Thiodiketopiperazines from the Marine-Derived Fungus Phoma sp

Dec 26, 2013 - OUCMDZ-1847, associated with the mangrove plant Kandelia candel. The structures including the absolute configurations of the new ...
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Thiodiketopiperazines from the Marine-Derived Fungus Phoma sp. OUCMDZ-1847 Fandong Kong, Yi Wang, Peipei Liu, Tianhan Dong, and Weiming Zhu* Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *

ABSTRACT: Three new thiodiketopiperazines, named phomazines A−C (1−3), along with 10 known analogues (4−13), were isolated from the fermentation broth of an endophytic fungus, Phoma sp. OUCMDZ-1847, associated with the mangrove plant Kandelia candel. The structures including the absolute configurations of the new compounds were unambiguously elucidated by spectroscopic, X-ray crystallographic, and Mosher’s methods along with quantum ECD and 13 C NMR calculations. Compounds 2, 4, 5, 11, and 12 showed cytotoxicities against the HL-60, HCT-116, K562, MGC-803, and A549 cell lines with IC50 values in the range 0.05 to 8.5 μM.

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RESULTS AND DISCUSSION Phomazine A (1) was obtained as an amorphous powder and an orthorhombic crystal from MeOH−CHCl3 (1:1, v/v). The molecular formula of 1 was determined to be C19H18N2O3S from the HRESIMS peak at m/z 355.1112 [M + H]+. The IR spectrum of 1 showed the presence of an amide (3365 and 1696 cm−1) and a benzene group (1649 cm−1). Analysis of the 1 H, 13C, and HSQC NMR data of 1 (Table 1) revealed the presence of one singlet methyl (δC/H 13.9/2.16), one methylene, two O- or N-substituted methines (δC/H 74.3/ 4.64, 69.6/4.87), one heteroatom-bonded sp3 quaternary carbon (δC 73.7), eight olefinic/aromatic methines, four olefinic/aromatic quarternary carbons, and two amide carbonyls (δC 165.1, 163.0). Apart from the additional phenyl moiety and the absence of an N-methyl moiety, these data are very similar to those of bis(dethio)-10a-methylthio-3a-deoxy-3,3adidehydrogliotoxin (14),8 indicating 1 as a TDKP. In addition, the COSY cross-peaks from H-5a (δH 4.87) through H-6 (δH 4.64) to H-7 (δH 5.64) and then to H-8 (δH 5.91) and H-9 (δH 5.97), along with the HMBC correlations (Figure 1) from H210 (δH 3.20/2.86) to C-5a (δC 69.6), C-9 (δC 119.3), C-9a (δC 134.2), C-10a (δC 73.7), and C-1 (δC 165.1), from H-5a to C-6 (δC 74.3), C-7 (δC 130.8), C-9, C-9a, C-10 (δC 39.4), and C-4 (δC 163.0), and from the thiomethyl protons (δH 2.16) to C10a, further demonstrated the presence of the same 6−5−6 TDKP nucleus as in 14.8 The remaining eight downfield carbon signals accounted for a styryl moiety located at C-3, which was demonstrated by HMBC correlations from H-11 (δH 6.84) to C-3 (δC 127.5), C-4, C-12 (δC 133.7), and C-13/17 (δC 130.2) and COSY data of H-13/17 (δH 7.60) to H-15 (δH 7.35) through H-14/16 (δH 7.43) (Figure 1). These data indicated

etabolites of marine organisms, especially marine fungi, have been a popular area of research for many years, and numerous structurally unique and biologically active natural products have been identified.1,2 Thiodiketopiperazines (TDKPs) such as the rostratins,3 epicoccins,4,5 epicorazine,6 emethacins,7 and gliotoxin and its analogues8 are an important class of compounds frequently isolated from fungi. Many TDKPs have shown antiproliferative,6 cytotoxic,3,8−10 antibacterial,11 and histone methyltransferase inhibitory activities.8 As a continuation of our previous investigations aimed at characterizing new and cytotoxic secondary metabolites of marine fungal origin,12−14 the endophytic fungus Phoma sp. OUCMDZ-1847 was isolated and identified from the fruit of the mangrove plant Kandelia candel (Rhizophoraceae) from Wenchan, Hainan Province, China. The EtOAc extract of the fermentation broth showed significant cytotoxicity against the HCT-116 cell line at 100 μg/mL. Chemical investigation led to the identification of three new TDPKs that we named phomazines A−C (1−3) as well as 10 known analogues, epicorazine A (4),15 epicorazine B (5),16 epicoccin D (6),4 epicoccin A (7),4 epicoccin C (8),4 epicoccin E (9),17 epicoccin B (10),4 epicorazine C (11),6 exserohilone A (12),18 and rostratin A (13)3 (Tables S2 and S3, Supporting Information). Compounds 2, 4, 5, 11, and 12 exhibited moderate to potent cytotoxicity against the HL-60, HCT-116, K562, MGC-803, and A549 cell lines. Although TDKPs are common, compounds biosynthesized from two phenylalanine residues with only one oxidized phenyl nucleus like 1 and 2 have not been reported. Herein we reported details of the isolation, structure elucidation, and bioactivities against human cancer cells of these compounds. © 2013 American Chemical Society and American Society of Pharmacognosy

Received: October 1, 2013 Published: December 26, 2013 132

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that the additional phenyl is located at C-11. The relative configuration of 1 was determined to be 5a,6-trans, and the thiomethyl group and H-6 are on the same face of the molecule by analysis of the NOESY data, as shown in Figure 2. This was further confirmed by single-crystal X-ray crystallographic analysis (Figure 3). The X-ray data also established the Zgeometry of the Δ3(11)-double bond. Although a sulfur atom was present in compound 1, the value of the Flack parameter, 0.10(12), was relatively too large to determine the absolute configuration. Thus, the modified Mosher’s method19 was used to determine the absolute configuration of C-6. Compound 1 was separately esterified with (S)- and (R)-MTPA chloride to give the (R)- and (S)-MTPA esters, 1a and 1b, respectively. The distribution of Δδ values between 1b and 1a indicated the 6S-configuration (Figure 4). As a consequence, C-5a and C-10a were assigned as the S- and R-configurations, respectively. Phomazine A (1) was thus identified as (3Z,5aS,6S,10aR)bis(dethio)-3a-deoxy-2-demethyl-10a-methylthio-3a-phenyl3,3a-didehydrogliotoxin. Phomazine B (2) was isolated as a brown oil, and its molecular formula was established as C20H22N2O3S2 by an HRESIMS peak at m/z 403.1151 [M + H]+, equivalent to 1 with an additional CH4S unit. The IR spectrum (3225, 1678, and 1641 cm−1) and NMR data of 2 were similar to those of 1, except that a thiomethyl signal (δC/H 14.1/2.34), a methylene signal (δC/H 45.5/3.49, 3.09), and a heteroatom-bonded quaternary carbon signal (δC 68.2) replace the corresponding trisubstituted ethylene signals (δC/H 118.0/6.84, 127.5) (Table 1). This deduction was further evidenced by HMBC Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Compounds 1−3 in DMSO-d6 1 position

δC

2 δH (J in Hz)

δC

1 (1′) 2 (2′) 3 (3′)

165.1, C

165.3, C

127.5, C

68.2, C

4 (4′) 5 (5′) 5a 6 (6′)

163.0, C

167.8, C

7 (7′) 8 (8′) 9 (9′) 9a 10

130.8, 124.0, 119.3, 134.2, 39.4,

10a 11

73.7, C 118.0, CH

6.84, s

12 13/17 14/16 15 10a-SCH3 3-SCH3 6-OH 7 (7′)-OH 8 (8′)-OH NH

133.7, 130.2, 129.3, 129.1, 13.9,

7.60, 7.43, 7.35, 2.16,

69.6, CH 74.3, CH CH CH CH C CH2

C CH CH CH CH3

4.87, d (13.4) 4.64, d (13.4) 5.64, d (9.8) 5.91, m 5.97, m α 3.20, d (15.7) β 2.86, d (15.7)

d (7.6) dd (7.6, 7.8) dd (7.6, 7.8) s

δH (J in Hz)

46.9, CH 209.0, C

CH CH CH C CH2

72.4, C 45.5, CH2 135.0, 131.0, 128.6, 127.9, 14.8, 14.1,

δC 162.7, C 76.6, C 32.8, CH2

69.0, CH 73.9, CH 131.1, 123.9, 120.4, 132.9, 38.3,

3

C CH CH CH CH3 CH3

6.17, s

4.32, d (13.7) 4.64, d (13.7)

44.2, CH2

5.60, d (9.4) 5.81, m 5.84, m

66.2, CH 66.5, CH 64.1, CH

δH (J in Hz)

2.96, dd (14,7, 8.3) 2.67, dd (14.7, 2.6) 3.13, ddd (8.3, 7.5, 2.6)

α 2.49, dd (16.3, 4.4) β 2.65, dd (16.3, 10.3) 3.65, m 4.71, dd (4.5, 4.1) 4.37, dd (7.5, 4.5)

α 2.71, d (15.3) β 1.64, d (15.3) 3.49 (d, 13.0) 3.09 (d, 13.0) 7.18 (d, 6.9) 7.27, dd (6.9, 7.4) 7.21, m 2.13, s 2.34, s 5.52, d (1.1) 5.16, br s 5.46, d (4.3) 9.16, s

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Figure 1. Key COSY and HMBC correlations of compounds 1−3.

Figure 2. Key NOESY correlations of compounds 1−3.

values between 2b and 2a indicated the 6S-configuration (Figure 4). Thus, the absolute configuration of compound 2 was determined to be 3R, 5aS, 6S, and 10aR. The structure of phomazine B (2) was therefore identified as (3R)-thiomethyl3,11-dihydrophomazine A. Phomazine B can be regarded as the phenyl-substituted derivative of bis(dethio)bis(methylthio)gliotoxin8 in which the 11-hydroxy (or 3a-hydroxy) was replaced by 11-phenyl. Phomazine C (3) was isolated as a white, amorphous powder. The molecular formula of 3 was determined to be C18H20N2O8S2 by HRESIMS, indicating 10 degrees of unsaturation. The similar IR (3225 and 1678 cm−1) and UV (λmax 205 nm) absorptions to compound 11 suggested that 3 is also a TDKP. The 1H and 13C NMR data (Table 1) displayed only half of the proton and carbon signals, which are two methylenes, four methines including three oxygenated or nitrogenated ones, an sp3 heteroatom-bonded quaternary carbon, an amide carbonyl carbon, and a carbonyl carbon, suggesting a symmetrical feature for 3. These NMR data were identical to those of the right half moiety (C-1′ to C-9′) of 11, indicating the double bond in 11 was hydrated to afford a CH2CH(OH) moiety with a β-oriented hydroxy (δH 5.16). This deduction was further supported by the key HMBC correlations from OH-7(7′) (δH 5.16) to C-7(7′) (δC 66.2), C-6(6′) (δC 44.2), and C-8(8′) (δC 66.5), along with the COSY data of H2-6(6′)/H-7(7′)/H-8(8′)/H-9(9′)/H-4(4′)/H23(3′), OH-7(7′)/H-7(7′), and OH-6(6′)/H-6(6′) (Figure 1). In addition, the key HMBC connectivities of H-9 (δH 4.37) to C-1′ (δC 162.7) and H-9′ (δH 4.37) to C-1 (δC 162.7) confirmed the symmetrical structure of 3. Cis-fused rings A and B could be deduced from the J value (7.5 Hz) between H-4(4′) and H-9(9′),3 and the large J value (10.3 Hz) between H-6(6′) (δH 2.65) and H-7(7′) must result from the coupling of two axial protons, assigning an α-orientation for H-7(7′). For the same reason, small J values of H-8(8′)/H-7(7′) (4.1 Hz) and H-8(8′)/H-9(9′) (4.5 Hz) indicated an α-oriented equatorial proton for H-8(8′). Furthermore, compound 3 showed the key NOESY correlations of H-4(4′)/Hβ-6(6′)/H-9(9′). These data revealed that H-4, HO-7, HO-8, and H-9 all are in the cisconfiguration relative to one another. The similarity of C-1′ to C-9′ of the 13C NMR shifts between 3 and 11 indicates the anti-orientation of the epithio group that gives the smallest

Figure 3. Molecular plot of compound 1 with the thermal ellipsoids at the 30% probability level. Only atom C10B of the disordered methyl group is represented for clarify.

Figure 4. Δδ (=δS − δR) values for (S)- and (R)-MTPA esters of 1 and 2.

correlations (Figure 1) from H2-11 (δH 3.49/3.09) and thiomethyl protons (δH 2.34) to C-3 (δC 68.2), from H2-11 to C-4 (δC 167.8), C-12 (δC 135.0), and C-13/17 (δC 131.0), and from H-13/17 (δH 7.18) to C-11 (δC 45.5). These data implied that compound 2 is the MeSH addition product of 1. The large J value (13.7 Hz) between H-5a (δH 4.32) and H-6 (δH 4.64) must be the result of the coupling of two axial protons, indicating the trans-orientation of H-5a and H-6. The NOESY connections (Figure 2) of 10a-SMe (δH 2.13) to H-6 and H-5a to H-13/17 indicated cis- and trans-orientations between the two thiomethyls and the thiomethyls with H-5a, respectively. That is to say that compound 2 has the same relative configuration as 1 except for the additional trans thiomethyl with H-5a. When reacted with (S)- and (R)-MTPA chloride, compound 2 gave the corresponding (R)- and (S)MTPA esters (2a and 2b), respectively. The distribution of Δδ 134

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Figure 5. Measured CD curves of 1−3 and 11 (a, b) and calculated ECD curves of 3 and ent-3 (c).

MGC-803, and A549 cell lines and 2 and 12 against the five human tumor cell lines are reported here for the first time. These results suggest that the α,β-unsaturated ketone moiety is required for cytotoxicity, which might serve as an attractive moiety for the development of potent thiodiketopiperazinoid anticancer agents.

steric interactions. This deduction was supported by 13C NMR chemical shift calculations of 3, 8(8′)-diepi-3, and 2(2′)-diepi-3. The results showed that structure 3 displayed the smallest relative errors for all nine carbons (≤2.1 ppm) among the three possible stereoisomers (Table S1, Supporting Information). The signs of the CD Cotton effects can be used to predict the absolute configuration of C-2 (2′), respectively.6,15,20,21 The similarity of the CD curve (Figure 5b) of 3 with that of 11 at 236 (Δε −1.7) and 266 (Δε +0.5) nm indicated the same Rconfigurations of C-2 (2′). This assignment was further confirmed by ECD calculations of 3 and ent-3 using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-31G(d) level.22 The preliminary conformational distribution search was performed by HyperChem 7.5 software. The corresponding minimum geometries were further fully optimized by using DFT at the B3LYP/6-31G(d) level as implemented in the Gaussian 03 program package. The ECD and 13C NMR chemical shift calculations were performed after optimization of the selected conformers at the B3LYP/631G(d) and B3LYP/6-311++G(2d,p)//B3LYP/6-31G(d) levels, respectively.23 The results showed that the measured CD curve matched well with the calculated ECD for 3 and was opposite that of ent-3 (Figure 5c), indicating the 2(2′)R,4(4′) R,7(7′)S,8(8′)R,9(9′)S-configuration. Thus, compound 3 was identified as (2(2′)R,4(4′)R,7(7′)S,8(8′)R,9(9′)S)-4-epi-7-hydroxy-6,7-dihydroepicorazine C. The cytotoxic activities of compounds 1−13 (Table 2) were examined against the K562, HL-60, and HCT-116 tumor cell

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Table 2. Cytotoxicities against Tumor Cells for 2, 4, 5, 11, and 12 (IC50, μM) HL-60 HCT-116 A549 K562 MGC-803

2

4

5

11

12

adriamycin

>10 >10 >10 >10 8.5

0.05 0.33 2.3 1.5 2.7

1.6 >10 >10 4.6 5.2

3.5 7.1 >10 >10 3.4

3.5 4.0 >10 >10 4.2

0.02 0.21 0.16 0.25 0.17

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter, and UV spectra were measured on a Beckman DU 640 spectrophotometer. CD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were taken on a Nicolet Nexus 470 spectrophotometer as KBr discs. 1 H NMR, 13C NMR, DEPT, HMQC, HMBC, COSY, NOESY, and ROESY spectra were acquired using a JEOL JNM-ECP 600 spectrometer or a Bruker Avance 500 spectrometer using TMS as an internal standard or residual solvent signals for referencing (DMSO δH/C 2.50/39.5). HRESIMS spectra were determined using the QTOF ULTIMA GLOBAL GAA076 LC mass spectrometer. Semipreparative HPLC was carried out using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min) and a πNAP column (COSMOSIL-pack, 10 × 250 mm, 5 μm, 4 mL/min). Sea salt used was made from the evaporation of seawater collected in Laizhou Bay (Weifang Haisheng Chemical Factory). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Biosciences), respectively. Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory). Fungal Material and Fermentation. The fungus Phoma sp. OUCMDZ-1847 was isolated from a mangrove fruit sample of Kandelia candel that was collected from Wenchang, Hainan Province, China, in August 2010. After it was ground into a powder, the fruit sample (1 g) was diluted to 10−2 g/mL with sterile water, 100 μL of which was deposited on a PDA (200 g potato, 20 g glucose, 20 g agar per liter of tap water) plate containing chloramphenicol (100 μg/mL) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified according to its morphological characteristics and 18S rRNA gene sequences (GenBank accession no. KF611891, Supporting Information). A reference culture of Phoma sp. OUCMDZ-1847 is maintained at −80 °C, and the voucher specimen of K. candel (no. KC20100821) is deposited in our laboratory. The isolate was cultured on slants of PDA medium at 28 °C for 5 days. Plugs of agar supporting mycelium growth were cut and transferred aseptically to 200 × 1000 mL Erlenmeyer flasks each containing 300 mL of liquid medium (20 g mannitol, 20 g maltose, 10 g glucose, 10 g monosodium glutamate, 3 g yeast extract, 0.5 g corn meal, 0.5 g KH2PO4, 0.3 g MgSO4, 17.5 g Na2HPO4·2H2O, 10.5 g C4H2O7·H2O, 33 g sea salt per liter of tap water, pH 7.0). The flask was incubated at room temperature under static conditions for 30 days. Extraction and Isolation. The cultures (60 L) were filtered through cheesecloth to separate the mycelial mass from the aqueous layer. The filtrate was then extracted three times by shaking with 3-fold

lines by the MTT method24 and against the A549 and MGC803 tumor cell lines by the SRB method.25 Adriamycin was used as the positive control. Compounds 2, 4, 5, 11, and 12 showed moderate to potent cytotoxicities against the HL-60, HCT-116, K562, MGC-803, and A549 cell lines, and compound 4 exhibited the most potent activity, whereas compounds 1, 3, 6−10, and 13 were inactive against the five tested cell lines. Although the cytotoxicities of 4, 5, and 11 against HeLa and K562 cell lines were reported previously,6 the cytotoxicities of 4, 5, and 11 against the HL-60, HCT-116, 135

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6.15 (m, 1H, H-8), 6.10 (m, 1H, H-9), 5.63 (d, J = 10.2 Hz, 1H, H-7), 5.25 (d, J = 13.8 Hz, 1H, H-5a), 3.30 (d, J = 15.6 Hz,1H, Ha-10), 2.83 (d, J = 15.6 Hz,1H, Hb-10), 2.27 (s, 3H, -SCH3); ESIMS m/z 571.1 [M + H]+. Compound 2a: 1H NMR (DMSO-d6, 600 MHz) δ 9.08 (s,1H, NH) 7.26 (d, J = 7.8 Hz, 2H, H-14/16), 7.22 (d, J = 7.8 Hz, 1H, H15), 7.16 (t, J = 7.8 Hz, 2H, H-13/17), 6.20 (d, J = 14.4 Hz, 1H, H11), 6.00 (m, 1H, H-8), 5.92 (m, 1H, H-9), 5.33 (d, J = 10.2 Hz, 1H, H-7), 4.89 (d, J = 14.4 Hz, 1H, H-5a), 3.46 (d, J = 12.6 Hz, 1H, Ha11), 3.00 (d, J = 12.6 Hz, 1H, Hb-11), 2.62 (d, J = 15.0 Hz, 1H, Ha10), 2.25 (s, 3H, 3-SCH3), 2.18 (s, 3H, 10a-SCH3), 1.22 (d, J = 15.0 Hz, 1H, Hb-10); ESIMS m/z 619.1 [M + H]+. Compound 2b: 1H NMR (DMSO-d6, 600 MHz) δ 9.05 (s, 1H, NH) 7.26 (d, J = 7.8 Hz, 2H, H-14/16), 7.22 (d, J = 7.8 Hz, 1H, H15), 7.13 (t, J = 7.8 Hz, 2H, H-13/17), 6.23 (d, J = 13.8 Hz, 1H, H11), 6.06 (m, 1H, H-8), 5.94 (m, 1H, H-9), 5.57 (d, J = 9.6 Hz, 1H, H7), 4.88 (d, J = 14.4 Hz, 1H, H-5a), 3.40 (d, J = 12.6 Hz, 1H, Ha-11), 2.96 (d, J = 12.6 Hz, 1H, Hb-11), 2.62 (d, J = 15.0 Hz, 1H, Ha-10), 2.26 (s, 3H, 3-SCH3), 2.18 (s, 3H, 10a-SCH3), 1.21 (d, J = 15.0 Hz, 1H, Hb-10); ESIMS m/z 619.1 [M + H]+. X-ray Crystal Data for Phomazine A (1). Compound 1 was obtained as a colorless orthorhombic crystal from MeOH: molecular formula C19H18N2O3S·CH3OH; space group P212121 with a = 10.5264(11) Å, b = 10.5286(12) Å, c = 17.9956(18) Å, V = 1994.4(4) Å3, Z = 4, Dcalcd = 1.287 mg/m3, μ = 0.190 mm−1, and F(000) = 816; crystal size 0.40 × 0.21 × 0.18 mm3. T = 298(2) K. A total of 10 052 unique reflections (2θ < 50°) were collected on a Bruker Smart CCD area detector diffractometer with graphitemonochromated Mo Kα radiation (λ = 0.71073 Å). The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of full-matrix leastsquares refinement was based on 3506 unique reflections (2θ < 50°) and 257 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0507 and wR2 = 0.0992 for I > 2σ(I) data. Disorder was found in the methyl group on the sulfur atom (C12B and C12A in 68:32 ratio). Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 921218. Copies of the data can be obtained, free of charge, on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax, +44 (0)-1223-336033; e-mail, [email protected]).

volumes of EtOAc, while the mycelium was extracted by acetone. After removing acetone by evaporation under vacuum, the obtained aqueous acetone solution was extracted three times with equal volumes of EtOAc. The combined EtOAc extracts were dried under vacuum to produce 30.3 g of extract. The EtOAc extract was subjected to a silica gel VLC column, eluting with a stepwise gradient of 0%, 20%, 40%, 60%, 80%, and 100% MeOH in CH2Cl2 (v/v), to give 12 fraction. Fraction 9 (306 mg) was subjected to Sephadex LH-20 chromatography (10 × 400 mm) and then further purified by HPLC over ODS (60% MeOH−H2O, v/v) to give compound 7 (tR 9.0 min; 30 mg). Fraction 8 (453 mg) was subjected to HPLC over ODS (50% MeOH−H2O, v/v) to yield 6 (tR 10.4 min, 16.9 mg). Fraction 11 was separated by Sephadex LH-20 column (20 × 800 mm), eluting with MeOH, to afford four subfractions. Subfraction 11-1 (16.6 mg) was further purified by HPLC over a πNAP column (60% MeOH−H2O, v/v) to afford 13 (tR 6.6 min, 8.3 mg). Subfraction 11-3 (34.5 mg) was subjected to HPLC over a πNAP column (40% MeOH−H2O, v/v) to yield 8 (tR 22.4 min, 15.3 mg) and 9 (tR 8.9 min, 5.8 mg). Fractions 5− 7 were combined and rechromatographed by Sephadex LH-20 (20 × 800 mm, MeOH) to afford four subfractions (Fr. 5-1−Fr. 5-4). Subfraction 5-2 (43.1 mg) was further purified by HPLC on ODS (60% MeOH−H2O, v/v) to yield 3 (tR 5.4 min, 3.6 mg), 4 (tR 6.1 min, 6.9 mg), and 12 (tR 8.1 min, 5.0 mg). Subfraction 5-3 (27 mg) was subjected to HPLC separation over ODS (40% MeOH−H2O, v/v) to yield 11 (tR 12.1 min, 12.3 mg). Recrystallization of subfraction 5-1 (18 mg) from MeOH gave 5 (9.5 mg). Subfraction 5-4 (87 mg) was separated by Sephadex LH-20 (10 × 400 mm) eluting with MeOH to afford three subfractions (Fr. 5-4-1−Fr. 5-4-3). Subfraction 5-4-3 (45 mg) was further purified by HPLC over a πNAP column (60% MeOH−H2O, v/v) to yield 1 (tR 17.6 min, 15.6 mg) and 2 (tR 23.1 min, 16.8 mg). Compound 10 (7.9 mg) was crystallized from a MeOH solution of subfraction 5-4-1 (19 mg). Phomazine A (1): colorless, orthorhombic crystal (MeOH); mp 176−177 °C; [α]25D +190 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 213 (3.87) and 308 (3.98) nm; CD (c 0.1, MeOH) λmax (Δε) 206 (+2.4), 224 (−3.0), and 283 (+7.2) nm; IR (KBr) νmax 3365, 1696, 1617,1431, 1375 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 355.1112 [M + H]+ (calcd for C19H19N2O3S, 355.1116). Phomazine B (2): brown oil; [α]25D +28 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.57), 213 (3.86), and 215 (3.82) nm; CD (c 0.1, MeOH) λmax (Δε) 204 (−1.6), 224 (−5.9), and 283 (+0.9) nm; IR (KBr) νmax 3365, 1678, 1641, 1411 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 403.1151 [M + H]+ (calcd for C20H23N2O3S2, 403.1150). Phomazine C (3): white, amorphous powder; [α]25D −58 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (3.45), 212 (3.58); CD (c 0.1, MeOH) λmax (Δε) 236 (−1.7), 266 (+0.5), and 298 (−0.06) nm; IR (KBr) νmax 3439, 2925, 1687, 1376, 1077 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 479.0547 [M + Na]+ (calcd for C18H20N2O8S2Na, 479.0559). Preparation of MTPA Esters of 1a and 1b. Compound 1 (1 mg for each) was reacted with either R-(−)- or S-(+)-MTPA chloride (10 μL) in 500 μL of anhydrous pyridine for 6 h. The reaction mixture was quenched with H2O and extracted with CH2Cl2 three times. The organic layers were combined and separated by semipreparative HPLC on ODS (80% MeOH−H2O, v/v) to afford the S-MTPA ester 1a (1.2 mg, tR 10.2 min) and R-MTPA ester 1b (0.8 mg, tR 10.3 min), respectively. By the same procedures, the S-MTPA ester 2a (0.9 mg, tR 14.4 min) and R-MTPA ester 2b (1.1 mg, tR 14.7 min) were prepared from compound 2 (1 mg for each), respectively. Compound 1a: 1H NMR (DMSO-d6, 600 MHz) δ 7.46 (d, J = 7.8 Hz, 2H, H-13/17), 7.44 (t, J = 7.8 Hz, 2H, H-14/16), 7.34 (d, J = 7.8 Hz, 1H, H-15), 6.58 (s, 1H, H-11), 6.15 (d, J = 14.4 Hz, 1H, H-6), 6.09 (m, 1H, H-8), 6.08 (m, 1H, H-9), 5.39 (d, J = 9.0 Hz, 1H, H-7), 5.28 (d, J = 15.0 Hz, 1H, H-5a), 3.30 (d, J = 15.0 Hz,1H, Ha-10), 2.84 (d, J = 15.0 Hz, 1H, Hb-10), 2.25 (s, 3H, -SCH3); ESIMS m/z 571.1 [M + H]+. Compound 1b: 1H NMR (DMSO-d6, 600 MHz) δ 7.46 (d, J = 7.8 Hz, 2H, H-13/17), 7.42 (t, J = 7.8 Hz, 2H, H-14/16), 7.34 (d, J = 7.8 Hz, 1H, H-15), 6.34 (s, 1H, H-11), 6.21 (d, J = 14.4 Hz, 1H, H-6),

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ASSOCIATED CONTENT

S Supporting Information *

Bioassay protocols used, the NMR spectra of compounds 1− 13, and the18S rRNA gene sequences of Phoma sp. OUCMDZ1847. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-532-82031268. Fax: +86-532-82031268. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from NSFC (Nos. 21172204, 41376148, and 81373298), from 973 and 863 programs of China (Nos. 2010CB833804 and 2012AA092104), and from the Special Fund for Marine Scientific Research in the Public Interest of China (No. 2010418022-3).

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