Cytotoxic Angucyclines from Amycolatopsis sp. HCa1, a Rare Actinobacteria Derived from Oxya chinensis

• Abstract
• Materials and Methods
• Supporting information
• Acknowledgements
• References

Abstract

Two new angucyclines, named (2R,3R)-2-hydroxy-8-O-methyltetrangomycin (1) and (2R,3R)-2-hydroxy-5-O-methyltetrangomycin (2), together with eight known compounds (3–10), were isolated from the culture of Amycolatopsis sp. HCa1, a rare actinobacteria isolated from the gut of Oxya chinensis. The new structures were elucidated through extensive spectroscopic data analysis, and their absolute configurations were assigned by application of the modified Mosher's method and CD spectrum comparison. Their in vitro cytotoxic activities against four cell lines including human cervical cancer cell line (HeLa), human gastric adenocarcinoma cell line (SGC-7901), human lung adenocarcinoma cell line (SPC-A-1), and mouse macrophage cell line (RAW264.7) were then investigated. Compounds 3, 4, 9, and 10 showed potent cytotoxic activities towards the HeLa cells with IC₅₀ values of 0.27, 0.11, 0.56, and 0.39 µM, respectively.

Actinobacteria associated with insect hosts are a very promising source of providing novel active small molecules that confer benefits to hosts [1], [2], [3], [4]. The insect inner gut system is an important natural environment harboring diverse microbes that may produce bioactive substances. Our previous investigation of microorganisms residing in the insect gut has reported unprecedented immunosuppressive natural products [5]. However, none of the biologically active metabolites produced by rare actinomycetes derived from the insect gut have been reported. Amycolatopsis, one group of rare actinomycetes, belonging to the family Pseudonocardiaceae, is a famous genus who can produce many novel bioactive metabolites [6], [7], [8]. In the course of searching for chemically new and biologically potent secondary metabolites produced by actinomycetes, a strain of Amycolatopsis sp. HCa1 was isolated from the gut of healthy Oxya chinensis, a kind of phytophagous insect, collected from the suburb of Nanchang, Jiangxi Province, China. The subsequent fractionation of the ethyl acetate extract from the culture broth of Amycolatopsis sp. HCa1 by a combination of chromatographic methods led to the isolation of two new angucyclines, (2R,3R)-2-hydroxy-8-O-methyltetrangomycin (1) and (2R,3R)-2-hydroxy-5-O-methyl-tetrangomycin (2), along with eight known metabolites identified as tetrangomycin (3) [9], [10], PD116779 (4) [11], tetrangulol (5) [10], [12], X-14881E (6) [13], sakyomicin B (7) [14], [15], tetracyclinone (8) [15], sakyomicin A (9) [15], and sakyomicin C (10) [15] ([Fig. 1]). The isolation, structural elucidation, and in vitro cytotoxic activities of these metabolites are reported herein.

Compound 1, obtained as a brown-yellow solid, was found to have the molecular formular C₂₀H₁₆O₆ as determined through HR-ESI-MS (positive mode, [M + Na]⁺ peak at m/z 375.0850, calcd. for C₂₀H₁₆O₆Na⁺, 375.0839), with 14 amu (CH₂) more than that of compound 4 [11]. The IR data obtained on 1 indicated the presence of ketone carbonyl at 1708.2 cm⁻¹ and non-chelated quinone carbonyl at 1667.6 cm⁻¹. The ¹H and ¹³C NMR spectra of compound 1 were very similar to those of 4, except for the chelated hydroxy group [δ _H 12.07] in 4 [11], which was replaced by an 8-methoxy group [δ _H 3.94, δ _C 56.4] in 1 ([Table 1]). The ³ J diagnostic HMBC correlations ([Fig. 2]) from the oxygenated methyl protons to C-8 (δ _C 159.4) and NOESY correlations ([Fig. 3]) of 8-OMe with H-9 [δ _H 7.55 (1H, d, 8.0)] allowed the methoxy group to be placed on C-8. Further comprehensive NMR analysis, utilizing data from HMQC, ¹H-¹H COSY, HMBC, and NOESY, allowed the full assignment of the structure for compound 1 as shown in [Fig. 1].

Compound 2, which appeared in an isolated form as an orange solid, gave a molecular formula of C₂₀H₁₆O₇ (13 degrees of unsaturation), as determined by HR-ESI-MS (m/z 391.0812 [M + Na]⁺) in combination with ¹H and ¹³C NMR data ([Table 1]). This formula showed the addition of an oxymethylene (-OCH₂) to the molecular formula of 4 [11]. The ¹H and ¹³C NMR data for 2 were very similar to those of compound 4, except for the new 5-methoxyl group signals [δ _H 4.05 (3H, s), δ _C 56.5] and a more upfield aromatic proton singlet at δ _H-6 7.71 (1H, s). A prominent NOESY NMR correlation ([Fig. 3]) between the methoxyl protons and H-6 (δ _H 7.71) could establish the position of the methoxyl group at C-5 (δ _C 160.2), which was also supported by the key three bond HMBC correlations ([Fig. 2]) of H-4 [δ _H 2.92 (2H, br s)] with C-5, and 5-OMe with C-5. The 1D and 2D NMR experiments permitted the assignment of the whole structure for compound 2 as shown in [Fig. 1].

The relative stereochemistry of 1 and 2 was established by the NOESY experiment. As illustrated in [Fig. 3], the observed NOESY correlations between H-2 and 3-CH₃, and between 2-OH and 3-OH in 1 and 2 confirmed the same relative configuration.

The absolute configuration of 1 was determined by the modified Mosher's method [16]. Treatment of 1 with (S)-MTPA Cl and (R)-MTPA Cl afforded the (S)-MTPA ester 1s and (R)-MTPA ester 1r, respectively. The difference in chemical shift values (Δδ = δ _S – δ _R) for all of the relevant signals except one (H-11) for the 1s and 1r ([Fig. 4]) suggested the R absolute configuration at C-2. Thus, the 2R and 3R absolute configuration was proposed for compound 1. The absolute configuration of 2 was accomplished from its CD spectrum, which was in good agreement with that of compound 1 ([Fig. 5]), highlighting their identity with the (2R,3R) absolute configuration.

It is possible that the methoxyl group of 1 or 2 was a result of a reaction with methanol in the procedure of extraction or isolation. An LC-MS experiment was performed to determine whether the two compounds were natural products or artefacts. The direct detection of extracts of fresh broth extracted by SPE C18 column demonstrated that 1 and 2 were natural products indeed.

The angucycline antibiotics [17], a large group of polycyclic aromatic polyketides mainly isolated from Streptomyces, exhibit a broad range of biological activities such as antibacterial [14], [18], [19], [20], antitumor [9], [11], and protease activation [12]. In the present research, the isolated compounds (1–10) were used to determine the in vitro cytotoxic activity against HeLa, SGC-7901, SPC-A-1, and RAW264.7 cell lines ([Table 2]) [21]. Compounds 3, 4, 9, and 10 showed potent cytotoxic activities toward the HeLa cells with IC₅₀ values of 0.27, 0.11, 0.56, and 0.39 µM, respectively. Compounds 2–4, 7, 9, and 10 displayed different cytotoxicity against the SGC-7901 cell line with the IC₅₀ values in a range from 4.41 to 93.46 µM. In RAW264.7 cells, compounds 3, 4, 7, 9, and 10 showed cytotoxic activity with IC₅₀ values of 32.27, 57.52, 56.14, 40.64, and 3.66 µM, respectively. Conversely, compounds 1, 5, 6, and 8 showed almost no cytotoxic activity toward the tested cell lines. The nonaromatic ring A may be responsible for the cytotoxicity of tetrangomycin (3) relative to tetrangulol (5). The hydroxyl at C-8 in PD116779 (4) seemed to play an important role because the methoxyl derivative 1 displayed almost no activity. The fact that sakyomicin A (9) and C (10) were more potent than their aglycone analogue (7) indicated that the presence of the glycone subunit is important for cytotoxicity. The absence of a hydroxyl group at C-2 significantly increased the IC₅₀ values from 10 to 9.

Materials and Methods

Strain: The actinobacteria, strain number HCa1, was isolated by one of the authors (Z. K. G.) from the gut of healthy Oxya chinensis collected in October 2008 from the suburb of Nanchang, Jiangxi Province, P. R. China. The isolate was identified by Dr. Y. C. Song as Amycolatopsis sp. by comparing its morphological characteristics and its 16S rDNA gene sequence with type strains of the genus Amycolatopsis. The voucher specimens of Oxya chinensis (IFB-SO10) and Amycolatopsis sp. HCa1 were deposited in the Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University.

(2R,3R)-2-hydroxy-8-O-methyltetrangomycin (1): brown-yellow solid, m. p. 123–125 °C; [α]_D ²⁵ = + 47.3° (c 0.23, MeOH); UV (MeOH) λ _max (nm) (log ε): 205 (4.47), 262 (4.40), 333 (3.63), 376 (3.66); CD (MeOH): Δε = 200 (+ 1.7), 211 (− 1.6), 228 (+ 1.6), 262 (− 0.5), 299 (+ 1.9), 315 (+ 0.6), 329 (+ 1.0), 349 (+ 0.5), 374 (− 0.2) nm; IR (KBr) ν _max (cm⁻¹): 3403.7, 2925.6, 2852.8, 1708.2, 1667.6, 1588.6, 1469.5, 1384.3, 1267.1, 1119.2, 1038.3, 961.1, 830.2, 715.3; HR-ESI-MS m/z 375.0850 [M + Na]⁺ (calcd. for C₂₀H₁₆O₆Na, 375.0839); ¹H and ¹³C NMR data, see [Table 1].

(2R,3R)-2-hydroxy-5-O-methyltetrangomycin (2): orange solid, m. p. 145–147  °C; [α]_D ²⁵ = + 85.9° (c 0.19, MeOH); UV (MeOH) λ _max (nm) (log ε): 219 (4.37), 280 (4.31), 391 (3.69); CD (MeOH): Δε = 200 (+ 2.9), 208 (− 2.2), 232 (+ 2.1), 264 (− 0.3), 297 (+ 2.3), 322 (+ 1.0), 331 (+ 1.2), 348 (+ 0.3), 369 (− 0.4) nm; IR (KBr) ν _max (cm⁻¹): 3428.6, 2923.5, 2852.0, 1714.5, 1667.9, 1640.4, 1582.6, 1562.0, 1460.1, 1384.1, 1371.1, 1291.8, 1229.5, 1083.6, 1001.9, 837.3, 767.9; HR-ESI-MS m/z 391.0812 [M + Na]⁺ (calcd. for C₂₀H₁₆O₇Na, 391.0788); ¹H and ¹³C NMR data, see [Table 1].

Supporting information

General experimental procedures, details on fermentation, extraction, isolation, and preparation of (S)-MTPA ester 1s and (R)-MTPA ester 1r, and the in vitro cytotoxicity test of compounds 1–10, as well as the 1D and 2D NMR spectra of compounds 1 and 2, and the ¹H-NMR spectra of 1s and 1r, are available as Supporting Information.

Acknowledgements

This work was cofinanced by grants from NSFC (90813036, 30821006, 20802035 and 21072092), JSNSF (BK2009010), MOST (2009ZX09501-013) and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

Conflict of Interest

All authors here declare that there are no conflicts of interest.

References

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Assoc. Prof. Dr. Hui Ming Ge

Institute of Functional Biomolecules
State Key Laboratory of Pharmaceutical Biotechnology
Nanjing University

22 Hankou Road

Nanjing 210093

China

Phone: +86 25 83 59 32 01

Fax: +86 25 83 59 32 01

Email: hmge@nju.edu.cn

References

• 1 Oh D C, Poulsen M, Currie C R, Clardy J. Dentigerumycin: a bacterial mediator of an ant-fungus symbiosis.  Nat Chem Biol. 2009;  5 391-393
  CrossrefPubMedSearch in Google Scholar
• 2 Scott J J, Oh D C, Yuceer M C, Klepzig K D, Clardy J, Currie C R. Bacterial protection of beetle-fungus mutualism.  Science. 2008;  322 63
  CrossrefPubMedSearch in Google Scholar
• 3 Currie C R, Scott J A, Summerbell R C, Malloch D. Fungus-growing ants use antibiotic-producing bacteria to control garden parasites.  Nature. 1999;  398 701-704
  CrossrefPubMedSearch in Google Scholar
• 4 Iwai K, Iwamoto S, Aisaka K, Suzuki M. Isolation of novel actinomycetes from spider materials.  Actinomycetologica. 2009;  23 8-15
  CrossrefPubMedSearch in Google Scholar
• 5 Zhang Y L, Ge H M, Zhao W, Dong H, Xu Q, Li S H, Li J, Zhang J, Song Y C, Tan R X. Unprecedented immunosuppressive polyketides from Daldinia eschscholzii, a mantis-associated fungus.  Angew Chem Int Ed. 2008;  47 5823-5826
  CrossrefPubMedSearch in Google Scholar
• 6 Kunimoto S, Lu J, Esumi H, Yamazaki Y, Kinoshita N, Honma Y, Hamada M, Ohsono M, Ishizuka M, Takeuchi T. Kigamicins, novel antitumor antibiotics. I. Taxonomy, isolation, physico-chemical properties and biological activities.  J Antibiot. 2003;  56 1004-1011
  CrossrefPubMedSearch in Google Scholar
• 7 Matsumoto N, Tsuchida T, Sawa R, Iinuma H, Nakamura H, Naganawa H, Sawa T, Takeuchi T. Epoxyquinomicins A, B, C and D, new antibiotics from Amycolatopsis. III. Physico-chemical properties and structure determination.  J Antibiot. 1997;  50 912-915
  CrossrefPubMedSearch in Google Scholar
• 8 Tsuchida T, Sawa R, Takahashi Y, Iinuma H, Sawa T, Naganawa H, Takeuchi T. Azicemicins A and B, new antimicrobial agents produced by Amycolatopsis. II. Structure determination.  J Antibiot. 1995;  48 1148-1152
  CrossrefPubMedSearch in Google Scholar
• 9 Zhu L L, Ostash B, Rix U, Nur-e-Alam M, Mayers A, Luzhetskyy A, Mendez C, Salas J A, Bechthold A, Fedorenko V, Rohr J. Identification of the function of gene lndM2 encoding a bifunctional oxygenase-reductase involved in the biosynthesis of the antitumor antibiotic landomycin E by Streptomyces globisporus 1912 supports the originally assigned structure for landomycinone.  J Org Chem. 2005;  70 631-638
  CrossrefPubMedSearch in Google Scholar
• 10 Kuntsmann M P, Mitscher L A. The structural characterization of tetrangomycin and tetrangulol.  J Org Chem. 1966;  31 2920-2925
  CrossrefPubMedSearch in Google Scholar
• 11 Kern D L, Schaumberg J P, Hokanson G C, French J C. PD 116 779, a new antitumor antibiotic of the benz[α]anthraquinone class.  J Antibiot. 1986;  39 469-470
  CrossrefPubMedSearch in Google Scholar
• 12 Yamashita N, Harada T, Shin-Ya K, Seto H. 6-Hydroxytetrangulol, a new CPP32 protease inducer produced by Streptomcyces sp.  J Antibiot. 1998;  51 79-81
  CrossrefPubMedSearch in Google Scholar
• 13 Maehr H, Liu C M, Liu M, Perrotta A, Smallheer J M, Williams T H, Blount J F. Microbial products. VI. Five novel metabolites related to benz[α]anthracene from an unidentified actinomycete designated X-14881.  J Antibiot. 1982;  35 1627-1631
  CrossrefPubMedSearch in Google Scholar
• 14 Gould S J, Cheng X C. New benz[α]anthraquinone secondary metabolites from Streptomyces phaeochromogenes.  J Org Chem. 1994;  59 400-405
  CrossrefPubMedSearch in Google Scholar
• 15 Irie H, Mizuno Y, Kouno I, Nagasawa T, Tani Y, Yamada H, Taga T, Osaki K. Structures of new antibiotic substances, Sakyomicin A, B, C and D; X-ray crystal and molecular structure of Sakyomicin A.  J Chem Soc Chem Commun. 1983;  174-175
  PubMedSearch in Google Scholar
• 16 Li E W, Tian R R, Liu S C, Chen X L, Guo L D, Che Y S. Pestalotheols A–D, bioactive metabolites from the plant endophytic fungus Pestalotiopsis theae.  J Nat Prod. 2008;  71 664-668
  CrossrefPubMedSearch in Google Scholar
• 17 Rohr J, Thiericke R. Angucycline group antibiotics.  Nat Prod Rep. 1992;  9 103-137
  CrossrefPubMedSearch in Google Scholar
• 18 Shigihara Y, Koizumi Y, Tamamura T, Homma Y, Isshiki K, Dobashi K, Naganawa H, Takeuchi T. 6-Deoxy-8-O-methylrabelomycin and 8-O-methylrabelomycin from a Streptomyces species.  J Antibiot. 1988;  41 1260-1264
  CrossrefPubMedSearch in Google Scholar
• 19 Gilpin M L, Balchin J, Box S J, Tyler J W. MM 47755, a new benz[α]anthracene antibiotic from a Streptomycete.  J Antibiot. 1989;  42 627-628
  CrossrefPubMedSearch in Google Scholar
• 20 Kesenheimer C, Groth U. Total synthesis of (-)-8-O-methyltetrangomycin (MM 47755).  Org Lett. 2006;  8 2507-2510
  CrossrefPubMedSearch in Google Scholar
• 21 Ge H M, Yu Z G, Zhang J, Wu J H, Tan R X. Bioactive alkaloids from endophytic Aspergillus fumigatus.  J Nat Prod. 2009;  72 753-755
  CrossrefPubMedSearch in Google Scholar

Assoc. Prof. Dr. Hui Ming Ge

Institute of Functional Biomolecules
State Key Laboratory of Pharmaceutical Biotechnology
Nanjing University

22 Hankou Road

Nanjing 210093

China

Phone: +86 25 83 59 32 01

Fax: +86 25 83 59 32 01

Email: hmge@nju.edu.cn

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