Sulfur-Containing Cytotoxic Curvularin Macrolides from - American

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Sulfur-Containing Cytotoxic Curvularin Macrolides from Penicillium sumatrense MA-92, a Fungus Obtained from the Rhizosphere of the Mangrove Lumnitzera racemosa Ling-Hong Meng,†,‡ Xiao-Ming Li,† Cui-Ting Lv,§ Chun-Shun Li,† Gang-Ming Xu,† Cai-Guo Huang,*,§ and Bin-Gui Wang*,† †

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, People’s Republic of China ‡ University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China § Department of Biochemistry and Molecular Biology, Second Military Medical University, Xiangyin Road 800, Shanghai 200433, People’s Republic of China S Supporting Information *

ABSTRACT: Sumalarins A−C (1−3), the new and rare examples of sulfur-containing curvularin derivatives, along with three known analogues (4−6), were isolated and identified from the cytotoxic extract of Penicillium sumatrense MA-92, a fungus obtained from the rhizosphere of the mangrove Lumnitzera racemosa. Their structures were established by detailed interpretation of NMR and MS data, and compound 1 was confirmed by X-ray crystallographic analysis. Compounds 1−3 and 5 showed potent cytotoxicity against some of the tested tumor cell lines. Sulfur substitution at C-11 or a double bond at C-10 significantly increased the cytotoxic activities of the curvularin analogues.

C

urvularin and its derivatives are produced by some fungal species mainly from the genera Curvularia,1 Aspergillus,2 Alternaria,3 and Penicillium.4 These compounds display various biological properties including phytotoxicity,3 cytotoxicity toward sea urchin embryogenesis,4 inhibition of cell division,4 inhibition of expression of human inducible nitric oxide synthase,5 and antifungal activities,6 as well as cytotoxicity against several human cancer cell lines.1 In continuation of our approach to identify new bioactive secondary metabolites from marine-derived fungi,7−10 we performed chemical investigations on the cytotoxic extract of the fungal strain Penicillium sumatrense MA-92, which was isolated from the rhizosphere of the marine mangrove plant Lumnitzera racemosa. As a result, three new curvularin derivatives, sumalarins A−C (1−3), rare examples of sulfur-containing curvularins, as well as three known analogues (4−6), were characterized from the culture extract of the fungus. The structures of compounds 1−6 were established on the basis of spectroscopic analysis and supported by the single-crystal X-ray diffraction analysis of 1. The cytotoxic activities against seven tumor cell lines were determined. This paper describes the isolation, structure determination, stereochemical assignment, and cytotoxicity of the isolated compounds. The rice solid culture of P. sumatrense MA-92 was exhaustively extracted with EtOAc to afford an extract which was further purified by a combination of column chromatography, including silica gel, Sephadex LH-20, Lobar LiChroprep © 2013 American Chemical Society and American Society of Pharmacognosy

RP-18, and semipreparative HPLC, to yield six curvularin derivatives (1−6). Compound 1 was obtained as colorless crystals and its molecular formula was determined as C20H26O8S on the basis of positive HR-ESI-MS, indicating eight degrees of unsaturation. Exhaustive analyses of the NMR data of 1 (Table 1) showed some similarity to the phenylacetic acid lactone moiety Received: July 28, 2013 Published: November 6, 2013 2145

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Table 1. NMR Spectroscopic Data for Compounds 1 and 2a 1

a

no.

δC

1 2 3 4 5 6 7 8 9 10

171.1, C 39.6, CH2 137.6, C 112.5, CH 158.3, C 102.8, CH 160.6, C 120.8, C 204.1, C 52.6, CH2

11 12

41.2, CH 33.2, CH2

13

23.7, CH2

14 15 16 17

32.6, 73.7, 21.6, 35.3,

18 19 20

71.8, CH 173.9, C 52.3, CH3

CH2 CH CH3 CH2

2 δH (J in Hz)

δC

δH (J in Hz)

171.1, C 39.8, CH2 137.8, C 112.5, CH 158.3, C 102.8, CH 160.6, C 120.5, C 204.1, C 53.0, CH2

3.71, m 6.35, d (1.8) 6.43, d (1.8)

α 2.76, br m β 3.71, m 3.31, m α 1.45, br m β 1.58, br m α 1.29, br m β 1.73, br m 1.52, m 4.83, br m 1.11, d (6.2) 2.95, d (5.6)

41.5, CH 33.3, CH2 24.0, CH2 32.6, 73.7, 21.6, 35.7,

4.36, t (5.6)

CH2 CH CH3 CH2

72.4, CH 173.6, C 67.5, CH2

3.69, s

21

66.8, CH2

22 23 24 25

172.0, C 20.9, CH3 171.4, C 20.5, CH3

3.68, m 6.33, br s 6.45, br s

α 2.72, br m β 3.68, m 3.37, m α 1.44, br m β 1.59, br m α 1.31, br m β 1.75, br m 1.52, m 4.82, br m 1.10, d (6.2) α 2.94, dd (13.6, 6.8) β 3.04, dd (13.6, 4.7) 4.38, dd (6.8, 5.0) α 4.24, m β 4.44, m α 4.15, dd (11.4, 5.9) β 4.34, m 1.96, s 2.00, s

Measured in acetone-d6 at 500 and 125 MHz for 1H and 13C, respectively.

of curvularin (4)11 but with an additional C4H7O3S unit. The primary difference in the NMR spectroscopic data was that the methylene signal at δH 1.52/1.73 and δC 23.4 for CH2-11 in 411 was replaced by a methine signal at δH 3.31 and δC 41.2 (CH11) in 1. In addition, NMR resonances corresponding to one methoxy, one ester carbonyl, one oxygenated methine, and one methylene were present in 1 (Table 1), implying that compound 1 should be a curvularin derivative with a substituent group at C-11. The observed COSY correlation from H-17 to H-18 as well as HMBC cross-peaks from H-17, H-18, and H-20 to the ester carbonyl carbon C-19 assigned the structure of the substituent group as shown in Supporting Information (SI) Figure S1. On the basis of the chemical shifts (Table 1), C-11 and C-17 were sulfur-substituted. The HMBC correlation (SI Figure S1) from H-17 to C-11 confirmed the substituent group at C-11. Unfortunately the relative configuration at C-11, C-15, and C-18 for compound 1 could not be solved either by J-coupling constants or by a NOESY experiment. However, upon slow evaporation of the solvent by storing the sample in a refrigerator, a single crystal of 1 was cultivated, making feasible an X-ray diffraction analysis (Figure 1) that unequivocally confirmed the structure of 1. The absolute configuration was determined based on measuring the anomalous dispersion effects by collecting Friedel pair reflections in the X-ray diffraction experiment. The heavy sulfur atom contained in the structure of 1 had a measurable

Figure 1. X-ray crystallographic structure of compound 1 (Note: A different numbering system is used for the structure in the text).

anomalous dispersion effect. The Flack parameter12 was 0.29(19) in the final refinement for all 3430 reflections with 264 Friedel pairs. The presence of the S-atom in the molecule 2146

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Table 2. Cytotoxicity of Compounds 1−5 against Seven Tumor Cell Lines (IC50, μM) 1 2 3 4 5 positive control

Du145a

HeLab

Huh 7c

MCF-7d

NCI-H460e

SGC-7901f

SW1990g

6.0 7.0 6.0 na 6.0 3.4h

6.0 8.0 6.0 na 6.0 12i

3.9 5.5 5.1 na 5.0 12j

4.4 4.7 4.4 na 4.0 3.1i

3.8 4.6 7.0 na 5.0 8.5h

5.5 6.8 8.5 na 6.0 0.011k

10 11 11 na 10 1.6l

a

Human prostate cancer. bHuman cervix carcinoma. cHuman hepatocarcinoma. dHuman breast carcinoma. eHuman large cell lung carcinoma. Human gastric carcinoma. gHuman pancreatic cancer. Positive controls used. hFluorouracil. iPaclitaxel. jCisplatin. kDoxorubicin. lGemcitabine. n.a.: no activity. f

allowed the assignment of S-configurations at C-11, C-15, and C-18. On the basis of the above evidence, the structure of 1 was determined and the trivial name sumalarin A was assigned to this compound. Compound 2 was assigned the molecular formula C25H32O11S (10 unsaturations), having one C5H6O3 unit more than 1, on the basis of HRESIMS data. The 1H NMR spectrum for 2 indicated that some impurities are present, and attempts to further purify this compound by HPLC and different CC steps using various solvent systems failed. Therefore, structure elucidation was carried out on the mixture, with a focus on the major component. Detailed comparison of the NMR data of 2 with that of 1 suggested that compound 2 had the same basic structure as 1. The NMR spectroscopic data (Table 1) differed from that of 1, mainly in the absence of the methoxy signal at δH 3.69 and δC 52.3 (C-20). Instead, two ester carbonyl signals at δC 172.0/171.4 (C-22/C-24), two methyl signals at δH 1.96/2.00 and δC 20.9/20.5 (C-23/C-25), as well as two methylene signals at δH 4.24 and 4.44/4.15 and δC 67.5/66.8(C-20/21), were observed in the NMR spectra of 2. The replacement of the methoxy at C-19 in 1 by an acetoxyethoxy in 2 was evidenced by the observed cross-peak from H-20 to H-21 in the COSY spectrum as well as by the correlation from H-21 to C-22 in the HMBC spectrum (SI Figure S1). The HMBC correlation from H-20 to C-19 placed the acetoxyethoxy group at C-19. As in the case of 1, the relative configuration for compound 2 could not be solved either by J-coupling constants or by a NOESY experiment, but from a biogenetic point of view it was tentatively assigned the same relative configuration as that of 1. The structure of 2 was assigned the trivial name sumalarin B. As might be expected, the CD spectrum of compound 2 was in full agreement with that of 1, which exhibited positive Cotton effects (CEs) at approximately 276 and 306 nm and negative CEs at 229 and 333 nm. Therefore, the absolute configuration of compound 2 was also assigned as 11S, 15S, and 18S. Compound 3 was assigned the molecular formula C19H24O8S (eight unsaturations), having one CH2 unit less than 1, on the basis of positive HRESIMS data. Its NMR spectroscopic data were very similar to 1. However, the signals of the methoxy group resonating at δH 3.69 (H-20) and δC 52.3 (C-20) in the NMR spectra of 1 were absent in that of 3, suggesting the replacement of an OCH3 in 1 by an OH in 3. This observation was further supported by the COSY and HMBC correlations (SI Figure S1). Compound 3 had an almost identical CD spectrum to those of 1 and 2. Consequently, the absolute configurations at C-11, C-15, and C-18 were all assigned as S. It should be mentioned that a compound with the same planar structure as 3 has the CAS Registry Number 1235379-39-1. This compound was very recently listed as being part of a

commercial compound library, but no stereostructure or spectroscopic data and source information were reported. We thus describe compound 3 as a naturally occurring product and name it as sumalarin C. A question immediately arises as to whether compounds 1 and 2 occurred naturally or were artifacts of the isolation procedures. Compounds 1 and 2, as well as 3 and 4, could be detected in the initial culture extract of the fungus by HPLC analysis (SI), suggesting that 1 and 2 were not formed during the chromatography steps. In addition, compound 3 did not degrade after stirring for two days at room temperature in EtOAc (0.6 mg of 3 dissolved in 1.0 mL EtOAc and mixed with 0.4 g Si gel). Although an artifact nature cannot be completely ruled out, this evidence supports a natural source for 1 and 2. In addition to compounds 1−3, the known curvularin (4),11 dehydrocurvularin (5),4 and curvularin-7-O-β-D-glucopyranoside (6)13 were also isolated and identified from the fungal strain P. sumatrense MA-92. Compounds 1 and 2 can be derived from 3 by esterification or esterification and acylation. Compound 3 is likely produced via Michael addition of the cysteine metabolite 3-mercaptolactate to the double bond of dehydrocurvularin (5, SI Figure S2)14 and thus is reminiscent of glutathione detoxification of Michael acceptors in mammalian metabolism. A similar condensation of mercaptolactate with a cyclohexenone was recently observed for the first time during the biotransformation of Sch-642305 (the 10-membered lactone) catalyzed by Aspergillus niger, which resulted in the formation of two unexpected sulfur-containing products14 and suggested that maybe some fungi can use mercaptolactate for detoxification reactions. Compounds 1−5 were assayed for their cytotoxic activities against seven tumor cell lines including Du145, HeLa, Huh 7, MCF-7, NCI-H460, SGC-7901, and SW1990 (Table 2). Compounds 1−3 and 5 displayed cytotoxic activities against each of the tested cell lines, with IC50 values ranging from 3.8 to 10 μM. In contrast, curvularin (4) was inactive toward all cell lines in our experiments. These data indicated that the sulfursubstitution at C-11 significantly increased the cytotoxic activities of the curvularin derivatives (1−3 vs 4), while for the curvularins lacking sulfur-substitution, the double bond at C-10 is essential for the cytotoxicity (5 vs 4).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined with an SGW X-4 micromelting-point apparatus. Optical rotations were measured on an Optical Activity AA-55 polarimeter. UV spectra were measured on a PuXi TU-1810 UV−visible spectrophotometer. CD spectra were acquired on a Chirascan spectropolarimeter. One-dimensional and two-dimensional NMR spectra were recorded at 500 and 125 MHz for 1H and 13C, 2147

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121.1 (C, C-8), 112.7 (CH, C-4), 103.1 (CH, C-6), 73.9 (CH, C-15), 71.9 (CH, C-18), 52.9 (CH2, C-10), 41.7 (CH, C-11), 39.9 (CH2, C2), 35.9 (CH2, C-17), 33.5 (CH2, C-12), 33.0 (CH2, C-14), 23.9 (CH2, C-13), 21.8 (CH3, C-16); ESIMS m/z 411.1 [M − H]−, 823.2 [2 M − H]−; HRESIMS m/z 413.1247 [M + H]+ (calcd for C19H25O8S, 413.1265). Curvularin (4). [α]25 D −34.9 (c 0.63, MeOH). Literature value [α]D −28.6 (c 0.4, EtOH).11 Dehydrocurvularin (5). [α]25 D −53.3 (c 0.30, MeOH). Literature 4 value [α]20 D −65.9 (c 1.8, EtOH). X-ray Crystallographic Analysis of Compound 1.16 All crystallographic data were collected on a Bruker Smart-1000 CCD diffractometer equipped with a graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å) at 293(2) K. The data were corrected for absorption by using the program SADABS.17 The structure was solved by direct methods with the SHELXTL software package.18 All nonhydrogen atoms were refined anisotropically. The H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during the structure refinement. The structure was refined by full-matrix least-squares techniques.19 Crystal data for compound 1. C20H26O8S, FW = 426.47, orthorhombic space group P212121, unit cell dimensions a = 9.2491(7)Å, b = 14.3509(13) Å, c = 15.8699(13) Å, V = 2106.5(3) Å3, α = β = γ = 90°, Z = 4, dcalcd = 1.345 mg/m3, crystal dimensions 0.18 mm × 0.11 mm × 0.08 mm, μ = 0.197 mm−1, F(000) = 904. The 3430 measurements yielded 2463 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0683 and wR2 = 0.1389 [I > 2σ(I)]. The absolute structure parameter was 0.29(19). Cytotoxicity Assay. The cytotoxic activities of the isolated compounds against seven tumor cell lines, including Du145 (human prostate cancer cell line), HeLa (human cervix carcinoma cell line), Huh 7 (human hepatocarcinoma cell line), MCF-7 (human breast carcinoma cell line), NCI-H460 (human large cell lung carcinoma cell line), SGC-7901(human gastric carcinoma cell line), and SW1990 (human pancreatic cancer cell line), and were determined according to previously reported methods.20

respectively, on a Bruker Avance 500 MHz spectrometer with TMS as internal standard. Mass spectra were determined on a VG Autospec 3000 or an API QSTAR Pulsar 1 mass spectrometer. Analytical and semipreparative HPLC were performed using a Dionex HPLC system equipped with P680 pump, ASI-100 automated sample injector, and UVD340U multiple wavelength detector controlled by Chromeleon software (version 6.80). Commercial available Si gel (200−300 mesh, Qingdao Haiyang Chemical Co.), Lobar LiChroprep RP−18 (40−63 μm, Merck), and Sephadex LH−20 (Pharmacia) were used for open column chromatography. Solvents for extraction and purification were distilled prior to use. Fermentation, Extraction, and Isolation. The fungus P. sumatrense MA-92 was isolated from the rhizosphere of the marine mangrove plant L. racemosa collected at WenChang in Hainan Island, P. R. China, in July 2010, using the protocol as described in our previous report.15 The fresh mycelia of P. sumatrense MA-92 were grown on PDA medium at 28 °C for 4 days and were then inoculated into 60 × 1000 mL conical flasks on rice solid medium (70 g rice, 0.3 g peptone, 0.1 g corn syrup, and 100 mL naturally sourced and filtered seawater that was obtained from the Huiquan Gulf of the Yellow Sea near the campus of the author’s institution) for 30 days at room temperature. The fermented cultures were exhaustively extracted with EtOAc (150 mL/flask) for one day. The combined EtOAc solution was concentrated under reduced pressure to give an extract (50 g), which was fractionated by silica gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield 11 fractions (fractions 1−11) based on TLC analysis. Fraction 5 (3.1 g) was further purified by reversed-phase column chromatography (CC) over Lobar LiChroprep RP-18 with a MeOH−H2O gradient (from 20: 80 to 100:0) to afford 4 (220.0 mg). Further purification of fraction 8 (5.1 g) by CC over Lobar LiChroprep RP-18 with a MeOH−H2O gradient (from 20:80 to 100:0) afforded two subfractions: fractions 8-1 and 8-2. Fraction 8-1 (332.5 mg) was further purified by CC on silica gel eluting with a CHCl3−MeOH gradient (from 40:1 to 2:1) on Sephadex LH−20 (MeOH) and then by semipreparative HPLC (Elite ODS-BP column, 10 μm; 10.0 mm × 300 mm; 70% MeOH−H2O, 3 mL/min) to afford compounds 2 (10.3 mg, tR 13.0 min) and 3 (33.0 mg, tR 18.1 min). Fraction 8-2 (137.6 mg) was also purified by CC on silica gel eluting with a CHCl3−MeOH gradient (from 50:1 to 1:1) to obtain compounds 1 (30.0 mg) and 5 (23.0 mg). Purification of fraction 9 (3.3 g) by CC over silica gel with a CHCl3−MeOH gradient (from 50:1 to 1:1), on Sephadex LH-20 (MeOH) and then by prep TLC (plate, 20 cm × 20 cm; developing solvents, CHCl3−MeOH, 10:1) yielded compound 6 (4.7 mg). Sumalarin A (1). Colorless crystals (MeOH); mp 169−171 °C; [α]25 D −22.5 (c 0.78, MeOH); UV (MeOH) λmax (log ε) 201 (4.30), 274 (3.72), 304 (3.65) nm; CD (0.40 mM, MeOH) λmax (Δε) 229 (−11.4), 276 (+4.48), 306 (+4.46), 333 (−3.65) nm; 1H and 13C NMR data, see Table 1; ESI-MS m/z 427 [M + H]+, 449 [M + Na]+, 465 [M + K]+; HRESIMS m/z 427.1402 [M + H]+ (calcd for C20H27O8S, 427.1421). Sumalarin B (2). Yellow oil; [α]25 D −5.55 (c 0.36, MeOH); UV (MeOH) λmax (log ε) 200 (4.31), 274 (3.78), 304 (3.70) nm; CD (0.54 mM, MeOH) λmax (Δε) 229 (−8.64), 275 (+3.27), 306 (+3.78), 334 (−3.03) nm; 1H and 13C NMR data, see Table 1; ESIMS m/z 541 [M + H]+; HRESIMS m/z 541.1792 [M + H]+ (calcd for C25H33O11S, 541.1744). Sumalarin C (3). Colorless oil; [α]25 D −6.49 (c 0.55, MeOH); UV (MeOH) λmax (log ε) 200 (4.30), 274 (3.80), 305 (3.72) nm; CD (0.44 mM, MeOH) λmax (Δε) 229 (−10.65), 277 (+4.15), 306 (+4.87), 334 (−3.51) nm; 1H NMR (500 MHz, acetone-d6) δH: 6.43 (1H, d, J = 1.9 Hz, H-6), 6.34 (1H, d, J = 1.9 Hz, H‑4), 4.82 (1H, m, H15), 4.37 (1H, t, J = 4.7 Hz, H-18), 3.73 (2H, br m, H-2), 3.71 (1H, m, Hb-10), 3.40 (1H, br m, H-11), 3.01 (1H, dd, J = 13.6, 4.7 Hz, Hb-17), 2.92 (1H, dd, J = 13.6, 4.7 Hz, Ha-17), 2.77 (1H, br m, Ha-10), 1.74 (1H, br m, Hb-13), 1.59 (1H, br m, Hb-12), 1.52 (2H, m, H-14), 1.47 (1H, br m, Ha-12), 1.28 (1H, br m, Ha-13), 1.10 (3H, d, J = 6.0 Hz, H16); 13C NMR (125 MHz, acetone-d6) δC: 204.4 (C, C-9), 174.8 (C, C-19), 171.4 (C, C-1), 160.9 (C, C-7), 158.6 (C, C-5), 137.7 (C, C-3),

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ASSOCIATED CONTENT

* Supporting Information S

Key HMBC and COSY correlations, possible pathway, and selected 1D and 2D NMR as well as CD spectra of compounds 1−3 as well as HPLC profiles detecting new compounds 1−3 in the initial extract, and X-ray crystallographic file of compound 1 (CIF). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: +86-532-82898553. Fax: +86-532-82880645. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Ministry of Science and Technology of China (2013AA092901 and 2010CB833800) and from the Natural Science Foundation of China (31270403 and 30910103914) is gratefully acknowledged.

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REFERENCES

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