Anti-Mycobacterial Nucleoside Antibiotics from a Marine-Derived Streptomyces sp. TPU1236A

Five new nucleoside antibiotics, named streptcytosines A–E (1–5), and six known compounds, de-amosaminyl-cytosamine (6), plicacetin (7), bamicetin (8), amicetin (9), collismycin B (10), and SF2738 C (11), were isolated from a culture broth of Streptomyces sp. TPU1236A collected in Okinawa, Japan. The structures of new compounds were elucidated on the basis of their spectroscopic data (HRFABMS, IR, UV, and 2D NMR experiments including 1H-1H COSY, HMQC, HMBC, and NOESY spectra). Streptcytosine A (1) belonged to the amicetin group antibiotics, and streptcytosines B–E (2–5) were derivatives of de-amosaminyl-cytosamine (6), 2,3,6-trideoxyglucopyranosyl cytosine. Compound 1 inhibited the growth of Mycobacterium smegmatis (MIC = 32 µg/mL), while compounds 2–5 were not active at 50 µg/disc. Bamicetin (8) and amicetin (9) showed the MICs of 16 and 8 µg/mL, respectively.


Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis is still one of the main infectious diseases all over the world including with the human immunodeficiency virus (HIV) and malaria [1][2][3]. It was OPEN ACCESS estimated that there were about 9 million clinical cases, 1.3 million deaths from TB, and 0.3 million deaths from HIV-co-infected TB in 2012 [4]. The development of multidrug-resistant TB (MDR-TB) has been observed as resistance against potent first-line drugs (rifampicin and isoniazid), and 450,000 patients were revealed to be infected by MDR-TB in 2012. Moreover, approximately 9.6% of these drug-resistant strains of T. tuberculosis were extensive drug-resistant TB (XDR-TB), which additionally shows resistance to some second-line drugs such as fluoroquinones, amikacin, kanamycin, and capreomycin. More recently, totally drug resistant TB (TDR-TB), which is resistant to all second-line drugs, has often been reported in India and other Asian countries [1][2][3][4][5]. Although a new drug, delmanid (Deltyba ® ), which inhibits the cell wall biosynthesis of mycobacteria, was approved in 2014 for the treatment of MDR-TB in EU and Japan [6,7], continuous efforts to discover new anti-TB agents with novel mechanisms of action and structural features are the emergent global demand.

Results and Discussion
The strain TPU1236A was isolated from a seawater sample collected at Iriomote Island in Okinawa, Japan. The partial sequence of the 16S rRNA gene used for identification showed 100% identity with those of Streptomyces badius and Streptomyces sindenensis. The bacterium was cultured by shaking in a liquid medium for 7 days at 25 °C in the dark.

Structure of Streptcytosine A (1)
Compound 1 was obtained as a colorless oil. Its molecular weight and formula (630, C30H42N6O9) were determined from HRFABMS and NMR data. 1 H and 13 C NMR signals were assigned by an analysis of 1 H-1 H COSY, HMQC, and HMBC data ( Table 1). Partial structures I-IV were established from 1 H-1 H COSY data, and connectivity between these partial structures were elucidated by the HMBC correlations ( Figure 3).
The chemical shifts and couplings of two sugar moieties (amosamine and amicetose units) and two aromatic rings (cytosine and PABA units) were very similar to those of amicetin (9) [9]. Differences in the molecular formulas and weights of 1 and 9 were only one carbon atom and 12 Da, which was detected at δ 61.1 in the 13 C NMR spectrum of 1. The protons attached to this carbon were observed as a broad singlet (2H) at δ 5.28, which showed HMBC correlations to C-16 and 17 ( Figure 3). HMBC correlations from H3-19 to C-16, 17, and 20 and from H2-20 to C-16 and 17 along with the above data allowed to assign the partial structure of 4-imidazolidinone. Consequently, the skeletal structure of compound 1 was assigned as Figure 3 and named streptcytosine A.   Since streptcytosine A (1) was obtained together with plicacetin (7), bamicetin (8), and amicetin (9) from the strain TPU1236A, the absolute configuration of the sugar moiety will be the same. The configuration of 1 at the C-17 position was also assigned to be the same as those of 8 and 9, since these compounds should be biosynthesized by the same pathway. Moreover, compounds 1 (+72), 7 (+96), 8 (+66), and 9 (+76) showed the same positive specific rotation. Therefore, streptcytosine A (1) may share the same configuration for whole asymmetric carbons as compounds 8 and 9, as depicted in Figures 1 and 2.
Streptcytosine B (2) had an S-Me group and 2-butenoyl unit. HMBC correlations from H3-11 to C-10, H-10 to C-8, and from H-9 to C-8 revealed the partial structure V as the amide moiety in 2. The orientation of the double bond was assigned as E from the coupling constant (14.6 Hz) between H-9 and 10.
The 2-methyl-2-butenoyl unit (partial structure VI) in streptcytosine C (3) was elucidated from 1 H-1 H COSY and HMBC data ( Figure 4). An NOE correlation was observed between H-10 and H3-11, but no cross peak was detected between H-10 and H3-12. These observations on NOEs were the same as cytosaminomycin D, which has a tiglic acid (2-methyl-2E-butenoic acid) moiety attached to the PABA unit by an amide bond [18]. Moreover, chemical shifts due to C-11 ( 14.7) and 12 (12.2) of 3 resembled those of cytosaminomycin D (14.5 and 12.2), which was compared the chemical shifts with those of tiglic acid (13.83 and 10.95) and angelic acid (15.93 and 20.22, 2-methyl-2Z-butenoic acid). Thus, the orientation of the double bond was assigned as E.

Anti-Mycobacterial Activity of Compounds 1-11
The antibacterial activities of compounds 1-11 against M. smegmatis NBRC 3207 were evaluated using the paper disc method [19], and MICs were determined by the liquid microdilution method using 96-well plastic plates (Table 3).
Compounds 1, 7-9, and 11 showed activity against M. smegmatis at 5 µg/disc. Amicetin (9) was reported to inhibit the growth of M. tuberculosis [20] and showed strong activity against M. smegmatis in our experiment. The inhibition activity of compounds 1 and 7 (MIC = 32 µg/mL) was about a half of that of compounds 8 (MIC = 16 µg/mL). Therefore, the 2-methylserine moiety attached to the PABA unit will be important for the anti-mycobacterial activities of these compounds.
On the other hand, compounds 2-6 were not active against M. smegmatis at 50 µg/disc. Consequently, the amino sugar (amosamine) and/or PABA moieties will be essential for anti-mycobacterial activity.

General Experimental Procedures
Optical rotations were measured with a JASCO P-2300 digital polarimeter (JASCO, Ltd., Tokyo, Japan). UV spectra were obtained on a Hitachi U-3310 UV-Visible spectrophotometer (Hitachi, Ltd., Tokyo, Japan) and IR spectra on a PerkinElmer Spectrum One Fourier transform infrared spectrometer (Waltham, MA, USA). NMR spectral data were obtained by a JEOL JNM-AL-400 NMR spectrometer (JEOL Ltd., Tokyo, Japan; 400 MHz for 1 H and 100 MHz for 13 C) in CD3OD (δH 3.31, δC 49.0). High-resolution FAB mass spectra were recorded on a JEOL JMS-MS 700 mass spectrometer (JEOL Ltd., Tokyo, Japan). Preparative HPLC was conducted using a Toyosoda CCPU instrument with a Tosoh UV-8010 detector.
The culture of strain TPU1236A was maintained on a Waksman agar slant (glucose 1%, peptone 0.5%, meat extract 0.5%, NaCl 0.3%, and agar 1.2% in deionized water) at 4 °C. The mycelia grown on the slant culture were inoculated in a 100-mL Erlenmeyer flask containing 40 mL of a seed medium (ASW-A medium: soluble starch 2%, glucose 1%, peptone 0.5%, yeast extract 0.5%, and CaCO3 0.3% in natural seawater, pH 7.0), and the flasks were shaken (150 rpm) at 25 °C for 3 days. The seed culture (2 mL) was transferred into 500-mL Erlenmeyer flasks, each containing 200 mL of the same medium, and incubated at 25 °C for 7 days at 150 rpm.

Anti-Mycobacterial Assay
The anti-mycobacterial assay was carried out using M. smegmatis NBRC 3207 with the paper disc method and liquid microdilution method. The strain NBRC 3207 was obtained from the Biological Resource Center (NBRC), NITE (Chiba, Japan) and maintained in 20% glycerol at −80 °C .
The test microorganism was cultured in Middlebook 7H9 broth (BD, Franklin Lakes, NJ, USA) containing 0.05% polysorbate 80 (BD), 0.5% glycerol, and 10% Middlebook OADC (BD) at 37 °C for 2 days and adjusted to 1.0 × 10 6 CFU/mL. The inoculum was spread on the above medium containing 1.5% agar in a square plate. Each sample in MeOH was adsorbed to a sterile filter disc (6 mm, Advantec, Tokyo, Japan), and, after evaporation of MeOH, the disc was placed in a plate and incubated for 2 days at 37 °C. Streptomycin sulfate (5 and 10 µg/disc) and MeOH were used as positive and negative controls, respectively. MICs were measured by the liquid microdilution method [22]. After 85 μL of Middlebrook 7H9 broth containing 0.05% polysorbate 80, 0.5% glycerol, and 10% Middlebook OADC was added to each well of a 96-well microplate (Corning Inc., Corning, NY, USA), test compound dissolved in MeOH (5.0 μL) was added to each well at the final concentration of 0.125 to 64 μg/mL, respectively. Finally, the test microorganism (10 μL) was added at a concentration of 1.0 × 10 6 CFU/mL. Microplates were incubated at 37 °C for 2 days. MIC was defined as the lowest concentration of test compound where the test microorganism cannot grow.

Conclusions
Five new pyrimidine nucleoside antibiotics, named streptcytosines A-E (1-5), and six known compounds 6-11 were obtained from a marine-derived actinomycete, Streptomyces sp. TPU1236A, isolated from seawater collected at Iriomote Island in Okinawa, Japan. The anti-mycobacterial activity of the extract of the culture broth was reproduced by new compound 1 (streptcytosine A), plicacetin (7), bamicetin (8), amicetin (9), and SF2738 C (11). Compounds 8 and 9 were about two-fold more active than the other three compounds.