ChemInform Abstract: A Novel Everninomicin Antibiotic Active Against Multidrug-Resistant Bacteria

Tetrahedron Letters 41 (2000) 6689±6693

A novel everninomicin antibiotic active against multidrug-resistant bacteria Min Chu,* Ronald Mierzwa, Mahesh Patel, John Jenkins, Pradip Das, Ben Pramanik and Tze-Ming Chan Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA Received 8 June 2000; accepted 3 July 2000

Abstract A novel oligosaccharide, Sch 58761 (1), was isolated from the fermentation broth of Micromonospora carbonaceae using diol-bonded/polyvinyl alcohol-functionalized silica gel (PVA-Sil) puri®cation. Structure determination of 1 was accomplished by extensive mass spectrometric and NMR studies. # 2000 Published by Elsevier Science Ltd. Keywords: novel oligosaccharide; puri®cation; 1H and 13C NMR data; structure elucidation.

Everninomicins are a class of oligosaccharide antibiotics1ÿ5 produced by Micromonospora carbonaceae,6 which possess highly potent activity against Gram-positive bacteria. More importantly, everninomicins demonstrate antibacterial activity both in vitro and in vivo against multiply resistant strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The major component, Ziracin, was under drug development in phase III clinical trials as an important antibiotic for the treatment of resistant bacterial infections. In the process of largescale puri®cation of Ziracin, a new member of the everninomicin family of compounds, Sch 58761 (1), was isolated from the fermentation broth by a tandem normal phase chromatographic approach. In this paper, we report the isolation, structure elucidation and biological activity of 1.

* Corresponding author. 0040-4039/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. PII: S0040-4039(00)01123-0

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An antibiotic complex (5 g) from the tail cut obtained during the puri®cation of Ziracin was dissolved in 20 mL of CH2Cl2:MeOH (96:4 v/v) and applied to 200 g of diol-bonded silica (YMC Co.). After sample application, the diol column was eluted with a mixed solvent of CH2Cl2:heptane:MeOH (60:40:2, v/v/v, 2.4 L) followed by the mobile phase of CH2Cl2:heptane:MeOH (75:25:2, 8 L) to yield an enriched complex containing compound 1 (200 mg). The enriched complex was further puri®ed by dissolving 40 mg of the sample in 0.5 mL CH2Cl2:MeOH (96:4 v/v) and injecting on a semi-preparative PVA-Sil column (25020 mm) eluted with CH2Cl2:MeOH (97.5:2.5 v/v). The pure 1 (5 mg, >98% purity) was collected with a ¯ow rate of 12 mL/min under UV detection at 265 nm. After four additional injections were made, a total of 24 mg of 1 was isolated as a white amorphous powder with mp=183±185 C and ‰Š20 D =^48.4 (c 0.1, MeOH). The molecular weight of 1 was determined to be 1663 based on FAB mass spectral data that showed a protonated molecular ion peak at m/z 1664 (M+H)+. The observation of a chlorine isotope cluster pattern in the FAB mass spectrum suggested three chlorine atoms in 1. The molecular formula was further deduced by elemental analysis as C70H96NO38Cl3 (anal.: C, 49.82%; H, 5.92%; N, 0.82%; Cl, 6.21%; calcd for C70H96NO38Cl3: C, 50.51%; H, 6.02%; N, 0.92%; Cl, 6.31%). The IR spectrum of 1 indicated the presence of hydroxyl, conjugated carbonyl and nitro functionalities due to strong absorptions at 3454, 1734 and 1544 cm^1, respectively. UV absorption at 211, 268 and 309 nm suggested the presence of an aromatic moiety. 1H and 13C NMR spectroscopic data are summarized and listed in Tables 1 and 2, Table 1 H NMR spectral data for Sch 58761(1)a

1

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respectively.8 As shown in Table 2, 13C NMR data indicated two distinctive ortho ester carbons at  118.93 and 120.17, as well as six anomeric methines at  92.29, 96.15, 97.57, 100.50, 100.90 and 104.09. 13

Table 2 C NMR spectral data for Sch 58761 (1)

A typical fragmentation pattern of an oligosaccharide was also observed in the FAB mass spectrum of 1. Assignments of individual fragments are summarized in Fig. 1 in comparison with Ziracin, which was well characterized and reported previously.5ÿ7 A total of eight sugar residues were recognized including the nitrosugar as A-ring. Two most distinctive fragments at m/z 969 and 695 Da indicated that the left side portion remains unchanged based on the observation of the same fragment of 695 Da in Ziracin. This assignment was further supported by sequential break down fragments at m/z 550, 363 and 233 Da. However, one of the major fragment at m/z 969 Da revealed a 34 mass units increase suggesting the presence of an extra chlorine atom due to its distinctive isotope distribution in comparison with Ziracin. Further fragmentation analysis of

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sequential peaks pointed out that the chlorine was located at the fragment of m/z 185 Da, 2,4-dihydroxy-6-methyl benzyl residue.

Figure 1. FAB-MS and NMR spectral data of Sch 58761 1

H NMR data con®rmed the chlorine replacement of proton on the benzyl ring because only one aromatic singlet at  6.34 was detected in 1. In order to determine the exact position, 2D NMR studies were conducted including NOESY and HMBC. As shown in Fig. 1, both HMBC and NOESY data reveal the strong correlation of methyl group and the proton at position-60. Therefore, the chlorine should be located at position-58. The stereochemistry of 1 was proposed to be the same as Ziracin9ÿ11 based on the NOESY spectral data analysis in comparison with Ziracin. Compound 1 exhibited potent antibacterial activity even slightly better than Ziracin against various pathogenic Gram-positive organisms,12 including Staphylococcus aureus (MIC: 0.25±1 mg/mL), Staphylococcus epidermidis (MIC: 1±2 mg/mL), Enterococcus faecium (MIC: 0.25±2 mg/mL) and Enterococcus faecalis (MIC: 1±2 mg/mL). Compound 1 also displayed better activity against methicillin-resistant Staphylococcus aureus (MRSA) strains (MIC90: 0.71 mg/mL) than vancomycin (MIC90: 1.28 mg/mL). Acknowledgements The authors thank Mr. S. Mittleman for IR data, Mr. G. Torraca for optical rotation and elemental analysis data, Dr. D. Loebenberg for biological activity data and Mrs. D. Scott for the preparation of this manuscript. References 1. Everninomicins are often referred to as orthosomycins. For a review, see: Wright, D. E. Tetrahedron 1979, 35, 1207±1221. 2. Girijavallabhan, V. M.; Ganguly, A. K. Kirk-Othmer Encyclopedia of Chemical Technology, 4th Ed.; John Wiley & Sons, 1992; Vol 3, pp. 259±266. 3. Ganguly, A. K.; Saksena, A. K. J. Antibiotics 1975, 28, 707±709. 4. Ganguly, A. K.; Sarre, O. Z.; Greeves, D.; Morton, J. J. Am. Chem. Soc. 1975, 97, 1982±1985.

6693 5. Ganguly, A. K.; Pramanik, B.; Chan, T. M.; Sarre, O. Z.; Liu, Y.-T.; Morton, J.; Girijavallabhan, V. Heterocycles 1989, 28, 83±88. 6. Loebenberg, D.; Cacciapuoti, A.; Naples, L.; Moss Jr., E. L.; Menzel Jr., F.; Hare, R. S.; Miller, G. H. In Programs and Abstracts of the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy; p. 202, Abstract 456. 7. Chan, T. M.; Osterman, R. M.; Morton, J. B.; Ganguly, A. K. Magn. Res. Chem. 1997, 35, 529±532. 8. Since compound 1 was recorded in CDCl3 with 10% CD3OD, a 0.05 ppm chemical shift should be added to all proton signals as an adjustment in order to compare with Ziracin, which was recorded in 100% CDCl3. In 13C NMR spectrum, a 0.20 ppm chemical shift should be added to all signals as well for the comparison. 9. Ganguly, A. K.; Sarre, O. Z.; McPhail, A. T.; Miller, R. J. Chem. Soc., Chem. Commun. 1979, 22±25. 10. Bean, J.-M.; Jaurand, G.; Esnault, J.; Sinay, P. Tetrahedron Lett. 1987, 1105±1109. 11. Ganguly, A. K.; McCormick, J. P.; Chan, T. M.; Saksena, A. K.; Das, P. R. Tetrahedron Lett. 1997, 7989±7992. 12. MIC range for everninomicin: Staphylococcus aureus (MIC: 1±4 mg/mL), Staphylococcus epidermidis (MIC: 0.5±4 mg/mL), Enterococcus faecium (MIC: 1±2 mg/mL) and Enterococcus faecalis (MIC: 0.5±2 mg/mL). A total of 24 strains were tested (six strains per species).