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Antimalarial and antimycobacterial agents from Streptomyces sp. BCC27095
Tetrahedron Letters 56 (2015) 6875–6877
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Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Antimalarial and antimycobacterial agents from Streptomyces sp. BCC27095 Chakapong Intaraudom, Nantiya Bunbamrung, Aibrohim Dramae, Kannawat Danwisetkanjana, Pranee Rachtawee, Pattama Pittayakhajonwut ⇑ National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Paholyothin Road, Klong Luang, Pathumthani 12120, Thailand
a r t i c l e
i n f o
Article history: Received 4 June 2015 Revised 7 September 2015 Accepted 28 October 2015 Available online 30 October 2015 Keywords: Streptomyces Antimalaria Antimycobacterial Anthraquinone Anthraquinone glycoside
a b s t r a c t Investigation of the terrestrial Streptomyces sp. BCC27095 led to the isolation of two new (1 and 2) and two known (3 and 5) anthraquinone derivatives along with the previously reported compound, steffimycin B (4). The chemical structures were determined on the basis of NMR spectroscopic data. These compounds exhibited significant antimalarial (against Plasmodium falciparum, K-1 multidrug resistant strain) and antimycobacterial (against Mycobacterium tuberculosis H37Ra) activities with IC50 values in the range of 0.32–3.20 lg/mL and MIC in a range of 0.0031–1.56 lg/mL, respectively. They were also evaluated for antibacterial activity against Bacillus cereus and for cytotoxicity against both malignant (MCF-7, KB, NCI-H187) and non-malignant (Vero) cells. Ó 2015 Elsevier Ltd. All rights reserved.
As a part of our continuing search for new bioactive compounds by employing bioassay-guided selection for further chemical investigation of secondary metabolites, the crude extract of Streptomyces sp. BCC27095 was found to exhibit strong antimalarial (IC50 2.72 lg/mL) and antimycobacterial (MIC 0.195 lg/mL) activities. Systematic isolation and purification led to the isolation of four compounds, two of which (1 and 2) were new and two (3 and 5) which were synthetically known and had been previously obtained from the reactions of steffimycin B and steffimycin, respectively.1,2 All compounds, except compound 2, were evaluated for antimalarial, antimycobacterial, and antibacterial (against Bacillus cereus) activities as well as cytotoxicity (both cancerous and non-cancerous cells). The actinomycetes strain was collected from the soil at Phu Hin Rong Kla National Park, Phitsanulok province, Thailand and identified by Dr. Chanwit Suriyachadkun as Streptomyces sp. from its morphological and 16S ribosomal DNA characteristics. The strain was deposited at BIOTEC Culture Collection (BCC), Thailand and registered as BCC27095. Streptomyces sp. was grown on ISP-2 agar (yeast extract-malt extract agar) and then the agar was cut into pieces. The pieces were then transferred into seven 250 mL Erlenmeyer flasks, each containing 100 mL of the BIO19 medium3, for 7 days at 28 °C on ⇑ Corresponding author. Tel.: +66 2 5646700x3559; fax: +66 2 5646632. E-mail address: [email protected] (P. Pittayakhajonwut). http://dx.doi.org/10.1016/j.tetlet.2015.10.098 0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.
a rotary shaker at 250 rpm. Fractions (25 mL) of the liquid medium were transferred into 1 L Erlenmeyer flasks, containing 250 mL of the production medium (LS10), consisting of (w/v): 2.0% mannitol, 2.0% soymeal, 0.03% trace element4, and 0.2% v/v vitamin B complex (Blackmore). The production scale (total volume 7 L) was cultivated for 7 days at 28 °C on a rotary shaker at 250 rpm. Next, the culture (broth and cells) was extracted three times with an equal volume of EtOAc before drying over Na2SO4 and evaporating to dryness, yielding a gum (1.4 g). The gum was chromatographed on a Sephadex LH20 column (3.5 cm 4.9 cm) to afford three main fractions as shown by TLC and HPLC analyses. The first fraction (0.52 g) was further purified by preparative HPLC, using a Sunfire C18 column from Waters (10 lm, diam. 19 mm 250 mm) eluting with a linear gradient system of 20–60% aqueous CH3CN over 20 min at a flow rate of 20 mL/min, to furnish compounds 3 (4.7 mg), 1 (5.2 mg), and 4 (0.14 g), respectively. The second fraction (0.32 g) was further purified by preparative HPLC, using a Sunfire C18 column from Waters (10 lm, diam. 19 mm 250 mm) eluting with a linear gradient system of 15–55% aqueous CH3CN over 20 min at a flow rate of 20 mL/min, to yield compounds 3 (6.7 mg) and 4 (45.2 mg). The third fraction (0.33 g) was further purified by preparative HPLC, using Sunfire C18 column from Waters (10 lm, diam. 19 mm 250 mm) eluted with a linear gradient system of 15–60% aqueous CH3CN over 20 min at a flow rate of 20 mL/min, to obtain compounds 3 (4.3 mg), 4 (19.0 mg), 5 (12.2 mg) and 2 (1.2 mg), respectively.
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Compound 15 (Table 1) was obtained as a red solid. The 1H NMR spectrum was almost identical to the reported data for steffimycin B (4)1,6 except for an additional hydroxyl proton (dH 13.90) and the absence of an aromatic proton, indicating the substitution of one of the aromatic protons in steffimycin B with a hydroxyl group. HRESIMS spectral data confirmed the molecular formula C29H32O14 (m/ z 627.1681 [M+Na]+), suggesting an extra oxygen atom. Moreover, the additional hydroxyl proton showed HMBC correlations to C10a (dC 126.6), C-11a (dC 114.1), and C-11 (dC 157.3), suggesting the position of this hydroxyl group to be at C-11. COSY spectral data and the coupling constants of the protons in a sugar unit (Table 1) indicated that H-10 and H-20 were situated at equatorial positions while H-30 , H-40 , and H-50 were at axial positions. The NOESY spectral data confirmed the relative configuration by cross-peak correlations from H-7 to H-10 and 8-OCH3; from H-40 to 50 -CH3; and from H-8 to 9-CH3. In addition, its specific rotation also gave the same sign as that of compound 4 ([a]D +91, CHCl3).7 On this evidence, compound 1 was determined to have the chemical structure as shown in Figure 1 and given the trivial name of steffimycin C. Compound 28 (Table 1) was obtained as an orange solid. The HRESIMS data established a mass ion peak at 327.0876 [MH], suggesting the molecular formula C18H16O6. The 1H NMR spectral data showed signals of two methyls at dH 1.63 (d, J = 6.8 Hz), 2.46 (s), a methoxy at dH 3.59, a sp3 methine at dH 5.14 (dq, J = 11.3, 6.8 Hz), three methines at dH 6.70 (d, J = 2.5 Hz), 7.38 (d, J = 2.5 Hz), and 7.63 (s), and two hydroxyl protons at dH 12.21 and 13.04. The 1H NMR spectrum showed no signal for the sugar
Table 1 H (500 MHz) and
1
Position
C (125 MHz) NMR assignments of compounds 1 and 2 in CDCl3 1
1
1 2 3 4 4a 5 5a 6 6a 7 8 8a 9 9a 10 10a 11 11a 12 12a 10 20 30 40 50 20 -OCH3 40 -OCH3 50 -CH3 2-OCH3 3-CH3 6-OCH3 8-OCH3 9-CH3 1-OH 4-OH 6-OH 8-OH 11-OH
13
H (mult., J in Hz)
7.47 (d, 2.5) — 6.77 (d, 2.5) — — — — — — 5.12 (d, 2.2) 3.71 (d, 2.2) — — — — — — — — — 5.57 (s) 3.52 (dd, 3.6, 1.4) 3.66 (br m) 3.07 (t, 9.4) 3.77 (m) 3.58 (s) 3.59 (s) 1.39 (d, 6.2) 3.97 (s) — — 3.56 (s) 1.55 (s) — 12.14 (s) 13.02 (s) — 13.90 (s)
2 13
C, type
108.9, CH 167.5, C 107.3, CH 165.9, C 110.2, C 189.2, C 116.4, C 155.2, C 139.9, C 72.8, CH 85.4, CH — 76.6, C — 197.5, C 126.6, C 157.3, C 114.1, C 185.8, C 135.0, C 101.0, CH 80.5, CH 71.3, CH 83.3, CH 69.1, CH 58.8, CH3 60.9, CH3 18.0, CH3 56.3, CH3 — — 60.1, CH3 22.5, CH3 — — — — —
1
H (mult., J in Hz)
— — — 7.63 (s) — 7.38 (d, 2.5) — — — 6.70 (d, 2.5) — — — — — — 5.14 (dq, 11.3, 6.8) — 1.63 (d, 6.8) — — — — — — — — — — 2.46 (s) 3.59 (s) — — 13.04 (s) — — 12.21 (s) 3.98 (d, 11.3)
13
C, type
160.5, C 138.0, C 144.4, C 122.8, CH 135.3, C 108.5, CH — 166.9, C — 106.8, CH 165.4, C 110.2, C 191.3, C 114.3, C 181.6, C 131.4, C 67.0, CH — 22.3, CH3 — — — — — — — — — — 20.5, CH3 56.1, CH3 — — — — — — —
Figure 1. Chemical structures of compounds 1–5.
moiety. The 13C NMR spectrum contained 18 signals comprising of two methyl, one methoxy, one oxymethine, three methine, and 11 quaternary carbons. The HMBC spectrum showed correlations from dH 2.46 (3-CH3) to C-2 (dC 138.0), C-3 (dC 144.4), and C-4 (dC 122.8); from dH 1.63 (H3-12) to C-11 (dC 67.0) and C-2; from H-4 (dH 7.63) to C-2, C-9a (dC 114.3), C-10 (dC 181.6), and 3-CH3 (dC 20.5); from H-5 (dH 7.38) to C-7 (dC 106.8), C-8a (dC 110.2), and C10; from H-7 (dH 6.70) to C-5, C-8a, and C-8 (dC 165.4); from 1-OH (dH 13.04) to C-1 (dC 160.5), C-2, and C-9a; from 8-OH (dH 12.21) to C-7, C-8, and C-8a. From this spectroscopic information, compound 2 was identified as 1,8-dihydroxy-2-(1-hydroxyethyl)-6-methoxy3-methylanthraquinone, which is the reduced form of 2-acetylphyscion, previously isolated from the roots of Berchemia floribunda.9 The 1H and 13C NMR spectral data of compounds 3–5 were identical to those given in the literature for 10-dihydrosteffimycin B, steffimycin B, and 7-deoxysteffimycinone, respectively.1,2,7 Compounds 3 and 5 have never been isolated from a natural source and were originally obtained from the chemical transformations of steffimycin B (4) and steffimycin, respectively.2 10-Dihydrosteffimycin B (3) from steffimycin B was biochemically converted using Chaetomium sp., UC-46342, while 7-deoxysteffimycinone (5) was obtained by microbial conversion from mixed organisms including Aeromonas hydrophila, Escherichia coli, and Citrobacter freundii.10 Moreover, compound 5 was also reported as the product obtained from the catalytic reduction of steffimycin.1 Due to the small quantity of compound 2 obtained, all compounds apart from compound 2, were evaluated for their antimalarial, antimycobacterial, and anti-Bacillus cereus activities as well as their cytotoxicity (against KB, MCF-7, NCI-H187, and Vero cells). The microculture radio isotope technique was used to study the antimalarial activity against the multidrug resistant strain Plasmodium falciparum K-1.11 The green fluorescent protein microplate assay (GFPMA) was performed to evaluate the growth inhibitory activity against Mycobacterium tuberculosis H37Ra and cytotoxicity against Vero cells (African green monkey kidney fibroblasts).12 The resazurin microplate assay (REMA) was employed to evaluate cytotoxicity against cancer cells including KB (human epidermoid carcinoma), MCF-7 (human breast cancer), NCI-H187 (human small cell lung cancer), antibacterial activity against Bacillus cereus.13,14 Standard reference(s) used as positive control(s) for each assay were also reported. Results showed that steffimycin B (4) exhibited the strongest antimycobacterial activity (MIC 0.0052 lM) which was equivalent to the standard reference, rifampicin (see Table 2). Compounds 1 and 3 also showed potential as antimycobacterial agents with MIC values of 0.32 and 0.66 lM, respectively, which were equivalent to the positive controls, streptomycin and isoniazid. Moreover, compound 1 displayed antimalarial activity
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C. Intaraudom et al. / Tetrahedron Letters 56 (2015) 6875–6877 Table 2 Biological activities of compounds 1, 3–5 Compound
Antimalariala IC50 (lM)
Anti-mycobacterialb MIC (lM)
Anti-Bacillus cereusc MIC, (lg/mL)
1 3 4 5
0.53 4.76 2.19 8.03
0.32 0.66 0.0052 3.92
6.25 50.0 1.56 >50
Cytotoxicityd IC50 (lM) MCF-7
KB
5.33 13.82 3.50 >125.5
10.45 16.12 6.75 >125.5
NCI-H187
Vero
4.33 10.62 3.28 >125.5
4.04 >84.7 10.50 51.21
a Dihydroartemisinin and mefloquine were used as positive controls and exhibited IC50 values of 1.98 103 and 0.08 lM, respectively. Maximum tested concentration was 10 lg/mL. b Rifampicin, streptomycin, isoniazid, ofloxacin, and ethambutol were used as positive controls and displayed MIC values of 0.004–0.030, 0.268–0.538, 0.168–0.343, 1.08– 2.16, and 1.15–2.30 lM, respectively. Maximum tested concentration was 50 lg/mL. c Vancomycin was used as the positive control and exhibited an IC50 value of 1.0 lg/mL. Maximum tested concentration was 50 lg/mL. d Ellipticine and doxorubicin were used as the positive controls for cytotoxicity against KB and NCI-H187 cell lines and exhibited IC50 values of 6.33, 0.76 lg/mL, and 1.41, 0.19 lM, respectively. Ellipticine was used as the positive control for cytotoxicity against the Vero cell line and showed an IC50 value of 6.54 lM. Doxorubicin and tamoxifen were used as the positive controls for cytotoxicity against the MCF-7 cell line and showed IC50 values of 15.71 and 24.39 lM, respectively. Maximum tested concentration for all cytotoxicity assays was 50 lg/mL.
against P. falciparum with IC50 value of 0.53 lM. Notably, all tested compounds showed both antimalarial and antimycobacterial activities with relatively low cytotoxicity against mammalian cancerous and non-cancerous cells (see Table 2). In addition, compounds 1 and 4 exhibited anti-B. cereus with IC50 values of 6.25 and 1.56 lg/mL, respectively, while compound 3 was active against B. cereus at the maximum tested concentration (50 lg/mL). Recently, anthracycline derivatives named aranciamycins, which are closely related to compounds 1, 3, and 4, were isolated from an Australian marine-derived Streptomyces sp. (CMB-M0150) and reported to inhibit the growth of Mycobacterium bovis BCG in vitro with IC50 values in a range of 0.7–1.7 lM).15 In conclusion, two new and two naturally new anthraquinone derivatives were isolated from terrestrial soil Streptomyces sp. BCC27095. Compounds 1, 3, and 4 had efficacy as antimycobacterial agents due to inhibition of M. tuberculosis H37Ra which was equivalent to reference compounds with relatively low cytotoxicity. Acknowledgement The National Center for Genetic Engineering and Biotechnology (BIOTEC) is acknowledged for the financial support. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.10. 098.
References and notes 1. Kelly, R. C.; Schletter, I.; Koert, J. M.; MacKellar, F. A.; Wiley, P. F. J. Org. Chem. 1977, 42, 3591. 2. Wiley, P. F.; Elrod, D. W.; Slavicek, J. M.; Marshall, V. P. J. Antibiot. 1980, 33, 819. 3. The BIO19 medium consisted of (w/v): 2% glucose, 0.5% peptone, 0.3% yeast, 0.5% meat extract, 0.05% NaCl, 0.3% (w/v) CaCO3, 0.1% (v/v) vitamin B complex (Blackmore, Australia). 4. Trace element consisted of (w/v): 0.4% CaCl22H2O, 0.2% ZnSO47H2O, 0.01% Na2B4O710H2O, 0.5% FeSO47H2O, 0.005% KI, 0.05% CoCl26H2O, 0.02% CuSO45H2O, 0.2% MnCl24H2O, 0.005% Na2MoO42H2O, 0.1% v/v H2SO4. 5. Steffimycin C (1): red solid; mp 190.2–191.0 °C; [a]24 D +179 (c 0.0175, CHCl3); UV kmax (MeOH) nm (log e) 248 (4.43), 285 (4.25), 505 (4.15); IR (ATR) cm1 3358, 2923, 2852, 1715, 1659, 1632, 1607, 1467, 1409, 1392, 1316, 1169, 1136, 1112, 1057, 1040, 972; HRESIMS m/z 627.1681[M+Na]+ (calcd for C29H32O4Na, 627.1684). 6. Wiley, P. F.; Elrod, D. W.; Harper, D. E. J. Antibiot. 1988, 41, 343. 7. Brodasky, T. F.; Reusser, F. J. Antibiot. 1974, 27, 809. 8. 1,8-Dihydroxy-2-(1-hydroxyethyl)-6-methoxy-3-methylanthraquinone (2): orange solid; [a]26 D 45 (c 0.0069, MeOH); UV kmax (MeOH) nm (log e) 227 1 (4.25), 268 (4.23), 444 (4.01); IR (ATR) cm 3393, 2924, 2852, 1622, 1600, 1457, 1394, 1317, 1271, 1249, 1213, 1164, 1101, 755; HRESIMS m/z 327.0876 [M-H]- (calcd for C18H15O6, 327.0874). 9. Wei, X.; Jiang, J. S.; Feng, Z. M.; Zhang, P. C. Chin. Chem. Lett. 2007, 18, 412. 10. Wiley, P. F.; Marshall, V. P. J. Antibiot. 1975, 28, 838. 11. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents Chemother. 1979, 16, 710. 12. Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents Chemother. 2003, 47, 3682. 13. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421. 14. Sarker, S. D.; Nahar, L.; Kumarasamy, Y. Methods 2007, 42, 321. 15. Khalil, Z. G.; Raju, R.; Piggott, A. M.; Salim, A. A.; Blumenthal, A.; Capon, R. J. J. Nat. Prod. 2015, 78, 949.
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Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Antimalarial and antimycobacterial agents from Streptomyces sp. BCC27095 Chakapong Intaraudom, Nantiya Bunbamrung, Aibrohim Dramae, Kannawat Danwisetkanjana, Pranee Rachtawee, Pattama Pittayakhajonwut ⇑ National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Paholyothin Road, Klong Luang, Pathumthani 12120, Thailand
a r t i c l e
i n f o
Article history: Received 4 June 2015 Revised 7 September 2015 Accepted 28 October 2015 Available online 30 October 2015 Keywords: Streptomyces Antimalaria Antimycobacterial Anthraquinone Anthraquinone glycoside
a b s t r a c t Investigation of the terrestrial Streptomyces sp. BCC27095 led to the isolation of two new (1 and 2) and two known (3 and 5) anthraquinone derivatives along with the previously reported compound, steffimycin B (4). The chemical structures were determined on the basis of NMR spectroscopic data. These compounds exhibited significant antimalarial (against Plasmodium falciparum, K-1 multidrug resistant strain) and antimycobacterial (against Mycobacterium tuberculosis H37Ra) activities with IC50 values in the range of 0.32–3.20 lg/mL and MIC in a range of 0.0031–1.56 lg/mL, respectively. They were also evaluated for antibacterial activity against Bacillus cereus and for cytotoxicity against both malignant (MCF-7, KB, NCI-H187) and non-malignant (Vero) cells. Ó 2015 Elsevier Ltd. All rights reserved.
As a part of our continuing search for new bioactive compounds by employing bioassay-guided selection for further chemical investigation of secondary metabolites, the crude extract of Streptomyces sp. BCC27095 was found to exhibit strong antimalarial (IC50 2.72 lg/mL) and antimycobacterial (MIC 0.195 lg/mL) activities. Systematic isolation and purification led to the isolation of four compounds, two of which (1 and 2) were new and two (3 and 5) which were synthetically known and had been previously obtained from the reactions of steffimycin B and steffimycin, respectively.1,2 All compounds, except compound 2, were evaluated for antimalarial, antimycobacterial, and antibacterial (against Bacillus cereus) activities as well as cytotoxicity (both cancerous and non-cancerous cells). The actinomycetes strain was collected from the soil at Phu Hin Rong Kla National Park, Phitsanulok province, Thailand and identified by Dr. Chanwit Suriyachadkun as Streptomyces sp. from its morphological and 16S ribosomal DNA characteristics. The strain was deposited at BIOTEC Culture Collection (BCC), Thailand and registered as BCC27095. Streptomyces sp. was grown on ISP-2 agar (yeast extract-malt extract agar) and then the agar was cut into pieces. The pieces were then transferred into seven 250 mL Erlenmeyer flasks, each containing 100 mL of the BIO19 medium3, for 7 days at 28 °C on ⇑ Corresponding author. Tel.: +66 2 5646700x3559; fax: +66 2 5646632. E-mail address: [email protected] (P. Pittayakhajonwut). http://dx.doi.org/10.1016/j.tetlet.2015.10.098 0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.
a rotary shaker at 250 rpm. Fractions (25 mL) of the liquid medium were transferred into 1 L Erlenmeyer flasks, containing 250 mL of the production medium (LS10), consisting of (w/v): 2.0% mannitol, 2.0% soymeal, 0.03% trace element4, and 0.2% v/v vitamin B complex (Blackmore). The production scale (total volume 7 L) was cultivated for 7 days at 28 °C on a rotary shaker at 250 rpm. Next, the culture (broth and cells) was extracted three times with an equal volume of EtOAc before drying over Na2SO4 and evaporating to dryness, yielding a gum (1.4 g). The gum was chromatographed on a Sephadex LH20 column (3.5 cm 4.9 cm) to afford three main fractions as shown by TLC and HPLC analyses. The first fraction (0.52 g) was further purified by preparative HPLC, using a Sunfire C18 column from Waters (10 lm, diam. 19 mm 250 mm) eluting with a linear gradient system of 20–60% aqueous CH3CN over 20 min at a flow rate of 20 mL/min, to furnish compounds 3 (4.7 mg), 1 (5.2 mg), and 4 (0.14 g), respectively. The second fraction (0.32 g) was further purified by preparative HPLC, using a Sunfire C18 column from Waters (10 lm, diam. 19 mm 250 mm) eluting with a linear gradient system of 15–55% aqueous CH3CN over 20 min at a flow rate of 20 mL/min, to yield compounds 3 (6.7 mg) and 4 (45.2 mg). The third fraction (0.33 g) was further purified by preparative HPLC, using Sunfire C18 column from Waters (10 lm, diam. 19 mm 250 mm) eluted with a linear gradient system of 15–60% aqueous CH3CN over 20 min at a flow rate of 20 mL/min, to obtain compounds 3 (4.3 mg), 4 (19.0 mg), 5 (12.2 mg) and 2 (1.2 mg), respectively.
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C. Intaraudom et al. / Tetrahedron Letters 56 (2015) 6875–6877
Compound 15 (Table 1) was obtained as a red solid. The 1H NMR spectrum was almost identical to the reported data for steffimycin B (4)1,6 except for an additional hydroxyl proton (dH 13.90) and the absence of an aromatic proton, indicating the substitution of one of the aromatic protons in steffimycin B with a hydroxyl group. HRESIMS spectral data confirmed the molecular formula C29H32O14 (m/ z 627.1681 [M+Na]+), suggesting an extra oxygen atom. Moreover, the additional hydroxyl proton showed HMBC correlations to C10a (dC 126.6), C-11a (dC 114.1), and C-11 (dC 157.3), suggesting the position of this hydroxyl group to be at C-11. COSY spectral data and the coupling constants of the protons in a sugar unit (Table 1) indicated that H-10 and H-20 were situated at equatorial positions while H-30 , H-40 , and H-50 were at axial positions. The NOESY spectral data confirmed the relative configuration by cross-peak correlations from H-7 to H-10 and 8-OCH3; from H-40 to 50 -CH3; and from H-8 to 9-CH3. In addition, its specific rotation also gave the same sign as that of compound 4 ([a]D +91, CHCl3).7 On this evidence, compound 1 was determined to have the chemical structure as shown in Figure 1 and given the trivial name of steffimycin C. Compound 28 (Table 1) was obtained as an orange solid. The HRESIMS data established a mass ion peak at 327.0876 [MH], suggesting the molecular formula C18H16O6. The 1H NMR spectral data showed signals of two methyls at dH 1.63 (d, J = 6.8 Hz), 2.46 (s), a methoxy at dH 3.59, a sp3 methine at dH 5.14 (dq, J = 11.3, 6.8 Hz), three methines at dH 6.70 (d, J = 2.5 Hz), 7.38 (d, J = 2.5 Hz), and 7.63 (s), and two hydroxyl protons at dH 12.21 and 13.04. The 1H NMR spectrum showed no signal for the sugar
Table 1 H (500 MHz) and
1
Position
C (125 MHz) NMR assignments of compounds 1 and 2 in CDCl3 1
1
1 2 3 4 4a 5 5a 6 6a 7 8 8a 9 9a 10 10a 11 11a 12 12a 10 20 30 40 50 20 -OCH3 40 -OCH3 50 -CH3 2-OCH3 3-CH3 6-OCH3 8-OCH3 9-CH3 1-OH 4-OH 6-OH 8-OH 11-OH
13
H (mult., J in Hz)
7.47 (d, 2.5) — 6.77 (d, 2.5) — — — — — — 5.12 (d, 2.2) 3.71 (d, 2.2) — — — — — — — — — 5.57 (s) 3.52 (dd, 3.6, 1.4) 3.66 (br m) 3.07 (t, 9.4) 3.77 (m) 3.58 (s) 3.59 (s) 1.39 (d, 6.2) 3.97 (s) — — 3.56 (s) 1.55 (s) — 12.14 (s) 13.02 (s) — 13.90 (s)
2 13
C, type
108.9, CH 167.5, C 107.3, CH 165.9, C 110.2, C 189.2, C 116.4, C 155.2, C 139.9, C 72.8, CH 85.4, CH — 76.6, C — 197.5, C 126.6, C 157.3, C 114.1, C 185.8, C 135.0, C 101.0, CH 80.5, CH 71.3, CH 83.3, CH 69.1, CH 58.8, CH3 60.9, CH3 18.0, CH3 56.3, CH3 — — 60.1, CH3 22.5, CH3 — — — — —
1
H (mult., J in Hz)
— — — 7.63 (s) — 7.38 (d, 2.5) — — — 6.70 (d, 2.5) — — — — — — 5.14 (dq, 11.3, 6.8) — 1.63 (d, 6.8) — — — — — — — — — — 2.46 (s) 3.59 (s) — — 13.04 (s) — — 12.21 (s) 3.98 (d, 11.3)
13
C, type
160.5, C 138.0, C 144.4, C 122.8, CH 135.3, C 108.5, CH — 166.9, C — 106.8, CH 165.4, C 110.2, C 191.3, C 114.3, C 181.6, C 131.4, C 67.0, CH — 22.3, CH3 — — — — — — — — — — 20.5, CH3 56.1, CH3 — — — — — — —
Figure 1. Chemical structures of compounds 1–5.
moiety. The 13C NMR spectrum contained 18 signals comprising of two methyl, one methoxy, one oxymethine, three methine, and 11 quaternary carbons. The HMBC spectrum showed correlations from dH 2.46 (3-CH3) to C-2 (dC 138.0), C-3 (dC 144.4), and C-4 (dC 122.8); from dH 1.63 (H3-12) to C-11 (dC 67.0) and C-2; from H-4 (dH 7.63) to C-2, C-9a (dC 114.3), C-10 (dC 181.6), and 3-CH3 (dC 20.5); from H-5 (dH 7.38) to C-7 (dC 106.8), C-8a (dC 110.2), and C10; from H-7 (dH 6.70) to C-5, C-8a, and C-8 (dC 165.4); from 1-OH (dH 13.04) to C-1 (dC 160.5), C-2, and C-9a; from 8-OH (dH 12.21) to C-7, C-8, and C-8a. From this spectroscopic information, compound 2 was identified as 1,8-dihydroxy-2-(1-hydroxyethyl)-6-methoxy3-methylanthraquinone, which is the reduced form of 2-acetylphyscion, previously isolated from the roots of Berchemia floribunda.9 The 1H and 13C NMR spectral data of compounds 3–5 were identical to those given in the literature for 10-dihydrosteffimycin B, steffimycin B, and 7-deoxysteffimycinone, respectively.1,2,7 Compounds 3 and 5 have never been isolated from a natural source and were originally obtained from the chemical transformations of steffimycin B (4) and steffimycin, respectively.2 10-Dihydrosteffimycin B (3) from steffimycin B was biochemically converted using Chaetomium sp., UC-46342, while 7-deoxysteffimycinone (5) was obtained by microbial conversion from mixed organisms including Aeromonas hydrophila, Escherichia coli, and Citrobacter freundii.10 Moreover, compound 5 was also reported as the product obtained from the catalytic reduction of steffimycin.1 Due to the small quantity of compound 2 obtained, all compounds apart from compound 2, were evaluated for their antimalarial, antimycobacterial, and anti-Bacillus cereus activities as well as their cytotoxicity (against KB, MCF-7, NCI-H187, and Vero cells). The microculture radio isotope technique was used to study the antimalarial activity against the multidrug resistant strain Plasmodium falciparum K-1.11 The green fluorescent protein microplate assay (GFPMA) was performed to evaluate the growth inhibitory activity against Mycobacterium tuberculosis H37Ra and cytotoxicity against Vero cells (African green monkey kidney fibroblasts).12 The resazurin microplate assay (REMA) was employed to evaluate cytotoxicity against cancer cells including KB (human epidermoid carcinoma), MCF-7 (human breast cancer), NCI-H187 (human small cell lung cancer), antibacterial activity against Bacillus cereus.13,14 Standard reference(s) used as positive control(s) for each assay were also reported. Results showed that steffimycin B (4) exhibited the strongest antimycobacterial activity (MIC 0.0052 lM) which was equivalent to the standard reference, rifampicin (see Table 2). Compounds 1 and 3 also showed potential as antimycobacterial agents with MIC values of 0.32 and 0.66 lM, respectively, which were equivalent to the positive controls, streptomycin and isoniazid. Moreover, compound 1 displayed antimalarial activity
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C. Intaraudom et al. / Tetrahedron Letters 56 (2015) 6875–6877 Table 2 Biological activities of compounds 1, 3–5 Compound
Antimalariala IC50 (lM)
Anti-mycobacterialb MIC (lM)
Anti-Bacillus cereusc MIC, (lg/mL)
1 3 4 5
0.53 4.76 2.19 8.03
0.32 0.66 0.0052 3.92
6.25 50.0 1.56 >50
Cytotoxicityd IC50 (lM) MCF-7
KB
5.33 13.82 3.50 >125.5
10.45 16.12 6.75 >125.5
NCI-H187
Vero
4.33 10.62 3.28 >125.5
4.04 >84.7 10.50 51.21
a Dihydroartemisinin and mefloquine were used as positive controls and exhibited IC50 values of 1.98 103 and 0.08 lM, respectively. Maximum tested concentration was 10 lg/mL. b Rifampicin, streptomycin, isoniazid, ofloxacin, and ethambutol were used as positive controls and displayed MIC values of 0.004–0.030, 0.268–0.538, 0.168–0.343, 1.08– 2.16, and 1.15–2.30 lM, respectively. Maximum tested concentration was 50 lg/mL. c Vancomycin was used as the positive control and exhibited an IC50 value of 1.0 lg/mL. Maximum tested concentration was 50 lg/mL. d Ellipticine and doxorubicin were used as the positive controls for cytotoxicity against KB and NCI-H187 cell lines and exhibited IC50 values of 6.33, 0.76 lg/mL, and 1.41, 0.19 lM, respectively. Ellipticine was used as the positive control for cytotoxicity against the Vero cell line and showed an IC50 value of 6.54 lM. Doxorubicin and tamoxifen were used as the positive controls for cytotoxicity against the MCF-7 cell line and showed IC50 values of 15.71 and 24.39 lM, respectively. Maximum tested concentration for all cytotoxicity assays was 50 lg/mL.
against P. falciparum with IC50 value of 0.53 lM. Notably, all tested compounds showed both antimalarial and antimycobacterial activities with relatively low cytotoxicity against mammalian cancerous and non-cancerous cells (see Table 2). In addition, compounds 1 and 4 exhibited anti-B. cereus with IC50 values of 6.25 and 1.56 lg/mL, respectively, while compound 3 was active against B. cereus at the maximum tested concentration (50 lg/mL). Recently, anthracycline derivatives named aranciamycins, which are closely related to compounds 1, 3, and 4, were isolated from an Australian marine-derived Streptomyces sp. (CMB-M0150) and reported to inhibit the growth of Mycobacterium bovis BCG in vitro with IC50 values in a range of 0.7–1.7 lM).15 In conclusion, two new and two naturally new anthraquinone derivatives were isolated from terrestrial soil Streptomyces sp. BCC27095. Compounds 1, 3, and 4 had efficacy as antimycobacterial agents due to inhibition of M. tuberculosis H37Ra which was equivalent to reference compounds with relatively low cytotoxicity. Acknowledgement The National Center for Genetic Engineering and Biotechnology (BIOTEC) is acknowledged for the financial support. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.10. 098.
References and notes 1. Kelly, R. C.; Schletter, I.; Koert, J. M.; MacKellar, F. A.; Wiley, P. F. J. Org. Chem. 1977, 42, 3591. 2. Wiley, P. F.; Elrod, D. W.; Slavicek, J. M.; Marshall, V. P. J. Antibiot. 1980, 33, 819. 3. The BIO19 medium consisted of (w/v): 2% glucose, 0.5% peptone, 0.3% yeast, 0.5% meat extract, 0.05% NaCl, 0.3% (w/v) CaCO3, 0.1% (v/v) vitamin B complex (Blackmore, Australia). 4. Trace element consisted of (w/v): 0.4% CaCl22H2O, 0.2% ZnSO47H2O, 0.01% Na2B4O710H2O, 0.5% FeSO47H2O, 0.005% KI, 0.05% CoCl26H2O, 0.02% CuSO45H2O, 0.2% MnCl24H2O, 0.005% Na2MoO42H2O, 0.1% v/v H2SO4. 5. Steffimycin C (1): red solid; mp 190.2–191.0 °C; [a]24 D +179 (c 0.0175, CHCl3); UV kmax (MeOH) nm (log e) 248 (4.43), 285 (4.25), 505 (4.15); IR (ATR) cm1 3358, 2923, 2852, 1715, 1659, 1632, 1607, 1467, 1409, 1392, 1316, 1169, 1136, 1112, 1057, 1040, 972; HRESIMS m/z 627.1681[M+Na]+ (calcd for C29H32O4Na, 627.1684). 6. Wiley, P. F.; Elrod, D. W.; Harper, D. E. J. Antibiot. 1988, 41, 343. 7. Brodasky, T. F.; Reusser, F. J. Antibiot. 1974, 27, 809. 8. 1,8-Dihydroxy-2-(1-hydroxyethyl)-6-methoxy-3-methylanthraquinone (2): orange solid; [a]26 D 45 (c 0.0069, MeOH); UV kmax (MeOH) nm (log e) 227 1 (4.25), 268 (4.23), 444 (4.01); IR (ATR) cm 3393, 2924, 2852, 1622, 1600, 1457, 1394, 1317, 1271, 1249, 1213, 1164, 1101, 755; HRESIMS m/z 327.0876 [M-H]- (calcd for C18H15O6, 327.0874). 9. Wei, X.; Jiang, J. S.; Feng, Z. M.; Zhang, P. C. Chin. Chem. Lett. 2007, 18, 412. 10. Wiley, P. F.; Marshall, V. P. J. Antibiot. 1975, 28, 838. 11. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents Chemother. 1979, 16, 710. 12. Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents Chemother. 2003, 47, 3682. 13. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421. 14. Sarker, S. D.; Nahar, L.; Kumarasamy, Y. Methods 2007, 42, 321. 15. Khalil, Z. G.; Raju, R.; Piggott, A. M.; Salim, A. A.; Blumenthal, A.; Capon, R. J. J. Nat. Prod. 2015, 78, 949.