Biosci. Biotechnol. Biochem., 69 (9), 1749–1752, 2005
Novel Relationship between the Antifungal Activity and Cytotoxicity of Marine-Derived Metabolite Xestoquinone and Its Family Mitsuhiro N AKAMURA,1; * Takahiko K AKUDA,1 Jianhua Q I,1 Masayuki H IRATA,1 Tomoaki S HINTANI,2 Yukio Y OSHIOKA,2 Tetsuji O KAMOTO,2 Yuichi O BA,1 Hideshi N AKAMURA,1; ** and Makoto O JIKA1; y 1
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan 2
Received May 9, 2005; Accepted June 10, 2005
Xestoquinone and related metabolites (the xestoquinone family) occur in marine sponges and are known to show a variety of biological activities. In this study, the first comprehensive evaluation of antifungal activity was performed for xestoquinone and nine natural and unnatural analogues in comparison with their cytotoxicity. The cytotoxicity against two human squamous cell carcinoma cell lines, A431 and Nakata, indicated that the terminal quinone structure of the polycyclic molecules was important (xestoquinone, etc.) and that the presence of a ketone group at C-3 of the opposite terminus dramatically diminished the activity (halenaquinone, etc.). In contrast, a ketone group at C-3 enhanced the antifungal activity against the plant pathogen, Phytophthora capsici, regardless of the presence of a quinone moiety. The cytotoxicity and antifungal activity of the xestoquinone family were negatively correlated with each other.
Key words:
marine natural product; xestoquinone; halenaquinone; cytotoxicity; antifungal activity
Marine sponges possess various bioactive metabolites, some of which have been regarded as defense substances. These bioactive compounds of marine origin are structurally unique and have attracted attention as candidates for medicinal drugs or biochemical tools. Xestoquinone (1) and halenaquinone (2) are polycyclic quinone-type metabolites that have been isolated from marine sponges of the Xestospongia genus (Fig. 1).1,2) Despite their structural similarity, these compounds show a variety of biological activities, such as cardiotonic activity,1) cytotoxicity,3–8) and inhibitory activity against enzymes such as protein tyrosine kinase,5,9) topoisomerases,8,10) myosin Ca2þ ATPase,11) etc. We have recently reported an effective preparation of xestoquinone (1) and its analogues, 2, 3, 5–8, 10, from
Fig. 1. Structures of Xestoquinone (1) and Related Compounds. y
To whom correspondence should be addressed. Tel/Fax: +81-52-789-4284; E-mail:
[email protected] Present address: RIKEN Harima Institute, Sayo-gun, Hyogo 679-5148, Japan ** Deceased November 9, 2000. *
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extracts of the marine sponge, Xestospongia sapra, and their inhibitory activity against myosin Ca2þ ATPase activity to reveal the role of the quinone structure for this activity (Fig. 1).12) We also designed and synthesized a biotinylated xestoquinone analog that exhibited stronger inhibitory activity than that of xestoquinone (1) to study the mechanism of action. Despite a number of such chemical and biological efforts, comprehensive studies on the structure-activity relationships, including an antifungal evaluation of the xestoquinone family, have never been performed, although fragmented knowledge has been accumulated so far. In this paper, we describe the preparation of two new members of the xestoquinone family and their structure-activity (antifungal activity and cytotoxicity) relationships by using a phytopathogenic microorganism and two human squamous cell carcinoma (SCC) cell lines.
Materials and Methods General. Thin-layer chromatography (TLC) was performed by using pre-coated silica gel 60 F254 plates (Art. 5715, Merck) or RP-18 F254 plates (Art. 15389, Merck). Open column chromatography was performed with silica gel 60 (230–400 Mesh ASTM, Merck). IR spectra were recorded by a Jasco FT/IR-8300 instrument, and UV spectra, by a Jasco V-530 spectrophotometer. NMR spectra were recorded by a Bruker ARX400 (400 MHz) instrument in a CDCl3 solution (99.9% atom enriched). NMR chemical shifts are referenced to the solvent peak of H 7.26 (residual CHCl3 ) or C 77.0 (CDCl3 ). High-resolution ESI-TOFMS were recorded by an Applied Biosystems Mariner mass spectrometer equipped with an electrospray ion source in the positive mode, using residual phthalates (m=z 279 and 391) as internal standards. Xestoquinone (1) and its analogues, 2, 3, 5–8, 10. These materials were prepared by the method described in the previous paper.12) (3R)-3-Acetoxyxestoquinone (4). A solution of 3 (1.0 mg, 3.0 mmol) in pyridine (0.5 ml) was stirred with acetic anhydride (0.5 ml) at room temperature for 2 h. The reaction mixture was concentrated, and the residue was chromatographed on silica gel (2.0 g) eluted with hexane–CHCl3 (2:8) to give 4 as a light brown powder (0.7 mg, 60%): []25 D þ84 (c 0.04, CHCl3 ); UV (MeCN) max : 218 (" 22000), 255 (23000), 288 (16000), 330 nm (sh, 7700); IR (KBr) max : 1734, 1676, 1604, 1322, 1244, 1137, 1037, 958, 852 cm1 ; 1 H-NMR (CDCl3 , 400 MHz) : 9.07 (1H, s), 8.24 (1H, s), 7.86 (1H, s), 7.07 (1H, d, J ¼ 10:4 Hz), 7.04 (1H, d, J ¼ 10:4 Hz), 5.86 (1H, t, J ¼ 8:0 Hz), 2.65 (1H, m), 2.58 (1H, m), 2.41 (1H, m), 2.16 (3H, s), 1.91 (1H, dt, J ¼ 4:4, 13.6 Hz), 1.65 (3H, s); 13 C-NMR (CDCl3 , 100 MHz) (partial data due to a limited sample amount) : 149.3, 139.4, 138.7, 127.2, 123.3, 121.4, 63.9, 32.5,
32.1, 25.9, 21.1; high-resolution ESI-TOF-MS: found, m=z 377.1014 ðM þ HÞþ ; calcd. for C22 H17 O6 , 377.1020. (3R)-3-Acetoxyxestoquinol dimethyl ether (9). Acetylation of alcohol 3 (3.7 mg, 10 mmol) was carried out under the same conditions as those used for 4. The crude material was chromatographed on silica gel (3.7 g), eluting with hexane–CHCl3 (4:6) to afford 9 as a yellow powder (4.0 mg, 96%): []25 D þ140 (c 0.25, CHCl3 ); UV (MeCN) max : 205 (" 16000), 225 (20000), 278 (9800), 305 (12000), 400 nm (2500); IR (KBr) max : 1734, 1675, 1629, 1618, 1345, 1274, 1243, 1148, 1092, 1046, 956 cm1 ; 1 H-NMR (CDCl3 , 400 MHz) : 9.28 (1H, s), 8.26 (1H, s), 7.78 (1H, s), 6.83 (1H, d, J ¼ 8:4 Hz), 6.71 (1H, d, J ¼ 8:4 Hz), 5.87 (1H, t, J ¼ 8:2 Hz), 3.98 (6H, s), 2.70 (1H, dt, J ¼ 13:2, 3.6 Hz), 2.55 (1H, m), 2.38 (1H, m), 2.16 (3H, s), 1.98 (1H, dt, J ¼ 4:4, 13.6 Hz), 1.64 (3H, s); 13 C-NMR (CDCl3 , 100 MHz) : 172.9, 171.2, 150.9, 148.8, 147.9, 146.1, 145.9, 144.6, 131.0, 127.5, 124.8, 124.4, 121.3, 117.7, 106.2, 103.5, 64.6, 55.7 (2C), 36.1, 33.2 (2C), 26.2, 21.2; high-resolution ESI-TOF-MS: found, m=z 407.1462 ðM þ HÞþ ; calcd. for C24 H23 O6 , 407.1489. Cell lines and cytotoxicity test. An A431 human vulval-derived epidermoid carcinoma cell line13) and Nakata oral squamous cell carcinoma cell line14) were used in this study. The cytotoxicity of each of these compounds was evaluated by a similar procedure to that reported.15,16) Briefly, the A431 or Nakata cell line was cultured in a serum-free RD medium [RPMI 1640 medium (Kyokuto, Tokyo Japan)-DMEM (Kyokuto) 1:1, vol/vol] containing five factors (10 mg/ml of insulin, 5 mg/ml of transferrin, 10 mM 2-mercaptoethanol, 10 mM 2-aminoethanol, and 10 nM selenite (all from Sigma Chemical Co.).17,18) In the proliferation assays, the cells were plated at 1:0 104 per well into 24-well tissue culture plates (BD Bioscience, NJ, U.S.A.) coated with type I collagen, and cultured in the same medium. The cells were allowed to attach and spread for 12 h prior to their incubation with xestoquinone (1) or its analogues at various concentrations (none, 0.4 ng/ml, 4 ng/ml, 40 ng/ml, 400 ng/ml, or 4000 ng/ml). On day 4, the cells were harvested by 0.05% trypsin/0.01% EDTA, and the cell numbers were counted with a Zf counter (Coulter Electronics Inc., Hialeah, FL, U.S.A.). IC50 values shown are the means of two replicates. Antifungal activity test. The antifungal activity was evaluated against plant pathogen Phytophthora capsici by using a paper disc assay as previously reported.19) Briefly, P. capsici was cultured on a synthetic agar medium in a 9-cm dish at 25 C for 2 days in the dark until the colony had grown to a size of about 3–4 cm in diameter. Each paper disc (8 mm in diameter) impregnated with a sample was placed 1 cm away from the front of the colony. After incubating for 30 h, the
Structure-Activity Relationships of the Xestoquinone Family
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Table 1. Cytotoxicity and Antifungal Activity of Xestoquinone (1) and Its Analogues
A431
IC50 (mM)a Nakata
25 mg/disc
Antifungal activity (mm)b 5 mg/disc
1 mg/disc
0.0091 4.7 0.28 0.0088 0.24 0.68 5.0 0.28 0.22 0.24 10
0.021 1.7 0.61 0.025 0.32 0.28 2.5 0.12 0.28 0.041 10
0 3 2 0.2 0 0 2 0 0 0 n.d.
0 1 0.2 0 0 0 1 0 0 0 n.d.
0 0.5 0 0 0 0 0.5 0 0 0 n.d.
Compound Xestoquinone (1) Halenaquinone (2) 3 4 5 6 7 8 9 10 Cisplatin a
Cytotoxicity against two human tumor cell lines. A431, American vulval-derived epidermoid carcinoma; Nakata, Japanese oral squamous cell carcinoma. Paper disc test against the plant pathogen Phytophthora capsici. The values indicate the distance between the paper disc edge and the fungal colony front based on zero for the control. n.d., not determined. b
distance between the edge of the colony and the paper disc was measured.
Results and Discussion Xestoquinone (1) and its analogues (2, 3, 5–8, and 10) were prepared from extracts of the marine sponge, Xestospongia sapra, as described previously.12) The structure and purity of each of these compounds were confirmed by 1 H-NMR spectra. The two acetates, 4 and 9, were prepared from the 3-hydroxy derivatives, 3 and 8, respectively, under the usual acetylation conditions. In the cytotoxicity test (Table 1), using two human squamous cell carcinoma cell lines, xestoquinone (1) and (3R)-3-acetoxyxestoquinone (4) were the most potent, the A431 cells being more sensitive than Nakata cells to these compounds. Halenaquinone (2) and halenaquinol dimethyl ether (7) were approximately 500 and 100 times less potent than 1 for A431 and Nakata cells, respectively, and roughly 10 times less potent than the other analogues. These results indicate that a carbonyl group at the C-3 position was unfavorable for cytotoxicity. Xestoquinone (1) and 4 were more potent than corresponding hydroquinone derivatives 6 and 9, suggesting that the presence of a quinone structure in the E ring could be important. The C-3 position (probably non-polar) is very important for cytotoxicity, and the effect of the quinone structure is secondary in contrast to the inhibition of myosin Ca2þ ATPase, in which the quinone structure was thought to be important.12) DNA cleavage could be one mechanism for the cytotoxicity8) and be interfered with by the presence of the C-3 ketone. In the antifungal activity test using the plant pathogen, P. capsici, however, quite the opposite tendency was observed (Table 1). Halenaquinone (2) and corresponding hydroquinone 7 were most active, suggesting that the presence of a ketone group at C-3 was very important. A polar hydroxyl group at C-3 in 3 also played a role in the antifungal activity. Since hydro-
Fig. 2. Correlation between Antifungal Activity and Cytotoxicity of the Xestoquinone Family. Antifungal activity at a dose of 25 mg/disc is plotted against two cytotoxicity values ( , A431 cell line; , Nakata cell line). The continuous and dotted lines respectively indicate approximated curves for the A431 and Nakata cell lines. See Table 1 for details of the biological activities.
quinone derivatives 7, 8 and 9 were all less active than corresponding quinone derivatives 2, 3 and 4, the quinone structure seems to have played an auxiliary factor in the antifungal activity. The opposite tendency in bioactivity between cytotoxicity and antifungal activity becomes obvious when both activities are plotted on a graph, indicating negative correlations (Fig. 2). The dipole moment at C-3 might have the opposite effect on the two biological activities. Although the mechanism for the antifungal activity is unclear, the -alkoxy-,-unsaturated ketone structure could be important because -methoxyacrylates are known as mitochondrial respiratory chain inhibitors against fungi.19,20) In conclusion, the antifungal evaluation of ten xestoquinone-related compounds revealed for the first time the importance of the C-3 ketone group in the halenaquinone-type compounds. A comparative evalua-
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tion of the cytotoxicity and antifungal activity of the xestoquinone family revealed an interesting correlation between these bioactivities and the importance of the C-3 position rather than the quinone moiety, contrary to the inhibition of myosin Ca2þ ATPase.
Acknowledgments
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11)
We are grateful to Akajima Marine Science Laboratory (Establishment of Tropical Marine Ecological Research, Tokyo) for their help in the collection of the marine animals. 12)
References 1)
2)
3)
4)
5)
6)
7)
8)
9)
Nakamura, H., Kobayashi, J., Kobayashi, M., Ohizumi, Y., and Hirata, Y., Physiologically active marine natural products from Porifera. VII. Xestoquinone. A novel cardiotonic marine natural product isolated from the Okinawan sea sponge Xestospongia sapra. Chem. Lett., 713–716 (1985). Roll, D. M., Scheuer, P. J., Matsumoto, G. K., and Clardy, J., Halenaquinone, a pentacyclic polyketide from a marine sponge. J. Am. Chem. Soc., 105, 6177–6178 (1983). Schmitz, F. J., and Bloor, S. J., Xesto- and halenaquinone derivatives from a sponge, Adocia sp., from Truk lagoon. J. Org. Chem., 53, 3922–3925 (1988). Fujiwara, H., Matsunaga, K., Saito, M., Hagiya, S., Furukawa, K., Nakamura, H., and Ohizumi, Y., Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells. Eur. J. Pharmacol., 413, 37–45 (2001). Lee, R. H., Slate, D. L., Moretti, R., Alvi, K. A., and Crews, P., Marine sponge polyketide inhibitors of protein tyrosine kinase. Biochem. Biophys. Res. Commun., 184, 765–772 (1992). Shen, Y.-C., Lo, K.-L., Kuo, Y.-H., and Chakraborty, R., Polycyclic quinones and hydroquinones, antitumor constituents from Taiwanese marine sponge Xestospongia sp. Chin. Pharm. J., 54, 207–213 (2002). Park, S. K., Oh, C., and Scheuer, P. J., The chemical constituents from the sponge Spongia sp. J. Kor. Chem. Soc., 39, 301–305 (1995). Concepcion, G. P., Foderaro, T. A., Eldredge, G. S., Lobkovsky, E., Clardy, J., Barrows, L. R., and Ireland, C. M., Topoisomerase II-mediated DNA cleavage by adocia- and xestoquinones from the Philippine sponge Xestospongia sp. J. Med. Chem., 38, 4503–4507 (1995). Alvi, K. A., Rodriguez, J., Diaz, M. C., Moretti, R., Wilhelm, R. S., Lee, R. H., Slate, D. L., and Crews, P., Protein tyrosine kinase inhibitory properties of planar polycyclics obtained from the marine sponge Xestospon-
13)
14)
15)
16)
17)
18)
19)
20)
gia cf. carbonaria and from total synthesis. J. Org. Chem., 58, 4871–4880 (1993). Bae, M. A., Tsuji, T., Kondo, K., Hirase, T., Ishibashi, M., Shigemori, H., and Kobayashi, J., Inhibition of mammalian topoisomerase I by xestoquinone and halenaquinone. Biosci. Biotechnol. Biochem., 57, 330–331 (1993). Sakamoto, H., Furukawa, K., Matsunaga, K., Nakamura, H., and Ohizumi, Y., Xestoquinone activates skeletal muscle actomyosin ATPase by modification of the specific sulfhydryl group in the myosin head probably distinct from sulfhydryl groups SH1 and SH2. Biochemistry, 34, 12570–12575 (1995). Nakamura, M., Kakuda, T., Oba, Y., Ojika, M., and Nakamura, H., Synthesis of biotinylated xestoquinone that retains inhibitory activity against Ca2þ ATPase of skeletal muscle myosin. Bioorg. Med. Chem., 11, 3077– 3082 (2003). Fabricant, R. N., De Larco, J. E., and Todaro, G., Nerve growth factor receptors on human melanoma cells in culture. Proc. Natl. Acad. Sci. U.S.A., 74, 565–569 (1977). Miyauchi, S., Moroyama, T., Kyoizumi, S., Asakawa, J., Okamoto, T., and Takada, K., Malignant tumor cell lines produce intereukin-1 like factor. In Vitro Cell. Dev. Biol., 24, 753–758 (1988). Michimukai, E., Kitamura, N., Zhang, Y., Wang, H., Hiraishi, Y., Sumi, K., Hayashido, Y., Toratani, S., Okamoto, T., and Takada, K., Mutations in the human homologue of the Drosophila segment polarity gene patched in oral squamous cell carcinoma cell lines. In Vitro Cell. Dev. Biol. Anim., 37, 459–464 (2001). Myoken, Y., Myoken, Y., Okamoto, T., Kan, M., Sato, J. D., and Takada, K., Release of fibroblast growth factor-1 by human squamous cell carcinoma correlates with autocrine cell growth. In Vitro Cell. Dev. Biol. Anim., 30A, 790–795 (1994). Sato, J. D., Kawamoto, T., and Okamoto, T., Cholesterol requirement of P3-X63-Ag8 and X63-Ag8.653 mouse myeloma cells for growth in vitro. J. Exp. Med., 165, 1761–1766 (1987). Zhang, Y., Wang, H., Toratani, S., Sato, J. D., Kan, M., McKeehan, W. L., and Okamoto, T., Growth inhibition by keratinocyte growth factor receptor of human salivary adenocarcinoma cells through induction of differentiation and apoptosis. Proc. Natl. Acad. Sci. U.S.A., 98, 11336–11340 (2001). Ojika, M., Suzuki, Y., Tsukamoto, A., Sakagami, Y., Fudou, R., Yoshimura, T., and Yamanaka, S., Cystothiazoles A and B, new bithiazole-type antibiotics from the myxobacterium Cystobacter fuscus. J. Antibiotics, 51, 275–281 (1998). Sauter, H., Steglich, W., and Anke, T., Strobilurins: Evolution of a new class of active substances. Angew. Chem. Int. Ed. Engl., 38, 1328–1349 (1999).
