Bioorganic & Medicinal Chemistry Letters 24 (2014) 2415–2419
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Synthesis and biological evaluation of (+)-neopeltolide analogues: Importance of the oxazole-containing side chain Haruhiko Fuwa ⇑, Takuma Noguchi, Masato Kawakami, Makoto Sasaki Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
a r t i c l e
i n f o
Article history: Received 18 March 2014 Revised 8 April 2014 Accepted 9 April 2014 Available online 18 April 2014 Keywords: Marine natural products Macrolides Antiproliferative activity Structure–activity relationships Suzuki–Miyaura coupling
a b s t r a c t We describe the synthesis and biological evaluation of (+)-neopeltolide analogues with structural modifications in the oxazole-containing side chain. Evaluation of the antiproliferative activity of newly synthesized analogues against A549 human lung adenocarcinoma cells and PANC-1 human pancreatic carcinoma cells have shown that the C19–C20 and C26–C27 double bonds within the oxazole-containing side chain and the terminal methyl carbamate group are essential for potent activity. Ó 2014 Elsevier Ltd. All rights reserved.
(+)-Neopeltolide (1, Fig. 1) is a 14-membered macrolide natural product that was isolated from a sponge of the Neopeltidae family, collected off the coast of Jamaica by Wright and co-workers.1 The gross structure including the relative configuration of 1 was proposed on the basis of 2D-NMR analyses and NOE correlations. Subsequently, Panek2 and Scheidt3 independently reassigned the relative configuration and unambiguously established the complete stereostructure of 1 through their total syntheses. The structure of 1 consists of a 14-membered macrocyclic backbone embedded with a tetrahydropyran ring and an oxazole-containing side chain attached to the tetrahydropyran. The oxazole-containing side chain of 1 is identical to that of a marine macrolide natural product, (+)-leucascandrolide A (2), previously reported by D’Ambrosio et al.4 These structurally related natural products 1 and 2 have been proposed to be the secondary metabolites of symbiotic cyanobacteria.1 Wright et al. have shown that 1 is a single-digit nanomolar antiproliferative agent against several cancer cell lines, including the A549 human lung adenocarcinoma, the NCI/ADR-RES ovarian sarcoma, and the P388 murine leukemia.1 They have also suggested that 1 may be cytostatic rather than cytotoxic to the PANC-1 human pancreatic carcinoma cell line and the DLD-1 colorectal adenocarcinoma cell line. Kozmin and co-workers have reported that 1 specifically binds to the complex III of the mitochondrial electron transport chain and inhibits mitochondrial ATP synthesis.5
⇑ Corresponding author. Tel./fax: +81 22 217 6214. E-mail address: [email protected] (H. Fuwa). http://dx.doi.org/10.1016/j.bmcl.2014.04.031 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
The structural and biological aspects of 1 have gained significant interests of the synthetic community; more than fifteen total and formal syntheses of 1 have been reported so far.6 Furthermore, the structure–activity relationships of 1 have been reported from several groups.7 Maier6f and Scheidt3b have independently synthesized some diastereomers of 1 to suggest the importance of the stereogenic centers along the macrocyclic backbone. Floreancig and coworkers have shown that structural alteration of the C8–C9 domain is possible without significant loss of potent activity.6p Very recently, we have disclosed the results of our detailed study on the stereostructure–activity relationships of the macrocyclic domain.6t Specifically, we have found that the axial orientation of the C5 oxazole-containing side chain relative to the 2,6-cis-substituted tetrahydropyran ring is imperative for nanomolar activity, and that the C11 and C13 stereogenic centers appear to control the orientation of the C13 n-propyl group that is essential for potent activity. Interestingly, the macrocyclic backbone could be rationally truncated while retaining nanomolar antiproliferative activity. The importance of the oxazole-containing side chain has also been demonstrated in previous studies. Scheidt and co-workers reported that replacement of the side chain with a benzoyl or n-octanoyl group resulted in greater than 1,000-fold loss of activity.3b Maier and coworkers showed that the Z-configuration of the C19–C20 double bond and the distance between the macrolactone domain and the oxazole ring were important for potent activity.6f The Floreancig group described that replacing the oxazole ring with a furan, benzene or pyridine ring led to approximately 30- to 3000-fold loss of activity against the HCT-116 colorectal carcinoma cell lines.6p Collectively, all these studies indicate that the oxazole ring would
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Figure 1. Structures of (+)-neopeltolide (1), (+)-leucascandrolide A (2), and ( )-8,9-dehydroneopeltolide (3).
be indispensable for 1 to show potent antiproliferative activity. However, the importance of the C19–C20 and C26–C27 double bonds and the terminal methyl carbamate group has not been fully addressed. Here we report the synthesis and biological evaluation of neopeltolide analogues to elucidate the structure–activity relationships of the oxazole-containing side chain. Our synthesis of the oxazole-containing side chain via Suzuki–Miyaura coupling8 facilitated the preparation of a series of side chain analogues. Evaluation of the antiproliferative activity of synthetic analogues against the A549 and PANC-1 cell lines showed that the C19–C20 and C26–C27 double bonds and the terminal methyl carbamate group were essential for low nanomolar activity. Our previous study has shown that ( )-8,9-dehydroneopeltolide (3, Fig. 1) is synthetically more easily available than 1 and that 3 is ca. 3-fold more active than 1 against the A549 cell line.6t Accordingly, structural analogues of 3 were synthesized and evaluated in the present study. To address the importance of the C19–C20 and C26–C27 double bonds and the terminal methyl carbamate group, we synthesized a series of side chain analogues 4–11 as shown in Schemes 1–4. The synthesis of the analogue 4, summarized in Scheme 1, started with tosylation of the known alcohol 12,9 followed by displacement with NaI, to give the iodide 13. Treatment of 13 with
dimethyl malonate (NaH, THF, 60 °C) provided the diester 14, which was decarboxylated under standard conditions10 to afford the ester 15. Hydrolysis of the methyl ester of 15 and subsequent esterification11 of the resultant carboxylic acid 16 with the alcohol 176t furnished the analogue 4.12 The synthesis of 5 and 6 commenced with hydrogenation of the alcohol 12 (Scheme 2). The resultant alcohol 18 was oxidized under Swern conditions13 to give the aldehyde 19, which was homologated using (CF3CH2O)2P(O)CH2CO2Me14 to deliver the a,b-unsaturated ester 20 as an inseparable 13:1 mixture of Z/E isomers. Hydrolysis of 20 gave the a,b-unsaturated carboxylic acid 21, and subsequent Mitsunobu esterification15 with the alcohol 226t afforded the analogue 5.12 The minor 19E isomer was removed by reverse-phase HPLC purification. Meanwhile, hydrogenation of the double bond of 21 led to the carboxylic acid 23. Esterification of 23 with the alcohol 176t under Yamaguchi conditions11 provided the analogue 6.12 The synthesis of the C19–C20 modified synthetic analogues 7–9 started from the known aldehyde 24 (Scheme 3).6f Takai methylenation of 24 gave the olefin 25.16 Suzuki–Miyaura coupling8,17 of 25 with a variety of readily accessible alkenyl/aryl iodides 26,18 27,19 and 28 provided the coupling products 29–31. Hydrolysis of 29–31 followed by Mitsunobu esterification15 with the alcohol 226f afforded the analogues 7–9.12
Scheme 1. Synthesis of analogue 4.
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2415–2419
Scheme 2. Synthesis of analogues 5 and 6.
Scheme 3. Synthesis of the C19–C20 modified analogues 7–9.
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Scheme 4. Synthesis of the C26–C28 modified analogues 10 and 11.
The C26–C28 modified synthetic analogues 10 and 11 were prepared from 2-butynoic acid (35) and 3-methyl-2-butenoic acid (36), respectively, as depicted in Scheme 4. Coupling of 35 and 36 with L-serine methyl ester hydrochloride by the action of DEPC20 gave the amides 37 and 38. Subsequent DAST-mediated cyclodehydration/bromination–elimination21 provided the oxazoles 39 and 40. Semi-hydrogenation of 39 led to the oxazole 41. DIBALH reduction of 41 and 40 was followed by Takai methylenation16 to deliver the olefins 44 and 45. Suzuki–Miyaura coupling8,17 of 44 and 45 with ethyl cis-b-iodoacrylate afforded the (Z)-a,b-unsaturated esters 46 and 47. After hydrolysis, the resultant carboxylic acids 48 and 49 were coupled with the alcohol 22 to furnish the analogues 10 and 11.12
Next, we evaluated the antiproliferative activity of the analogues 4–11 along with the parent compound 3 against A549 and PANC-1 cells, and the results are summarized in Table 1.22 Strikingly, the analogues 4 and 5 showed ca. 10–800-fold decrease in activity, and the analogue 6 was only marginally active at micromolar concentrations. Thus, it turned out that the C19–C20 and C26–C27 double bonds were essential for 3 to exert low nanomolar activity. The C19 methyl analogue 7 showed essentially the same potency with 3, whereas the C20 methyl analogue 8 was slightly less active than 3. Meanwhile, the antiproliferative activity of the benzoate analogue 9 dropped to the same level as that of 4. These results suggested that the C19–C20 double bond would be modifiable with an additional substituent without significant loss of
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2415–2419 Table 1 Antiproliferative activity of 3–11a
References and notes
Compound
3 4 5 6 7 8 9 10 11
2419
IC50 value A549
PANC-1
0.50 nMb 110 nM 400 nM >10 lM 2.5 nM 6.3 nM 500 nM 740 nM 3.9 lM
2.4 nM 26 nM 200 nM 3.2 lM 2.6 nM 6.4 nM 75 nM 440 nM 2.8 lM
a Antiproliferative activity of each compound was determined by WST-8 assay (n = 3).22 For details, see the Supplementary data. b Taken from Ref. 6t.
Figure 2. Structure of analogue with truncated side chain reported by Floreancig and co-workers.
activity, but its role as a conjugate acceptor might be essential for low nanomolar activity.23 Floreancig and co-workers reported that synthetic analogue 50 (Fig. 2), lacking the terminal methyl carbamate and the C26–C27 double bond, was ca. 140-fold less active than 3 against HCT-116 cells.6p However, the question whether both the terminal methyl carbamate group and the C26–C27 double bond are important for antiproliferative activity has not been addressed. Here, evaluation of the analogue 10 has clearly indicated that the terminal methyl carbamate group plays an important role in exerting potent antiproliferative activity. In addition, the analogue 11 suffered from further loss of activity, illustrating the difficulties in modifying the structure around the C26–C28 domain. In summary, we have established that the C19–C20 and C26– C27 double bonds and the terminal methyl carbamate group are indispensable structural elements for low nanomolar antiproliferative activity of (+)-neopeltolide. Coupled with our previous study,6t we have now fully elucidated the structure–activity relationships of this intriguing natural product. Further studies on the cellular effects of 3 are currently underway in our laboratory and will be reported in due course.
1. Wright, A. E.; Botelho, J. C.; Guzmán, E.; Harmody, D.; Linley, P.; McCarthy, P. J.; Pitts, T. P.; Pomponi, S. A.; Reed, J. K. J. Nat. Prod. 2007, 70, 412. 2. Youngsaye, W.; Lowe, J. T.; Pohlki, F.; Ralifo, P.; Panek, J. S. Angew. Chem., Int. Ed. 2007, 46, 9211. 3. (a) Custar, D. W.; Zabawa, T. P.; Scheidt, K. A. J. Am. Chem. Soc. 2008, 130, 804; (b) Custar, D. W.; Zabawa, T. P.; Hines, J.; Crews, C. M.; Scheidt, K. A. J. Am. Chem. Soc. 2009, 131, 12406. 4. D’Ambrosio, M.; Guerriero, A.; Debitus, C.; Pietra, F. Helv. Chim. Acta 1996, 79, 51. 5. Ulanovskaya, O. A.; Janjic, J.; Suzuki, M.; Sabharwal, S. S.; Schumacker, P. T.; Kron, S. J.; Kozmin, S. A. Nat. Chem. Biol. 2008, 4, 418. 6. For total and formal syntheses of neopeltolide, see: (a) Woo, S. K.; Kwon, M. S.; Lee, E. Angew. Chem., Int. Ed. 2008, 47, 3242; (b) Vintonyak, V. V.; Maier, M. E. Org. Lett. 2008, 10, 1239; (c) Fuwa, H.; Naito, S.; Goto, T.; Sasaki, M. Angew. Chem., Int. Ed. 2008, 47, 4737; (d) Paterson, I.; Miller, N. A. Chem. Commun. 2008, 4708; (e) Kartika, R.; Gruffi, T. R.; Taylor, R. E. Org. Lett. 2008, 10, 5047; (f) Vintonyak, V. V.; Kunze, B.; Sasse, F.; Maier, M. E. Chem. Eur. J. 2008, 14, 11132; (g) Tu, W.; Floreancig, P. E. Angew. Chem., Int. Ed. 2009, 48, 4567; (h) Kim, H.; Park, Y.; Hong, J. Angew. Chem., Int. Ed. 2009, 48, 7577; (i) Guinchard, X.; Roulland, E. Org. Lett. 2009, 11, 4700; (j) Fuwa, H.; Saito, A.; Naito, S.; Konoki, K.; YotsuYamashita, M.; Sasaki, M. Chem. Eur. J. 2009, 15, 12807; (k) Yadav, J. S.; Kumar, G. G. K. S. N. Tetrahedron 2010, 66, 480; (l) Cui, Y.; Wangyang, T.; Floreancig, P. E. Tetrahedron 2010, 66, 4867; (m) Martinez-Solorio, D.; Jennings, M. P. J. Org. Chem. 2010, 75, 4095; (n) Fuwa, H.; Saito, A.; Sasaki, M. Angew. Chem., Int. Ed. 2010, 49, 3041; (o) Yang, Z.; Zhang, B.; Zhao, G.; Yang, J.; Xie, X.; She, X. Org. Lett. 2011, 13, 5916; (p) Cui, Y.; Balachandran, R.; Day, B. W.; Floreancig, P. E. J. Org. Chem. 2012, 77, 2225; (q) Gangavaram, S.; Reddy, S.; Kallaganti, V. S. R. Org. Biomol. Chem. 2012, 10, 3689; (r) Raghavan, S.; Samanta, P. K. Org. Lett. 2012, 14, 2346; (s) Athe, S.; Chandrasekhar, B.; Roy, S.; Pradhan, T. K.; Ghosh, S. J. Org. Chem. 2012, 77, 9840; (t) Fuwa, H.; Kawakami, M.; Noto, K.; Muto, T.; Suga, Y.; Konoki, K.; YotsuYamashita, M.; Sasaki, M. Chem. Eur. J. 2013, 19, 8100; (u) Ghosh, A. K.; Shurrush, K. A.; Dawson, Z. L. Org. Biomol. Chem. 2013, 11, 7768. and Refs. 2,3,5.; For synthetic studies on neopeltolide, see: (v) Florence, G. J.; Cadou, R. F. Tetrahedron Lett. 2010, 51, 5761; (w) Hartmann, E.; Oestreich, M. Angew. Chem., Int. Ed. 2010, 49, 6195. 7. See Refs. 3b,6f,j,p,t. 8. For reviews, see: (a) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457; (b) Suzuki, A. Chem. Commun. 2005, 4759; (c) Suzuki, A. Angew. Chem., Int. Ed. 2011, 50, 6722; (d) Suzuki, A.; Yamamoto, Y. Chem. Lett. 2011, 40, 894. 9. Hornberger, K. R.; Hamblett, C. L.; Leighton, J. L. J. Am. Chem. Soc. 2000, 122, 12894. 10. Krapcho, A. P.; Jahngen, E. G. E., Jr.; Lovey, A. J. Tetrahedron Lett. 1974, 1091. 11. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn. 1979, 52, 1989. 12. All neopeltolide analogues were purified by preparative HPLC prior to biological testing, and their purity was determined by HPLC analysis. For details, see the Supplementary data. 13. Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43, 2480. 14. Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405. 15. For reviews of Mitsunobu reaction, see: (a) Mitsunobu, O. Synthesis 1981, 1; (b) But, T. Y. S.; Toy, P. H. Chem. Asian J. 2007, 2, 1340; (c) Swamy, K. C. K.; Kumar, N. N. B.; Balaraman, E.; Kumar, K. V. P. P. Chem. Rev. 2009, 109, 2551. 16. Okazoe, T.; Hibino, J.; Takai, K.; Nozaki, H. Tetrahedron Lett. 1985, 26, 5581. 17. (a) Johnson, C. R.; Braun, M. P. J. Am. Chem. Soc. 1993, 115, 11014; (b) Sasaki, M.; Fuwa, H.; Inoue, M.; Tachibana, K. Tetrahedron Lett. 1998, 39, 9027. 18. Larock, R. C.; Doty, M. J.; Han, X. J. Org. Chem. 1999, 64, 8770. 19. Zhang, W.; Xu, H.; Xu, H.; Tang, W. J. Am. Chem. Soc. 2009, 131, 3832. 20. Ikota, N.; Shioiri, T.; Yamada, S.; Tachibana, S. Chem. Pharm. Bull. 1980, 28, 3347. 21. Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org. Lett. 2000, 2, 1165. 22. Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. Anal. Commun. 1999, 36, 47. 23. We have confirmed that the C19–C20 double bond actually acts as a conjugate acceptor, by reacting A24 as a model compound with methyl thioglycolate. The thiol adduct B was isolated in 75% yield, along with recovered A in 13% yield. Partial isomerization of the C19–C20 double bond (Z/E = 2.8:1) was observed in the 1H NMR spectrum of the recovered A, indicating that the conjugate addition was reversible.
Acknowledgments This work was supported in part by a Grant-in-Aid for Young Scientists (A) (No. 23681045) and by Grant-in-Aids for Scientific Research on Innovative Areas ‘Chemical Biology of Natural Products’ (Nos. 24102517 and 26102708). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 04.031.
24. Wang, Y.; Janjic, J.; Kozmin, S. A. J. Am. Chem. Soc. 2002, 124, 13670.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of (+)-neopeltolide analogues: Importance of the oxazole-containing side chain Haruhiko Fuwa ⇑, Takuma Noguchi, Masato Kawakami, Makoto Sasaki Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
a r t i c l e
i n f o
Article history: Received 18 March 2014 Revised 8 April 2014 Accepted 9 April 2014 Available online 18 April 2014 Keywords: Marine natural products Macrolides Antiproliferative activity Structure–activity relationships Suzuki–Miyaura coupling
a b s t r a c t We describe the synthesis and biological evaluation of (+)-neopeltolide analogues with structural modifications in the oxazole-containing side chain. Evaluation of the antiproliferative activity of newly synthesized analogues against A549 human lung adenocarcinoma cells and PANC-1 human pancreatic carcinoma cells have shown that the C19–C20 and C26–C27 double bonds within the oxazole-containing side chain and the terminal methyl carbamate group are essential for potent activity. Ó 2014 Elsevier Ltd. All rights reserved.
(+)-Neopeltolide (1, Fig. 1) is a 14-membered macrolide natural product that was isolated from a sponge of the Neopeltidae family, collected off the coast of Jamaica by Wright and co-workers.1 The gross structure including the relative configuration of 1 was proposed on the basis of 2D-NMR analyses and NOE correlations. Subsequently, Panek2 and Scheidt3 independently reassigned the relative configuration and unambiguously established the complete stereostructure of 1 through their total syntheses. The structure of 1 consists of a 14-membered macrocyclic backbone embedded with a tetrahydropyran ring and an oxazole-containing side chain attached to the tetrahydropyran. The oxazole-containing side chain of 1 is identical to that of a marine macrolide natural product, (+)-leucascandrolide A (2), previously reported by D’Ambrosio et al.4 These structurally related natural products 1 and 2 have been proposed to be the secondary metabolites of symbiotic cyanobacteria.1 Wright et al. have shown that 1 is a single-digit nanomolar antiproliferative agent against several cancer cell lines, including the A549 human lung adenocarcinoma, the NCI/ADR-RES ovarian sarcoma, and the P388 murine leukemia.1 They have also suggested that 1 may be cytostatic rather than cytotoxic to the PANC-1 human pancreatic carcinoma cell line and the DLD-1 colorectal adenocarcinoma cell line. Kozmin and co-workers have reported that 1 specifically binds to the complex III of the mitochondrial electron transport chain and inhibits mitochondrial ATP synthesis.5
⇑ Corresponding author. Tel./fax: +81 22 217 6214. E-mail address: [email protected] (H. Fuwa). http://dx.doi.org/10.1016/j.bmcl.2014.04.031 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
The structural and biological aspects of 1 have gained significant interests of the synthetic community; more than fifteen total and formal syntheses of 1 have been reported so far.6 Furthermore, the structure–activity relationships of 1 have been reported from several groups.7 Maier6f and Scheidt3b have independently synthesized some diastereomers of 1 to suggest the importance of the stereogenic centers along the macrocyclic backbone. Floreancig and coworkers have shown that structural alteration of the C8–C9 domain is possible without significant loss of potent activity.6p Very recently, we have disclosed the results of our detailed study on the stereostructure–activity relationships of the macrocyclic domain.6t Specifically, we have found that the axial orientation of the C5 oxazole-containing side chain relative to the 2,6-cis-substituted tetrahydropyran ring is imperative for nanomolar activity, and that the C11 and C13 stereogenic centers appear to control the orientation of the C13 n-propyl group that is essential for potent activity. Interestingly, the macrocyclic backbone could be rationally truncated while retaining nanomolar antiproliferative activity. The importance of the oxazole-containing side chain has also been demonstrated in previous studies. Scheidt and co-workers reported that replacement of the side chain with a benzoyl or n-octanoyl group resulted in greater than 1,000-fold loss of activity.3b Maier and coworkers showed that the Z-configuration of the C19–C20 double bond and the distance between the macrolactone domain and the oxazole ring were important for potent activity.6f The Floreancig group described that replacing the oxazole ring with a furan, benzene or pyridine ring led to approximately 30- to 3000-fold loss of activity against the HCT-116 colorectal carcinoma cell lines.6p Collectively, all these studies indicate that the oxazole ring would
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Figure 1. Structures of (+)-neopeltolide (1), (+)-leucascandrolide A (2), and ( )-8,9-dehydroneopeltolide (3).
be indispensable for 1 to show potent antiproliferative activity. However, the importance of the C19–C20 and C26–C27 double bonds and the terminal methyl carbamate group has not been fully addressed. Here we report the synthesis and biological evaluation of neopeltolide analogues to elucidate the structure–activity relationships of the oxazole-containing side chain. Our synthesis of the oxazole-containing side chain via Suzuki–Miyaura coupling8 facilitated the preparation of a series of side chain analogues. Evaluation of the antiproliferative activity of synthetic analogues against the A549 and PANC-1 cell lines showed that the C19–C20 and C26–C27 double bonds and the terminal methyl carbamate group were essential for low nanomolar activity. Our previous study has shown that ( )-8,9-dehydroneopeltolide (3, Fig. 1) is synthetically more easily available than 1 and that 3 is ca. 3-fold more active than 1 against the A549 cell line.6t Accordingly, structural analogues of 3 were synthesized and evaluated in the present study. To address the importance of the C19–C20 and C26–C27 double bonds and the terminal methyl carbamate group, we synthesized a series of side chain analogues 4–11 as shown in Schemes 1–4. The synthesis of the analogue 4, summarized in Scheme 1, started with tosylation of the known alcohol 12,9 followed by displacement with NaI, to give the iodide 13. Treatment of 13 with
dimethyl malonate (NaH, THF, 60 °C) provided the diester 14, which was decarboxylated under standard conditions10 to afford the ester 15. Hydrolysis of the methyl ester of 15 and subsequent esterification11 of the resultant carboxylic acid 16 with the alcohol 176t furnished the analogue 4.12 The synthesis of 5 and 6 commenced with hydrogenation of the alcohol 12 (Scheme 2). The resultant alcohol 18 was oxidized under Swern conditions13 to give the aldehyde 19, which was homologated using (CF3CH2O)2P(O)CH2CO2Me14 to deliver the a,b-unsaturated ester 20 as an inseparable 13:1 mixture of Z/E isomers. Hydrolysis of 20 gave the a,b-unsaturated carboxylic acid 21, and subsequent Mitsunobu esterification15 with the alcohol 226t afforded the analogue 5.12 The minor 19E isomer was removed by reverse-phase HPLC purification. Meanwhile, hydrogenation of the double bond of 21 led to the carboxylic acid 23. Esterification of 23 with the alcohol 176t under Yamaguchi conditions11 provided the analogue 6.12 The synthesis of the C19–C20 modified synthetic analogues 7–9 started from the known aldehyde 24 (Scheme 3).6f Takai methylenation of 24 gave the olefin 25.16 Suzuki–Miyaura coupling8,17 of 25 with a variety of readily accessible alkenyl/aryl iodides 26,18 27,19 and 28 provided the coupling products 29–31. Hydrolysis of 29–31 followed by Mitsunobu esterification15 with the alcohol 226f afforded the analogues 7–9.12
Scheme 1. Synthesis of analogue 4.
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2415–2419
Scheme 2. Synthesis of analogues 5 and 6.
Scheme 3. Synthesis of the C19–C20 modified analogues 7–9.
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Scheme 4. Synthesis of the C26–C28 modified analogues 10 and 11.
The C26–C28 modified synthetic analogues 10 and 11 were prepared from 2-butynoic acid (35) and 3-methyl-2-butenoic acid (36), respectively, as depicted in Scheme 4. Coupling of 35 and 36 with L-serine methyl ester hydrochloride by the action of DEPC20 gave the amides 37 and 38. Subsequent DAST-mediated cyclodehydration/bromination–elimination21 provided the oxazoles 39 and 40. Semi-hydrogenation of 39 led to the oxazole 41. DIBALH reduction of 41 and 40 was followed by Takai methylenation16 to deliver the olefins 44 and 45. Suzuki–Miyaura coupling8,17 of 44 and 45 with ethyl cis-b-iodoacrylate afforded the (Z)-a,b-unsaturated esters 46 and 47. After hydrolysis, the resultant carboxylic acids 48 and 49 were coupled with the alcohol 22 to furnish the analogues 10 and 11.12
Next, we evaluated the antiproliferative activity of the analogues 4–11 along with the parent compound 3 against A549 and PANC-1 cells, and the results are summarized in Table 1.22 Strikingly, the analogues 4 and 5 showed ca. 10–800-fold decrease in activity, and the analogue 6 was only marginally active at micromolar concentrations. Thus, it turned out that the C19–C20 and C26–C27 double bonds were essential for 3 to exert low nanomolar activity. The C19 methyl analogue 7 showed essentially the same potency with 3, whereas the C20 methyl analogue 8 was slightly less active than 3. Meanwhile, the antiproliferative activity of the benzoate analogue 9 dropped to the same level as that of 4. These results suggested that the C19–C20 double bond would be modifiable with an additional substituent without significant loss of
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2415–2419 Table 1 Antiproliferative activity of 3–11a
References and notes
Compound
3 4 5 6 7 8 9 10 11
2419
IC50 value A549
PANC-1
0.50 nMb 110 nM 400 nM >10 lM 2.5 nM 6.3 nM 500 nM 740 nM 3.9 lM
2.4 nM 26 nM 200 nM 3.2 lM 2.6 nM 6.4 nM 75 nM 440 nM 2.8 lM
a Antiproliferative activity of each compound was determined by WST-8 assay (n = 3).22 For details, see the Supplementary data. b Taken from Ref. 6t.
Figure 2. Structure of analogue with truncated side chain reported by Floreancig and co-workers.
activity, but its role as a conjugate acceptor might be essential for low nanomolar activity.23 Floreancig and co-workers reported that synthetic analogue 50 (Fig. 2), lacking the terminal methyl carbamate and the C26–C27 double bond, was ca. 140-fold less active than 3 against HCT-116 cells.6p However, the question whether both the terminal methyl carbamate group and the C26–C27 double bond are important for antiproliferative activity has not been addressed. Here, evaluation of the analogue 10 has clearly indicated that the terminal methyl carbamate group plays an important role in exerting potent antiproliferative activity. In addition, the analogue 11 suffered from further loss of activity, illustrating the difficulties in modifying the structure around the C26–C28 domain. In summary, we have established that the C19–C20 and C26– C27 double bonds and the terminal methyl carbamate group are indispensable structural elements for low nanomolar antiproliferative activity of (+)-neopeltolide. Coupled with our previous study,6t we have now fully elucidated the structure–activity relationships of this intriguing natural product. Further studies on the cellular effects of 3 are currently underway in our laboratory and will be reported in due course.
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Acknowledgments This work was supported in part by a Grant-in-Aid for Young Scientists (A) (No. 23681045) and by Grant-in-Aids for Scientific Research on Innovative Areas ‘Chemical Biology of Natural Products’ (Nos. 24102517 and 26102708). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 04.031.
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