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ÿþAvailable online at www.sciencedirect.com Bioorganic & Medicinal Chemistry Letters 18 (2008) 2585 2589 Synthesis and antimicrobial properties of Monensin A esters Adam HuczyDski,a Joanna StefaDska,b Piotr Przybylski,a Bogumil Brzezinskia,* and Franz Bartlc a Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland b Medical University, Department of Pharmaceutical Microbiology, Oczki 3, 02-007 Warsaw, Poland c Institute of Medical Physics and Biophysics Charité, Universitätsmedizin Berlin Campus Charité Mitte, Ziegelstr. 5/9, 10117 Berlin, Germany Received 6 February 2008; revised 12 March 2008; accepted 14 March 2008 Available online 16 March 2008 Abstract The esters (2 10) of the ionophore antibiotic Monensin (1) were synthesized by four different methods, which are discussed in detail. These new esters were characterized by various spectroscopic techniques and subsequently tested in the face of their antimicrobial properties. Three derivatives (3, 8 and 10) showed activity against Gram-positive bacteria. Additionally derivative (10) exhibited a relatively low antifungal activity against Candida in contrast to Monensin A. Ó 2008 Elsevier Ltd. All rights reserved. Monensin A (1, Fig. 1) isolated from Streptomyces related with a strong antifungal and/or antibacterial cinnamonensis is a well-known polyether antibiotic, activity.10,11 Furthermore, our previous results demon- capable to transport monovalent and divalent metal cat- strated that the esterification of Monensin A influences ions across lipid membranes. Therefore, it belongs to a its physicochemical properties and consequently these group of highly bioactive molecules.1,2 Monensin exhib- Monensin derivates have the potential to change the its antibiotic,3 coccidiostatic,4 cardiovascular5 and other mode of action.12 These interesting results have moti- important biological and medical properties.6 Only vated our group to optimize the synthesis methods and recently it was shown that Monensin A is also a highly to study the biological activities of these new com- effective ionophore for Li+, Rb+ as well as for Pb2+ pounds. In the present contribution, we compare four cations.7 These properties are the basis of many biolog- methods of Monensin A ester synthesis. Furthermore, ical and pharmaceutical fields of application of this we compare the antimicrobial activity of the new ester compound and new ones can be expected for the future. with the activity of unmodified Monensin A. Up to now various derivatives of Monensin were synthe- The synthesis of carboxylic esters is one of the most fun- sized in order to reduce its toxicity and to extend its damental methods in organic chemistry to obtain useful fields of application.8 In previous publications we natural and synthetic compounds. However, most ester- reported on the synthesis and the physicochemical prop- ification procedures require rather harsh conditions such erties of several new Monensin A esters.9 The complex- as the presence of strong acids, bases or other catalysts. ation of mono- and divalent metal cations by Monensin Furthermore, the reactions of this type often proceed A esters and the properties of these complexes have been only at high temperatures. However, Monensin A is very described in detail. These esters show especially high sensitive to acidic conditions and heating. For this rea- affinity towards Na+ and Ca2+ cations.9 son we attempted to choose the reaction conditions for the esterification as mild as possible. The new esters of From the literature data it is known that the complexa- Monensin A (2 10) were prepared according to four dif- tion ability towards Na+ and Ca2+ cations is often ferent methods. All esters used for our experiments including the acyloxy esters are stable under the experi- mental conditions. This was checked by several spectro- Keywords: Ionophores; Monensin; Esters; Synthesis; Antimicrobial scopic and spectrometric methods such as FT-IR, NMR activity. and ESI-MS. * Corresponding author. E-mail: bbrzez@amu.edu.pl 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.03.038 2586 A. HuczyDski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585 2589 30-31 monensin A (1): R = H (2): R = CH2CH3 29 32 13 (4): R = H2C C CH2 H2C (3): R = H 17 O O 9 O 28 (6): R = H2C (5): R = H2C O 21 34 O 5 OH 25 HO IV 36 33 X 27 Me 26 O O OMe (7): R = (8): R = HO H2C O O H2C O 35 Me 1 XI O OR O (9): R = H2C NO2 (10): R = H2C N Figure 1. Chemical structure of Monensin A and its esters. These synthesis pathways summarized in Scheme 1 are the presence of DCC, PPy and p-TSA (p-toluenesulfonic the only efficient ones, albeit with different yields. acid monohydrate). This method was quite efficient and esters (3), (7) and (8) could be obtained in high yields In the first method DCC (N,N0-dicyclohexylcarbodiim- (up to 70%). However, under the same reaction condi- ide) was used as a coupling agent (Scheme 1, method tions, the Monensin benzyl ester (4) was only obtained a).13,14 This procedure results in low to moderate chem- with a yield of 35%.14 ical yields, for example, the yield of Monensin ethyl ester (2) was only 20%. The fourth reliable strategy of Monensin (1) esterifica- tion is based on the direct alkylation of carboxylate ions. When PPy (4-pyrrolidinopyridine), a very effective acyl- This method uses the corresponding alkyl bromides with ation catalyst, was additionally used in catalytic 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as an effective amounts, the yield of compound (2) could be drastically nucleophilic catalysts. Under these reaction conditions increased to 71%. However, for the synthesis of the the yield was above 75% (4, 6). Note that the use of alkyl other Monensin esters such as (3), (7) and (8), this ester- bromides instead of alkyl chlorides significantly ification method was unsatisfactory because only low increases the yields of the respective esters. Interestingly, yields of these compounds could be achieved (Scheme this esterification method shows also a remarkable 1, method b).14 solvent dependence. Toluene was the most appropriate of all solvents tested, probably because of an optimal The third method for the synthesis of new Monensin solubility of reactants and products in this solvent esters (Scheme 1, method c) is based on the reaction (Scheme 1, method d).14 between Monensin A and the appropriate alcohol in All esters (2 10) can easily be purified by column chro- matography on silica gel. The structures of the esters were determined on the basis on elemental analysis, 1 13 (4) (89%*); (5) (30%**); FT-IR, H, C NMR, ESI-MS and semiempirical (PM5) methods.14,15 (6) (80%*); (9) (45%**); (10) (76%*) Monensin A (1) as well as the new esters of Monensin A (2 10) were tested in vitro in the face of their antibacte- d) rial and antifungal activity. The Gram-positive cocci, Gram-negative rods and yeasts-like micro-organisms c) used in the tests are collected in Table 1. (3) (67%); (4) (35%); a) monensin A (2) (20%) (1) (7) (72%); (8) (69%) Hospital strains of S. aureus were isolated from different biological materials of patients of the Warsaw Medical b) University Hospital. 10 of these strains were methicil- lin-susceptible (MSSA) and 10 other strains were methi- (2) (71%); (3) (32%); cillin-resistant (MRSA). Due to this resistance these strains were tested concerning their sensitivity towards (7) (37%); (8) (15%) the new Monensin A esters. The other micro-organisms Scheme 1. Synthetic access to Monensin A ester derivatives. Reagents used here were provided by the Department of Pharma- and conditions: (a) EtOH, DCC (N,N0-dicyclohexylcarbodiimide), ceutical Microbiology, Medical University of Warsaw, CH2Cl2, 0 °C to rt; (b) R-OH (corresponding alcohols), DCC, PPy (4- Poland.16,17 pyrrolidinopyridine), CH2Cl2, 0 °C to rt; (c) R-OH, DCC, PPy, p-TSA (p-toluenesulfonic acid monohydrate), CH2Cl2, 0 °C to rt; (d) R-X The data concerning the antimicrobial activity of the (bromides* or chlorides**), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), compounds are summarized in Table 1. toluene, 90 100 °C, 5 h. A. HuczyDski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585 2589 2587 Table 1. Antimicrobial activity of Monensin A (1) and its esters: (3), (8), (10); diameter of the growth inhibition zone (GIZ) [mm] and minimal inhibitory concentration (MIC) [lg/ml]16,17 Growth inhibition zone (GIZ) [mm] and Minimal inhibitory concentration (MIC) [lg/ml] (1)(3)(8)(10) GIZ MIC GIZ MIC GIZ MIC GIZ MIC S. aureus NCTC 4163 22 2 13 100 19 100 23 12.5 S. aureus ATCC 25923 22 1 11 100 19 50 20 6.25 S. aureus ATCC 6538 20 2 13 100 17 100 23 12.5 S. aureus ATCC 29213 18 1 13 100 17 50 25 6.25 S. epidermidis ATCC 12228 15 2  100 13 100 24 12.5 B. subtilis ATCC 6633 22 1 15 12.5 20 25 27 6.25 B. cereus ATCC 11778 18 2 14 12.5 19 50 25 6.25 E. hirae ATCC 10541  12.5  >400  >400 13 50 M. luteus ATCC 9341 12 4  100 11 200 22 25 M. luteus ATCC 10240 12 2 11 50 13 50 18 12.5 E. coli ATCC 10538 na na na na E. coli ATCC 25922 na na na na E. coli NCTC 8196 na na na na P. vulgaris NCTC 4635 na na na na P. aeruginosa ATCC 15442 na na na na P. aeruginosa NCTC 6749 na na na na P. aeruginosa ATCC 27853 na na na na B. bronchiseptica ATCC 4617 na na na na C. albicans ATCC 10231 na na na 18 200 C. albicans ATCC 90028 na na na 17 200 C. parapsilosis ATCC 22019 na na na 14 400 na, no activity in disc diffusion test denotes lack of the growth inhibition zone. Among the tested compounds (Table 1), only Monensin ring moiety, since it was previously observed that vari- A (1) as well as three ester derivatives (3, 8 and 10) ous compounds containing this substituent show moder- showed detectable but different activities against ate antibacterial and strong antifungal activities.18 The Gram-positive bacteria. Only one of the derivatives low antibacterial activity of compound 3 is probably (10) exhibited relatively low antifungal activity against connected with the presence of allyl group, which has Candida while Monensin A was inactive against these a significant influence on the antimicrobial activity.19 fungi. Compounds (2, 4 7 and 9) were inactive towards all micro-organisms tested.16 Drug-resistant Gram-positive bacterial pathogens including methicillin-resistant S. aureus (MRSA) cause The interactions between the oxygen atoms of Monensin serious chemotherapeutic problems in hospitals.20 Our A or of the Monensin A esters with mono- and di-valent studies reveal that Monensin A clearly shows antimicro- metal cations lead to the formation of pseudo-cyclic struc- bial activity against both MRSA (methicillin-resistant) tures which are additionally stabilized by intramolecular and MSSA (methicillin-susceptible) strains of Staphylo- hydrogen bonds.2,7,9 In previous investigations we could coccus aureus at doses MIC = 1 2 lg/ml, whereas from show that the mode of complex formation with Na+ cat- among the Monensin A esters studied only compound ions is very similar for the majority of Monensin A deriv- 10 shows interesting activity at the doses MIC = 6.25 atives and rather independent of the nature of the 12.5 lg/ml (Supplementary material, Table 1S). respective ester groups. This suggests that the ester groups are not engaged in the coordination process.7,9 Thus, the In the present work, we synthesized nine new esters differences in the biological activities between Monensin (2 10) of Monensin A using four different synthesis A and the Monensin A derivatives described here are pathways. We provide evidence that three Monensin not based on a different capability of complex formation esters (3, 8 and 10) show antibacterial activity against but on other parameters such as size and chemical nature human pathogenic bacteria, including antibiotic-resis- of the substituent. One of these parameters is potentially tant S. aureus. Concerning the Monensin A esters, only the lipophilic character of the substituent, which evokes compound (10) shows relatively low antifungal activity. lower solubility in aqueous solutions. Furthermore, the presence of aromatic substituent in the ester group, such as phenyl (compounds 4 and 9) or naphthalene rings Acknowledgments (compounds 5 and 6), might decrease the mobility of Monensin A esters in the lipid bilayers. Adam HuczyDski thanks the Foundation for Polish Science for fellowship. Financial assistance of the Polish The highest antibacterial activity of compound 10 Ministry of Science and Higher Education Grant No. among the ester derivates and its slight antifungal activ- N204 056 32/1432 is gratefully acknowledged by ity is probably related to the presence of the morpholine P. Przybylski. 2588 A. HuczyDski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585 2589 Nagatsu, A.; Mizukami, H.; Ogihara, Y.; Sakakibara, J. Supplementary data Chem. Pharm. Bull. 2001, 49, 711; (e) Tsukube, H.; Sohmiya, H. J. Org. Chem 1991, 56, 875; (f) Maruyama, Supplementary data associated with this article can be K.; Sohmiya, H.; Tsukube, H. J. Chem. Soc., Chem. found, in the online version, at doi:10.1016/j.bmcl.2008. Commun. 1989, 864; (g) Nagatsu, A.; Tabunoki, Y.; 03.038. Nagai, S.; Ueda, T.; Sakakibara, J.; Hidaka, H. Chem. Pharm. Bull. 1997, 45, 966; (h) Nagatsu, A.; Sakakibara, J. Yakugaku Zasshi. 1997, 117, 583. 9. (a) HuczyDski, A.; Przybylski, P.; Brzezinski, B.; Bartl, F. References and notes Biopolymers 2006, 81, 282; (b) HuczyDski, A.; Przybylski, P.; Brzezinski, B.; Bartl, F. Biopolymers 2006, 82, 491; (c) 1. (a) Ferdani, R.; Gokel, G. W. Encyclopedia of Supramo- HuczyDski, A.; Michalak, D.; Przybylski, P.; Brzezinski, lecular Chemistry: Ionophores; Marcel Dekker Inc., 2004, B.; Bartl, F. J. Mol. Struct. 2006, 797, 99; (d) HuczyDski, pp. 760 766; (b) Westley, J. W. In Polyether Antibiotics. A.; Michalak, D.; Przybylski, P.; Brzezinski, B.; Bartl, F. Naturally Occurring Acid Ionophores; Marcel Dekker Inc.: J. Mol. Struct. 2007, 828, 130; (e) HuczyDski, A.; Aowicki, New York, 1982; vol. 1, pp. 1 20; (c) Pressman, B. C. D.; Brzezinski, B.; Bartl, F. J. Mol. Struct. doi:10.1016/ Antibiotics and Their Complexes; Marcel Dekker Inc.: j.molstruc.2007.08.004; (f) HuczyDski, A.; Przybylski, P.; New York, 1985, pp. 1 18; (d) Westley, J. W. In Polyether Brzezinski, B. J. Mol. Struct. 2006, 788, 176; (g) Antibiotics. Naturally Occurring Acid Ionophores; Marcel HuczyDski, A.; Przybylski, P.; Schroeder, G.; Brzezinski, Dekker Inc.: New York, 1983; vol. 2, pp. 51 86; (e) B. J. Mol. Struct. 2007, 29, 111; (h) HuczyDski, A.; Dutton, C. J.; Banks, B. J.; Cooper, C. B. Nat. Prod. Rep. Przybylski, P.; Brzezinski, B. Tetrahedron 2007, 63, 8831. 1995, 12, 165. 10. (a) Rao, S.; Venkateswerlu, G. Curr. Microbiol. 1989, 19, 2. (a) Riddell, F. G. Chirality 2002, 14, 121; (b) Martinek, T.; 253; (b) Dashper, S. G.; O Brien-Simpson, N. M.; Cross, Riddell, F. G.; Wilson, C.; Weller, C. T. J. Chem. Soc. K.; Paolini, R. A.; Hoffman, B.; Catmull, D.; Malkoski, Perkin Trans. 2 2000, 35; (c) Ben-Tal, N.; Sitko, D.; M.; Reynolds, E. C. Antimicrob. Agents Chemother. 2005, Bransburg-Zabary, S.; Nachliel, E.; Gutman, M. Biochim. 49, 2322. Biophys. Acta 2000, 1466, 221; (d) Pinkerton, M.; Stein- 11. Pankiewicz, R.; Remlein-Starosta, D.; Schroeder, G.; rauf, L. K. J. Mol. Biol. 1970, 49, 533; (e) Mollenhauer, H. Brzezinski, B. J. Mol. Struct. 2006, 783, 136. H.; Morre, D. J.; Rowes, R. D. Biochim. Biophys. Acta 12. HuczyDski, A.; Przybylski, P.; Brzezinski, B.; Bartl, F. 1990, 1031, 225. J. Phys. Chem. B 2006, 110, 15615. 3. (a) Pressman, B. C. Annu. Rev. Biochem. 1976, 45, 501; (b) 13. Synthesis of Monensin A (1): Monensin A sodium salt Shumard, R. F.; Callender, M. E. Antimicrobial Agents (Fluka) was dissolved in dichloromethane and stirred Chemother. 1967, 7, 369. vigorously with a layer of aqueous sulphuric acid (pH 1.5). 4. Nebbia, C.; Ceppa, L.; Dacasto, M.; Nachtmann, C.; The organic layer containing MONA was washed with Carletti, M. J. Vet. Pharmacol. Ther. 2001, 24, 399. distilled water, and dichloromethane was evaporated 5. Fahim, M.; Pressman, B. C. Life Sci. 1981, 29, 1959. under reduced pressure to dryness. 6. (a) Jenkins, T. C.; Fellner, V.; Mcuffey, R. K. J. Dairy Sci. 14. (a) General procedure for the synthesis of Monensin A 2003, 86, 324; (b) Yamazaki, Y.; Sejima, H.; Yuguchi, M.; esters (2 10): (method a, Scheme 1): To a mixture of (1) Shinozuka, K.; Isokawa, K. J. Oral Sci. 2007, 49, 107; (c) (500 mg, 0.75 mmol) in dichloromethane (15 ml) the Clark, M. R.; Mohandas, N.; Shohet, S. B. J. Clin. Invest. following compounds were added: DCC (206 mg, 1982, 70, 1074; (d) Ramanzin, M.; Bailoni, L.; Schiavon, 1.0 mmol), EtOH (5 mmol). The mixture was first stirred S.; Bittante, G. J. Dairy Sci. 1997, 80, 1136; (e) Bergen, W. at a temperature below 0 °C for 24 h and then for further G.; Bates, D. B. J. Anim. Sci. 1984, 58, 1465; (f) Cardona, 24 h at room temperature. Subsequently, the solvent was C. J.; Galey, F. D.; Bickford, A. A.; Charlton, B. R.; evaporated under reduced pressure to dryness. The residue Cooper, G. L. Avian. Dis. 1993, 37, 107; (g) Johnson, D. was then suspended in hexane and filtered off. The filtrate C.; Spear, P. G. J. Virol. 1982, 43, 1102; (h) Schlegel, R.; was evaporated under reduced pressure and the residue Willingham, M.; Pastan, I. Biochem. Biophys. Res. Com- purified chromatographically on silica gel (Fluka type 60) mun. 1981, 102, 992; (i) Marsh, M.; Wellsteed, J.; Kern, to give (2) (20% yield) as a colourless oil showing a H.; Harms, E.; Helenius, A. Proc. Natl. Acad. Sci. U.S.A. tendency to form the glass state; (b) (method b, Scheme 1): 1982, 79, 5297; (j) Iacoangeli, A.; Melucci-Vigo, G.; To a mixture of MONA (500 mg, 0.75 mmol) in dichlo- Risuleo, G. Biochimie 2000, 82, 35; (k) Tanabe, K. Blood romethane (15 ml) the following compounds were added: Cells 1990, 16, 437; (l) Adovelande, J.; Schrével, J. Life DCC (206 mg, 1.0 mmol), PPy (50 mg, 0.33 mmol), cor- Sci. 1996, 59, 309 . responding alcohol (5.0 mmol). The mixture was first 7. (a) HuczyDski, A.; Ratajczak-Sitarz, M.; Katrusiak, A.; stirred at a temperature below 0 °C for 24 h and then for Brzezinski, B. J. Mol. Struct. 2007, 871, 92; (b) HuczyDski, further 24 h at room temperature. After this time the A.; Ratajczak-Sitarz, M.; Katrusiak, A.; Brzezinski, B. solvent was evaporated under reduced pressure to dryness. J. Mol. Struct. doi:10.1016/j.molstruc.2007.12.005; (c) The residue was suspended in hexane and filtered off. The Hamadinia, S. A.; Shimelis, O. G.; Tan, B.; Erdahl, W. filtrate was evaporated under reduced pressure and the L.; Chapman, C. J.; Renkes, G. D.; Taylor, R. W.; residue was purified chromatographically on silica gel Pfeiffer, D. R. J. Biol. Chem. 2002, 277, 38113; (d) (Fluka type 60) to give corresponding esters (2 3, 7 8) Hamadinia, S. A.; Tan, B.; Erdahl, W. L.; Chapman, C. J.; (yield from 15% to 71%) as a colourless oil showing a Taylor, R. W.; Pfeiffer, D. R. Biochemistry 2004, 43, tendency to form the glass state; (c) (method c, Scheme 1): 15956. To a mixture of MONA (500 mg, 0.75 mmol) in dichlo- 8. (a) Nagatsu, A.; Tanaka, R.; Hashimoto, M.; Mizukami, romethane (15 ml) the following compounds were added: H.; Ogihara, Y.; Sakakibara, J. Tetrahedron Lett. 2000, DCC (206 mg, 1.0 mmol), PPy (50 mg, 0.33 mmol), cor- 41, 2629; (b) Tanaka, R.; Nagatsu, A.; Mizukami, H.; responding alcohol (5 mmol) and p-TSA (28.5 mg, Ogihara, Y.; Sakakibara, J. Tetrahedron 2001, 57, 3005; (c) 0.15 mmol). The mixture was first stirred at a temperature Nagatsu, A.; Takahashi, T.; Isomura, M.; Nagai, S.; below 0 °C for 24 h and then for further 24 h at room Ueda, T.; Murakami, N.; Sakakibara, J.; Hatano, K. temperature. The solvent was subsequently evaporated Chem. Pharm. Bull. 1994, 42, 2269; (d) Tanaka, R.; A. HuczyDski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585 2589 2589 under reduced pressure to dryness. The residue was For the disc diffusion method, sterile filter paper discs suspended in hexane and filtered off. The filtrate (9 mm diameter, Whatman No. 3 chromatography paper) was evaporated under reduced pressure and the residue were dripped with the compound solutions tested (in was purified chromatographically on silica gel (Fluka type ethanol) to load 400 lg of a given compound per disc. Dry 60) to give (3 4, 7 8) (yield from 35% to 73%) as a discs were placed on the surface of an appropriate agar colourless oil showing a tendency to form the glass state; medium. The results (diameter of the growth inhibition (d) (method d, Scheme 1): A mixture of alkyl bromide or zone) were read after 18 h of incubation at 35 °C. chloride (1.45 mmol), MONA (500 mg, 0.75 mmol), and For MICs determination, all compounds were dissolved in DBU (175 mg, 1.15 mmol) and 40 ml toluene was heated DMSO. Concentrations of the agents tested in solid at 90 °C for 5 h. After cooling, the precipitate DBU- medium ranged from 3.125 to 400 lg/ml. The final hydrohalide (DBUHX) was filtered and washed hexane. inoculum of all organisms studied was 104 CFU mL 1 The filtrate and the washing were combined and evapo- (colony forming units per ml), except the final inoculums rated under reduced pressure. The residue was purified by for E. hiraeATCC 10541, which was 105 CFU mL 1. A chromatography on silica gel (Fluka type 60) to give the control test was also performed for DMSO which was corresponding ester (4 6, 9 10) (yield from 30% to 89%) as found inactive in the culture medium. Minimal inhibitory a colourless oil showing a tendency to form the glass concentrations were read off after 18 h (for bacteria) and state. 24 h (for yeasts) of incubation at 35 °C. Ionophore 15. Selected spectra data for (5): ESI-MS (m/z): 834 (M+Na+); antibiotic Monensin A was used as a control for bacteria 1 H NMR (d ppm in CD3CN): 2.69 (1H, m, 2-H), 2.78 (1H, and fluconazole (for the disc diffusion method 25 lg per t, J = 6.6 Hz, O(11)-H), 3.11 (3H, s, 35-H), 3.33(2H, lt, disc has been used) for yeast (C. albicans ATCC 10231 J = 6.6 Hz, 26-H), 3.50 (1H, t, J = 4.3 Hz, 3-H), 3.58 (1H, GIZ = 22 mm, MIC = 1 lg/ml; C. albicans ATCC 90028 overlapped, 3-H), 3.60 (1H, overlapped, 13-H), 3.62 (1H, GIZ = 32 mm, MIC = 1 lg/ml; C. parapsilosis ATCC overlapped, 21-H), 3.84 (1H, d, J = 4.4 Hz, 17-H), 3.92 22019 GIZ = 22 mm, MIC = 2 lg/ml). (1H, s, O(10)-H), 4.0 (1H, dd, J = 2.2 Hz, 7.1 Hz, 5-H), 17. (a) Clinical and Laboratory Standards Institute. Perfor- 4.18 (1H, d, J = 8.8 Hz, O(4)-H), 4.21 (1H, m, 20H), 5.57, mance Standards for Antimicrobial Disc Susceptibility 5.64 (each 1H, both d, J = 12.6 Hz, OCH2-Ar), 7.45 8.11 Tests; Approved Standard M2-A9. Clinical and Labora- 13 (9H, Ar), 0.70 2.28 pattern of 45 protons; C NMR (d tory Standards Institute, Wayne, PA, USA, 2006; Clinical ppm in CD3CN): 175.9, 134.6, 132.8, 132.4, 129.9, 129.4, and Laboratory Standards Institute. Methods for Dilution 128.3, 127.5, 126.9, 126.3, 127.7, 108.5, 97.9, 88.1, 86.9, Antimicrobial Susceptibility Tests for Bacteria That Grow 86.4, 84.4, 81.8, 77.9, 77.3, 71.9, 68.7, 67.5, 65.2, 58.2, 41.5, Aerobically; Approved Standard M7-A7. Clinical and 39.7, 37.7, 36.8, 35.9, 35.1, 34.9, 34.6, 34.0, 32.6, 32.0, 30.1, Laboratory Standards Institute, Wayne, PA, USA, 2006. 28.4, 26.3, 17.8, 16.5, 16.1, 12.5, 12.2, 11.1, 8.3; IR(KBr): 18. (a) Mercer, E. I. Biochem. Soc. Trans. 1991, 19, 788; (b) 1734 cm 1 (mC@O); Elemental analysis: (%): Calcd for Hiratani, T.; Asagi, Y.; Matsusaka, A.; Uchida, K.; C47H70O11: C, 69.60; H, 8.70; Found: C, 69.40; H, 8.89. Yamaguchi, H. Jpn. J. Antibiot. 1991, 44, 993; (c) Isenring, Compounds (3 4 and 7) as well as their complexes with H. P. In Recent Trends in the Discovery, Development and monovalent cations were characterized by us in Refs. 9c e, Evaluation of Antifungal Agents; Fromtling, R. A., Ed.; respectively. Prous Science J.R. Publishers: S.A., 1987; pp 543 554. 16. Antimicrobial activity was examined by the disc diffusion 19. (a) Sehi, L.; Woo, Y.; Kyung, K. H. J. Microbiol. and MIC method under standard conditions using Muel- Biotechnol. 2006, 16, 1236; (b) Choi, K.; Kyung, K. H. ler Hinton II agar medium (Becton Dickinson) for bac- J. Food Sci. 2005, 70, 305. teria and RPMI agar with 2% glucose (Sigma) for yeasts, 20. (a) Athanassa, Z.; Siempos, I. I.; Falagas, M. E. Eur. according to CLSI (previously NCCLS) guidelines.17 Respir. J. 2008, 31, 625; (b) Izumida, M.; Nagai, M.; Ohta, The compounds giving some growth inhibition zone in A.; Hashimoto, S.; Kawado, M.; Murakami, Y.; Tada, Y.; disc diffusion assay were tested by the twofold serial agar Shigematsu, M.; Yasui, Y.; Taniguchi, K. J. Epidemiol. dilution technique to determine their minimal inhibitory 2007, 17, S42; (c) Klein, E.; Smith, D. L.; Laxminarayan, concentration (MIC) values. R. Emerg. Infect. Dis. 2007, 13, 1840.

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