A New Pleuromutilin Derivative: Synthesis,Crystal Structure and Antibacterial Evaluation①

YI Yun-Peng YANG Gun-Zhou GUO Zhi-Ting AI Xin SHANG Ruo-Feng② LIANG Jin-Ping②

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A New Pleuromutilin Derivative: Synthesis,Crystal Structure and Antibacterial Evaluation①

YI Yun-PengaYANG Guan-ZhoubGUO Zhi-TingaAI XinaSHANG Ruo-Fenga②LIANG Jian-Pinga②

a(730050)b(730020)

A new pleuromutilin derivative, 14-O-[(4,6-diaminopyrimidine-2-yl) thioacetate] mutilin, was synthesized and structurally characterized by IR, NMR spectra and single-crystal X-ray diffraction. This compound contains a 5-6-8 tricyclic carbon skeleton and a pyrimidine ring. Its crystal is of orthorhombic system, space group21with= 10.0237(6),= 12.6087(7),= 10.3749(8) ?,= 101.48(1)°,= 1284.99(14) ?3,= 2,(000) = 540,D(Mg/m3) = 1.299,= 0.165 mm-1,= 0.0649 and= 0.0797. Theantibacterial activity study using Oxford cup assay showed this compound displayed more potent activity than pleuromutilin and similar antibacterial activity to that of tiamulin.

pleuromutilin, single-crystal structure, antibacterial activities;

1 INTRODUCTION

Pleuromutilin (1) was first isolated in a crys- talline form from cultures of two species of basidio- mycetes,andin 1951[1]. Pleuromutilin is a diterpene, constituted of a rather rigid 5-6-8 tricyclic carbon skeleton with eight stereogenic centers[2,3]. The derivatives of pleuromutilin have received much investigative attention due to their high activities against drug- resistant Gram-positive bacteria and mycoplasmasand[4], pharmacodynamic proper- ties[5], and no target-specific cross-resistance to other antibiotics[6,7]. The molecular modifications of pleuromutilin were focused essentially on the C-14 glycolic acid chain, especially with a thioether group and a basic group together[8]. Further altera- tions within this group led to the development of three drugs: tiamulin, valnemulin, and retapamulin. The first two drugs were used as veterinary drugs[9], while retapamulin became the first pleuromutilin approved for human use in 2007[10]. Footprinting analysis showed that pleuromutilin derivatives inhibited the bacterial protein synthesisa specific interaction with the 23S rRNA of the 50S bacterial ribosome subunit[4, 11].

Herein, we prepare the title compound, 14-O- [(4,6-diaminopyrimidine-2-yl) thioacetate] mutilin (3), which was characterized by IR,1H NMR and13C NMR spectral analysis, and report its single- crystal structure. The synthesis route is depicted in Scheme 1.

Scheme 1. Synthetic route of the title compound

2 EXPERIMENTAL

2. 1 Reagents and instruments

All reagents obtained from commercial sources were of AR grade and used without further puri- fication. Melting points were determined on a Tianda Tianfa YRT-3 apparatus (China) with open capillary tubes and were uncorrected. IR spectra were obtained on a Thermo Nicolet NEXUS-670 spectrometer and recorded as KBr thin film and the absorptions were reported in cm-1. NMR spectra were recorded on Bruker-400 MHz spectrometers in appropriate solvents. Chemical shifts () in1H NMR were expressed in parts per million (ppm) relative to the tetramethylsilane. Multiplicities of signals are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad),.13C NMR spectra were recorded on 100 MHz spectrometers. The single-crystal structure of the title compound was determined on an Agilent SuperNova X-diffractometer. All reactions were monitored by TLC on 0.2 mm thick silica gel GF254 pre-coated plates. After elution, plates were visualized under UV illumination at 254 nm for UV active materials. Further visualizations were achieved by staining with 0.05% KMnO4aqueous solution. Column chromatography was carried out on silica gel (200～300 mesh). The products were eluted in appropriate solvent mixture under air pressure. Concentration and evaporation of the solvent after reaction or extraction were carried out on a rotary evaporator.

2. 2 Synthesis and characterization of the title compound

2. 2. 1 Synthesis of intermediate 2

Forty percent NaOH solution (5 mL, 50 mmol) was added dropwise to a mixture of pleuromutilin (7.57 g, 20 mmol) and-toluenesulfonyl chloride (4.2 g, 22 mmol) in t-butyl methyl ether (20 mL) and water (5 mL). The mixture was stirred vigorously under reflux for 1 h, then diluted with water (50 mL) and stirred under an ice bath for 15 min, followed by washing with water (50 mL) and cold t-butyl methyl ether (20 mL). Filtration afforded compound 2 as white solid. It was used in the next step without further purification. Yield: 93%. IR (KBr): 3446(OH, s), 2924(CH2, s), 2863(CH3, s), 1732(C=O, vs), 1597(C=C-C=, m), 1456(CH2, s), 1371(CH3, s), 1297(C=S, s), 1233(S=O, s), 1117(C=O, s), 1035(C-OH, s), 832(C=C-C=, m), 664(CH2, m) cm-1.1H NMR (400 MHz, CDCl3)7.80～7.82 (d, 2H, J = 4.0 Hz), 7.35～7.37 (d, 2H, J = 4.0 Hz), 6.43(q, 1H, J = 17.2 Hz, 10.8 Hz), 5.75～5.78 (d, 1H, J = 4.2 Hz), 5.31～5.34 (d, 1H, J = 6.4 Hz), 5.17～5.21 (d, 1H, J = 8.8 Hz), 4.48 (s, 2H), 3.34 (d, 1H, J = 6.4 Hz), 2.45(s, 3H), 2.21～2.29 (m, 3H), 2.01～2.08(m, 3H), 1.63～1.65 (dd, 2H, J1= 10Hz, J2= 7.2 Hz), 1.46～1.50 (m, 5H), 1.41～1.44 (m, 1H), 1.33～1.36 (m, 1H), 1.22～1.26 (s, 5H), 1.11～1.15 (m, 1H), 0.87 (d, 3H, J = 6.8Hz), 0.63 (d, 3H, J = 6.8 Hz);13C NMR(100 MHz, CDCl3)216.7, 164.8, 145.2, 138.6, 132.5, 129.9, 127.9, 117.2, 74.4, 70.2, 64.9, 57.9, 45.3, 44.4, 43.9, 41.7, 36.4, 35.9, 34.3, 30.2, 26.7, 26.3, 24.7, 21.6, 16.4, 14.6, 11.4.

2. 2. 2 Synthesis of the title compound 3

A solution of 30% aqueous NaOH (2.2 mmol) was added to a stirred mixture of 4,6-diamino-2-mercap- topyrimidine hydrate (2.8 mg, 2.0 mmol) in 5 mL CH3OH, and the resulting reaction mixture was stirred at room temperature for 30 min. Compound 2 (1.17 g, 2.2 mmol) in 15 mL CH2Cl2was added dropwise to the reaction mixture and stirred at 0oC for 36 h. Then the reaction mixture was concentrated in vacuo, followed by the addition of EtOAc and washing with brine and water. The organic layer was dried over anhydrous MgSO4, filtered, and con- centrated in vacuo to give the residue that was puri- fied by column chromatography using silica gel to give the desired product[12]. White solid; yield: 95%, IR (KBr): 3475(NH, m), 3377(NH, m), 2933(CH2,s), 1728(C=O, s), 1619(C=O, vs), 1582(C=C-C=, s), 1547(C=C-C=, s), 1465(CH2, s), 1309(OH, s), 1153(C-S, m), 1117(C=O, m), 1019(C-O, m) cm-1.1H NMR (400 MHz, DMSO)1H NMR (400 MHz, DMSO)6.14 (d,= 11.2 Hz, 1H), 6.11～5.97 (m, 3H), 5.52 (d,= 7.8 Hz, 1H), 5.12 (d,= 15.7 Hz, 1H), 5.01 (dd,= 26.9, 15.7 Hz, 1H), 4.50 (d,= 5.8 Hz, 1H), 4.17～3.92 (m, 1H), 3.80 (q,= 16.0 Hz, 1H), 3.42 (s, 1H), 2.39 (s, 1H), 2.29～2.14 (m, 1H), 2.12～1.88 (m, 4H), 1.75～1.54 (m, 2H), 1.54～1.11 (m, 9H), 1.12～0.89 (m, 4H), 0.82 (d,= 6.5 Hz, 3H), 0.62 (d,= 6.4 Hz, 3H).13C NMR (101 MHz, DMSO)217.6 , 168.3 , 167.3, 163.8, 141.2, 115.8, 79.6, 73.1, 70.0, 60.2, 57.7, 45.4, 44.5, 44.0, 42.0, 36.8, 34.5, 33.3, 30.6, 29.0, 27.1, 24.9, 21.2, 16.6, 15.0, 12.0.

2. 3 Crystal data and structure determination

The crystals of the title compound suitable for X-ray structure determination were obtained by slowly evaporating a mixed solvent of ethanol and acetone for about twenty days at room temperature. A colorless single crystal with dimensions of 0.31mm × 0.35mm × 0.36mm was selected and mounted in air onto thin glass fibers. X-ray intensity data were collected at 291.73(10) K on an Agilent SuperNova-CCD diffractometer equipped with a mirror-monochromatic Mo(= 0.71073 ?) radiation. A total of reflections were collected in the range of 3.55≤≤26.02o (index ranges: –12≤≤12, –14≤≤15, –7≤≤12) by using anscan mode with 4161 independent ones (int= 0.0493), of which 2223 with> 2() were considered as observed and used in the succeeding refinements. The structure was solved by direct methods using SUPERFLIP program[13]and refined with SHELXL program[14, 15]by full-matrix least-squares techni- ques on2. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms were deter- mined with theoretical calculations. A full-matrix least-squares refinement gave the final= 0.0649,= 0.0797 (= 1/[2(F2) + (0.000)2], where= (F2+ 2F2)/3), (Δ/)max= 0.000,= 0.947, (Δ)max= 0.213, and (Δ)min= –0.276 e/?3.

2. 4 Antibacterial activity measurement

Oxford cup assay was performed to evaluate the inhibition rate of the title compound in the growth of bacteria. Inoculum was prepared in 0.9% saline using McFarland standard and spread uniformly on nutrient agar plates. The title compound, as well aspleuromutilin and tiamulin fumarate used as reference drugs, was weighed 12800 μg accurately and dissolved with 3 mL ethanol, followed by diluting to 10 mL with distilled water. Then all the solutions were diluted with distilled water by two folds. The resulting solutions were added indivi- dually to the Oxford cups which were placed at equidistance on the above agar surfaces. The zone of inhibition for each concentration was measured after 24 h incubation at 37oC. The same procedure was repeated in triplicate.

3 RESULTS AND DISCUSSION

The title compound was prepared according to Scheme 1. Almost all pleuromutilin derivatives were synthesized from 22-O-tosylpleuromutilin (Com- pound 2) which was obtained by the reaction of pleuromutilin and-toluenesulfonyl chloride to activate the 22-hydroxyl of pleuromutilin. The title compound was then obtained in good yield by the nucleophilic attack of 4,6-diamino-2-mercaptopyri- midine hydrate on compound 2 under alkaline conditions. The IR,1H NMR and13C NMR for the obtained compounds are all in good agreement with the assumed structure. A single crystal of the title compound was cultured for X-ray diffraction analysis to confirm the configuration. The selected bond lengths and bond angles are shown in Table 1, and hydrogen bonding parameters in Table 2.

Table 1. Selected Bond Lengths (?) and Bond Angles (°)

Table 2. Hydrogen Bond Lengths (?) and Bond Angles (°)

Symmetry codes: (a) 1,,; (b) –1–, –0.5+, –1–; (c) –1+,,

The crystal structure in Fig. 1 shows that the whole molecule of the complex consists of a 5-6-8 tricyclic carbon skeleton and a pyrimidine ring, in which all bond lengths are in normal ranges. Five chiral carbons can be found in the molecule and the absolute configurations of C(2), C(3), C(4), C(5), C(9) are S, R, R, R and S. The five-membered ring (C(3), C(4), C(11), C(12), C(14)) is not planar and the dihedral angles formed by C(3)–C(12)–C(13)– C(14) and C(1)–C(4)–C(14) is 38.133o. The bond lengths of C(3)–C(12) and C(12)–C(13) are shorter than that of C(3)–C(2) and C(13)–C(14), which may be caused by the conjugation with carbonyl (C=O). The eight-membered ring (C(2), C(3), C(4), C(5), C(6), C(7), C(8), C(1)) exhibits a boat conformation, while the six-membered ring (C(3), C(4), C(11), C(10), C(9), C(2), C(3)) exhibits a chair conforma- tion and the dihedral angles formed by C(3)–C(4)– C(11), C(3)–C(11)–C(10)–C(2) and C(10)–C(9)– C(2) are 46.674 and 47.847o, respectively.

Fig. 1. Molecular structure of the title compound (Hydrogen atoms were omitted for clarity)

The side chain of C(14) exhibits a zig-zag con- formation. The bond length of O(2)–C(15) is shorter than that of O(1)–C(1) which may be caused by the conjugation with carbonyl (C=O).Four intramole- cular hydrogen bonds are formed in the structure at C(21)–H(21A)···O(1), C(21)–H(21B)···O(3), C(22)–H(22C)···O(1) and C(26)–H(26A)···O(4) via C atoms of tricyclic skeleton and the O atoms of ester, hydroxyl group and carbonyl group, respect- tively. At the same time, three intermolecular hydrogen bonds are formed between two molecules of the title compound at N(3)–H(1)···O(4),N(3)– H(3B)···O(2), and O(4)–H(4)···N(1). One of the amino groups at the terminal side chain takes part in two hydrogen bonds and its N atom acts as two hydrogen-bond donors to the O atom of hydroxyl group and carbonyl group. Meanwhile, the O atom of hydroxyl group also acts as a hydrogen-bond donor to the N atom of the other amino group at the terminal side chain. These intermolecular hydrogen bonds link the molecular network structure and play key roles in stabilizing the crystal packing (Fig. 2).

Fig. 2. Molecular packing of the title compound

The synthesized compound 3 was screened for theirantibacterial activity against MRSA, andusing Oxford cup assay. The zones of inhibition for different concentrations of com- pound 3, pleuromutilin and tiamulin fumaratewere measured. The results of antibacterial activi- ties are given in Table 3. Compound 3 showed higher antibacterial activities than pleuromutilin but simi- lar antibacterial activity against MRSA andto that of tiamulin fumarate. Thedata of Oxford cup assay provide important information for further activity studies.

Table 3. Zone of Inhibition of the Compounds in mm

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①Supported by Basic Scientific Research Funds in Central Agricultural Scientific Research Institutions (No. 1610322014003) and the Agricultural Science and Technology Innovation Program (ASTIP)

. Shang Ruo-Feng, born in 1974, doctor in medicinal chemistry. E-mail: shangrf1974@163.com;Liang Jian-Ping, born in 1963, doctor in animal drugs. E-mail: liangjp100@sina.com

10.14102/j.cnki.0254-5861.2011-0750

2 April 2015; accepted27 May 2015 (CCDC 1400655)