29.65, 29.44, 27.29, 25.87, 25.44, 23.88, 18.29, 16.59, 15.88, 13.95, 10.53. IR (KBr) cm⁻¹: 3443, 3268, 2931, 1716, 1557, 1328, 1161, 1017, 732. FAB-MS m/z: 704.3421 [M−H]⁻ (Calcd for C₃₆H₅₄N₃O₇S₂: 704.3403).

14-O-(Boc-2-amino-3-phenyl)propanoyl-thiazolyl)-4-methyl Thioether Mutilin (14o)

White foam solid; yield: 54%; mp 92.2–94.0°C. ¹H-NMR (CDCl₃) δ: 7.24–7.12 (m, 3H), 7.08 (d, J=6.7 Hz, 2H), 6.69 (s, 1H), 6.41 (dd, J=17.4, 11.0 Hz, 1H), 5.69 (d, J=8.4 Hz, 1H), 5.28 (dd, J=11.0, 1.3 Hz, 1H), 5.15 (dd, J=17.4, 1.4 Hz, 1H), 4.60 (s, 1H), 3.77–3.62 (m, 2H), 3.33 (dd, J=10.2, 6.5 Hz, 1H), 3.17 (d, J=11.5 Hz, 1H), 3.07–2.88 (m, 3H), 2.35–2.21 (m, 1H), 2.21–2.08 (m, 2H), 2.08–1.95 (m, 2H), 1.70 (dd, J=14.4, 2.4 Hz, 1H), 1.65–1.42 (m, 4H), 1.41–1.34 (m, 4H), 1.34–1.16 (m, 11H), 1.14–1.00 (m, 4H), 0.82 (d, J=7.0 Hz, 3H), 0.65 (d, J=6.9 Hz, 3H). ¹³C-NMR (CDCl₃) δ: 216.09, 169.18, 167.66, 154.51, 145.29, 138.16, 135.00, 128.19, 127.73, 126.11, 116.21, 110.35, 79.81, 73.63, 68.31, 57.17, 54.73, 52.43, 44.45, 43.83, 42.91, 40.74, 36.90, 35.76, 35.01, 33.45, 31.79, 30.63, 29.42, 27.18, 25.85, 25.43, 23.86, 15.85, 13.93, 10.51. IR (KBr) cm⁻¹: 3348, 2932, 1717, 1541, 1279, 1164, 1017, 731. FAB-MS m/z: 752.3403 [M−H]⁻ (Calcd for C₄₀H₅₄N₃O₇S₂: 752.3403).

RESULTS AND DISCUSSION

Tested Strains

Methicillin-sensitive Staphylococcus aureus ATCC25923 (MSSA), methicillin-sensitive Staphylococcus epidermidis ATCC12228 (MSSE), Streptococcus agalactiae ATCC13813 (S. aga), Pyogeniccoccus (Pyogens) ATCC19615 and methicillin-resistant S. aureus (MRSA) were purchased from Weilu Co., Ltd. (Shanghai, China). MG was obtained from the Department of Avian Infectious Disease of the Shanghai Veterinary Research Institute.

Bioassay

The antibacterial properties of the pleuromutilin derivatives 13a–o were evaluated in vitro against MSSA, MSSE, MSRA, S. aga, Pyogens and MG via the broth dilution method according to the National Committee for Clinical Laboratory Standards (NCCLS). The minimum inhibitory concentration (MIC, µg/mL) results are listed in Table 1. Erythomycin, tiamulin and valnemulin were used as reference drugs; tylosin was used instead of erythomycin for MG. All of the tested compounds exhibited moderate to good antibacterial activity against all strains. The MICs of derivatives against MSSA, MSSE, MRSA, S. aga, Pyogens and MG in vitro were in range of >1.78−0.00085, >4−0.0017, >1.78−0.0039, >16−0.25, >1.78−0.056 and >0.25−0.0039 µg/mL, respectively. The antibacterial effects for most aromatic acyl derivatives and the intermediate 9 were better than those of tiamulin and erythromye against MSSA, MSSE, MRSA and MG. Compounds 13j or 13a, c and e moderately inhibited the growth of S. aga, which were equal to those of tiamulin or valnemulin respectively. Compounds 13i and l showed potent activity against MSSE and MRSA, which were better than that of valnemulin. However, all the tested compounds exhibited weak activity against Pyogens compared with the reference drugs.

Table 1. Antibacterial Activity (MIC, µg/mL) in Vitro

Compound	(MIC, µg/mL)
13a–g	MSSA	MSSE	MRSA	S. aga	Pyogens	MG
13a	0.014	0.028	0.063	0.5	0.11	0.063
13b	0.056	0.22	0.25	8	0.89	0.031
13c	0.056	0.056	0.125	0.5	0.44	0.031
13d	0.056	0.11	0.25	4	>1.78	0.016
13e	0.056	0.056	0.063	0.5	>1.78	0.0078
13f	0.11	0.11	0.5	>16	>1.78	0.25
13g	0.11	0.44	0.5	>16	>1.78	0.125
13h	0.0017	0.0069	0.0078	0.25	0.056	0.016
13i	0.00085	0.0035	0.0078	0.25	0.056	0.0078
13j	0.028	0.056	0.063	1	1.78	0.13
13k	>1.78	>1.78	>4	>16	>1.78	>0.25
13l	0.00085	0.0017	0.0039	2	0.22	0.0078
13m	0.11	0.22	0.5	8	0.22	0.031
13n	0.44	0.44	1	8	0.44	0.13
13o	0.22	0.44	0.5	2	0.44	0.031
9	0.028	0.056	0.125	2	0.22	0.0078
Erythromycin	0.11	0.22	>4	>16	0.0017	0.031*
Tiamulin (2)	0.11	0.11	0.5	1	0.028	0.031
Valnemulin (3)	0.0035	0.028	0.063	0.5	0.0035	0.0039

* The MIC of Tylosin.

From the MIC data, we can conclude that the structural modification of pleuromutilin can markedly affect the activity and the results further confirm that modification on the side chain at C-14 position of pleuromutilin derivatives with thiazole group can indeed improve antibacterial activity of the prototype compound against organisms.¹⁵⁾ Amino substitution in the benzene ring can improve activity in comparison with the reference drugs except against Pyogens and MG, and para substitution (13i) is better for antibacterial activity than meta substitution (13h) with being found 3–10-fold more potent than erythromye and tiamulin against MSSA and MSSE. The result is consistent with the previous research results^16,17) which amino group could increase the antibacterial effect whether linking with benzene ring or not. Nitro, methoxy, hydroxy and dichloro substituents contribute little to the increased activity compared with the analogue (13a) that possesses unsubstitution benzoyl group. The para nitro-substitution derivative (13d) shows a little better antibacterial activity against MSSA and MSSE than the ortho substitution derivative (13e). Increasing a methylene between benzene moiety and carbonyl group decreases the bioactivity of derivate (13b). Introducing pyridine ring to C14 side chain can significantly improve activity (13l) and the effect is better than that of all the controls against MSSA, MSSE and MRSA, but in contrast with the previous study,¹⁵⁾ modification by pyridine group not shows satisfactory bioactivity, even not as good as modified by piperidinyl group. We think it may be caused by the methylene group between piperidinyl and carbonyl and needs more detail research in future. Compound modified by furancarbonyl group (13j) presents antibacterial activity, whereas derivative possessing thiophenecarbonyl (13k) group almost does not have bioactivity. The amino acid analogues (13m–o) showed moderate activity. The derivative (13g) possessing structure similar to NTZ doesn’t show outstanding activity as expected and displays even worse than 13a which bears phenyl group, it illustrates that simple association of antibacteria agent with pleuromutilin cannot increase bioactivity, and ortho-hydroxyl group substituent contributed little to the increased antibacterial activity, furthermore, more detail work about the position of hydroxyl group substituent maybe need to reveal.

In a word, some of the designed pleuromutilin derivatives showed the improvement antibacterial activity against Gram-positive, which indicates that the side chain of pleuromutilin derivatives indeed has great effect on the properties of the pleuromutilin derivatives and further on the bioactivity. The results will give new ideas for modification of novel pleuromutilin derivatives on the position C14, and be served as the foundation for screening the promising antibiotics for clinical use.

CONCLUSION

We designed and synthesized a series of new pleuromutilin derivatives that incorporated the acylthiazolyl moiety. The antibacterial activity of these new pleuromutilin derivatives were evaluated in vitro against five kinds of bacteria and the mycoplasma MG. All the derivatives exhibited moderate to good inhibitory characteristics against sensitive and resistant Gram-positive bacteria. Compounds 13i and l showed better activities among the synthesized analogues. The MICs of 13i, bearing a 4-aminobenzamide-thiazolyl group on the C-14 glycolic acid side chain, and 13l, bearing a 4-pyridinecarbonyl-thiazolyl group on the C-14 glycolic acid side chain, had better acitities against MSSA (MIC=0.0017 µg/mL), MSSE (MIC=0.0017, 0.0035 µg/mL) and MRSA (MIC=0.0078 µg/mL). The MICs of synthesized new pleuromutilin derivatives against S. aga, Pyogens and MG were weak.

Acknowledgments

This work was completed at the Key Laboratory of Veterinary Drug Safety Evaluation and Residues Research. We gratefully acknowledge the financial support from the Special Fund for Agro-scientific Research “Development and application of new type of special animal drugs” (No. 201303038).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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