OH -COONa THE STRUCTURES OF PLURACIDOMYCINS, NEW CARBAPENEM ANTIBIOTICS

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1 536 THE JOURNL OF NTIIOTIS PR THE STRUTURES OF PLURIDOMYINS, NEW RPENEM NTIIOTIS Sir: New -lactam antibiotics which have broad antibacterial and strong 8-lactamase inhibitory activities were detected among the metabolites of a new strain of streptomycete named Streptomyces pluracidomyceticus nov. sp. (P-41746). On paper electrophoresis at ph 7.0, certain of these components migrated toward the anode with greater mobility than that of olivanic acids1,2) (Table 1). From these preliminary experiments we assumed that these components should be new carbapenem compounds presumably having two or three acidic functions, and named them pluracidomycins (). Pluracidor,iycins were extracted from the fermentation filtrate using a phase-transfer reagent, separated by ion-exchange resin to factors, and, and purified by reverse-phase chromatography and gel filtration in the forms of sodium salts. In a typical batch, 200 liters of the filtrate gave about 4 mg of factor, 100 mg of factor and 1 mg of factor. Pluracidomycins are fairly stable n the ph range of Their UV and 1H NMR spectra are shown in Figs. I and 2, respectively. esides pluracidomycins, penicillin N, cephamycin, deacetoxycephalosporin, epithienamycin and MM17880 were detectec in the broth, and also the sulfoxides of epithienarlycin and D which were characterized as new compounds. From the S H NMR spectra it was easily pointed out that the pluracidomycins all have the sulfated olivanic acid structure1~3). However, they lack the N-acetyl proton signal found in olivanic acids such as MM45 (4), MM13902 (5) and MM (6) and have neither -H = H- nor -H2- H2- functions. Therefore, pluracidomycins can be distirguished from the sulfated olivanic Fig. 1. UV spectra of pluracidomycins in H2O. acids in the substituents at -3. This conclusion was also confirmed by the 13 NMR data listed in Table 2. The main component, pluracidomycin ( ) shows the highest mobility on paper electrophoresis (Table 1) and has no 1H and 13 signals attributable to the -3 substituent, which may be an acidic function. The IR spectrum of exhibited broader and stronger absorption bands at cm-1 and cm-1 than those of MM45, therefore, the substituent at -3 was concluded to be a sulfonate as shown in the structure (1), which was also agreeable with the elemental analysis. is probably identical with antibiotic SF 2103 found by the Meiji-Seika group4). Pluracidomycin ( ) as well as pluracidomycin ( ) has 1H signals essentially Table 1. Paper electrophoresis (1130m phosphate buffer ph 7.0; 12 V/cm; 1.5 hours). ntibiotics Epithienamycin MM45 M M arbon No OONa 8-H3 S(0)-H2- S(O)-H- OH -OONa Table NMR in D2Oa. b (s) 129.0(s) 32.8 (t) 55.1 (d) 58.7 (d) (s) 73.7 (d) (s) 19.4 (q) c 140.0(s) (s) 30.5 (t) 54.7 (d) 59.1 (d) (s) 74.0 (d) (s) 19.3 (q) 60.2 (t) (s) Relative mobility c (s) (s) 30.4 (t) 54.7 (d) 59.2 (d) (s) 73.9 (d) (s) 19.3 (q) 86.5 (d) a ppm from DSS (internal H3N, 5=1.7). b Recorded with a Varian XL spectro - meter. c Recorded with a Varian XL-200 spectrometer.

2 VOL. XXXV NO. 4 THE JOURNL OF NTIIOTIS 537 Fig. 2. 1H NMR spectra of pluracidomycins, and in D!O at 100 MHz (o ppm from external TMS).

3 538 THE JOURNL OF NTIIOTIS PR Fig. 3. Structures of pluracidomycins and related compounds. (I) R=S03Na 0 (2) R=S-H2000Na (3) R=S-H. (3') R=S-HO 0 } (4) R=S-H=HNHc MM 45 (_) R=S-H=HNHc MM (6) R=S-H2H2NHc MM (7) R=S-H2OH dihydro (8) R=S-H2OONa deoxy 8 identical with those of MM451~3) except the signals due to the substituents at -3. Moreover, since and exhibit the 13 chemical shifts of -3 almost identical to that of MM- 451,3), both compounds should also have the sulfoxide function adjacent to the -3 substituent as in MM45. Different from MM45, has a methylene group (o 1H; 4.31, 2H, s., 6 13; 60.2 t.) and a carbonyl function (6 13; s.) in the -3 substituent. Taking account of the paper electrophoresis and the elemental analysis the carbonyl group is probably a constituent of a carboxyl function. has much stronger IR bands assignable as OO- at 1600 cm-1 and 1400 cm-1 in comparison with those of and or MM45. This evidence suggests that has the structure (2) as shown in Fig. 3. has a methine group (o 1H; 5.86, 1H, s., 6 13; 86.5, d.) next to the sulfoxide on -3, and no additional 13 signals could be observed. From the 111 and 13 chemical shifts the presence of at least one hydroxyl group, on this methine is likely. The 1H NMR of in DMSO-de dramatically changed this methine signal from 5.86 ppm to 9.62 ppm (1 H, broad singlet. inextinguishable by D,O-addition), but the 1H NMR of the recovered sample in D,O regenerated the original methine signal at 5.86 ppm. This change suggests the conversion, - and the structure of is concluded to be (3) or (3'). mong pluracidomycins, only was positive to 2,4-dinitrophenyl hydrazine and the formation of hydrazone or semicarbazone was confirmed by HPL. Further, was reduced by sodium borohydride to the alcohol (7), which also has strong -lactamase inhibitory activity. The relative configurations of the -5, -6, -8 and the sulfoxide of and should be identical with those of MM45 because of the good correlation of 1H chemical shifts and coupling constantsl-3). MM45 has been related biosynthetically to MM223805) (epithienamycin ), the stereochemistry of which was elucidated as 5-R, 6-R, 8-S6). With respect to the sulfoxide of MM45, the configuration is inferred to be R from the D spectrum1) which closely resembles that of ) (carpetimycin) whose structure has been established by X-ray analysis8,9) s the chromophores of pluracidomycins and MM145 are different, the direct comparison of Table 3. ntibacterial activity of pluracidomycins, and. Organism Staphylococcus 209P J-1 Staphylococcus -146 Streptococcus -203 Escherichria coli NIHJ J-2 Escherichia Klebsiella SRL-1 Klebsiella aureus aureus pyogene.s coli 377a pnenmoniae sp. 363b Proteus mirabilis PR-4 Proteus vulgaris N-32~ Enterobacter cloacae 23 3 Serratia rnarcescen.s T Pseudomonas aeruginosc T 619 >100 MI (ug/ml)a 100 > > 100 Determined by agar dilution method in sensitivity-disc agar and inoculated by one loopful of ca. 10 cells per ml. b Penicillinase producing strain. ephalosporinase producing strain.

4 VOL. XXXV NO. 4 THE JOURNL OF NTIIOTIS 539 Table 4. Inhibition of 8-lactamases produced by NOKI Gram-negative TSUJI bacteria by pluracidomycins. Source of p-lactamasea Escherichia coli 6 Enterobacter cloacae 92 Proteus vulgaris 31 Escherichia coli W3110 RTEM Klebsiella sp.363 Enterobacter cloacae 53 lass' - Ib la Ic IIIa IV IVa their OTTON-effects is of no use for the determination of the absolute configuration. Therefore, was reduced to the corresponding sulfide (8) by Til310), and the D spectrum was compared with that of epithienamycin. oth spectra showed the same OTTON-effects, accordingly, pluracidomycins should have the configuration identical with that of MM45 as shown in Fig. 3. The sulfoxides of and are arranged spatially in the same direction as those of MM45, carpetimycins and asparenomycins10), but in the case of only the sulfoxide should be designated by the S-configuration according to the sequence rule. The biological properties of pluracidomycins are summarized in Tables 3 and 4*. The details will be reported separately. cknowledgements Minimum effective concentration (µg/ml)e ` a Enzyme preparations used were partially purified. blassification of RIHMOND and SYKES. e Inhibitor was incubated with enzyme at room temperature for 10 minutes prior to adding nitrocefin ( icg/ml) and minimum concentration to inhibit color change was determined. The authors wish to thank to Dr. K. TNK for his interest. Thanks are also due to Messrs. S. KOHZUKI, Y. TKHSHI, N. UOTNI and T. TNIMOTO for technical assistance. * The in vitro data were obtained by Dr. T. YOSHID and his colleague. KZUO NGSHIM MSKI KOYSHI YOSHIHIRO TERUI KOIHI MTSUMOTO EIJI KONDO Shionogi Research Laboratories Shionogi & o., Ltd. Fukushima-ku, Osaka 553, Japan (Received January 14, 1982) References 1) ROWN,. G.; D. F. ORETT,. J. EGLINGTON & T. T. HOWRTH: Structures of olivanic acid derivatives MM45 and MM13902, two new, fused 48-lactams isolated from Streptomyces olivaceus. J. hem. Soc., hem. omm. 1977: 523-5, ) HOOD, J. D.; S. J. ox & M. S. VERRLL: Olivanic acids, a family of 8-lactam antibiotics with 8-lactamase inhibitory properties produced by Streptomyces species. IL Isolation and characterization of the olivanic acids MM45, MM and MM17880 from Streptomyces olivaceus. J. ntibiotics 32: , ) MED, K.; S. TKHSHI, M. SEZKI, K. IINUM, H. NGNW, S. KONDO, M. OHNO & H. UMEZW: Isolation and structure of a Q- lactamase inhibitor from Streptomyces. J. ntibiotics 30: , ) EZKI, N.; K. OH, S. MNO, Y. KONDO, M. SEZKI, T. NIW, K. WTNE, S. INOUE, T. ITO & T. NIID: SF-2103, a novel i?-lactamase inhibitor. II. Isolation, physico-chemical properties and structure. The 2th Scientific Meeting of Japan ntibiotics Research ssociation, Sept., ) ox, S. J.; J. D. HOOD & S. R. SPER: Four further antibiotics related to olivanic acid produced by Streptomyces olivaceus: Fermentation, isolation, characterization and biosynthetic studies. J. ntibiotics 32: , ) SSIDY, P. J.; G. LERS-SHONERG, R. T. GOEGELMN, T. MILLER,. RISON, E. 0. STPLEY & J. IRNUM: Epithienamycins. II. Isolation and structure assignment. J. ntibiotics 34: , ) HRD, S.; S. SHINGW, Y. NOZKI, M. SI & T. KIsru: S: and H2, new carbapenem antibiotics. II. Isolation and structures. J. ntibiotics 33: , ) NKYM, M.;. IWSKI, S. KIMUR, T. MIZOGUHI, S. TNE,. MURKMI, I. WT-

5 540 THE JOURNL OF NTIIOTIS PR NE, M. OKUHI, H. ITOH, Y. SINO, F. KO- YSHI & T. MORI: arpetimycins and, new -lactam antibiotics. J. ntibiotics 33: ~1980 9) NKYM, M.; S. KIMUR, S. TNE, T. MIZO- GUHI, I. WTNE, T. MORI, K. MIYHR & T. KWSKI: Structures and absolute configurations of carpetimycins and. J. ntibiotics 34: , ) TSUJI, N.; K. NGSHIM, M. KOYSHI, J. SHOJI, T. KTO, Y. TERUI, H. NKI & M. SHIRO: sparenomycins, and, new carbapenern antibiotics. III. Structures. J. ntibiotics 35: 24~32, 1982