New Pyrrolobenzodiazepine Antibiotics, RK-1441A and B II. Isolation and Structure Hiroyuki Osada, MasakazuUramoto, Jun Uzawa,

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1 Agric. Biol. Chem., 54 (ll), , New Pyrrolobenzodiazepine Antibiotics, RK-1441A and B II. Isolation and Structure Hiroyuki Osada, MasakazuUramoto, Jun Uzawa, Kaichiro Kajikawa,* and Kiyoshi Isono1" RIKEN {The Institute of Physical and Chemical Research), Wako, Saitama , Japan *Ageo Seiken, Yadzu, Ageo, Saitama 362, Japan Received May 7, 1990 Antibacteriophage antibiotics, RK-1441Aand B, related to neothramycin were isolated from the culture broth of Streptomyces sp. and their structures were deduced from spectroscopic analyses. The structure of RK-1441Awas 8,l l-dihydroxy-3,7-dimethoxy-5-oxo-l^-pyrr61o[2,l-c:l,4]benzodiazepine. RK-1441Bis a tautomeric mixture at C-3 of the structure, 3,8,1 1-trihydroxy-7-methoxy-5-oxol //-pyrrolo [2, l -c: l,4] benzodiazepine. Newmembers of the pyrrolo[l,4]benzodiazepine group of antibiotics, RK-1441A and B (Fig. 1) have been isolated from the culture broth of Streptomyces sp. RK } The antibiotics showed antibacteriophage activity. In this paper, the isolation and structural study of RK-1441A and B are reported. Results and Discussion Isolation Fermentation was done as described in our Fig. 1. Structures ofrk-1441a and B. previous paper.1} The broth filtrate (3.1 1) was put onto a charcoal column, which was washed with water and 30%aqueous EtOH and eluted with 70% acetone. Active fractions were collected, concentrated in vacuo, and then put onto a silica gel column, which was developed stepwise with a solvent system, EtOAc-EtOH (100:1, 10:1,4:1,2:1,and 1:1).RK-1441A appeared in fractions eluted by 10 : 1 EtOAc-EtOH. RK-1441B was eluted by the solvent EtOAc-EtOH, 4 : 1. Each active fraction was collected and further purified by HPLCusing a reverse phase column (Senshu Pak N5251: solvent, 30%aqueous acetonitrile; flow rate, 6ml/min; detection, UV220nm). These procedures ars summarized in Scheme 1. Physico-chemicalproperties ofrk-1441a and B RK-1441A was obtained as a yellow amorphous powder: mp C with decomposition, [a]^ (c 0.52, MeOH), m/z (M-H2O)+ by high resolution mass spectrometry (HR-MS). The molecular formula C14H18N2O5 was established by HR-MSand supported by 13C- and ^-NMR. The UV spectrum (Fig. 2a) of RK-1441A was similar to those of neothramycins A and B.2) Corresponding author.

2 2884 H. Osada et al. Scheme 1. Purification Procedures. RK-1441A was purified as a single peak in HPLC but RK-1441B was obtained as a tautomeric mixture. Immediately after the separation, RK-1441B gave a single peak in HPLCanalysis, however, a counter peak appeared and increased gradually. The ratio of the two peaks in the mixture reached approximately 5:3 after 24hr. RK-1441B has a molecular formula, C13H14N2O5found by HR-MS (M+, m/z ). As shown in Fig. 2b, the UV spectrum of the antibiotic was different from that of RK-1441A. Structure elucidation of RK-1441A A close similarity in the UV, ^-NMR, and 13C-NMR spectra of RK-1441A to neothramycins A and B suggested that the skeletons of the antibiotics are similar. The analyses of 1H-1H homoscalar and the XH-13C heteroscalar correlated (COSY) spectra of RK-1441Aindicated that the substitutions at C-3 and C-l 1 are different from neothramycins A and B. The spectra of RK-1441Awere similar to those of methylneothramycin A, which was derived from neothramycin A3) (Table I). The hydroxyl group on C-ll was confirmed by the correlations between H-l l (<5H 4.38 d, /=8.8Hz) and H-lla (dh 3.60 q, /=8.8Hz). The position of two methoxy groups were identified as C-3 and C-7 based

3 Structures of RK-1441 A and B 2885 Fig. 2. UV Absorption Profile in 50% EtOH Solution, a, RK-1441A; b, RK-1441B. Table I. Comparison of 1U- and 13C-NMRData of RK-1441A and Methylneothramycin A2) RK A" Methylneothramycin Ab 5H (ppm) (multiplicity, /: Hz) Sc (ppm) dn (ppm) (multiplicity, /: Hz) 5C (ppm) (m) (m) (m) (m) (d, 4.9) (d, 4.2) OCH (s) (s) a (s) (s) OCH (s) (s) (s) (s) a H 4.38 (d, 8.8) (d, 4.5) Ha 3.60 (dd, 8.8) (m, 9.0, 8.5) 54.0 a Measuredin MeOD. b Measured in dioxane (data from ref. 2).

4 2886 H. Osada et al. on the correlation between C-3 (dc 87.8) and O-CH3 (SH 3.35) and also between C-7 (<5C 143.5) and O-CH3 (<5H 3.83) by heteronuclear multiple bond correlation (HMBC) spectroscopy. In the nuclear Overhauser effect (NOE) difference spectra irradiated at 3-OCH3 protons, NOEswere observed for H-3 and H-ll protons. This indicated that 3-OCH3 and H-l l were on the same side of the structure of RK-1441A, therefore, H-3 and H-l la were on the same side of the pyrrolidine ring. The proton signal of H-3 appeared as a doublet with a/value of4.9hz and the signal ofh-l la appeared as a quartet with a Jvalue of 8.8Hz to both H-la and H-lb. These results suggested that the relative configuration of the fivemembered ring of RK-1441Ais the same as the 3-OCH3 derivative of neothramycin A.3) In the case of tomaymycin4) and chicamycin A5) which have 1 l-r and 1 la-s configurations, the coupling between H-ll and H-lla is not observed. However, in the case of RK-1441A, the proton (H-ll) of the carbinolamine appeared as a doublet (J=8.8 Hz) at 4.38ppm coupling with a methin proton (H-lla). The coupling constant suggested that the relationship between H-l1 and H-lla ofrk-1441a is in trans configuration which is different from that of tomaymycin and chicamycin A. Moreover, the dihedral angle between H-ll and H-l la is calculated as approximately 160 from the coupling constant (/H-n,H-iia^8.8 Hz) by the Karplus equation.6) Thus, the relative configuration of RK-1441A was identified as shown in Fig. 1. It was reported2) that the 3-hydroxy residue of neothramycin was easily methylated by treatment with anhydrous methanol under acidic conditions. However, RK-1441A was isolated without using methanol as described in Scheme1. Moreover, when the fermentation broth of RK-1441strain was extracted with EtOAc, the peak derived from RK-1441A was observed by HPLC analysis (Senshu Pak N5251, 30% aqueous acetonitrile) even though the peaks of neothramycins A and B were not observed. Therefore, it is considered that RK-1441A is not an artifact but a fermentation product of the strain RK Structure elucidation of RK-1441B Purified RK-1441B was used for the measurement of ^-NMR. The spectrum showed a pair of signals due to the pyrrolidine ring corresponding to the tautomers at C-3 as in the case of neothramycins A and B.2) All carbon and proton signals of RK-1441B were assigned as shown in Table II by analyses of ^^H homoscalar COSY and ^-^C heteroscalar COSYspectra. Most of the signals were observed at similar chemical shifts to those of RK-1441A. However, the signal of H-ll was not observed in the ^-NMR spectrum of RK-1441B. The proton on C-lla appeared at SH 4.12 as a doublet, which was 0.52ppm down field from the corresponding signal in the spectrum of RK-1441A(H-lla, SH 3.60). In the 13C-NMR spectrum of RK-1441B, a carbon signal, C-ll, appeared at Sc 164.9ppm, on the other hand, the corresponding carbon signal of RK-1441A appeared at Sc 97.9ppm in the spectrum of RK-1441A. From these results, the structure ofrk-1441b was deduced as shown in Fig. 1. The observation of two double doublet Table II. 2H- and 13C-NMR Data of RK-1441B <5H (ppm) (multiplicity, /: Hz) Sc (ppm) (m) (m), (d, 4.9) OH 3.40 (s) 5 6a (s) 7 7-OCH (s) 8 8-OH 5.92 (s) (s) 9a 10NH (s) ll lla 4.12 (d, 8.0)

5 Structures of RK-1441 A and B 2887 signals assigned as H-3 (SH 5.91) and H-l la (<SH 4.12) in the ^-NMR spectrum of RK-1441B indicated that the relation between H-3 and H-lla is cis, which is the same to that of neothramycin B. Oxotomaymycin, which is the C-l l keto form of tomaymycin, was reported to be biologically inactive.7) Hurley8) reported that the C-ll position in the pyrrolo[l,4]- benzodiazepine group antibiotics was important for the biological activity. In the case of RK-1441B, which showed the antibacteriophage activity in vivo but not in vitro, the C-ll position of the structure is a keto form. It was suggested that host cells converted RK- 144IB to the active form in the cell as described in our preceding paper.1} Experimental RK-1441A. The compound was obtained as an amorphous yellow powder (22.5mg). mp C with decomposition; HR-MS m/z: (M-H2O)+. Calcd for C14H16N2O4 ( ); FD-MS m/z: 276 (M -H2O)+; Md (c 0.52, methanol); IR vmax (KBr) cm"1: 3,400, 1,680, 1,620; UV Amax nm (50% aqueous EtOH) (e): 235 (31,000), 264 (ll,900), 318 (5,900). RK-1441B. The compound was obtained as an amorphous pale yellow powder (42.3 mg). mp C; HR-MS m/z: Calcd for C13H14N2O5 ( ); FD-MS m/z: 278 (M+); [a]^ (c 0.54, ethanol); IR vmax(kbr) cm"1: 3,400, 1,620; UV Amax (50% aqueous EtOH) nm (e): 235 (55,000), 267 (sh 15,400), 300 (8,600), 310 (sh 6,600). Instrumental analyses. Optical rotation was measured on a Perkin-Elmer 24I MCpolarimeter. The melting point was taken on a Yanagimotomicro melting point apparatus. UVand IR spectra were measured on a Hitachi 220A spectrophotometer and a Shimadzu IR27G recording IR spectrophotometer, respectively. A Hitachi M-80 mass spectrometer wasused to measurethe molecularweight. ^-NMRand 13C-NMRspectra were obtained using JMN GSX-500 and Jeol FX-100 FI-NMR spectrometers, respectively. For NMRmeasurement, CDC13 was used as a solvent and TMSwas used as an internal standard. Acknowledgments. We thank Mr. Y. Esumi and Ms. T. Chijimatsu for MS and NMR measurements, respectively. Weare grateful to Dr. S. Kondo (Institute of Microbial Chemistry) for his gift of neothramycin. References 1) H. Osada, K. Ishinabe, T. Yano, K. Kajikawa, H. Kusakabe and K. Isono, Agric. Biol. Chern., 54, 2875 (1990). 2) T. Takeuchi, M. Miyamoto, M. Ishizuka, H. Nakagawa, S. Kondo, M. Hamada and H. Umezawa, /. Antibiot., 29, 93 (1976). 3) M. Miyamoto, S. Kondo, H. Naganawa, K. Maeda, M. Ohno and H. Umezawa, /. Antibiot., 30, 340 (1977). 4) K. Kariyone, H. Yazawa and M. Kohsaka, Chem. Pharm. Bull, 19, 2289 (1971). 5) M. Konishi, H. Ohkuma, N. Naruse and H. Kawaguchi, /. Antibiot., 37, 200 (1984). 6) M. Karplus, /. Phys. Chem., 30, ll (1959). 7) L. Hurley, C. Gairola and N. Das, Biochemistry, 15, 3760 (1976). 8) L. Hurley, /. Antibiot., 30, 349 (1977).