Campechic Acids A and B: Anti-invasive ... - American Chemical Society

Mar 4, 2014 - Unamuno, 37007 Salamanca, Spain. §. Department of Bioscience, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyam...
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Campechic Acids A and B: Anti-invasive Polyether Polyketides from a Soil-Derived Streptomyces Linkai Yu,† Martha E. Trujillo,‡ Satoshi Miyanaga,§ Ikuo Saiki,§ and Yasuhiro Igarashi*,† †

Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan ‡ Departmento de Microbiologia y Genetica, Edificio Departamental Lab, Universidad de Salamanca, 205, Campus Miguel de Unamuno, 37007 Salamanca, Spain § Department of Bioscience, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan S Supporting Information *

ABSTRACT: Campechic acids A (1) and B (2), two new polyketides, were isolated from the culture extract of Streptomyces sp., and their structures were determined by NMR and MS spectroscopic analysis. Campechic acids are polyether-polyketides functionalized by two tetrahydrofuran rings, an enolized 1,3-diketone, and multiple methyl substitutions. Absolute configuration of nine stereogenic centers in 1, except for four chiral centers in the cyclic ether moieties, was determined by the 1H NMR anisotropy method in combination with chemical degradation. Campechic acids exhibited potent inhibitory effects on tumor cell invasion with IC50 values in the nanomolar to submicromolar range.

A

extract, resulting in the isolation of campechic acids A (1) and B (2), two new members of the polyether class of polyketides. Herein, we report the isolation, structure determination, and biological activity of 1 and 2.

ctinomycetes, saprophytic Gram-positive bacteria residing widely in nature from terrestrial to marine environments, play a pivotal role in the production of bioactive secondary metabolites useful for pharmacological application.1 About 40% of the known bioactive compounds of microbial origin are derived from actinomycetes.2 Among this promising taxon, members of Streptomyces are recognized as the most commercially and medicinally important producers, as they alone account for 75% of total bioactive actinomycete metabolites reported.3 Owing to this unparalleled ability in secondary metabolism, significant efforts are still being directed toward the chemistry and biology of Streptomyces.4 Metastasis is the process by which a tumor cell leaves the primary tumor, disseminates via the blood or lymphatic vessels, and grows at a distant site to establish a secondary tumor.5 During this process, translocation of tumor cells across extracellular barriers, namely, invasion, is a crucial step for tumor cells to accomplish the metastasis. Since metastasis is the leading cause of treatment failure and mortality in cancer patients, clinical efficacy of invasive inhibitors has been extensively exploited in cancer chemotherapy.6 As a part of our continuing search for novel bioactive compounds with therapeutic potential, Streptomyces sp. CHI93 was found to produce unknown metabolites displaying UV absorption maximum at 278 nm that were not present in our in-house metabolite database. As these metabolites also exhibited inhibitory activity in a tumor cell invasion assay, HPLC/UV-guided purification was conducted from the culture © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The producing strain CHI93 was isolated from a rock soil sample collected in Campeche, Mexico. The isolate was identified as Streptomyces by phylogenetic analysis based on the 16S rRNA genes. This strain was cultured in A-16 medium, and the whole culture broth was extracted with 1-butanol. The crude extract was subjected to silica gel column chromatography, followed by HPLC purification and subsequent LH-20 gel filtration, yielding 1 (18.2 mg) and 2 (2.9 mg) from 2 L of culture. Campechic acid A (1) was obtained as an optically active, pale yellow oil. Analysis by HR-ESITOFMS gave a [M − H]− Received: December 19, 2013 Published: March 4, 2014 976

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Figure 1. COSY and key HMBC correlations for 1.

Scheme 1. Oxidative Degradation of 3 to 6 and 7

From the 1H−1H COSY spectrum, three isolated spin systems were deduced for the left half of 1 (substructure B): H29/H3-30, H-25/H2-26/H2-27, and a 10-carbon fragment from H-15 to H2-23 containing a double bond at C-16/C-17. HMBC correlations from a methyl H3-40 to C-27, C-28, and C-29 connected these carbons as C-27/C-28/C-29 and placed a methyl group on C-28. Another methyl H3-39 showed correlations to C-23, C-24, and C-25, establishing the connectivity for C-23/C-24/C-25 and the methyl substitution at C-24. The carbon chemical shifts indicated the attachment of an oxygen atom to C-19, C-21, C-24, C-25, C-28, and C-29. Because two carbonyl carbons (C-1, C-9) and two double bonds (C-10/C-11, C-16/C-17) accounted for four of the six unsaturation degrees, two of the aforementioned six oxygenated carbons should have a hydroxy group, and the remaining four carbons could be connected by an oxygen bridge forming two ether rings. The positions of hydroxylated carbons were confirmed to be C-19 and C-29 by analyzing the COSY and HMBC spectra of bis-(R)-MTPA ester (4a) prepared from 3. The ether linkage between C-25 and C-28 was indicated by ROESY correlations for H3-40/H-25 and H-25/H3-39, and another ether linkage between C-21 and C-24 as well as a cis relative configuration of H3-40 to H-25 was established (Figure 2A). The geometry of the double bond between C-16 and C-17

peak at m/z 677.4996 corresponding to a molecular formula of C40H70O8 (Δ −0.2 mmu, calcd for C40H69O8) requiring six double bond equivalents. The IR spectrum displayed absorption bands for hydroxy and carbonyl functional groups at 3425 and 1707 cm−1, respectively. The presence of 40 carbons including 5 quaternary, 14 methine, 11 methylene, and 10 methyl carbons was confirmed by 13C NMR and HSQC spectral analysis. Detailed 2D NMR analysis revealed that 1 was composed of two partial structures: the right portion from C-1 to C-14 bearing a 1,3-diketone and a terminal carboxylic functionality and the left portion from C-15 to C-30 containing two cyclic ether rings (Figure 1). In the right half of the molecule (substructure A), five proton-bearing fragments, H3-31/H-2/ H2-3/H-4/H3-32, H3-33/H-6, H3-35/H2-34, H3-36/H-12/H213, and H3-37/H-14, were identified from the 1H−1H COSY spectrum. These fragments were joined on the basis of HMBC correlations. First, long-range couplings from H3-31, H-2 and H2-3 to C-1 connected a carbonyl carbon C-1 to C-2. This carbon was assigned as a carboxyl group on the basis of the NMR assignment of methyl ester 3, which was prepared by methylation of 1 using TMSCHN2 (Scheme 1). Two methyl groups H3-32 and H3-33 were correlated to C-5, and H2-5 in turn showed correlations to C-3, C-4, C-6, and C-7, connecting the H3-33/H-6 fragment to C-4 through the C-5 methylene carbon. Furthermore, HMBC correlations were observed from H3-33, H2-34, and H2-5 to C-7, H2-7 and H2-34 to C-8, and H27, H-8, and H2-34 to the carbonyl carbon C-9 (δC 197.2). These correlation data established the connectivity from C-6 to C-9 and an ethyl substitution at C-8. The remaining two proton-bearing fragments were joined between C-13 and C-14 on the basis of HMBC correlations from H3-37 and H-14 to C13. This fragment was expanded to include the oxygenated sp2 C-11 (δC 198.7) at C-12 by HMBC correlations from H3-36 and H-12 to C-11. Finally, HMBC correlations from H-10 (δH 5.49) to C-9 and C-11 completed the assignment of the right fragment. It was not possible to determine the location of the enolic double bond in the enolized 1,3-diketone.

Figure 2. Key ROESY correlations for 1. 977

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was assigned as E on the basis of ROESY correlations of H-16 to H-14, H3-37, H-18, and H3-38 (Figure 2B) to complete the substructure B. Finally, substructures A and B were joined between C-14 and C-15 by HMBC correlations of H-14 and H3-37 to C-15, providing the full planar structure of 1. The absolute configurations of the secondary hydroxy groups at C-19 and C-29 were assigned by applying the modified Mosher’s method.7 The methyl ester 3 was treated with (S)and (R)-MTPA chlorides to give the bis-(R)- and (S)-MTPA esters (4a and 4b), respectively. The signs of ΔδS−R (δS − δR) values for the protons from H-16 to H3-38 and H3-30 were positive, while those for H2-20 to H3-40 were negative, establishing the 19S and 29R configurations (Figure 3). The

Figure 5. ΔδS−R values for mono-PGME derivaties (8a and 8b) of 6 and bis-PGME derivaties (9a and 9b) of 7.

by the 1H NMR chemical shift difference of geminal methylene protons.9 If the chemical shift difference (Δδ) between the geminal methylene protons is small (0.0−0.1 ppm), the two methyl groups are placed in anti-relationship. If the Δδ value is larger than 0.4 ppm, the two methyl groups are placed in synrelationship. On the basis of this empirical rule, the relative configurations were assigned syn for C-2/C-4 (Δδ = 0.54 ppm) and anti for C-4/C-6 (Δδ = 0.03 ppm). Therefore, the absolute configurations of C-4 and C-6 were determined as 4S and 6S, respectively (Figure 6A). The cis relative configuration for the

Figure 3. ΔδS−R values for bis-MTPA derivatives (4a and 4b) of 3.

small vicinal coupling constant (J = 4.9 Hz) between H-18 and H-19 suggested a syn relationship of the methyl group at C-18 to the 19-OH group, namely, the 18S configuration. The absolute configuration of C-2 was determined by applying the PGME method. Phenylglycine methyl ester (PGME) is a chiral anisotropic reagent for the determination of the absolute configuration of 2-substituted chiral carboxylic acids.8 1H NMR analysis of (R)- and (S)-PGME amides (5a and 5b) prepared from 1 showed positive ΔδS−R values for the protons from H-2 to H3-33 and a negative value for H3-31, confirming the 2S configuration (Figure 4).

Figure 6. Relative configurations of 1,3-n-methyl-branched system in 1 and 2. Assignments for δHa and δHb are interchangeable. Figure 4. ΔδS−R values for PGME derivatives (5a and 5b) of 1.

H3-40 methyl and H-25 was suggested by ROESY correlation, but the absolute configurations of four stereocenters in the cyclic ether moieties remain to be clarified. Campechic acid B (2) was obtained as a pale yellow oil. HRESITOFMS analysis gave a [M − H]− peak at m/z 663.4841 in the negative ion mode appropriate for a molecular formula of C39H68O8 (Δ 0.0 mmu, calcd for C39H67O8), corresponding to the loss of a methylene fragment (CH2) from 1. Comparing to the spectral data for 1, resonances for a triplet methyl group had disappeared and those for a doublet methyl group were observed in the 1H and 13C NMR spectra of 2, suggesting that an ethyl group at C-8 was replaced by a methyl group (Supplementary Figure S1). This was supported by the observation of HMBC correlations from the doublet methyl H3-34 to C-7, C-8, and C-9 (Supplementary Figure S1). The planar structure of 2 was identical to BE-39891, a cytotoxic substance from Streptomyces described in a patent.10 Although the NMR data have not been fully assigned to BE-39891, these two compounds might be identical because the 1H and 13C

In order to assign the absolute configurations of the remaining methyl and ethyl branches in the right half of the molecule, the methyl ester 3 was subjected to oxidative degradation employing RuCl3 as catalyst to provide a diacid monomethyl ester 6 and a diacid 7 (Scheme 1). These carboxylic acids were converted to mono-(R)- and (S)-PGME amides (8a and 8b) and bis-(R)- and (S)-PGME amides (9a and 9b), respectively. In the 1H NMR spectra of 8a and 8b, positive ΔδS−R values were observed for H3-32, H3-33 and the protons from H-3 to H-8, whereas negative ΔδS−R values were shown for H2-34 and H3-35. The 1H NMR spectra of 9a and 9b revealed a positive ΔδS−R value for H3-36 and a negative value for H3-37 (Figure 5). On the basis of these results, the absolute configurations of C-8, C-12, and C-14 were determined as 8S, 12R, and 14S, respectively. It is proposed that relative configurations of methyl groups in 1,3-n-methyl-branched carbon chain systems can be predicted 978

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Table 1. 1H and 13C NMR Data for 1 and 2 in CDCl3 campechic acid A (1) position

δCa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

180.4 36.8 41.8 27.9 45.3 27.9 40.6 47.7 197.2 98.9 198.7 40.1 41.2 30.8 40.2 129.3 133.5 42.9 74.4 40.2 80.7 31.9 27.5 85.1 83.8 34.0 30.4 86.9 73.2 17.4 17.8 19.2 19.2 26.6 11.9 18.5 19.0 16.0 25.7 23.9

campechic acid B (2)

δH, mult (J in Hz)b

HMBCa,c

2.54, md 1.66/1.12, md 1.54, md 1.06/1.03, md 1.45, md 1.70/1.16, md 2.20, md

1, 1, 6 3, 4 5, 7,

5.49, s

8, 9, 11, 12

2.44, md 1.63/1.15, md 1.45, md 1.99/1.84, md 5.36, md 5.35, md 2.21, md 3.59, ddd (6.0, 6.0, 6.0) 1.63, md 4.15, md 2.07/1.67, md 1.81/1.69, md

10, 11, 13, 13, 15, 16, 16, 17, 18, 19, 21, 21,

3.98, dd (6.5, 8.5) 1.93/1.86, md 2.17/1.51, md

24, 26, 39 24, 25, 27 25, 26, 28, 29, 40

3.78, q (6.8) 1.13, d (6.8) 1.15, d (7.0) 0.826, d (6.5) 0.829, d (6.5) 1.61/1.45, md 0.88, t (7.5) 1.12, d (6.5) 0.86, d (6.6) 1.00, d (6.9) 1.27, s 1.14, s

27, 28, 30, 40 28, 29 1, 2, 3 3, 4, 5 5, 6, 7 7, 8, 9, 35 8, 34 11, 12, 13 13, 14, 15 17, 18, 19 23, 24, 25 27, 28, 29

3, 31 2, 4, 5, 31, 32 4, 6, 7, 32, 33 6, 8, 9, 33, 34 9, 34, 35

11, 12, 15 14, 17, 18, 17, 18, 19, 22, 23 22,

13, 36 14, 36, 37 16, 18 19, 19, 21, 21, 23

17, 37 38 38 38 22

24, 39

δCa 179.6 36.4 42.0 27.9 44.5 27.9 40.7 39.7 198.0 97.2 198.2 39.7 41.5 30.4 39.7 129.0 133.2 42.5 73.9 39.8 80.2 31.5 27.2 84.7 83.4 33.6 30.0 86.5 72.8 18.0 17.4 18.9 18.9 18.1 17.0 18.9 15.7 25.3 23.5

δH, mult (J in Hz)b

HMBCa,c

2.54, md 1.65/1.15, md 1.56, md 1.04, md 1.48, md 1.71/1.11, md 2.43, md

1, 1, 6 3, 4 5, 7,

5.49, s

8, 9, 11, 12

2.43, md 1.67/1.14, md 1.46, md 1.99/1.86, md 5.36, md 5.35, md 2.21, md 3.59, ddd (6.0, 6.0, 6.0) 1.63, md 4.15, md 2.08/1.68, md 1.82/1.70, md

10, 11, 13, 13, 15, 16, 16, 17, 18, 19, 21, 21,

3.98, dd (7.0, 8.5) 1.92/1.87, md 2.17/1.51, md

24, 26, 39 24, 25, 27 25, 26, 28, 29, 40

3.79, 1.12, 1.15, 0.84, 0.84,

q d d d d

(6.8) (6.8) (7.0) (6.5) (6.5)

27, 28, 30, 40 28, 29 1, 2, 3 3, 4, 5 5, 6, 7

1.12, 1.13, 0.87, 1.00, 1.28, 1.14,

d d d d s s

(7.5) (6.5) (6.6) (6.9)

7, 8, 9 11, 12, 13, 14, 17, 18, 23, 24, 27, 28,

3, 31 2, 4, 5, 31, 32 4, 6, 7, 32, 33 6, 8, 9, 33, 34 9, 34, 35

11, 12, 15 14, 17, 18, 17, 18, 19, 22, 23 22,

13, 36 14, 36, 37 16, 18 19, 19, 21, 21, 23

17, 37 38 38 38 22

24, 39

13 15 19 25 29

a

Recorded at 100 MHz. bRecorded at 500 MHz. cProton showing HMBC correlation to indicated carbon. dCoupling constant could not be determined due to signal overlapping.

MIC values of 6.25 μg/mL but were inactive against Escherichia coli and Candida albicans. Prior to conducting the invasion assay, the cytotoxicity of 1 and 2 was assessed against murine colon carcinoma 26-L5 cells. Compounds 1 and 2 displayed cytotoxicity with IC50 values of 0.3 and 4.5 μM, respectively. The in vitro inhibition of tumor cell invasion was evaluated using Transwell chambers with Matrigel/fibronectin-coated filters at noncytotoxic concentrations.11 Compound 1 displayed the most potent inhibitory activity among the inhibitors we have reported with an IC50 value of 5.5 nM,12 whereas compound 2 was ca. 60-fold less active with an IC50 of 0.3 μM. In summary, we have identified two polyether-class polyketides, campechic acids A (1) and B (2), from the culture extract of a soil-derived Streptomyces sp. CHI93. These compounds displayed potent inhibitory activity against the in

NMR data for 2 and BE-39891 were similar to each other. Absolute configurations of the α-methine of the carboxyl group (C-2) and two hydroxy groups (C-19 and C-26) in 2 were determined as 2S, 19S, and 29R by the PGME and the modified Mosher’s method in a similar way as described for campechic acid A (1) (Supplementary Figures S2 and S3). Relative configurations between the four methyl groups at C-2, C-4, C6, and C-8 were assigned as syn for C-2/C-4 (Δδ = 0.50 ppm), anti for C-4/C-6 (Δδ = 0.0), and syn for C-6/C-8 (Δδ = 0.60), thereby establishing the 4S, 6S, and 8S configurations (Figure 6B). Degradation study was hampered by the scarcity of the material, but 12R and 14R absolute configurations are inferred from the biosynthetic relationship. Bioactivity of 1 and 2 were evaluated in antimicrobial, cytotoxic, and anti-invasive assays. Both compounds showed modest antibacterial activity against Micrococcus luteus with 979

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mixture was stirred for 10 min at 0−5 °C. The reaction mixture was concentrated in vacuo, and the residue was chromatographed on a silica gel column (n-hexane−EtOAc, 10:1−1:2) to give 3 (3.8 mg) in 93% yield: 1H NMR (500 MHz) δ 0.82 (3H, d, J = 6.4 Hz, H-32), 0.83 (3H, d, J = 6.4 Hz, H-33), 0.88 (3H, t, J = 7.0 Hz, H-35), 1.03 (2H, m, H-5), 1.13 (3H, d, J = 7.0 Hz, H-31), 1.16 (1H, m, H-7), 1.16 (1H, m, H-3), 1.47 (1H, m, H-34), 1.47 (1H, m, H-6), 1.47 (1H, m, H-4), 1.59 (1H, m, H-34), 1.61 (1H, m, H-3), 1.70 (1H, m, H-7), 2.21 (1H, m, H-8), 2.54 (1H, m, H-2), 3.66 (3H, s, OMe); 13C NMR (100 MHz) δ 11.9 (C-35), 17.8 (C-31), 19.4 (C-33), 19.5 (C-32), 26.7 (C-34), 27.9 (C-6), 28.0 (C-4), 37.2 (C-2), 41.1 (C-7), 41.4 (C-3), 45.2 (C-5), 47.8 (C-8), 51.5 (OMe), 177.4 (C-1), 197.3 (C-9); HR-ESITOFMS m/z 691.5155 [M − H]− (calcd for C41H71O8 691.5154). Bis-(R)- and (S)-MTPA Esters of 3 (4a and 4b). To a solution of 3 (1.0 mg, 1.4 μmol) in dry pyridine (50 μL) was added (S)-MTPA chloride (7 μL, 37 μmol) at room temperature. After standing for 1 h, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane− EtOAc, 8:1−1:1) to give bis-(R)-MTPA ester 4a (1.4 mg, 86% yield): 1 H NMR (500 MHz) δ 0.88 (3H, d, J = 7.0 Hz, H-38), 1.14 (3H, s, H39), 1.16 (3H, s, H-40), 1.26 (3H, d, J = 6.5 Hz, H-30), 1.53 (1H, m, H-22), 1.61 (1H, m, H-23), 1.62 (1H, m, H-20),1.65 (1H, m, H-27), 1.65 (1H, m, H-26), 1.83 (1H, m, H-27), 1.88 (1H, m, H-26), 2.01 (1H, m, H-20), 2.03 (1H, m, H-23), 2.04 (1H, m, H-22), 2.49 (1H, m, H-18), 3.93 (1H, m, H-25), 3.96 (1H, m, H-21), 4.99 (1H, q, J = 6.5 Hz, H-29), 5.12 (1H, dt, J = 8.5, 4.5 Hz, H-19), 5.24 (1H, dd, J = 15.0, 8.0 Hz, H-17), 5.38 (1H, dt, J = 15.0, 7.0 Hz, H-16); HR-ESITOFMS m/z 1147.5916 [M + Na]+ (calcd for C61H86F6O12Na 1147.5916). In the same manner as described for 4a, 4b was prepared from 3 and (R)MTPA chloride: 1H NMR (500 MHz) δ 0.99 (3H, d, J = 6.9 Hz, H38), 1.10 (3H, s, H-39), 1.12 (3H, s, H-40), 1.34 (3H, d, J = 6.3 Hz, H-30), 1.46 (1H, m, H-22), 1.54 (1H, m, H-27), 1.56 (1H, m, H20),1.57 (1H, m, H-23), 1.57 (1H, m, H-26), 1.72 (1H, m, H-27), 1.82 (1H, m, H-23), 1.83 (1H, m, H-26), 1.94 (1H, m, H-22), 1.99 (1H, m, H-20), 2.55 (1H, m, H-18), 3.79 (1H, m, H-21), 3.86 (1H, dd, J = 8.0, 6.5 Hz, H-25), 5.00 (1H, q, J = 6.3 Hz, H-29), 5.10 (1H, dt, J = 8.5, 4.5 Hz, H-19), 5.27 (1H, dd, J = 15.5, 8.0 Hz, H-17), 5.38 (1H, dt, J = 15.5, 8.0 Hz, H-16); HR-ESITOFMS m/z 1147.5916 [M + Na]+ (calcd for C61H86F6O12Na 1147.5916). (R)- and (S)-PGME Amides of 1 (5a and 5b). To a solution of 1 (1.0 mg, 1.4 μmol) in dry DMF (40 μL) and triethylamine (40 μL) were added (R)-PGME (0.6 mg, 3.0 μmol), PyBOP (1.5 mg, 3.0 μmol), and HOAT (0.4 mg, 3.0 μmol) at room temperature. After standing for 3 h, the reaction mixture was adsorbed on an ODS column, and the column was washed with 5% HCO2H and eluted with MeOH. The eluent was concentrated, and the residue was purified by preparative HPLC (Nacalai Tesque, 5C-18 AR-II, 10 mm × 250 mm; MeCN−0.1% HCO2H, 50:50; 4.0 mL/min). Evaporation of the collected fraction and extraction with EtOAc gave (R)-PGME amide (5a, 1.1 mg, 95% yield): 1H NMR (500 MHz) δ 0.72 (3H, d, J = 6.5 Hz, H-33), 0.78 (3H, d, J = 6.5 Hz, H-32), 0.87 (3H, t, J = 7.5 Hz, H35), 0.98 (2H, m, H-5), 1.13 (1H, m, H-3), 1.14 (3H, d, J = 7.0 Hz, H31), 1.16 (1H, m, H-7), 1.40 (1H, m, H-6), 1.40 (1H, m, H-4), 1.43 (1H, m, H-34), 1.59 (1H, m, H-3), 1.60 (1H, m, H-34), 1.61 (1H, m, H-7), 2.18 (1H, m, H-8), 2.40 (1H, m, H-2), 5.48 (1H, m, H-10); HRESITOFMS m/z 824.5682 [M − H]− (calcd for C49H78NO9 824.5682). In the same manner as described for 5a, 5b was prepared from 1 and (S)-PGME: 1H NMR (500 MHz) δ 0.81 (3H, d, J = 6.5 Hz, H-33), 0.84 (3H, d, J = 6.5 Hz, H-32), 0.87 (3H, d, J = 6.8 Hz, H35), 1.03 (2H, m, H-5), 1.13 (3H, d, J = 7.0 Hz, H-31), 1.15 (1H, m, H-3), 1.18 (1H, m, H-7), 1.42 (1H, m, H-34), 1.45 (1H, m, H-6), 1.55 (1H, m, H-4), 1.55 (1H, m, H-34), 1.61 (1H, m, H-7), 1.62 (1H, m, H-3), 2.20 (1H, m, H-8), 2.41 (1H, m, H-2), 5.49 (1H, m, H-10); HRESITOFMS m/z 824.5682 [M − H]− (calcd for C49H78NO9 824.5682). Oxidative Degradation of 3 To Yield 6 and 7. To a solution of 3 (6.0 mg, 8.7 μmol) in MeCN/CCl4/H2O (1.5 mL; each 0.5 mL) was added NaIO4 (60 mg, 0.28 mmol) and a solution of RuCl3 in 0.1 M Na2HPO4 solution (1 mg/mL, 0.6 mL, 2.9 μmol) at 0−5 °C. After stirring overnight, a saturated solution of Na2S2O3 (1 mL) was added

vitro tumor cell invasion into the reconstituted extracellular matrix with IC50 in the nanomolar to submicromolar range.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a JASCO DIP-3000 polarimeter. UV spectra were recorded on a Hitachi U-3210 spectrophotometer. IR spectra were measured on a Perkin-Elmer Spectrum 100. NMR spectra were obtained on a Bruker AVANCE 400 or a Bruker AVANCE 500 spectrometer in CDCl3 and referenced to residual solvent signals (δH 7.27, δC 77.0). HR-ESITOFMS were recorded on a Bruker microTOF focus. Cosmosil 75C18-PREP (Nakalai Tesque Inc., 75 μm) was used for ODS column chromatography. Microorganism. Strain CHI93 was isolated from a rock sample collected in Campeche, Mexico. Sampling of the rock was carried out under collaboration with Dr. L. Maldonado, Universidad Autonoma Nacional de Mexico, Instituto de Ciencias del Mar y Limnologı ́a on November 15 in 2002. The strain was identified as a member of the genus Streptomyces on the basis of 99.5% similarity in 16S rRNA gene sequence (1413 nucleotides; GenBank accession number HF565369) to Streptomyces davawensis JCM 4913 (accession number HE971709).13 Fermentation. Strain CHI93 cultured on a slant agar medium consisting of soluble starch 0.5%, glucose 0.5%, meat extract (Kyokuto Pharmaceutical Industrial Co., Ltd.) 0.1%, yeast extract (Difco Laboratories) 0.1%, NZ-case (Wako Chemicals USA, Inc.) 0.2%, NaCl 0.2%, CaCO3 0.1%, and agar 1.5% was inoculated into 500-mL K-1 flasks each containing 100 mL of the V-22 seed medium consisting of soluble starch 1%, glucose 0.5%, NZ-case 0.3%, yeast extract 0.2%, tryptone (Difco Laboratories) 0.5%, K2HPO4 0.1%, MgSO4·7H2O 0.05%, and CaCO3 0.3% (pH 7.0). The flasks were placed on a rotary shaker (200 rpm) at 30 °C for 4 days. The seed culture (3 mL) was transferred into 500-mL K-1 flasks each containing 100 mL of the A16 production medium consisting of glucose 2.0%, Pharmamedia (Traders Protein) 1.0%, CaCO3 0.3%, and Diaion HP-20 (Mitsubishi Chemical Co.) 1%. The pH of the medium was adjusted to 7.0 before sterilization. The inoculated flasks were placed on a rotary shaker (200 rpm) at 30 °C for 6 days. Extraction and Isolation. At the end of the fermentation period, 100 mL of 1-butanol was added to each flask, which were agitated on a rotary shaker for 1 h. The mixture was centrifuged at 6000 rpm for 10 min, and the organic layer was separated from the aqueous layer containing the mycelium. Evaporation of the solvent gave approximately 1.1 g of extract from 2 L of culture. The crude extract (1.1 g) was subjected to silica gel column chromatography with a step gradient of CHCl3−MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v). Fraction 4 (4:1) was concentrated to provide 0.44 g of brown oil, which was then purified by preparative HPLC (Waters, XTerra RP18, 7 μm, 300 mm × 19 mm; MeCN−10 mM NH4HCO3, a linear gradient from 15:85 to 85:15 over 35 min; 15 mL/min). Evaporation of the collected fractions and extraction with EtOAc gave semipure materials, which were further purified by Sephadex LH-20 gel filtration with CH2Cl2−MeOH (50:50) to afford campechic acids A (1, 18.2 mg) and B (2, 2.9 mg). Campechic Acid A (1). Pale yellow oil; [α]22D −10.5 (c 0.90, CHCl3); UV (MeOH) λmax (log ε) 278 (4.02) nm; (0.01 N HCl− MeOH) 278 (4.04); (0.01 N NaOH−MeOH) 294 (4.12); IR νmax 3424, 2965, 2929, 2872, 1707, 1604 cm−1; for 1H and 13C NMR data, see Table 1; HR-ESITOFMS m/z 677.4996 [M − H]− (calcd for C40H69O8 677.4998). Campechic Acid B (2). Pale yellow oil; [α]22D +0.9 (c 1.0, CHCl3); UV (MeOH) λmax (log ε) 278 (3.93) nm; (0.01 N HCl− MeOH) 278 (3.96); (0.01 N NaOH−MeOH) 295 (4.12); IR νmax 3425, 2964, 2930, 2873, 1707, 1603 cm−1; for 1H and 13C NMR data, see Table 1; HR-ESITOFMS m/z 663.4841 [M − H]− (calcd for C39H67O8 663.4841). Methylation of 1 To Yield Methyl Ester 3. To a solution of 1 (4.0 mg, 5.9 μmol) in MeOH (1 mL) was added a n-hexane solution of (trimethylsilyl)diazomethane (2.0 M, 0.46 mL, 8.1 μmol), and the 980

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to quench the reaction, and 1 N HCl (1 mL) was then added and extracted with EtOAc (3 mL × 3). The organic layer was concentrated, and the residue was subjected to silica gel column chromatography (CHCl3/MeOH, 1:0−10:1) to give 6 (1.5 mg) and 7 (0.8 mg). For diacid monomethyl ester 6: 1H NMR (500 MHz) δ 0.85 (3H, d, J = 6.5 Hz, H-32), 0.87 (3H, d, J = 6.5 Hz, H-33), 0.95 (3H, t, J = 7.5 Hz, H-35), 1.05 (2H, m, H-5), 1.14 (3H, d, J = 7.0 Hz, H-31), 1.14 (1H, m, H-3), 1.19 (1H, m, H-34), 1.50 (1H, m, H-4), 1.52 (1H, m, H-6), 1.53 (1H, m, H-7), 1.62 (1H, m, H-7), 1.65 (1H, m, H-34), 1.69 (1H, m, H-3), 2.40 (1H, m, H-8), 2.57 (1H, m, H-2), 3.68 (3H, s, OMe); 13 C NMR (100 MHz) δ 11.8 (C-35), 17.9 (C-31), 19.3 (C-32), 19.5 (C-33), 26.1 (C-7), 28.1 (C-6), 28.2 (C-4), 37.2 (C-2), 40.0 (C-34), 42.0 (C-3), 44.7 (C-8), 45.1 (C-5), 51.5 (OMe), 177.7 (C-1), 179.83 (C-9); HR-ESITOFMS m/z 271.1906 [M − H]− (calcd for C15H27O4 271.1915). For diacid 7: 1H NMR (500 MHz) δ 1.03 (3H, d, J = 6.5 Hz, H-37), 1.19 (3H, d, J = 7.0 Hz, H-36), 1.19 (1H, m, H-13), 1.81 (1H, m, H-13), 1.99 (1H, m, H-14), 2.26 (2H, d, J = 7.0 Hz, H-15), 2.60 (1H, m, H-12); 13C NMR (100 MHz) δ 18.4 (C-36), 20.1 (C37), 28.8 (C-14), 37.8 (C-12), 41.0 (C-13), 42.0 (C-15), 178.2 (C16), 182.6 (C-11); HR-ESITOFMS m/z 197.0784 [M + Na]+ (calcd for C8H14O4Na 197.0784). (R)- and (S)-PGME Amides of 6 (8a and 8b). In the same manner as described for 5a, 8a and 8b were prepared from 6. (R)-PGME Amide 8a. 1H NMR (500 MHz) δ 0.71 (3H, d, J = 6.5 Hz, H-32), 0.80 (3H, d, J = 6.5 Hz, H-33), 0.93 (3H, t, J = 7.0 Hz, H35), 0.98 (2H, m, H-5), 1.08 (1H, m, H-3), 1.11 (3H, d, J = 7.0 Hz, H31), 1.13 (1H, m, H-7), 1.39 (1H, m, H-6), 1.39 (1H, m, H-4) 1.45 (1H, m, H-34), 1.58 (1H, m, H-7), 1.60 (1H, m, H-3),1.62 (1H, m, H34) 2.18 (1H, m, H-8), 2.51 (1H, m, H-2) 3.65 (3H, s, OMe); HRESITOFMS m/z 442.2564 [M + Na]+ (calcd for C24H37NO5Na 442.2564). (S)-PGME Amide 8b. 1H NMR (500 MHz) δ 0.82 (3H, d, J = 6.5 Hz, H-32), 0.83 (3H, t, J = 7.5 Hz, H-35), 0.86 (3H, d, J = 6.5 Hz, H33), 1.03 (2H, m, H-5), 1.10 (3H, d, J = 7.0 Hz, H-31), 1.12 (1H, m, H-3), 1.17 (1H, m, H-7), 1.44 (1H, m, H-34), 1.48 (1H, m, H-4), 1.52 (1H, m, H-6), 1.58 (1H, m, H-34), 1.62 (1H, m, H-7), 1.63 (1H, m, H-3), 2.20 (1H, m, H-8), 2.54 (1H, m, H-2), 3.67 (3H, s, OMe); HRESITOFMS m/z 442.2564 [M + Na]+ (calcd for C24H37NO5Na 442.2564). Bis-(R)- and (S)-PGME Amides of 7 (9a and 9b). In the same manner as described for 5a, 9a and 9b were prepared from 7. (R)-PGME Amide 9a. 1H NMR (500 MHz) δ 1.01 (3H, d, J = 6.5 Hz, H-37), 1.13 (3H, d, J = 6.5 Hz, H-36), 1.27 (1H, m, H-13), 1.81 (1H, m, H-13), 2.10 (1H, m, H-14), 2.25 (2H, m, H-15), 2.39 (1H, m, H-12); HR-ESITOFMS m/z 491.2153 [M + Na]+ (calcd for C26H32N2O6Na 491.2153). (S)-PGME Amide 9b. 1H NMR (500 MHz) δ 0.97 (3H, d, J = 6.5 Hz, H-37), 1.20 (3H, d, J = 7.0 Hz, H-36), 1.24 (1H, m, H-13), 1.86 (1H, m, H-13), 2.02 (1H, m, H-14), 2.16 (2H, m, H-15), 2.49 (1H, m, H-12); HR-ESITOFMS m/z 491.2153 [M + Na]+ (calcd for C26H32N2O6Na 491.2153). Methylation of 2 To Yield Methyl Ester 10. In the same manner as described for the preparation of 3, the methyl ester of campechic acid B (10) was prepared from 2. 1H NMR (500 MHz) δ 0.82 (3H, d, J = 6.5 Hz, H-32), 0.84 (3H, d, J = 6.0 Hz, H-33), 1.04 (2H, m, H-5), 1.11 (1H, m, H-7), 1.12 (3H, d, J = 6.0 Hz, H-34), 1.13 (3H, d, J = 7.0 Hz, H-31), 1.15 (1H, m, H-3), 1.49 (1H, m, H-6), 1.49 (1H, m, H-4), 1.61 (1H, m, H-3), 1.70 (1H, m, H-7), 2.43 (1H, m, H-8), 2.55 (1H, m, H-2), 3.67 (3H, s, OMe); 13C NMR (100 MHz) δ 15.5 (C-31), 16.4 (C-34), 17.1 (C-33), 17.2 (C-32), 25.4 (C-6), 25.7 (C-4), 34.9 (C-2), 37.8 (C-8), 38.7 (C-7), 40.1 (C-3), 42.3 (C-5), 49.2 (OMe), 175.2 (C-1), 196.2 (C-9); HR-ESITOFMS m/z 701.4966 [M + Na]+ (calcd for C40H70O8Na 701.4963). Bis-(R)- and (S)-MTPA Esters of 10 (11a and 11b). In the same manner as described for 4a, 11a and 11b were prepared from 10. Bis-(R)-MTPA Ester 11a. 1H NMR (500 MHz) δ 0.88 (3H, d, J = 7.0 Hz, H-37), 1.14 (3H, s, H-38), 1.16 (3H, s, H-39), 1.26 (3H, d, J = 6.5 Hz, H-30), 1.54 (1H, m, H-22), 1.61 (1H, m, H-23), 1.63 (1H, m, H-20),1.65 (1H, m, H-27), 1.65 (1H, m, H-26), 1.83 (1H, m, H-27), 1.89 (1H, m, H-26), 1.97 (1H, m, H-23), 2.02 (1H, m, H-20), 2.03

(1H, m, H-22), 2.50 (1H, m, H-18), 3.92 (1H, m, H-25), 3.95 (1H, m, H-21), 5.00 (1H, q, J = 6.0 Hz, H-29), 5.12 (1H, m, H-19), 5.25 (1H, dd, J = 15.0, 7.5 Hz, H-17), 5.39 (1H, dt, J = 15.0, 7.0 Hz, H-16); HRESITOFMS m/z 1109.5784 [M − H]− (calcd for C60H83F6O12 1109.5794). Bis-(R)-MTPA Ester 11b. 1H NMR (500 MHz) δ 0.99 (3H, d, J = 7.0 Hz, H-37), 1.10 (3H, s, H-38), 1.12 (3H, s, H-39), 1.34 (3H, d, J = 6.5 Hz, H-30), 1.46 (1H, m, H-22), 1.54 (1H, m, H-27), 1.56 (1H, m, H-20),1.57 (1H, m, H-23), 1.62 (1H, m, H-26), 1.72 (1H, m, H-27), 1.81 (1H, m, H-26), 1.83 (1H, m, H-23), 1.93 (1H, m, H-22), 1.99 (1H, m, H-20), 2.56 (1H, m, H-18), 3.79 (1H, m, H-21), 3.86 (1H, t, J = 7.5 Hz, H-25), 5.00 (1H, q, J = 6.5 Hz, H-29), 5.10 (1H, m, H-19), 5.28 (1H, dd, J = 15.2, 8.0 Hz, H-17), 5.42 (1H, dt, J = 15.2, 7.0, H16); HR-ESITOFMS m/z 1109.5795 [M − H]− (calcd for C60H83F6O12 1109.5794). (R)- and (S)-PGME Amides of 2 (12a and 12b). In the same manner as described for 5a, 12a and 12b were prepared from 2. (R)-PGME Amide 12a. 1H NMR (500 MHz) δ 0.73 (3H, d, J = 6.5 Hz, H-33), 0.79 (3H, d, J = 6.5 Hz, H-32), 0.98 (2H, m, H-5), 1.09 (1H, d, J = 6.5 Hz, H-34), 1.12 (1H, m, H-7), 1.13 (1H, m, H-3), 1.15 (3H, d, J = 7.0 Hz, H-31), 1.42 (1H, m, H-6), 1.42 (1H, m, H-4), 1.54 (1H, m, H-7), 1.60 (1H, m, H-3), 2.39 (1H, m, H-8), 2.40 (1H, m, H2), 5.47 (1H, m, H-10); HR-ESITOFMS m/z 834.5490 [M + Na]+ (calcd for C48H77NO9Na 834.5491). (S)-PGME Amide 12b. 1H NMR (500 MHz) δ 0.83 (3H, d, J = 6.5 Hz, H-33), 0.85 (3H, d, J = 6.5 Hz, H-32), 1.03 (2H, m, H-5), 1.09 (1H, d, J = 6.5 Hz, H-34), 1.13 (3H, d, J = 7.0 Hz, H-31), 1.13 (1H, m, H-7), 1.15 (1H, m, H-3), 1.43 (1H, m, H-6), 1.52 (1H, m, H-4), 1.58 (1H, m, H-7), 1.62 (1H, m, H-3), 2.40 (1H, m, H-8), 2.43 (1H, m, H2), 5.49 (1H, m, H-10); HR-ESITOFMS m/z 834.5490 [M + Na]+ (calcd for C48H77NO9Na 834.5491). Antimicrobial Assay. Antimicrobial assays were carried out using Eschcerichia coli NIH-JC2, Micrococcus luteus ATCC9343, Bacillus subtilis PCI219, Staphylococcus aureus IFO12732, and Candida albicans IFO1594. Mueller Hinton broth (DIFCO) was used for bacteria, and yeast nitrogen base (DIFCO) supplemented with 2% glucose was used for C. albicans. Test microorganisms were inoculated into a 32-mL test tube containing 8 mL of the liquid medium. After incubation on a reciprocal shaker for 20 h at 30 °C, the cells were collected by centrifugation (3000 rpm, 5 min), and the cell suspension (1 × 105 cells/mL) was prepared in saline. Then, the liquid medium (135 μL), the cell suspension (15 μL), and the sample solution in DMSO (0.5−1 μL) were added into the wells of a 96-well culture plate, and the plate was agitated gently to mix the solution. After incubation for 20 h at 37 °C (E. coli) or 30 °C (other bacteria and C. albicans), the absorbance at 650 nm was measured using a microplate reader. Cytotoxicity Assay. Cytotoxicity assay was carried out using murine colon 26-L5 carcinoma cells. Murine colon 26-L5 carcinoma cells (5 × 103) were suspended in 100 μL of RPMI containing 1% FCS and various concentrations of (R)- or (S)-1 and 2 and seeded into the wells of a 96-well culture plate. After 24 h of incubation in a humidified 5% CO2 incubator at 37 °C, the wells were washed with PBS, and the cells were fixed with 20% formalin. Then, the wells were washed with PBS, and the cells were stained with crystal violet for 30 min. After washing with PBS, the crystal violet dye was extracted with 30% acetic acid, and the absorbance at 590 nm was measured. Invasion and Migration Assays. Invasion of tumor cells through the reconstituted basement membrane, Matrigel, was assessed in Transwell cell culture chambers (see Table 2). In brief, polyvinylpyrrolidone-free carbonate filters of 8.0-μm pore size (Nucleopore, Pleasanton, CA, USA) were mounted in the Transwell chambers

Table 2. Inhibition of Tumor Cell Invasion by 1 and 2 IC50 (μM) 1 2 981

cytotoxicity

anti-invasion

SI (cytotoxicity/anti-invasion)

0.4 4.5

0.0055 0.3

73 15

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(Costar 3422, Cambridge, MA, USA). The lower surface of the filters was coated with 20 μg of fibronectin, and the upper surface was coated with 5 μg of Matrigel (Collaborative Research Inc., Bedford, MA, USA). Exponentially growing colon 26-L5 cells were harvested and resuspended in RPMI containing 1% FCS and various concentrations of (R)- or (S)-1 and 2. Then, 100 μL of cell suspension (2 × 104 cells/ chamber) was then added to the upper chamber and incubated in a 24well culture plate at 37 °C for 6−8 h. At the end of incubation, the cells on the filters were fixed with methanol and stained with hematoxylin for 3 min and eosin for 10 s. After gentle rinsing with water, the cells remaining in the Matrigel were removed by wiping the upper chamber with a cotton swab. The filters containing the stained cells that had invaded or migrated through the lower surface were removed from the Transwell chambers. The number of the invaded or migrated cells was counted in five different fields of each well under the microscope. Doxorubicin, an anthracycline antitumor drug, was used as a positive control in this assay. It displayed the inhibitory activity with an IC50 value of 2 μM.

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Saito, N.; Sakurai, H.; Saiki, I.; Furumai, T.; Igarashi, Y. J. Antibiot. 2006, 59, 698−703. (c) Igarashi, Y.; Shimasaki, R.; Miyanaga, S.; Oku, N.; Onaka, H.; Sakurai, H.; Saiki, I.; Kitani, S.; Nihira, T.; Wimonsiravude, W.; Panbangred, W. J. Antibiot. 2010, 63, 563−565. (d) Igarashi, Y.; Kim, Y.; In, Y.; Ishida, T.; Kan, Y.; Fujita, T.; Iwashita, T.; Tabata, H.; Onaka, H.; Furumai, T. Org. Lett. 2010, 12, 3402− 3405. (e) Miyanaga, S.; Sakurai, H.; Saiki, I.; Onaka, H.; Igarashi, Y. Bioorg. Med. Chem. Lett. 2010, 20, 963−965. (13) Nakagawa, Y.; Kawasaki, H. 16S rRNA-based analysis. In Identification Manual of Actinomycetes; The Society for Actinomycetes Japan, Eds.; Business Center for Academic Societies Japan: Tokyo, 2001; pp 88−117.

ASSOCIATED CONTENT

S Supporting Information *

Copies of NMR spectra for campechic acids A and B and their derivatives. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-766-56-7500. Fax: +81-766-56-2498. E-mail: yas@ pu-toyama.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Prof. T. Okuda and Ms. Y. Sudoh at Tamagawa University for assistance with antimicrobial assays. REFERENCES

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