Bioorganic & Medicinal Chemistry Letters 23 (2013) 6234–6238
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Anti-cancer evaluation of carboxamides of furano-sesquiterpene carboxylic acids from the soft coral Sinularia kavarattiensis Singanaboina Rajaram a, Udugu Ramulu a, Dasari Ramesh a, Dudem Srikanth b, Papri Bhattacharya a, Peddikotla Prabhakar a, Shasi V. Kalivendi b, Katragadda Suresh Babu a,⇑, Yenamandra Venkateswarlu a,⇑, Suryakiran Navath a,c,⇑ a b c
Division of Natural Products Chemistry, Toxicology Laboratory, CSIR-IICT, Hyderabad 500 007, India Division of Biology, Toxicology Laboratory, CSIR-IICT, Hyderabad 500 007, India Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States
a r t i c l e
i n f o
Article history: Received 27 August 2013 Revised 25 September 2013 Accepted 30 September 2013 Available online 10 October 2013 Keywords: Sinularia kavarattiensis Cytotoxic activity Furano-sesquiterpene carboxylic acid Semi-synthesis
a b s t r a c t The chemical investigation of soft coral Sinularia kavarattiensis is described. It yielded furano-sesquiterpene carboxylic acids 1 and 2 and their methyl esters 3 and 4. Semi-synthesis of furano-sesquiterpene carboxylic acid 1 gave amide derivatives 5–12. Structures of all the compounds were established by IR, NMR and mass spectral analysis. Interestingly all compounds are selectively potent on leukemia cell line. All these compounds were screened for cytotoxic activity against five human cancer cell lines (leukemia, prostate, lung, breast and cervix). Among these compounds 9 and 10 showed promising activity against leukemia and prostate cancer cell lines. Ó 2013 Elsevier Ltd. All rights reserved.
The furano-sesquiterpenes constitute a large and ever-growing class of natural products that have been derived from both terrestrial and marine organisms.1–4 Highly substituted furans are found as key structural elements in many bioactive natural products, pharmaceuticals, agrochemical, and other fine-chemicals.5,6 They also represent versatile building blocks for the synthesis of more elaborate heterocyclic compounds.7 The associated furan ring has been found linked with or fused to a wide range of terpenoid frameworks including the bisabolane, cadinane, eudesmane, elemane, eremophilane, germacrane, lindenane, and farnesane skeletons.8,9 These furano-sesquiterpenes have been found to possess antibacterial, antifungal, cytotoxic, antiinflammatory, antitubercular, antiviral, anti HIV, apoptosis induction, and, antileukemic activities.10,11 Furan ring containing compound Lapatinib is used in the treatment of breast cancer. Cancer is one of the leading causes of death worldwide. The discovery and development of active, selective, and less toxic compounds for the treatment of malignancy are one of the most important goals in medicinal chemistry. The understanding of the biology of cancer has improved to a great extent in recent years and has strongly impacted both experimental and clinical tumor therapy. Natural products are gaining importance and attention from chemists and pharmacologists to discover lead molecules ⇑ Corresponding authors. Tel.: +1 5206212396; fax: +1 5206218407 (S. Navath). E-mail address: [email protected] (S. Navath). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.09.093
due to their selectivity. Marine organisms have provided natural product chemists with a rich source of unusual secondary metabolites.12 Most of the medicinal and biological agents used on a world-wide basis are either natural products or modified natural products, for example, soft corals, sponges, tunicates, mollusks and bryozoans, and are currently in advanced preclinical evaluation.13 Irciformonin analogs (irciformonin B, F, 15-acetylirciformonin B, 10-acetylirciformonin B) are triterpenoid-derived metabolites that were isolated from the marine sponge Ircinia sp.14 Their chemical structures are similar, and they exhibit high cytotoxicity against a panel of cancer cell lines and K562, DLD-1, HepG2 and Hep3B cells, with IC50 values ranging from 0.03 to 10.2 lM.15 It appears that the furan moiety is highly implicated in likely responsible for their cytotoxic effects. Soft corals of the genus Sinularia (Alcyoniidae) have been well recognized as a rich source of furano-sesquiterpenes (Fig 1).16 Chemical or biochemical transformation of an active molecule is a tool to obtain a more active molecule than its natural counterpart. Commercially available or late stages of clinical trials today are of natural origin.17 As part of our investigations of bio-active compounds18–22 from marine organisms, we describe here the isolation of compounds 1–3 from the soft coral Sinularia kavarattiensis and the semi-synthesis of a series of analogues of compound 1 with moderate to potent cytotoxic activity against a panel of human cancer cell lines. All the derivatives were screened for cancer activity against five human cancer cell lines (leukemia, prostate, lung, breast and cervix). It
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O
O
HO
HO O
O
1
O
O
2
O
O
O
O 3 R
O
4
N R O 5-12
Figure 1. Furano-sesquiterpene carboxylic acids and their amides.
O
O OH + R-NH-R O
HATU, DIPEA THF, 0 0C-RT, 3h
1
R N R
O 5-12
Scheme 1. Semi-synthesis of furano-sesquiterpene carboxamides 5–12.
was observed that most of the derivatives displayed significant activity as compared to their parent compound. The structures of the target compounds were established by IR, 1H NMR, 13C NMR and mass spectral analysis. The soft coral Sinularia kavarattiensis (IIC-1008) was collected at a depth of 30 feet by SCUBA diving near the Mandapam Coast (9° 160 N, 79° 120 E), Tamilnadu, India. Freshly collected soft coral were placed in ethyl acetate at the site of collection until workup. The initial ethyl acetate from the soft coral specimens (1.5 kg wet weight) was removed and re-extracted with 1:1 dichloromethane–ethyl acetate at room temperature. The combined extract including initial ethyl acetate extract was concentrated under reduced pressure to a predominantly aqueous extract which was extracted into ethyl acetate. The crude ethyl acetate extract of the soft coral Sinularia kavarattiensis was subjected to gel filtration chromatography on Sephadex LH-20 using 1:1 dichloromethane– ethyl acetate, followed by silica gel chromatography eluting with hexane through hexane–ethyl acetate mixtures to ethyl acetate to afford compounds 1–4 in good quantities. Compound 1 is a white solid, non-hygroscopic and stable at room temperature. From the forgoing spectral data the structure of compound 1 was established as 5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3carboxylic acid, a known furano-sesquiterpene carboxylic acid, previously isolated from the Australian soft coral Sinularia gonatodes.23 It was found to inactivate bee venom phospholipase A2 (bvPLA2), a promising target for the development of anti-inflammatory agents, with an IC50 of 0.5 mM.24 Several furano-sesquiterpenes, including methylfurans, furan methyl esters, and furan carboxylic acids 2–4, were isolated from the Australian soft coral Sinularia capillosa.25 We found that there is more compound 1 in Sinularia kavarattiensis. Recently, the bio-evaluation of its geometrical isomer (compound 4)26,27 which induced apoptosis via the mitochondrial-mediated caspase dependent pathway in THP-1, leukemia cell line has been reported. These results prompted us to derivatize compound 1. There are no reports of the synthesis or semi-synthetic derivatives of compound 1, but, its geometrical isomer (compound 2) was synthesized.28 Amide containing natural
and synthetic compounds display a broad range of pharmacological activities.29,30 However, it is interesting to observe that in the last decade, a number of piperazine31–33 derivatives have been synthesized to exploit their chemotherapeutic potential. Hence the present work has been taken up. The isolation of crude ethyl acetate extract of the soft coral Sinularia kavarattiensis was subjected to gel filtration chromatography on Sephadex LH-20 using 1:1 dichloromethane–methanol, followed by silica gel chromatography eluting with hexane through hexane–ethyl acetate mixtures to ethyl acetate to afford compounds 1–4.34–39 Furano-sesquiterpene carboxylic acid 1, was coupled with different amines HATU (O-(7-azabenzotriazol1-yl)-N,N,N0 ,N0 -tetramethyluronium hexafluorophosphate) and DIPEA (N,N-diisopropylethylamine) in THF for 4 h at room temperature to yield amides followed by workup and purification by silica gel column chromatography,40 gave the corresponding amides as shown in Scheme 1. All reactions were clean and obtained near quantitative yields. The synthesized compounds were well characterized by spectral data41–48 and documented in Table 1. Furano-sesquiterpene carboxylic acids and their corresponding amide derivatives were tested for anticancer activity in vitro against five human cancer cell lines: DU145 (human prostate cancer), A549 (human epithelial lung carcinoma), HeLa (human epithelial cervical cancer), MCF-7 (human breast adenocarcinoma) and THP 1 (human monocytic leukemia). The inhibition concentrations (IC50 values) are summarized in Table 2.49,50 As is evident from Table 2, all derivatives of furano-sesquiterpenes showed better activity against all the four cell lines relative to the furano-sesquiterpene carboxylic acid. Compound 3 is only half active as its geometrical isomer on THP-1 but doubly active on HeLa cell lines. Analysis of MTT assay results with the furanosesquiterpenes that 9 and 10 are the most potent against the THP1 and DU145 cell lines. Compound 10 also inhibits A549, MCF7 and HeLa cell lines growth, whereas 6 inhibits growth of the DU145 cell line. Except for 8 and 11 all furano-sesquiterpenes shows good activity against the THP1 cell line. It is interesting that in general the compounds prepared from piperazine analogues 5–
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Table 1 Semi-synthesis of furano-sesquiterpene carboxamides 5–14 S. No.
Productb
R-NH-R
HN
Yielda (%)
O
N
N
5
86
N
O
O
N
HN
N
6
88
N
O HN
O
N
N
7
HN
O
N
N
8
91
N
O
HN
93
N
O
O
N
O
N
9
O
N
O
84
O
O O
HN 10
N
N
N
NH 2
O N H
11
O
H 2N
O N H
O b
90
O
NH 2
12
a
88
O
N H
85
O
Isolated yields after column chromatography. Products were characterized by NMR and mass spectral data.
Table 2 Cytotoxicities of furano-sesquiterpene with prostate, lung, cervix, breast, and leukemiaa S. No.
DU145 (IC50 in lM)
A549 (IC50 in lM)
HeLa (IC50 in lM)
Mcf7 (IC50 in lM)
THP1 (IC50 in lM)
1 2 3 4 5 6 7 8 9 10 11 12
60.1 ± 1.9 38.9 ± 2.7 59.3 ± 2.3 25.1 ± 1.6 24.1 ± 1.7 20.4 ± 2.1 30.3 ± 2.6 45.3 ± 3.1 17.9 ± 3.1 16.8 ± 4.3 56.5 ± 1.6 41.3 ± 3.6
89.3 ± 3.4 89.7 ± 2.6 89.6 ± 2.6 28.6 ± 1.7 23.6 ± 1.8 27.9 ± 2.4 28.3 ± 1.9 50.7 ± 2.4 45.5 ± 2.4 24.6 ± 3.1 63.2 ± 2 69.7 ± 1.6
81 ± 2.6 80.4 ± 3.5 59.4 ± 3.4 35.5 ± 2.5 30.5 ± 2.4 35.9 ± 1.8 32.1 ± 3.6 59.4 ± 2.7 44.2 ± 1.8 28.5 ± 1.5 59.5 ± 2.7 73.4 ± 3.1
61.9 ± 1.5 74.5 ± 1.6 75.4 ± 1.9 28.9 ± 3.8 27.9 ± 3.5 30.3 ± 2.5 39.8 ± 2.8 53.7 ± 3.5 50.8 ± 2.1 26.7 ± 2.8 65.3 ± 1.7 57.7 ± 1.9
56.6 ± 3.1 39.8 ± 2.1 51.4 ± 2.4 25.8 ± 4.5 20.8 ± 4.2 26.5 ± 2.4 28.6 ± 2.8 50.9 ± 2.1 15.9 ± 2.8 17.8 ± 1.6 60.4 ± 1.6 59 ± 2.8
a Cell lines were treated with different concentrations of compounds for 48 h as described under Materials and methods. Cell viability was measured employing MTT assay. IC50 values are indicated as the mean ± SD of three independent experiments. NA denotes activity >100 lM.
10 are the potential impact in improving the activity compared to aromatic and aliphatic amines 11 and 12. Among all of the furano-sesquiterpenes tested, (4-(benzo[d][1,3]dioxol-5ylmethyl)piperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)
furan-3-yl)methanone 9 inhibited the growth of the THP1 and DU145 cell lines most effectively, with an IC50 values of 15.9 and 17.9 lM, respectively. On the other hand (4-benzhydrylpiperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-yl)meth-
S. Rajaram et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6234–6238
anone (10) inhibited the growth of the THP1 and DU145 cell lines effectively, with IC50 values of 17.5 and 16.8 lM, respectively. In conclusion, isolation of furano-sesquiterpene carboxylic acids 1 and 2 and their methyl esters 3 and 4 from soft coral Sinularia kavarattiensis and semi-synthesis of a series of new amide derivatives of compound 1 and bio-evaluation of their anticancer activity in vitro against five human cancer cell lines (DU-145, A-549, HeLa, MCF-7 and THP 1) was carried out. Among these compounds 9 and 10 showed promising anticancer activity against leukemia and lung cancer. Interestingly all compounds are selectively potent in leukemia. This has laid a solid foundation for further optimization of this class of compounds by a systematic refinement including the synthesis of compounds to improve their overall pharmaceutical properties. Acknowledgements The authors thank to MoES and SMiLE, CSIR, New Delhi, India, for financial assistance and the Director, Indian Institute of Chemical Technology (IICT) for his constant encouragement and Dr. R. Bates, University of Arizona, for editing this manuscript. References and notes 1. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2011, 28, 196–268. 2. Gaspar, H.; Gavagnin, M.; Calado, G.; Castelluccio, F.; Mollo, E.; Cimino, G. Tetrahedron 2005, 61, 11032–11037. 3. Banerjee, R.; Kumar, H. K. S.; Banerjee, M. Int. J. Rev. Life Sci. 2012, 2, 7–16. 4. Held, C.; Fröhlich, R.; Metz, P. Angew. Chem., Int. Ed. 2001, 40, 1058–1060. 5. Metz, P.; Stölting, J.; Läge, M.; Krebs, B. Angew. Chem., Int. Ed. Engl. 1994, 33, 2195–2197. 6. Hou, X. L.; Cheung, H. Y.; Hon, T. Y.; Kwan, P. L.; Lo, T. H.; Tong, S. Y. T.; Wong, H. N. C. Tetrahedron 1998, 54, 1955–2020. 7. Lipshutz, B. H. Chem. Rev. 1986, 86, 795. 8. Fraga, B. M. Nat. Prod. Rep. 2005, 22, 465–486. 9. Allen, A. J.; Vaillancourt, V.; Albizati, K. F. Org. Prep. Proced. Int. 1994, 26, 1–84. 10. Kamel, H. N.; Slattery, M. Pharm. Biol. 2005, 43, 253–269. 11. Cheng, S. Y.; Huang, K. J.; Wang, S. K.; Wen, Z. H.; Chen, P. W.; Duh, C. Y. J. Nat. Prod. 2010, 73, 771–775. 12. Faulkner, D. J. Nat. Prod. Rep. 2001, 18, 1–49. 13. Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P.; Gerwick, W. H. Mol. Cancer Ther. 2005, 4, 333–342. 14. Shen, Y.-C.; Lo, K.-L.; Lin, Y.-C.; Khalil, A. T.; Kuo, Y.-H.; Shih, P.-S. Tetrahedron Lett. 2006, 47, 4007–4010. 15. Su, J. H.; Tseng, S. W.; Lu, M. C.; Liu, L. L.; Chou, Y.; Sung, P. J. J. Nat. Prod. 2011, 74, 2005–2009. 16. Lakshmi, V.; Kumar, R. Nat. Prod. Res. 2009, 23, 801–850. 17. Cragg, G. N.; Newmann, D. J.; Snades, K. M. J. Nat. Prod. 1997, 60, 52–60. 18. Reddy, V. R. M.; Rama Rao, M.; Rhodes, D.; Hansen, M. S. T.; Rubins, K.; Bushman, F. D.; Venkateswarlu, Y.; Faulkner, D. J. J. Med. Chem. 1999, 42, 1901–1907. 19. Reddy, T. S.; Suryakiran, N.; Narasimhulu, M.; Ramesh, D.; Mahesh, K. C.; Sai Krishna, A.; Kavitha, P.; Rao, J. V.; Venkateswarlu, Y. Bioorg. Med. Chem. Lett. 2012, 22, 4900–4906. 20. Ramesh, P.; Reddy, N. S.; Venkateswarlu, Y. Tetrahedron Lett. 1998, 39, 8217– 8220. 21. Venkateswarlu, Y.; Sridevi, K. V.; Rama Rao, M. J. Nat. Prod. 1999, 62, 756–758. 22. Ravinder, K.; Vijender Reddy, A.; Krishnaiah, P.; Ramesh, P.; Ramakrishna, S.; Laatsch, H.; Venkateswarlu, Y. Tetrahedron Lett. 2005, 46, 5475–5478. 23. Coll, J. C.; Mitchell, S. J.; Stokie, G. J. Tetrahedron Lett. 1977, 18, 1539–1542. 24. Grace, K. J. S.; Zavortink, D.; Jacobs, R. S. Biochem. Pharmacol. 1994, 47, 1427– 1434. 25. Bowden, B. F.; de Coll, J. C.; de Silva, E. D.; Costa, M. S. L.; Djura, P. J.; Mahendran, M.; Tapiolas, D. M. Aust. J. Chem. 1983, 36, 371–376. 26. Arepalli, S. K.; Sridhar, V.; Rao, J. V.; Kennady, P. K.; Venkateswarlu, Y. Apoptosis 2009, 14, 729–740. 27. Limna Mol, V. P.; Raveendran, T. V.; Parameswaran, P. S.; Kunnath, R. J.; Sathyan, N. Ind. J. Mar. Sci. 2010, 39, 270–273. 28. Williams, D. H.; Faulkner, D. J. Tetrahedron 1996, 52, 4245–4256. 29. Fang, G.; Xue, M.; Su, M.; Hu, D.; Li, Y.; Xiong, B.; Maa, L.; Meng, T.; Chen, Y.; Li, J.; Li, J.; Shen, J. Bioorg. Med. Chem. Lett. 2012, 22, 4540–4545. 30. Burkhard, J. A.; Wagner, B.; Fischer, H.; Schuler, F.; Muller, K.; Carreira, E. M. Angew. Chem., Int. Ed. 2010, 49, 3524–3527. 31. Hatnapure, D. G.; Keche, A. P.; Rodge, A. H.; Birajdar, S. S.; Tale, R. H.; Kamble, V. M. Bioorg. Med. Chem. Lett. 2012, 22, 6385–6390. 32. Song, J.; Lee, H.; Kim, Y. J.; Kim, S. Y.; Kim, D.; Min, K. H. Bioorg. Med. Chem. Lett. 2012, 22, 6943–6946.
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33. Dong, M.; Lu, L.; Li, H.; Wang, X.; Lu, H.; Jiang, S.; Dai, Q. Bioorg. Med. Chem. Lett. 2012, 22, 3284–3286. 34. Freshly collected soft coral were placed in ethyl acetate at the site of collection until workup. The initial ethyl acetate from the soft coral specimens (1.5 kg wet weight) was removed and re-extracted with 1:1 dichloromethane–ethyl acetate (3 2 L) at room temperature. The combined extract including initial ethyl acetate extract was concentrated under reduced pressure to a predominantly aqueous extract which was extracted into ethyl acetate (3 500 mL). The crude ethyl acetate extract of the soft coral Sinularia kavarattiensis was subjected to gel filtration chromatography on Sephadex LH 20 using 1:1 dichloromethane–ethyl acetate, followed by silica gel chromatography eluting with hexane through hexane–ethyl acetate mixtures to ethyl acetate to afford compounds 1–4 in good quantities. Compounds 1–4 were identified as a known furano-sesquiterpenoid carboxylic acids by their spectral data. 35. 5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-carboxylic acid (1): Yielded 500 mg; 1H NMR (300 MHz, CDCl3) d: 7.92 (1H, s), 6.70 (1H, dd, J = 10.5 and 16.5 Hz), 6.45 (1H, s), 6.01 (1H, br s), 5.30 (1H, t, J = 6.2 Hz), 5.16 (1H, d, J = 16.5 Hz), 5.05 (1H, d, J = 10.5 Hz), 2.19–2.39 (4H, m), 1.94 (3H, s), 1.78 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.6, 146.8, 141.0, 140.7, 133.4, 129.7, 120.1, 113.8, 113.5, 106.6, 40.7, 25.9, 19.7, 18.7. 36. 5-((1E,5E)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-carboxylic acid (2): Yielded 50 mg; 1H NMR (300 MHz, CDCl3) d: 7.95 (1H, s), 6.71 (1H, dd, J = 10.5 and 16.5 Hz), 6.40 (1H, s), 6.01 (1H, br s), 5.35 (1H, t, J = 7.1 Hz), 5.26 (1H, d, J = 17.0 Hz), 5.05 (1H, d, J = 11.0 Hz), 2.19–2.39 (4H, m), 1.95 (3H, s), 1.76 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.5, 146.8, 141.1, 140.7, 133.5, 129.7, 120.1, 113.9, 113.5, 106.7, 40.7, 25.9, 19.5, 18.5. 37. Methyl 5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-carboxylate (3): Yielded 300 mg; 1H NMR (300 MHz, CDCl3) d: 7.92 (1H, s), 6.72 (1H, dd, J = 10.5 and 16.5 Hz), 6.46 (1H, s), 6.01 (1H, br s), 5.32 (1H, t, J = 6.2 Hz), 5.16 (1H, d, J = 16.5 Hz), 5.05 (1H, d, J = 10.5 Hz), 4.1 (s, 3H), 2.19–2.39 (4H, m), 1.94 (3H, s), 1.78 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.6, 146.8, 141.0, 140.7, 133.4, 129.7, 120.1, 113.8, 113.5, 106.6, 51.5, 40.7, 25.9, 19.7, 18.7. 38. Methyl 5-((1E,5E)-2,6-dimethylocta-1,5,7-trienyl)furan-3-carboxylate (4): Yielded 95 mg; 1H NMR (300 MHz, CDCl3) d: 7.95 (1H, s), 6.71 (1H, dd, J = 10.5 and 16.5 Hz), 6.40 (1H, s), 6.01 (1H, br s), 5.35 (1H, t, J = 7.1 Hz), 5.26 (1H, d, J = 17.0 Hz), 5.05 (1H, d, J = 11.0 Hz), 4.1 (s, 3H), 2.19–2.39 (4H, m), 1.95 (3H, s), 1.76 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.5, 146.8, 141.1, 140.7, 133.5, 129.3, 120.2, 113.8, 113.5, 106.6, 51.5, 40.9, 25.9, 19.6, 18.6. 39. Sinularia kavarattiensis (Specimen No. IIC-1008) was taxonomically identified by Dr. P.A. Thomas of CFMRI, Trivandrum, India, was collected by hand using scuba off the coast of Mandapam, Tamilnadu, India, in April 2011, at a depth of 5–10 m. A voucher sample was deposited at the NIO Goa. 40. Typical experimental procedure for the preparation of amide derivatives of furanosesquiterpene acid: To a solution of furano-sesquiterpene carboxylic acid (1 mmol) in THF was added HATU (1.5 mmol) and DIPEA (2.5 mmol) at 0 °C, stirred at same temp for 30 min, then amine (1.5 mmol) was added. The mixture was stirred at rt for 4 h. After completion of the reaction, the reaction mixture was poured into ice water and washed with ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel (60–120 mesh) to give the corresponding amide derivatives 5–12. (Compound 12 prepared by using the amine (0.5 mmol)). 41. (5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-yl)(4-methylpiperazin-1-yl) methanone (5): 1H NMR (300 MHz, CDCl3) d: 7.52 (1H, s), 6.70 (1H, dd, J = 10.7 and 17.2 Hz), 6.23 (1H, s), 5.98 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.15 (1H, d, J = 17.2 Hz), 5.03 (1H, d, J = 10.7 Hz), 3.64–3.76 (4H, m), 2.50–2.39 (4H, m), 2.34–2.25 (5H, m), 2.12–2.20 (2H, m), 1.91 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 141.2 (2C), 133.4, 132.7, 129.7, 121.8, 113.7, 113.4, 107.1, 54.5 (4C), 45.4, 40.6, 25.7, 19.7, 18.6. ESI-MS (m/z) 329 [M+H]+. 42. (5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-yl)(4-ethylpiperazin-1-yl) methanone (6): 1H NMR (300 MHz, CDCl3) d: 7.51 (1H, s), 6.70 (1H, dd, J = 10.7 and 17.2 Hz), 6.24 (1H, s), 5.99 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.14 (1H, d, J = 17.2 Hz), 5.03 (1H, d, J = 10.7 Hz), 3.73–3.63 (4H, m), 2.51–2.39 (6H, m), 2.35–2.10 (4H, m), 1.91 (3H, s), 1.75 (3H, s), 1.06 (3H, t, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 141.2, 140.3, 133.5, 132.8, 129.7, 122.1, 113.7, 113.5, 107.2, 52.2 (4C), 40.7, 38.6, 25.8, 19.7, 18.6, 11.4. ESI-MS (m/z) 343 [M+H]+. 43. (5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-yl)(4-phenylpiperazin-1-yl) methanone (7): 1H NMR (300 MHz, CDCl3) d: 7.55 (1H, s), 7.27–7.18 (3H, m), 6.96–6.82 (2H, m), 6.70 (1H, dd, J = 10.2 and 17.2 Hz), 6.26 (1H, s), 6.00 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.15 (1H, d, J = 17.2 Hz), 5.04 (1H, d, J = 10.2 Hz), 3.86– 3.75 (4H, m), 3.19–3.09 (4H, m), 2.36–2.23 (2H, m), 2.21–2.12 (2H, m), 1.92 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 150.7, 141.2, 140.4, 133.4, 132.8, 129.7, 129.3 (2C), 121.9, 120.7, 116.7 (2C), 113.7, 113.5, 107.3, 49.7 (4C), 40.6, 25.8, 19.7, 18.6. ESI-MS (m/z) 391 [M+H]+. 44. (4-Benzylpiperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-yl) methanone (8): 1H NMR (300 MHz, CDCl3) d: 7.53 (1H, s), 7.41–7.20 (5H, m), 6.70 (1H, dd, J = 10.2 and 17.2 Hz), 6.23 (1H, s), 5.99 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.14 (1H, d, J = 17.2 Hz), 5.04 (1H, d, J = 10.2 Hz), 3.86–3.75 (4H, m), 3.51 (2H, s), 3.19–3.09 (4H, m), 2.36–2.12 (4H, m), 1.92 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 141.2, 140.4, 137.4, 133.4, 132.8, 129.7, 128.6 (2C), 127.7 (2C), 126.4, 120.7, 113.7, 113.5, 107.3, 63.1, 49.7 (4C), 40.6, 25.8, 19.7, 18.6. ESI-MS (m/z) 405 [M+H]+. 45. (4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1, 5,7-trienyl)furan-3-yl)methanone (9): 1H NMR (300 MHz, CDCl3) d: 7.49 (1H, s),
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7.19 (1H, s), 6.79 (1H, s), 6.68 (2H, s), 6.22 (1H, s), 5.97 (1H, s), 5.88 (2H, s), 5.31 (1H, t, J = 7.6 Hz), 5.14 (1H, d, J = 6.0 Hz), 5.00 (1H, d, J = 11.3 Hz), 3.70–3.59 (4H, m), 3.42 (2H, s), 2.47–2.34 (4H, m), 2.32–2.10 (4H, m), 1.98 (3H, s), 1.74 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.8, 147.7, 146.9, 141.3, 141.1, 133.4, 131.7, 130.6, 129.7, 122.4, 113.7, 113.5, 110.8, 109.4, 107.9, 107.3, 100.9, 62.4, 52.7 (4C), 40.6, 25.8, 19.7, 18.6. ESI-MS (m/z) 449 [M+H]+. 46. (4-Benzhydrylpiperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-yl) methanone (10): 1H NMR (300 MHz, CDCl3) d: 7.46 (1H, s), 7.38–7.30 (4H, m), 7.26–7.08 (6H, m), 6.69 (1H, dd, J = 10.7 and 17.3 Hz), 6.20 (1H, s), 5.95 (1H, s), 5.29 (1H, t, J = 7.17 Hz), 5.13 (1H, d, J = 17.3 Hz), 5.02 (1H, d, J = 10.7 Hz), 4.18 (1H, s), 3.71–3.54 (4H, m), 2.41–2.20 (6H, m), 2.18–2.09 (2H, m), 1.87 (3H, s), 1.73 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.8, 153.7, 142.0 (2C), 141.1, 140.2, 133.4 (2C), 129.7, 128.6 (4C), 127.8 (4C), 127.2 (2C), 117.8, 113.7, 113.5, 107.3, 75.9, 51.9 (4C), 40.64, 25.7, 19.7, 18.6. ESI-MS (m/z) 481 [M+H]+. 47. 5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)-N-p-tolylfuran-3-carboxamide (11): 1 H NMR (300 MHz, CDCl3) d: 7.84 (1H, s), 7.45 (1H, br s), 7.40 (2H, d, J = 8.309 Hz), 7.07 (2H, d, J = 8.309), 6.71 (1H, dd, J = 10.3 and 17.3 Hz), 6.40 (1H, s), 5.99 (1H, s), 5.31 (1H, t, J = 6.9 Hz), 5.15 (1H, d, J = 17.3 Hz), 5.04 (1H, d, J = 10.3 Hz), 2.33–2.10 (7H, m), 1.92 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 165.9, 154.8, 142.7, 140.7, 135.0, 134.1, 133.4, 132.8, 129.7, 129.5 (2C), 122.4, 120.3 (2C), 113.7, 113.5, 105.3, 40.6, 25.7, 20.8, 19.7, 18.7. ESI-MS (m/z) 336 [M+H]+. 48. N,N0 -(Propane-1,3-diyl)bis(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-carboxamide) (12): 1H NMR (300 MHz, CDCl3) d: 7.82 (1H, s), 7.19 (1H, s), 6.71 (1H, dd, J = 11.3 and 17.3 Hz), 6.40 (1H, s), 6.0 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.14 (1H, d, J = 17.3 Hz), 5.04 (1H, d, J = 11.3 Hz), 3.47–3.34 (4H, m), 2.35–2.23 (2H, m), 2.21–2.08 (2H, m), 1.92 (3H, s), 1.74 (3H, s), 1.71–1.59 (2H, m). 13C NMR (75 MHz, CDCl3) d: 163.6 (2C), 154.7 (2C), 142.6 (2C), 140.4, 133.5, 132.8,
129.7, 123.6, 113.7 (4C), 105.4 (2C), 40.7 (2C), 38.6 (4C), 35.7 (2C), 25.8 (2C), 19.7 (2C), 18.7 (2C). ESI-MS (m/z) 531 [M+H]+. 49. All cell lines used in this study were purchased from the American Type Culture Collection (ATCC). A549 (human lung carcinoma epithelial), and HeLa (human epithelial cervical cancer) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing non essential amino acids and 10% FBS. MCF-7 (human breast adenocarcinoma), DU 145 (human prostate carcinoma epithelial) cells were cultured in Eagle’s minimal essential medium (MEM) containing nonessential amino acids, 1 mM sodium pyruvate, 10 mg/mL bovine insulin, and 10% FBS. THP 1 (human monocytic leukemia) cells were cultured in RPMI-1640 containing 0.05 2-mercaptoethanol and 10% FBS. All cell lines maintained in humidified atmosphere of 5% CO2 at 37 °C. Cells were trypsinized when subconfluent from T75 flasks/90 mm dishes and seeded in 96 well plates at a concentration of 1 104 cells/mL in complete medium, treated with compounds at desired concentrations for 48 h, and harvested as required. 50. This assay is a quantitative colorimetric method for determination of cell survival and proliferation. The assessed parameter is the metabolic activity of viable cells. Metabolically active cells reduce pale yellow tetrazolium salt (MTT) to a dark blue water-insoluble formazan, which can be directly quantified after solubilisation with DMSO. The absorbance of the formazan directly correlates with the number of viable cells. The cells were plated in 96-well plates at a density of 1 104 cells in 200 lL of medium per well of 96-well plate. Cultures were incubated with different concentrations of test compounds and incubated for 48 h. The assay was terminated with the addition of 100 lg/mL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 1 h. The supernatant was aspirated and plates were air dried. MTT-formazon crystals dissolved in 100 lL DMSO. The optical density (O.D.) was measured at 570 nm using TECAN multi mode reader.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Anti-cancer evaluation of carboxamides of furano-sesquiterpene carboxylic acids from the soft coral Sinularia kavarattiensis Singanaboina Rajaram a, Udugu Ramulu a, Dasari Ramesh a, Dudem Srikanth b, Papri Bhattacharya a, Peddikotla Prabhakar a, Shasi V. Kalivendi b, Katragadda Suresh Babu a,⇑, Yenamandra Venkateswarlu a,⇑, Suryakiran Navath a,c,⇑ a b c
Division of Natural Products Chemistry, Toxicology Laboratory, CSIR-IICT, Hyderabad 500 007, India Division of Biology, Toxicology Laboratory, CSIR-IICT, Hyderabad 500 007, India Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States
a r t i c l e
i n f o
Article history: Received 27 August 2013 Revised 25 September 2013 Accepted 30 September 2013 Available online 10 October 2013 Keywords: Sinularia kavarattiensis Cytotoxic activity Furano-sesquiterpene carboxylic acid Semi-synthesis
a b s t r a c t The chemical investigation of soft coral Sinularia kavarattiensis is described. It yielded furano-sesquiterpene carboxylic acids 1 and 2 and their methyl esters 3 and 4. Semi-synthesis of furano-sesquiterpene carboxylic acid 1 gave amide derivatives 5–12. Structures of all the compounds were established by IR, NMR and mass spectral analysis. Interestingly all compounds are selectively potent on leukemia cell line. All these compounds were screened for cytotoxic activity against five human cancer cell lines (leukemia, prostate, lung, breast and cervix). Among these compounds 9 and 10 showed promising activity against leukemia and prostate cancer cell lines. Ó 2013 Elsevier Ltd. All rights reserved.
The furano-sesquiterpenes constitute a large and ever-growing class of natural products that have been derived from both terrestrial and marine organisms.1–4 Highly substituted furans are found as key structural elements in many bioactive natural products, pharmaceuticals, agrochemical, and other fine-chemicals.5,6 They also represent versatile building blocks for the synthesis of more elaborate heterocyclic compounds.7 The associated furan ring has been found linked with or fused to a wide range of terpenoid frameworks including the bisabolane, cadinane, eudesmane, elemane, eremophilane, germacrane, lindenane, and farnesane skeletons.8,9 These furano-sesquiterpenes have been found to possess antibacterial, antifungal, cytotoxic, antiinflammatory, antitubercular, antiviral, anti HIV, apoptosis induction, and, antileukemic activities.10,11 Furan ring containing compound Lapatinib is used in the treatment of breast cancer. Cancer is one of the leading causes of death worldwide. The discovery and development of active, selective, and less toxic compounds for the treatment of malignancy are one of the most important goals in medicinal chemistry. The understanding of the biology of cancer has improved to a great extent in recent years and has strongly impacted both experimental and clinical tumor therapy. Natural products are gaining importance and attention from chemists and pharmacologists to discover lead molecules ⇑ Corresponding authors. Tel.: +1 5206212396; fax: +1 5206218407 (S. Navath). E-mail address: [email protected] (S. Navath). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.09.093
due to their selectivity. Marine organisms have provided natural product chemists with a rich source of unusual secondary metabolites.12 Most of the medicinal and biological agents used on a world-wide basis are either natural products or modified natural products, for example, soft corals, sponges, tunicates, mollusks and bryozoans, and are currently in advanced preclinical evaluation.13 Irciformonin analogs (irciformonin B, F, 15-acetylirciformonin B, 10-acetylirciformonin B) are triterpenoid-derived metabolites that were isolated from the marine sponge Ircinia sp.14 Their chemical structures are similar, and they exhibit high cytotoxicity against a panel of cancer cell lines and K562, DLD-1, HepG2 and Hep3B cells, with IC50 values ranging from 0.03 to 10.2 lM.15 It appears that the furan moiety is highly implicated in likely responsible for their cytotoxic effects. Soft corals of the genus Sinularia (Alcyoniidae) have been well recognized as a rich source of furano-sesquiterpenes (Fig 1).16 Chemical or biochemical transformation of an active molecule is a tool to obtain a more active molecule than its natural counterpart. Commercially available or late stages of clinical trials today are of natural origin.17 As part of our investigations of bio-active compounds18–22 from marine organisms, we describe here the isolation of compounds 1–3 from the soft coral Sinularia kavarattiensis and the semi-synthesis of a series of analogues of compound 1 with moderate to potent cytotoxic activity against a panel of human cancer cell lines. All the derivatives were screened for cancer activity against five human cancer cell lines (leukemia, prostate, lung, breast and cervix). It
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O
O
HO
HO O
O
1
O
O
2
O
O
O
O 3 R
O
4
N R O 5-12
Figure 1. Furano-sesquiterpene carboxylic acids and their amides.
O
O OH + R-NH-R O
HATU, DIPEA THF, 0 0C-RT, 3h
1
R N R
O 5-12
Scheme 1. Semi-synthesis of furano-sesquiterpene carboxamides 5–12.
was observed that most of the derivatives displayed significant activity as compared to their parent compound. The structures of the target compounds were established by IR, 1H NMR, 13C NMR and mass spectral analysis. The soft coral Sinularia kavarattiensis (IIC-1008) was collected at a depth of 30 feet by SCUBA diving near the Mandapam Coast (9° 160 N, 79° 120 E), Tamilnadu, India. Freshly collected soft coral were placed in ethyl acetate at the site of collection until workup. The initial ethyl acetate from the soft coral specimens (1.5 kg wet weight) was removed and re-extracted with 1:1 dichloromethane–ethyl acetate at room temperature. The combined extract including initial ethyl acetate extract was concentrated under reduced pressure to a predominantly aqueous extract which was extracted into ethyl acetate. The crude ethyl acetate extract of the soft coral Sinularia kavarattiensis was subjected to gel filtration chromatography on Sephadex LH-20 using 1:1 dichloromethane– ethyl acetate, followed by silica gel chromatography eluting with hexane through hexane–ethyl acetate mixtures to ethyl acetate to afford compounds 1–4 in good quantities. Compound 1 is a white solid, non-hygroscopic and stable at room temperature. From the forgoing spectral data the structure of compound 1 was established as 5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3carboxylic acid, a known furano-sesquiterpene carboxylic acid, previously isolated from the Australian soft coral Sinularia gonatodes.23 It was found to inactivate bee venom phospholipase A2 (bvPLA2), a promising target for the development of anti-inflammatory agents, with an IC50 of 0.5 mM.24 Several furano-sesquiterpenes, including methylfurans, furan methyl esters, and furan carboxylic acids 2–4, were isolated from the Australian soft coral Sinularia capillosa.25 We found that there is more compound 1 in Sinularia kavarattiensis. Recently, the bio-evaluation of its geometrical isomer (compound 4)26,27 which induced apoptosis via the mitochondrial-mediated caspase dependent pathway in THP-1, leukemia cell line has been reported. These results prompted us to derivatize compound 1. There are no reports of the synthesis or semi-synthetic derivatives of compound 1, but, its geometrical isomer (compound 2) was synthesized.28 Amide containing natural
and synthetic compounds display a broad range of pharmacological activities.29,30 However, it is interesting to observe that in the last decade, a number of piperazine31–33 derivatives have been synthesized to exploit their chemotherapeutic potential. Hence the present work has been taken up. The isolation of crude ethyl acetate extract of the soft coral Sinularia kavarattiensis was subjected to gel filtration chromatography on Sephadex LH-20 using 1:1 dichloromethane–methanol, followed by silica gel chromatography eluting with hexane through hexane–ethyl acetate mixtures to ethyl acetate to afford compounds 1–4.34–39 Furano-sesquiterpene carboxylic acid 1, was coupled with different amines HATU (O-(7-azabenzotriazol1-yl)-N,N,N0 ,N0 -tetramethyluronium hexafluorophosphate) and DIPEA (N,N-diisopropylethylamine) in THF for 4 h at room temperature to yield amides followed by workup and purification by silica gel column chromatography,40 gave the corresponding amides as shown in Scheme 1. All reactions were clean and obtained near quantitative yields. The synthesized compounds were well characterized by spectral data41–48 and documented in Table 1. Furano-sesquiterpene carboxylic acids and their corresponding amide derivatives were tested for anticancer activity in vitro against five human cancer cell lines: DU145 (human prostate cancer), A549 (human epithelial lung carcinoma), HeLa (human epithelial cervical cancer), MCF-7 (human breast adenocarcinoma) and THP 1 (human monocytic leukemia). The inhibition concentrations (IC50 values) are summarized in Table 2.49,50 As is evident from Table 2, all derivatives of furano-sesquiterpenes showed better activity against all the four cell lines relative to the furano-sesquiterpene carboxylic acid. Compound 3 is only half active as its geometrical isomer on THP-1 but doubly active on HeLa cell lines. Analysis of MTT assay results with the furanosesquiterpenes that 9 and 10 are the most potent against the THP1 and DU145 cell lines. Compound 10 also inhibits A549, MCF7 and HeLa cell lines growth, whereas 6 inhibits growth of the DU145 cell line. Except for 8 and 11 all furano-sesquiterpenes shows good activity against the THP1 cell line. It is interesting that in general the compounds prepared from piperazine analogues 5–
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Table 1 Semi-synthesis of furano-sesquiterpene carboxamides 5–14 S. No.
Productb
R-NH-R
HN
Yielda (%)
O
N
N
5
86
N
O
O
N
HN
N
6
88
N
O HN
O
N
N
7
HN
O
N
N
8
91
N
O
HN
93
N
O
O
N
O
N
9
O
N
O
84
O
O O
HN 10
N
N
N
NH 2
O N H
11
O
H 2N
O N H
O b
90
O
NH 2
12
a
88
O
N H
85
O
Isolated yields after column chromatography. Products were characterized by NMR and mass spectral data.
Table 2 Cytotoxicities of furano-sesquiterpene with prostate, lung, cervix, breast, and leukemiaa S. No.
DU145 (IC50 in lM)
A549 (IC50 in lM)
HeLa (IC50 in lM)
Mcf7 (IC50 in lM)
THP1 (IC50 in lM)
1 2 3 4 5 6 7 8 9 10 11 12
60.1 ± 1.9 38.9 ± 2.7 59.3 ± 2.3 25.1 ± 1.6 24.1 ± 1.7 20.4 ± 2.1 30.3 ± 2.6 45.3 ± 3.1 17.9 ± 3.1 16.8 ± 4.3 56.5 ± 1.6 41.3 ± 3.6
89.3 ± 3.4 89.7 ± 2.6 89.6 ± 2.6 28.6 ± 1.7 23.6 ± 1.8 27.9 ± 2.4 28.3 ± 1.9 50.7 ± 2.4 45.5 ± 2.4 24.6 ± 3.1 63.2 ± 2 69.7 ± 1.6
81 ± 2.6 80.4 ± 3.5 59.4 ± 3.4 35.5 ± 2.5 30.5 ± 2.4 35.9 ± 1.8 32.1 ± 3.6 59.4 ± 2.7 44.2 ± 1.8 28.5 ± 1.5 59.5 ± 2.7 73.4 ± 3.1
61.9 ± 1.5 74.5 ± 1.6 75.4 ± 1.9 28.9 ± 3.8 27.9 ± 3.5 30.3 ± 2.5 39.8 ± 2.8 53.7 ± 3.5 50.8 ± 2.1 26.7 ± 2.8 65.3 ± 1.7 57.7 ± 1.9
56.6 ± 3.1 39.8 ± 2.1 51.4 ± 2.4 25.8 ± 4.5 20.8 ± 4.2 26.5 ± 2.4 28.6 ± 2.8 50.9 ± 2.1 15.9 ± 2.8 17.8 ± 1.6 60.4 ± 1.6 59 ± 2.8
a Cell lines were treated with different concentrations of compounds for 48 h as described under Materials and methods. Cell viability was measured employing MTT assay. IC50 values are indicated as the mean ± SD of three independent experiments. NA denotes activity >100 lM.
10 are the potential impact in improving the activity compared to aromatic and aliphatic amines 11 and 12. Among all of the furano-sesquiterpenes tested, (4-(benzo[d][1,3]dioxol-5ylmethyl)piperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)
furan-3-yl)methanone 9 inhibited the growth of the THP1 and DU145 cell lines most effectively, with an IC50 values of 15.9 and 17.9 lM, respectively. On the other hand (4-benzhydrylpiperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-yl)meth-
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anone (10) inhibited the growth of the THP1 and DU145 cell lines effectively, with IC50 values of 17.5 and 16.8 lM, respectively. In conclusion, isolation of furano-sesquiterpene carboxylic acids 1 and 2 and their methyl esters 3 and 4 from soft coral Sinularia kavarattiensis and semi-synthesis of a series of new amide derivatives of compound 1 and bio-evaluation of their anticancer activity in vitro against five human cancer cell lines (DU-145, A-549, HeLa, MCF-7 and THP 1) was carried out. Among these compounds 9 and 10 showed promising anticancer activity against leukemia and lung cancer. Interestingly all compounds are selectively potent in leukemia. This has laid a solid foundation for further optimization of this class of compounds by a systematic refinement including the synthesis of compounds to improve their overall pharmaceutical properties. Acknowledgements The authors thank to MoES and SMiLE, CSIR, New Delhi, India, for financial assistance and the Director, Indian Institute of Chemical Technology (IICT) for his constant encouragement and Dr. R. Bates, University of Arizona, for editing this manuscript. References and notes 1. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2011, 28, 196–268. 2. Gaspar, H.; Gavagnin, M.; Calado, G.; Castelluccio, F.; Mollo, E.; Cimino, G. Tetrahedron 2005, 61, 11032–11037. 3. Banerjee, R.; Kumar, H. K. S.; Banerjee, M. Int. J. Rev. Life Sci. 2012, 2, 7–16. 4. Held, C.; Fröhlich, R.; Metz, P. Angew. Chem., Int. Ed. 2001, 40, 1058–1060. 5. Metz, P.; Stölting, J.; Läge, M.; Krebs, B. Angew. Chem., Int. Ed. Engl. 1994, 33, 2195–2197. 6. Hou, X. L.; Cheung, H. Y.; Hon, T. Y.; Kwan, P. L.; Lo, T. H.; Tong, S. Y. T.; Wong, H. N. C. Tetrahedron 1998, 54, 1955–2020. 7. Lipshutz, B. H. Chem. Rev. 1986, 86, 795. 8. Fraga, B. M. Nat. Prod. Rep. 2005, 22, 465–486. 9. Allen, A. J.; Vaillancourt, V.; Albizati, K. F. Org. Prep. Proced. Int. 1994, 26, 1–84. 10. Kamel, H. N.; Slattery, M. Pharm. Biol. 2005, 43, 253–269. 11. Cheng, S. Y.; Huang, K. J.; Wang, S. K.; Wen, Z. H.; Chen, P. W.; Duh, C. Y. J. Nat. Prod. 2010, 73, 771–775. 12. Faulkner, D. J. Nat. Prod. Rep. 2001, 18, 1–49. 13. Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P.; Gerwick, W. H. Mol. Cancer Ther. 2005, 4, 333–342. 14. Shen, Y.-C.; Lo, K.-L.; Lin, Y.-C.; Khalil, A. T.; Kuo, Y.-H.; Shih, P.-S. Tetrahedron Lett. 2006, 47, 4007–4010. 15. Su, J. H.; Tseng, S. W.; Lu, M. C.; Liu, L. L.; Chou, Y.; Sung, P. J. J. Nat. Prod. 2011, 74, 2005–2009. 16. Lakshmi, V.; Kumar, R. Nat. Prod. Res. 2009, 23, 801–850. 17. Cragg, G. N.; Newmann, D. J.; Snades, K. M. J. Nat. Prod. 1997, 60, 52–60. 18. Reddy, V. R. M.; Rama Rao, M.; Rhodes, D.; Hansen, M. S. T.; Rubins, K.; Bushman, F. D.; Venkateswarlu, Y.; Faulkner, D. J. J. Med. Chem. 1999, 42, 1901–1907. 19. Reddy, T. S.; Suryakiran, N.; Narasimhulu, M.; Ramesh, D.; Mahesh, K. C.; Sai Krishna, A.; Kavitha, P.; Rao, J. V.; Venkateswarlu, Y. Bioorg. Med. Chem. Lett. 2012, 22, 4900–4906. 20. Ramesh, P.; Reddy, N. S.; Venkateswarlu, Y. Tetrahedron Lett. 1998, 39, 8217– 8220. 21. Venkateswarlu, Y.; Sridevi, K. V.; Rama Rao, M. J. Nat. Prod. 1999, 62, 756–758. 22. Ravinder, K.; Vijender Reddy, A.; Krishnaiah, P.; Ramesh, P.; Ramakrishna, S.; Laatsch, H.; Venkateswarlu, Y. Tetrahedron Lett. 2005, 46, 5475–5478. 23. Coll, J. C.; Mitchell, S. J.; Stokie, G. J. Tetrahedron Lett. 1977, 18, 1539–1542. 24. Grace, K. J. S.; Zavortink, D.; Jacobs, R. S. Biochem. Pharmacol. 1994, 47, 1427– 1434. 25. Bowden, B. F.; de Coll, J. C.; de Silva, E. D.; Costa, M. S. L.; Djura, P. J.; Mahendran, M.; Tapiolas, D. M. Aust. J. Chem. 1983, 36, 371–376. 26. Arepalli, S. K.; Sridhar, V.; Rao, J. V.; Kennady, P. K.; Venkateswarlu, Y. Apoptosis 2009, 14, 729–740. 27. Limna Mol, V. P.; Raveendran, T. V.; Parameswaran, P. S.; Kunnath, R. J.; Sathyan, N. Ind. J. Mar. Sci. 2010, 39, 270–273. 28. Williams, D. H.; Faulkner, D. J. Tetrahedron 1996, 52, 4245–4256. 29. Fang, G.; Xue, M.; Su, M.; Hu, D.; Li, Y.; Xiong, B.; Maa, L.; Meng, T.; Chen, Y.; Li, J.; Li, J.; Shen, J. Bioorg. Med. Chem. Lett. 2012, 22, 4540–4545. 30. Burkhard, J. A.; Wagner, B.; Fischer, H.; Schuler, F.; Muller, K.; Carreira, E. M. Angew. Chem., Int. Ed. 2010, 49, 3524–3527. 31. Hatnapure, D. G.; Keche, A. P.; Rodge, A. H.; Birajdar, S. S.; Tale, R. H.; Kamble, V. M. Bioorg. Med. Chem. Lett. 2012, 22, 6385–6390. 32. Song, J.; Lee, H.; Kim, Y. J.; Kim, S. Y.; Kim, D.; Min, K. H. Bioorg. Med. Chem. Lett. 2012, 22, 6943–6946.
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33. Dong, M.; Lu, L.; Li, H.; Wang, X.; Lu, H.; Jiang, S.; Dai, Q. Bioorg. Med. Chem. Lett. 2012, 22, 3284–3286. 34. Freshly collected soft coral were placed in ethyl acetate at the site of collection until workup. The initial ethyl acetate from the soft coral specimens (1.5 kg wet weight) was removed and re-extracted with 1:1 dichloromethane–ethyl acetate (3 2 L) at room temperature. The combined extract including initial ethyl acetate extract was concentrated under reduced pressure to a predominantly aqueous extract which was extracted into ethyl acetate (3 500 mL). The crude ethyl acetate extract of the soft coral Sinularia kavarattiensis was subjected to gel filtration chromatography on Sephadex LH 20 using 1:1 dichloromethane–ethyl acetate, followed by silica gel chromatography eluting with hexane through hexane–ethyl acetate mixtures to ethyl acetate to afford compounds 1–4 in good quantities. Compounds 1–4 were identified as a known furano-sesquiterpenoid carboxylic acids by their spectral data. 35. 5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-carboxylic acid (1): Yielded 500 mg; 1H NMR (300 MHz, CDCl3) d: 7.92 (1H, s), 6.70 (1H, dd, J = 10.5 and 16.5 Hz), 6.45 (1H, s), 6.01 (1H, br s), 5.30 (1H, t, J = 6.2 Hz), 5.16 (1H, d, J = 16.5 Hz), 5.05 (1H, d, J = 10.5 Hz), 2.19–2.39 (4H, m), 1.94 (3H, s), 1.78 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.6, 146.8, 141.0, 140.7, 133.4, 129.7, 120.1, 113.8, 113.5, 106.6, 40.7, 25.9, 19.7, 18.7. 36. 5-((1E,5E)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-carboxylic acid (2): Yielded 50 mg; 1H NMR (300 MHz, CDCl3) d: 7.95 (1H, s), 6.71 (1H, dd, J = 10.5 and 16.5 Hz), 6.40 (1H, s), 6.01 (1H, br s), 5.35 (1H, t, J = 7.1 Hz), 5.26 (1H, d, J = 17.0 Hz), 5.05 (1H, d, J = 11.0 Hz), 2.19–2.39 (4H, m), 1.95 (3H, s), 1.76 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.5, 146.8, 141.1, 140.7, 133.5, 129.7, 120.1, 113.9, 113.5, 106.7, 40.7, 25.9, 19.5, 18.5. 37. Methyl 5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-carboxylate (3): Yielded 300 mg; 1H NMR (300 MHz, CDCl3) d: 7.92 (1H, s), 6.72 (1H, dd, J = 10.5 and 16.5 Hz), 6.46 (1H, s), 6.01 (1H, br s), 5.32 (1H, t, J = 6.2 Hz), 5.16 (1H, d, J = 16.5 Hz), 5.05 (1H, d, J = 10.5 Hz), 4.1 (s, 3H), 2.19–2.39 (4H, m), 1.94 (3H, s), 1.78 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.6, 146.8, 141.0, 140.7, 133.4, 129.7, 120.1, 113.8, 113.5, 106.6, 51.5, 40.7, 25.9, 19.7, 18.7. 38. Methyl 5-((1E,5E)-2,6-dimethylocta-1,5,7-trienyl)furan-3-carboxylate (4): Yielded 95 mg; 1H NMR (300 MHz, CDCl3) d: 7.95 (1H, s), 6.71 (1H, dd, J = 10.5 and 16.5 Hz), 6.40 (1H, s), 6.01 (1H, br s), 5.35 (1H, t, J = 7.1 Hz), 5.26 (1H, d, J = 17.0 Hz), 5.05 (1H, d, J = 11.0 Hz), 4.1 (s, 3H), 2.19–2.39 (4H, m), 1.95 (3H, s), 1.76 (3H, s), 13C NMR (75 MHz, CDCl3) d: 168.7, 156.5, 146.8, 141.1, 140.7, 133.5, 129.3, 120.2, 113.8, 113.5, 106.6, 51.5, 40.9, 25.9, 19.6, 18.6. 39. Sinularia kavarattiensis (Specimen No. IIC-1008) was taxonomically identified by Dr. P.A. Thomas of CFMRI, Trivandrum, India, was collected by hand using scuba off the coast of Mandapam, Tamilnadu, India, in April 2011, at a depth of 5–10 m. A voucher sample was deposited at the NIO Goa. 40. Typical experimental procedure for the preparation of amide derivatives of furanosesquiterpene acid: To a solution of furano-sesquiterpene carboxylic acid (1 mmol) in THF was added HATU (1.5 mmol) and DIPEA (2.5 mmol) at 0 °C, stirred at same temp for 30 min, then amine (1.5 mmol) was added. The mixture was stirred at rt for 4 h. After completion of the reaction, the reaction mixture was poured into ice water and washed with ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel (60–120 mesh) to give the corresponding amide derivatives 5–12. (Compound 12 prepared by using the amine (0.5 mmol)). 41. (5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-yl)(4-methylpiperazin-1-yl) methanone (5): 1H NMR (300 MHz, CDCl3) d: 7.52 (1H, s), 6.70 (1H, dd, J = 10.7 and 17.2 Hz), 6.23 (1H, s), 5.98 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.15 (1H, d, J = 17.2 Hz), 5.03 (1H, d, J = 10.7 Hz), 3.64–3.76 (4H, m), 2.50–2.39 (4H, m), 2.34–2.25 (5H, m), 2.12–2.20 (2H, m), 1.91 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 141.2 (2C), 133.4, 132.7, 129.7, 121.8, 113.7, 113.4, 107.1, 54.5 (4C), 45.4, 40.6, 25.7, 19.7, 18.6. ESI-MS (m/z) 329 [M+H]+. 42. (5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-yl)(4-ethylpiperazin-1-yl) methanone (6): 1H NMR (300 MHz, CDCl3) d: 7.51 (1H, s), 6.70 (1H, dd, J = 10.7 and 17.2 Hz), 6.24 (1H, s), 5.99 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.14 (1H, d, J = 17.2 Hz), 5.03 (1H, d, J = 10.7 Hz), 3.73–3.63 (4H, m), 2.51–2.39 (6H, m), 2.35–2.10 (4H, m), 1.91 (3H, s), 1.75 (3H, s), 1.06 (3H, t, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 141.2, 140.3, 133.5, 132.8, 129.7, 122.1, 113.7, 113.5, 107.2, 52.2 (4C), 40.7, 38.6, 25.8, 19.7, 18.6, 11.4. ESI-MS (m/z) 343 [M+H]+. 43. (5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)furan-3-yl)(4-phenylpiperazin-1-yl) methanone (7): 1H NMR (300 MHz, CDCl3) d: 7.55 (1H, s), 7.27–7.18 (3H, m), 6.96–6.82 (2H, m), 6.70 (1H, dd, J = 10.2 and 17.2 Hz), 6.26 (1H, s), 6.00 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.15 (1H, d, J = 17.2 Hz), 5.04 (1H, d, J = 10.2 Hz), 3.86– 3.75 (4H, m), 3.19–3.09 (4H, m), 2.36–2.23 (2H, m), 2.21–2.12 (2H, m), 1.92 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 150.7, 141.2, 140.4, 133.4, 132.8, 129.7, 129.3 (2C), 121.9, 120.7, 116.7 (2C), 113.7, 113.5, 107.3, 49.7 (4C), 40.6, 25.8, 19.7, 18.6. ESI-MS (m/z) 391 [M+H]+. 44. (4-Benzylpiperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-yl) methanone (8): 1H NMR (300 MHz, CDCl3) d: 7.53 (1H, s), 7.41–7.20 (5H, m), 6.70 (1H, dd, J = 10.2 and 17.2 Hz), 6.23 (1H, s), 5.99 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.14 (1H, d, J = 17.2 Hz), 5.04 (1H, d, J = 10.2 Hz), 3.86–3.75 (4H, m), 3.51 (2H, s), 3.19–3.09 (4H, m), 2.36–2.12 (4H, m), 1.92 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.9, 141.2, 140.4, 137.4, 133.4, 132.8, 129.7, 128.6 (2C), 127.7 (2C), 126.4, 120.7, 113.7, 113.5, 107.3, 63.1, 49.7 (4C), 40.6, 25.8, 19.7, 18.6. ESI-MS (m/z) 405 [M+H]+. 45. (4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1, 5,7-trienyl)furan-3-yl)methanone (9): 1H NMR (300 MHz, CDCl3) d: 7.49 (1H, s),
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7.19 (1H, s), 6.79 (1H, s), 6.68 (2H, s), 6.22 (1H, s), 5.97 (1H, s), 5.88 (2H, s), 5.31 (1H, t, J = 7.6 Hz), 5.14 (1H, d, J = 6.0 Hz), 5.00 (1H, d, J = 11.3 Hz), 3.70–3.59 (4H, m), 3.42 (2H, s), 2.47–2.34 (4H, m), 2.32–2.10 (4H, m), 1.98 (3H, s), 1.74 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.9, 153.8, 147.7, 146.9, 141.3, 141.1, 133.4, 131.7, 130.6, 129.7, 122.4, 113.7, 113.5, 110.8, 109.4, 107.9, 107.3, 100.9, 62.4, 52.7 (4C), 40.6, 25.8, 19.7, 18.6. ESI-MS (m/z) 449 [M+H]+. 46. (4-Benzhydrylpiperazin-1-yl)(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-yl) methanone (10): 1H NMR (300 MHz, CDCl3) d: 7.46 (1H, s), 7.38–7.30 (4H, m), 7.26–7.08 (6H, m), 6.69 (1H, dd, J = 10.7 and 17.3 Hz), 6.20 (1H, s), 5.95 (1H, s), 5.29 (1H, t, J = 7.17 Hz), 5.13 (1H, d, J = 17.3 Hz), 5.02 (1H, d, J = 10.7 Hz), 4.18 (1H, s), 3.71–3.54 (4H, m), 2.41–2.20 (6H, m), 2.18–2.09 (2H, m), 1.87 (3H, s), 1.73 (3H, s). 13C NMR (75 MHz, CDCl3) d: 163.8, 153.7, 142.0 (2C), 141.1, 140.2, 133.4 (2C), 129.7, 128.6 (4C), 127.8 (4C), 127.2 (2C), 117.8, 113.7, 113.5, 107.3, 75.9, 51.9 (4C), 40.64, 25.7, 19.7, 18.6. ESI-MS (m/z) 481 [M+H]+. 47. 5-((1E,5Z)-2,6-Dimethylocta-1,5,7-trienyl)-N-p-tolylfuran-3-carboxamide (11): 1 H NMR (300 MHz, CDCl3) d: 7.84 (1H, s), 7.45 (1H, br s), 7.40 (2H, d, J = 8.309 Hz), 7.07 (2H, d, J = 8.309), 6.71 (1H, dd, J = 10.3 and 17.3 Hz), 6.40 (1H, s), 5.99 (1H, s), 5.31 (1H, t, J = 6.9 Hz), 5.15 (1H, d, J = 17.3 Hz), 5.04 (1H, d, J = 10.3 Hz), 2.33–2.10 (7H, m), 1.92 (3H, s), 1.75 (3H, s). 13C NMR (75 MHz, CDCl3) d: 165.9, 154.8, 142.7, 140.7, 135.0, 134.1, 133.4, 132.8, 129.7, 129.5 (2C), 122.4, 120.3 (2C), 113.7, 113.5, 105.3, 40.6, 25.7, 20.8, 19.7, 18.7. ESI-MS (m/z) 336 [M+H]+. 48. N,N0 -(Propane-1,3-diyl)bis(5-((1E,5Z)-2,6-dimethylocta-1,5,7-trienyl)furan-3-carboxamide) (12): 1H NMR (300 MHz, CDCl3) d: 7.82 (1H, s), 7.19 (1H, s), 6.71 (1H, dd, J = 11.3 and 17.3 Hz), 6.40 (1H, s), 6.0 (1H, s), 5.31 (1H, t, J = 6.8 Hz), 5.14 (1H, d, J = 17.3 Hz), 5.04 (1H, d, J = 11.3 Hz), 3.47–3.34 (4H, m), 2.35–2.23 (2H, m), 2.21–2.08 (2H, m), 1.92 (3H, s), 1.74 (3H, s), 1.71–1.59 (2H, m). 13C NMR (75 MHz, CDCl3) d: 163.6 (2C), 154.7 (2C), 142.6 (2C), 140.4, 133.5, 132.8,
129.7, 123.6, 113.7 (4C), 105.4 (2C), 40.7 (2C), 38.6 (4C), 35.7 (2C), 25.8 (2C), 19.7 (2C), 18.7 (2C). ESI-MS (m/z) 531 [M+H]+. 49. All cell lines used in this study were purchased from the American Type Culture Collection (ATCC). A549 (human lung carcinoma epithelial), and HeLa (human epithelial cervical cancer) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing non essential amino acids and 10% FBS. MCF-7 (human breast adenocarcinoma), DU 145 (human prostate carcinoma epithelial) cells were cultured in Eagle’s minimal essential medium (MEM) containing nonessential amino acids, 1 mM sodium pyruvate, 10 mg/mL bovine insulin, and 10% FBS. THP 1 (human monocytic leukemia) cells were cultured in RPMI-1640 containing 0.05 2-mercaptoethanol and 10% FBS. All cell lines maintained in humidified atmosphere of 5% CO2 at 37 °C. Cells were trypsinized when subconfluent from T75 flasks/90 mm dishes and seeded in 96 well plates at a concentration of 1 104 cells/mL in complete medium, treated with compounds at desired concentrations for 48 h, and harvested as required. 50. This assay is a quantitative colorimetric method for determination of cell survival and proliferation. The assessed parameter is the metabolic activity of viable cells. Metabolically active cells reduce pale yellow tetrazolium salt (MTT) to a dark blue water-insoluble formazan, which can be directly quantified after solubilisation with DMSO. The absorbance of the formazan directly correlates with the number of viable cells. The cells were plated in 96-well plates at a density of 1 104 cells in 200 lL of medium per well of 96-well plate. Cultures were incubated with different concentrations of test compounds and incubated for 48 h. The assay was terminated with the addition of 100 lg/mL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 1 h. The supernatant was aspirated and plates were air dried. MTT-formazon crystals dissolved in 100 lL DMSO. The optical density (O.D.) was measured at 570 nm using TECAN multi mode reader.
