Drug Discoveries & Therapeutics. 2013; 7(6):

Transcription

1 Drug Discoveries & Therapeutics. 0; ():-. Brief Report DI: 0./ddt.0.v.. xoprothracarcin, a novel pyrrolo[,]benzodiazepine antibiotic from marine Streptomyces sp. M0 Yong an, Yaoyao Li, Yan Shen, Jie Li, Wenjun Li, Yuemao Shen, * Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Ji'nan, Shandong, China; Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, China. ABSTRACT: A novel pyrrolo[,]benzodiazepine antibiotic, designated oxoprothracarcin (), was isolated from the marine strain Streptomyces sp. M0 along with three known secondary metabolites, cyclo(d)-pro-(d)-val (), cyclo(d)-pro- (D)-Leu (), and limazepine A (). The chemical structures of these substances were elucidated by spectroscopic analyses, including D- and D-MR and ESI-MS. Antitumor and antibacterial assays indicated that compounds weakly inhibit the growth of MDA-MB- and A cells. The isolation of compound with a high yield ( mg/0 L) indicated that this marine S. sp. M0 may provide new lead compounds for structural modification and drug screening. Keywords: Marine Streptomyces, cyclo(d)-pro-(d)-val, cyclo(d)-pro-(d)-leu, oxoprothracarcin, limazepine A. Introduction Traditionally, secondary metabolites produced by microorganisms, and especially terrestrial actinomycetes, are remarkable sources of lead compounds for drug discovery (). owever, the rate at which new metabolites from terrestrial actinomycetes are discovered has decreased, and the rate of reisolation of known compounds has increased due to the replication of isolating microbial strains (). Compared to terrestrial actinomycetes, marine actinomycetes are relatively unexploited sources (). The fact that marine Streptomyces in particular are rich in novel bioactive metabolites is just becoming apparent (-). Moreover, genetic analysis has indicated that some marine-derived *Address correspondence to: Dr. Yuemao Shen, School of Pharmaceutical Sciences of Shandong University, o. West Wenhua Road, Ji'nan, Shandong 00, China. yshen@sdu.edu.cn Streptomyces form an independent clade (). The present study obtained four metabolites, i.e. cyclo(d)- Pro-(D)-Val () (), cyclo(d)-pro-(d)-leu () (), oxoprothracarcin () (-), and limazepine A () () (Figure ), from the metabolites of marine Streptomyces sp. M0. Reported here are the isolation, structural determination, and antitumor and antibacterial activities of compounds -.. Materials and Methods.. General MR spectra were recorded on a Bruker Advance 00 spectrometer (Bruker, Fällanden, Switzerland) at 00/0 Mz. Mass spectra were obtained on ABI-000 mass spectrometers (AB SCIEX, Framingham, MA, USA). An Agilent 0 PLC system (Agilent, Santa Clara, CA, USA) with a C- column (. 0 mm, μm) was also used. Column chromatography included a RP- (Merck, Darmstadt, Germany) column and a Sephadex L-0 column (GE healthcare, Uppsala, Sweden). TLC analyses were performed with precoated silica gel GF plates ( mm, Qingdao Marine Chemical Factory, Qingdao, China). All general chemical reagents were purchased from Sinopharm Chemical Reagent Company (Beijing, China). Figure. Chemical structures of compounds.

2 Drug Discoveries & Therapeutics. 0; ():-... Microorganism sample The strain Streptomyces sp. M0 was isolated from mangrove sediment collected from ut Bay and grown on medium containing trehalose as the sole carbon source [trehalose 0g, ( ) S. g, K P. g, K P. g, MgS.0 g, CuS 0.00 g, FeS 0.00 g, MnCl 0.00 g, ZnS 0.00 g, distilled water L, p.-.] at ºC. This strain was identified as Streptomyces sp. by partial S rra gene sequencing analysis... Tumor cell lines The MDA-MB- human breast cancer cell line and A human lung cancer cell line were purchased from the American Type Culture Collection (ATCC). All cells were maintained in DMEM with 0% fetal bovine serum (FBS, Gibco) in a humidified C incubator in % C at C... Fermentation and isolation Fermentation took place for d on YMG (0 L) agar plates at C. The culture was diced and extracted with AcEt/Me/Ac (0::). The organic solution was collected through filtration and the remaining agar residue was extracted exhaustively with the same solvent until the filtrate was colorless. Upon evaporation, the combined filtrate yielded a crude extract. The crude extract was partitioned between water and EtAc (:, v/v) until the EtAc layer was colorless. The EtAc extract was partitioned between Me and petroleum ether. The Me layer was concentrated in a vacuum to yield a brown syrupy extract (. g). The extract was subjected to MPLC (0 g RP- silica gel; 0%, 0%, 0%, and 00% Me, L for each gradient) to yield fractions, Fr. a d. Fraction a ( mg) was separated by column chromatography (CC) over Sephadex L-0 (in Me) to yield one subfraction (Fr.a.). Fr.a. ( mg) was subjected to PLC (C- column,. 0 mm, μm; 0% Me) to yield ( mg) and ( mg). Fraction b (0 mg) was subjected to CC over Sephadex L-0 (in Me) to yield two subfractions (Fr.b. and Fr.b.). Fr.b. (mg) was purified by Sephadex L-0 (in Me) to yield ( mg). Fr.b. ( mg) was purified by recrystallization (Me) to yield (0 mg)... Biological study The cytotoxicity of compounds - was assessed using a -(,-dimethylthiazol--yl),-diphenyltetrazolium bromide (MTT) cell survival assay (), Briefly, MDA-MB- and A cells were seeded in -well plates overnight and treated three times with increasing concentrations of compounds. After the cells were treated for h, a 0-μL of aliquot of MTT solution ( mg/ml) was added and cells were incubated for h at C. Two hundred μl of DMS was then added to dissolve formazan crystals. The color density was measured with a microplate reader (M-0, Bio- Rad) at 0 nm. Growth inhibition rates were calculated as follows: (A0 control cells A0 treated cells )/A0 control cells 00%. The antibacterial activity of compounds - was tested against Bacillus subtilis (CMCC (B) 0), Bacillus pumilus (CMCC (B) 0), and Penicillium avellaneum (UC ) using the filter paper method. Growth inhibition was calculated as the radius of the inhibition zone.. Results and Discussion. Elucidation of the structures of compounds ESI-MS revealed the molecular weight of compound to be Da. The C-MR spectrum of (Table ) displayed 0 signals. The -MR spectrum of (Table ) displayed signals. -MR signals at δ. (t,. z, ) and.0 (s, ) and C-MR signals at δc. and. revealed the presence of two acylamino groups, indicating that is a cyclic dipeptide. -MR signals at δ.-. (m, ),.0 (d, J =., ), and 0. (d, J =., ) and C-MR signals at δc.,., and. indicated the presence of an isopropyl group. The MBC correlation from C(0) to C(), along with the - CSY correlation between C() C (0) C (), indicated the presence of fragment A (Figure ), which was a valine residue. -MR signals at δ.-. (m, ),.-. (m, ),.0-.0 (m, ), and.-. (m, ) and C-MR signals at δ.,., Table. -MR and C-MR spectroscopic data for compounds and (MeD) 0 δ in ppm. J in z. Compound Compound Pro.-. (m, ).-. (m, ).0-.0 (m).-. (m).0 (m). (t, J =.).-. (m).0 (d, J =., ) 0. (d, J =., ).t.t.t 0.0d.s Val.d.d.q.q.s Pro.0-. (m, ).-. (m).-.0 (m).-.0 (m).-. (m). (t, J =.).-. (m).-. (m, ).0-. (m) 0. (d, J =., ) 0. (d, J =., ).t.t.t 0.d.s Leu.d.t.d.q.q.s

3 Drug Discoveries & Therapeutics. 0; ():-. 0 A 0 and. indicated the presence of three C groups. Furthermore, the MBC spectrum showed that C () was correlated with C(). In combination with the - CSY correlation between C () C() C(), these findings indicated the presence of fragment B (Figure ), which was a proline residue (Figure ). The spectral data for were consistent with those reported in the literature (). Thus, compound was determined to be cyclo(d)-pro-(d)-val. ESI-MS revealed the molecular weight of compound to be 0 Da. The chemical structure of was determined by comparing MR data for with those for. Both compounds had similar spectroscopic data (Table ), except for C(), C(), and C(). -MR signals at δ.-. (m, ),.0-. (m, ), 0. (d, J =., ), and 0. (d, J =., ) and C-MR signals at δc.,.,., and. indicated the presence of an isobutyl group. The MBC spectrum showed that C() was correlated with C(). Along with the - CSY correlation between C() C (0) C() C (), these findings indicated that had a leucine residue. The spectral data of were consistent with those reported in the literature (). Therefore, compound was determined to be cyclo(d)-pro-(d)-leu. ESI-MS data revealed the molecular weight of compound to be Da. The -MR spectrum of (Table ) displayed signals. The C-MR spectrum of (Table ) displayed signals for one methyl, two methylenes, six methines, and five quaternary carbon atoms, including two carbonyl groups (with one at δc. C() and the other at δc. C()). This suggested the presence of two acylamino groups as in compounds and. The -MR signal at δ.0 (, m) and C-MR signals at δc. and. suggested a trisubstituted Compound B Figure. Selected MBC ( C) and - CSY ( ) correlations, and the structures of fragments A and B of compound. Table. -MR and C-MR spectroscopic data for compounds and (DMS-d ) Position a a / a Me.-. (m).-. (m) δ in ppm. J in z. Compound Compound.0 (d, J =.). (d, J =.). (d, J =.). (t, J =.). (t, J =.). (d, J =.) 0. (s). (dd, J =.,.).-.0 (m). (d, J =., ) A.t.s.t.s.s.d.d.d.d.s.s.d.d.q.-. (m).-. (m). (s).0 (d, J =.). (d, J =.). (s, ). (dd, J = 0.,.). (q, J =., ).0 (t, J =., ). (s, ).0t.s.d.s 0.s.d 0.d 0.s.s.s.0s.d.t.q.q double bond. The - CSY correlation between C() C() C() C() indicated that had a disubstituted benzene. ext, the MBC correlation from C() to C() indicated the presence of fragment A (Figure ). The double bond was assigned at C() and C() in accordance with the MBC correlation (Figure ) of δ. C () with δc. C() and δ., δ. C () and that of δ.0, δ. C () with δc. C(). The MBC correlation from C(a) and C () to C(), in combination with the - CSY correlation between C () C(a), indicated the presence of fragment B (Figure ). Therefore, compound was determined a a B Compound Figure. Selected MBC ( C) and - CSY ( ) correlations, and the structures of fragments A and B of compound.

4 Drug Discoveries & Therapeutics. 0; ():-. to be (S,E)--ethylidene-,-dihydro--benzo[e] pyrrolo[,-a][,]diazepine-,(0,a)-dione, a new natural product that has been registered (CAS o.: 0-- ). After consulting the names of reported analogues (-), compound was designated oxoprothracarcin. ESI-MS data revealed the molecular weight of compound to be Da. The -MR and C-MR spectral data for (Table ) were similar to those for. The -MR spectrum of (Table ) displayed signals. The C-MR spectrum of (Table ) displayed signals for two methyls, two methylenes, four methines, and seven quaternary carbon atoms, including two carbonyl groups (with one at δc.0 C() and the other at δc. C()). The -MR signal at δ. (, s) and C-MR signals at δc. and. suggested a trisubstituted double bond like that in compound. owever, the MBC correlation from C (), C (), C(a), C (), and C () to C() and the - CSY correlation between C () C () and C () C(a) indicated a C=C bond between C() and C(). The - CSY correlation between C() C(), and the C-MR signals at δc 0. C() and. C() indicated the presence of a methoxyl group at C() and a hydroxyl group at C(). The spectral data for compound were identical to those of limazepine A reported in the literature ()... Biological study At a concentration of 0 μm, compounds - inhibited the growth of MDA-MB- cells at rates of.%,.%, 0.%, and.%, and they inhibited the growth of A cells at rates of.%,.%,.%, and 0.%. The antibacterial activity of compounds - was tested against Bacillus subtilis (CMCC (B) 0), Bacillus pumilus (CMCC (B) 0), and Penicillium avellaneum (UC ) using the filter paper method. Activity of each compound was tested twice at a concentration of.0 mg/ml with a loading volume of 0 μl. Results indicated that compounds - had no effect on the growth of the bacteria tested at 0 μg/disc... Conclusions and perspectives Genetic analysis indicated that some marine Streptomyces form an independent clade (), so marine Streptomyces was surmised to potentially be rich in novel secondary metabolites. The present study succeeded in isolating only one new compound, which suggests that fermentation medium screening and/or genetic manipulation are needed to encourage the production of secondary metabolites in marine Streptomyces. owever, compound was isolated with a high yield ( mg/0 L) and could be used as a lead compound for structural modifications. Acknowledgement This work was financially supported by the Program (the ational Basic Research Program of China, grant no. 00CB0). References. Zheng Y, Shen Y. Clavicorolides A and B, sesquiterpenoids from the fermentation products of edible fungus Clavicorona pyxidata. rg Lett. 00; :0-.. Fenical W, Jensen PR. Developing a new resource for drug discovery: marine actinomycete bacteria. ature Chem Bio. 00; :-.. Bull AT, Stach JEM. Marine actinobacteria: ew opportunities for natural product search and discovery. Trends Microbiol. 00; :-.. Williams DE, Dalisay DS, Patrick B, Matainaho T, Andrusiak K, Deshpande R, Myers CL, Piotrowski JS, Boone C, Yoshida M. Padanamides A and B, highly modified linear tetrapeptides produced in culture by a Streptomyces sp. isolated from a marine sediment. rg Lett. 0; :-.. Sun P, Maloney K, am SJ, aste M, Raju R, Aalbersberg W, Jensen PR, izet V, ensler ME, Fenical W. Fijimycins A-C, three antibacterial etamycin-class depsipeptides from a marine-derived Streptomyces sp. Bioorg Med Chem. 0; :-.. Matsuo Y, Kanoh K, Jang J, Adachi K, Matsuda S, Miki, Kato T, Shizuri Y. Streptobactin, a tricatecholtype siderophore from marine-derived Streptomyces sp. YM-. J at Prod. 0; :-.. Xu Y, e J, Tian XP, Li J, Yang LL, Xie Q, Tang SK, Chen YG, Zhang S, Li WJ. Streptomyces glycovorans sp. nov., Streptomyces xishensis sp. nov. and Streptomyces abyssalis sp. nov. isolated from marine sediments. Int J Syst Evol Microbiol. 0; :-.. Fdhila F, Vázquez V, Sánchez JL, Riguera R. dd- Diketopiperazines: Antibiotics active against Vibrio anguillarum isolated from marine bacteria associated with cultures of Pecten maximus. J at Prod. 00; :-0.. Shimizu K, Kawamoto I, Tomita F, Morimoto M, Fujimoto K. Prothracarcin, a novel antitumor antibiotic. J Antibiotics. ; :. 0. Arima K, Kosaka M, Tamura G, Imanaka, Sakai. Studies on tomaymycin, a new antibiotic. I. Isolation and properties of tomaymycin. J Antibiotics. ; :.. Kariyone K, Yazawa, Kohsaka M. The structure of tomaymycin and oxotomaymycin. Chem Pharm Bull. ; :-.. Fotso S, Zabriskie TM, Proteau PJ, Flatt PM, Santosa DA, Mahmud T. Limazepines A F, pyrrolo [, ] benzodiazepine antibiotics from an Indonesian Micrococcus sp. J at Prod. 00; :0-.. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. ; :-. (Received March, 0; Revised December 0, 0; Accepted December, 0)

5 Drug Discoveries & Therapeutics. 0; ():-. Appendix Cyclo[Val-Pro] (). Colorless crystal. - and C-MR spectral data: see Table. ESI-MS: m/z =. ([M + ] + ) and m/z =.( [M + a] + ). Cyclo[Leu-Pro] (). Colorless crystal. - and C-MR spectral data: see Table. ESI-MS: m/z =. ([M + ] + ) and m/z =.( [M + a] + ). xoprothracarcin (-ethylidene-,-dihydro-- benzo[e]pyrrolo[,-a][,]diazepine-,(0,a)- dione, ). White crystal. - and C-MR spectral data: see Table. ESI-MS: m/z =. ([M + ] + ) and m/z =.( [M + a] + ). Limazepine A (). White crystal. - and C-MR spectral data: see Table. ESI-MS: m/z =. ([M + ] + ) and m/z =. ([M + a] + ).