- Email: [email protected]
Synthesis and cytotoxicity evaluation of highly functionalized pyranochromenes and pyranopyrans
Bioorganic & Medicinal Chemistry Letters 22 (2012) 7261–7264
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxicity evaluation of highly functionalized pyranochromenes and pyranopyrans Narender Reddy Emmadi a, Krishnaiah Atmakur a,⇑, Ganesh Kumar Chityal b,⇑, Sujitha Pombala b, Jagadeesh Babu Nanubolu c a
Crop Protection Chemicals Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India Chemical Biology Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India c Laboratory of X-ray crystallography, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India b
a r t i c l e
i n f o
Article history: Received 15 May 2012 Revised 21 August 2012 Accepted 5 September 2012 Available online 23 September 2012 Keywords: 4-Hydroxy chromenes Ethyltrifluoro acetoacetate Aromatic aldehydes Tetrahydropyrano[3,2-c]chromenes Dihydropyrano[3,2-b]pyrans
a b s t r a c t A series of fluorinated tetrahydropyrano[3,2-c]chromenes and dihydropyrano[3,2-b]pyran derivatives have been synthesized and their in vitro cytotoxic activities have been determined in cervical cancer cell line (HeLa), human breast adenocarcinoma cell line (MDA-MB-231 and MCF-7) and human alveolar adenocarcinoma cell line (A549). Compounds 4g, 4k, 4p showed a very potent activity against MDA-MB-231, and 4c, 4p showed promising activity against MCF-7, while compounds 4c, 4g, 4p showed moderate activity against HeLa. Ó 2012 Elsevier Ltd. All rights reserved.
Pyranochromenes also known as pyranobenzopyrans are an important class of oxygen containing heterocycles that have attracted significant importance in the field of organic and natural product chemistry.1,2 Chromene, the basic skeleton of pyranochromenes is the isomer of coumarins and also noted to be present as an essential core moiety in numerous natural and biologically active compounds.3–7 Further, pyranochromenes and pyrano pyran scaffolds are one of the most commonly encountered heterocycles, which forms the important component of pharmacophores for a number of compounds having medicinal significance8 including anticancer, antiviral, antibacterial, antioxidant, anti-HIV,9–11 antihyperglycemic and anti-dyslipidemic activities.12 Meanwhile the presence of fluorine, especially trifluoromethyl group at a specified position in a molecule alters the reactivity because of its strong electron withdrawing character, lipophilicity and metabolic stability. The special character of fluorine has been thoroughly exploited in the design of novel targets for pharmaceutical, agrochemical and material science industries.13–16 In the recent past, a number of methods have been reported for the synthesis of dihydropyranochromenes17–22 and benzo[h]chromenes.23–29 However, few reports have appeared in the literature on the synthesis of tetrahydropyrano[3,2-c]chromenes30–32 and no re-
ports are available on trifluoromethyl substituted pyrano[3,2-c] chromenes and dihydropyrano[3,2-b]pyrans. With this background and also as a part of our continuous interest on the synthesis of trifluoromethyl substituted heterocycles of biological interest,33,34 herein we report the synthesis and cytotoxicity evaluation of hitherto unreported fluorinated tetrahydropyrano [3,2-c]chromenes and dihydropyrano[3,2-b]pyran derivatives. Initially, a model reaction was tried to prepare the fluorinated tetrahydropyrano[3,2-c]chromenes starting with simple benzaldehyde (1) ethyltrifluoro acetoacetate (2) and 4-hydroxy-2H-chromene-2-one (3a) (Scheme 1), in water at room temperature followed by at reflux temperature using a series of catalysts. Though the water mediated reaction method for the synthesis of pyranochromenes using various catalysts, that is, diammonium hydrogen phosphate17 TBAB,20 DBU21 and basic alumina24 are well documented in the literature, it did not work for our substrates. In a second attempt, the same reaction was conducted in ethanol
CHO
E-mail addresses: [email protected] (K. Atmakur), [email protected] (G.K. Chityal). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.09.018
O
F3C 1
⇑ Corresponding authors. Tel.: +91 40 27191436; fax: +91 40 27193382.
O
OH OEt
2
O 3a
Catalyst
O
Ethanol, reflux
Scheme 1.
F3C O O
OH COOEt O 4a
7262
N. R. Emmadi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7261–7264
As the optimum parameters were established, the generality of the reaction has been studied with varying substituent’s on (1), (3) (Scheme 2) and observed that the methodology was compatible to all the substituent’s. However, high yields were observed when electron withdrawing groups were present in the Para position (4d, 4e, 4m and 4o) to aldehyde functional group in 1 (Table 2). The same reaction (Scheme 1) when refluxed in ethanol without any catalyst, very poor yields were obtained (15% in 24 h, Table 1). The methodology was extended to the preparation of dihydro pyrano[3,2-b]pyrans (4s–w) by reacting compounds (1), (2) with 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (3b) and the desired products 4s-w were obtained in high yields. Structure of the products 4 were well characterized by their proton 1H NMR spectroscopy, where C-3 and C-4 protons were found to be trans to each other based on their chemical shifts (doublet) and coupling constants (J = 11.52 and 11.33). This data is in agreement with the earlier report35. In order to find out the absolute configuration of 4, (as there are three stereogenic centers in the molecule and possible for isomers) X-ray crystallography studies36 have been conducted on 4a. Based on the crystallography data (Fig. 1), it was observed that the
Table 1
S.No
Catalyst (mol %)
Time (h)
Yield (%)
1 2 3 4 5 6 7
TEA(30) DBU(10) DBU(20) DBU(30) DABCO(30) Piperdine(30) NH4OAc(30) N-Methy Imidazole(30) —
12 12 12 6 10 12 12 12 12 24
25 52 63 70 56 64 35 40 40 15
8 9
with various catalysts. Though the solvent role is not clear, interesting results were obtained in ethanol at reflux temperature with DBU as the catalyst. Encouraged by the results, the reaction was carried out with different mole ratios (Table 1) with different catalysts. However 30 mol % DBU proved to be an effective and high yielding parameter within 6 h.
OH
R2
O 3a
CHO
O 1
R
R
O
F3C
1
OEt
2
R
F3C O O
O
OH COOEt R1 O 4 a-r
R
DBU(30%) Ethanol, reflux 6h
O
2 HO
O OH
HO
O 3b
O
O
CF3 OH COOEt
R1 R 4 s-w
Scheme 2.
Table 2
S. No
(4)
R
R1
R2
Time (h)
Yield (%)
Melting point (°C)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q
H Cl F NO2 CN Me OMe OH OH Iso propyl OMe H NO2 F CN OH H
H H H H H H H H OMe H OMe H H H H H H
H H H H H H H H H H H F F F F F Me
6 6 7 6 6 7 8 7 8 6 7 6 6 6 7 8 6
70 65 67 72 74 64 68 65 58 67 61 68 75 66 70 62 67
115–116 138–140 107–108 112–114 114–115 144–146 134–136 163–165 110–112 145–147 158–160 145–146 118–120 142–144 109–111 170–171 172–174
18
4r
—
H
8
60
186–188
H H H H OMe
— — — — —
6 6 8 7 8
68 65 67 58 64
150–152 112–114 173–174 170–171 159–160
CHO
N
19 20 21 22 23
4s 4t 4u 4v 4w
H Me OMe OH OMe
N. R. Emmadi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7261–7264 Table 3 Cytotoxicity data, IC50 (lM) Entry
Compound
HeLa
MDA-MB-231
MCF-7
A549
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
4c 4e 4g 4h 4i 4j 4k 4m 4n 4o 4p 4r 4t 4u 4v Doxorubicin
11.01 — 11.23 — — — 87.01 — — — 11.45 — — — — <1
11.27 — 2.20 — — — 7.71 — — — 6.61 — — — — <1
7.67 — 71.7 — — — 53.89 — — — 6.54 — — — — 1
— — 82.37 — — — 34.28 — — — 33.86 — — — — 1
-No activity. HeLa- Cervical cancer cell line. MDA-MB-231- Human breast adenocarcinoma cell line. MCF-7- Human breast adenocarcinoma cell line. A549- Human alveolar adenocarcinoma cell line.
Figure 1. (a) The molecular structure of 4a. (b) The molecular structure of 4a with the atom-numbering scheme. The compound crystallized in the monoclinic space group P21/c with two molecules in the asymmetric unit. Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radius.
7263
Further, compounds in 4 series were taken-up to evaluate the cytotoxicity, since they are lipophilic in nature due to the presence of trifluoromethyl group and expected to penetrate into the membrane in considerable concentration and suppose to exhibit potent activity. Therefore, the cytotoxicity was determined against a panel of human tumor cell lines such as cervical cancer cell line (HeLa), human breast adenocarcinoma cell lines (MDA-MB-231 and MCF-7), human alveolar adenocarcinoma cell line (A549) and the results are tabulated in (Table 3) and human alveolar adenocarcinoma cell line (A549). In the series of pyranochromene derivatives, compounds 4c, 4g, 4k, 4p having fluorine, methoxy, di-methoxy and hydroxy functional groups on C-4 phenyl ring had the distinct tendency to increase the in vitro antitumor potency and exhibited an excellent cytotoxicity against MDA-MB-231, MCF-7 and HeLa. Compounds 4g, 4k, and 4p showed potent activity against MDA-MB-231 with IC50 value of 2.20, 7.71 and 6.61 lM (Table 2, entries 3, 7 and 11). Compounds 4c, 4p showed very promising activity against MCF-7 with IC50 value of 7.67 and 6.54 lM (Table 2, entries 1, 11) respectively and compounds 4c, 4g, 4p showed moderate activity with IC50 value of 11.01, 11.23 and 11.45 lM against HeLa (Table 2, entries 1,3,11). Cytotoxicity data was assessed on the basis of measurement of in vitro growth of tumor cell lines in 96 well plates by cellmediated reduction of tetrazolium salt to water insoluble formazan crystals using doxorubicin as a standard. The compounds were tested for cytotoxicity against a panel of four different tumor cell lines: A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), HeLa derived from human cervical cancer cells (ATCC No. CCL-2), MDA-MB-231 derived from human breast adenocarcinoma cells (ATCC No. HTB-26) and MCF7 derived from human breast adenocarcinoma cells (ATCC No. HTB-22) using the MTT assay.38 The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the doseresponse curves. IC50 values (in lM) are expressed as the average of two independent experiments. In conclusion, we prepared hitherto unreported fluorinated tetrahydro pyrano[3,2-c]chromenes,39 dihydropyrano[3,2-b]pyran derivatives using simple reaction conditions in a single pot and evaluated their in vitro cytotoxicity against a panel of human tumor cell lines. In the series of pyranochromene derivatives, four compounds 4c, 4g, 4k and 4p showed very potent activity and the data is helpful to further exploit these compounds for anticancer activity mechanistic studies and also helpful to design and synthesize more such derivatives to be taken-up for further studies. Acknowledgments Authors are thankful to Director IICT for constant support and ENR is thankful to CSIR New Delhi for SRF.
compound exists as a racemic mixture (enantiomers and are non separable) comprising the SSR and RRS configuration at C-2, C-3 and C-4 asymmetric centers. These studies support the 1H NMR data of 4a. Attempts to prepare the dehydrated product of 4 by reported protocols especially for the trifluoromethyl substituted heterocycles were unsuccessful in spite of refluxing them in toluene in presence of several reagents such as concentrated H2SO4, P2O5, PTSA, TFA and also PPTS and TPP independently. This may be attributed to the fact that the weaker electron donating character of oxygen in the pyran ring, as the dehydration is an elimination reaction where the activated complex stability is more dependent on the involvement of lone pair electrons on the heteroatom that is present in the heterocyclic ring.37
Supplementary data Supplementary data (X-ray crystallographic data, experimental procedure and spectroscopic data of all compounds) associated with this article can be found, in the online version, at http:// dx.doi.org/10.1016/j.bmcl.2012.09.018. References and notes 1. Hepworth, J. In Comprehensive Heterocyclic Chemistry; Katrizky, A. R., Rees, C. W., Eds.; Pergamon: Oxford, UK, 1984; Vol. 3, p 737. 2. Ellis, G. P. In Chromenes, Chromanones, and Chromones in the Chemistry of Heterocyclic Compounds; Wiley: New York, 1977; Vol. 31, p 11. 3. Mandal, T. K.; Kuznetsov, V. V.; Soldatenkov, A. T. Chem. Heterocycl. Compd. 1994, 30, 867.
7264
N. R. Emmadi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7261–7264
4. Kulkarni, M. V.; Kulkarni, G. M.; Lin, C.-H.; Sun, C.-M. Curr. Med. Chem. 2006, 13, 2795. 5. Santana, L.; Uriarte, E.; Roleira, F.; Milhazes, N.; Borges, F. Curr. Med. Chem. 2004, 11, 3239. 6. Murray, D. H.; Mendez, J.; Brown, S. A. The Natural Coumarins: Occurrence, Chemistry and Biochemistry; J. Wiley & Sons: New York, 1982. 7. O’Kennedy, R.; Thornes, R. D. Coumarins: Biology, Applications and Mode of Action; J. Wiley & Sons: Chichester, 1997. 8. Comprehensive Medicinal Chemistry; Hansch, C., Sammes, P. G., Taylor, J. B., Eds.; Pergamon: New York, 1990. Vol. 6. 9. Cardellina, J. H.; Bokesch, H. R.; McKee, T. C.; Boyd, M. R. Bioorg. Med. Chem. Lett. 1995, 5, 1011. 10. McKee, T.; Fuller, R. W.; Covington, C. D.; Cardellina, J. H., II; Gulakowski, R. J.; Krepps, B. L.; McMahon, J. B.; Boyd, M. R. J. Nat. Prod. 1996, 59, 754. 11. Galinis, D. L.; Fuller, R. W.; McKee, T. C.; Cardellina, J. H., II; Gulakowski, R. J.; McMahon, J. B.; Boyd, M. R. J. Med. Chem. 1996, 39, 4507. 12. Kumar, A.; Maurya, R. A.; Sharma, S.; Ahmad, A.; Singh, A. B.; Bhatia, G.; Srivastava, A. K. Bioorg. Med. Chem. Lett. 2009, 19, 6447. 13. Filler, R.; Banks, R. E. Organofluorine Chemicals and Their Industrial Applications; Ellis Horwood: Chichester, UK, 1979. 14. Frezza, M.; Balestrino, D.; Soulere, L.; Reverchon, S.; Queneau, Y.; Forestier, C.; Doutheau, A. Eur. J. Org. Chem. 2006, 4731. 15. Welch, J. T. Tetrahedron 1987, 43, 3123. 16. Buscemi, S.; Pace, A.; Piccionello, A.; Macaluso, G.; Vivona, N. J. Org. Chem. 2005, 70, 3288. 17. Abdolmohammadi, S.; Balalaie, S. Tetrahedron Lett. 2007, 48, 3299. 18. Cravotto, G.; Nano, G. M.; Tagliapietra, S. Synthesis 2001, 1, 49. 19. Ren, Q.; Gao, Y.; Wang, J. Chem. Eur. J. 2010, 16, 13594. 20. Khurana, J. M.; Kumar, S. Tetrahedron Lett. 2009, 50, 4125. 21. Khurana, J. M.; Nand, B.; Saluja, P. Tetrahedron 2010, 66, 5637. 22. Heravi, M. M.; Sadjadi, S.; Haj, N. M.; Oskooie, H. A.; Bamoharram, F. F. Catal. Commun. 2009, 10, 1643. 23. Kumar, D.; Reddy, V. B.; Mishra, B. G.; Rana, R. K.; Mallikarjuna, N.; Nadagouda; Verma, R. S. Tetrahedron 2007, 63, 3093. 24. Maggi, R.; Ballini, R.; Sartori, G.; Sartorio, R. Tetrahedron Lett. 2004, 45, 2297. 25. Bloxham, J.; Dell, C. P.; Smith, C. W. Heterocycles 1994, 38, 399. 26. Balalaie, S.; Ramezanpour, S.; Bararjanian, M.; Gross, J. H. Synth. Commun. 2008, 38, 78. 27. Ballini, R.; Bosica, G.; Conforti, M. L.; Maggi, R.; Mazzacani, A.; Righi, P.; Sartori, G. Tetrahedron 2001, 57, 1395. 28. Wang, X.; ShiShi, D.; Yu, H.; Wang, G.; Tu, S. Synth. Commun. 2004, 34, 509.
29. Elagamey, A. G. A.; El-Taweel, F. M. A. A.; Khodeir, M. N. M.; Elnagdi, M. H. Bull. Chem. Soc. Jpn. 1993, 66, 464. 30. Cravotto, G.; Nano, G. M.; Palmisano, G.; Tagliapietra, S. Synthesis 2003, 8, 1286. 31. Dong, Z.; Feng, J.; Cao, W.; Liu, X.; Lin, L.; Feng, X. Tetrahedron Lett. 2011, 52, 3433. 32. Cravotto, G.; Nano, G. M.; Palmisano, G.; Pilati, T.; Tagliapietra, S. J. Heterocycl. Chem. 2001, 38, 965. 33. Krishnaiah, A.; Narsaiah, B. J. Fluorine Chem. 2001, 109, 183. 34. Krishnaiah, A.; Reddy, E. N.; Sridhar, B. J. Hetercycl. Chem. Accepted and is in press. 35. Li, D.; Song, L.; Song, S.; Zhu, S. Z. J. Fluorine Chem. 2007, 128, 952. 36. Crystal data for compound 4a: C22H17F3O6, M = 434.36, colorless block, 0.40 0.28 0.13 mm3, monoclinic, space group P21/c (No. 14), a = 13.8545(12), b = 12.5537(11), c = 24.054(2) Å, b = 93.980(2)°, V = 4173.5(6) Å3, Z = 8, Dc = 1.383 g/cm3, F0 0 0 = 1792, CCD area detector, MoKa radiation, k = 0.71073 Å, T = 294(2)K, 2hmax = 50.0°, 39308 reflections collected, 7349 unique (Rint = 0.0254). Final GooF = 1.021, R1 = 0.0436, wR2 = 0.1170, R indices based on 5622 reflections with I > 2r(I) (refinement on F2), 607 parameters. l = 0.118 mm 1. CCDC 892543 contains supplementary crystallographic data for the structure. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 (0)1223 336 033; email: [email protected]]. 37. Li, D.; Song, L.; Li, X. F.; Xing, C. H.; Peng, W. M.; Zhu, S. Z. Eur. J. Org. Chem. 2007, 3520. 38. Mosmann, T. J. Immunol. Methods 1983, 65, 55. 39. General procedure for preparation of compounds 4: A mixture of 4-hydroxy coumarine (3a) or kojicacid (3b) (1.2 mmol), ethyl trifluoacetoacetate (2) (1.3 mmol), appropriate aldehyde (1) (1.3 mmol) in ethanol (10 ml) in presence of 30 mol % DBU was refluxed for a stipulated period by monitoring the reaction progress by TLC. After completion of the reaction, ethanol was evaporated under reduced pressure and the residue was purified by flash column chromatography using hexane and ethyl acetate as the eluent to get compound 4. Ethyl 2-hydroxy-5-oxo-4-phenyl-2-(trifluoro methyl)-2,3,4,5-tetra hydro pyrano chromene-3-carboxylate[3,2-c] 4a: mp = 115–116 °C; 1H NMR (CDCl3, 300 MHz): d 7.85 (d, J = 7.55 Hz, 1H), 7.55 (t, J = 7.55 Hz, 1H), 7.35–7.20 (m, 5H), 7.16–7.12 (m, 2H), 4.20 (d, J = 11.33 Hz, 1H), 4.08 (q, J = 7.17 Hz, 2H), 3.15 (d, J = 11.52 Hz, 1H), 1.04 (t, J = 7.17 Hz, 3H) ppm; 19F NMR (500 MHz, CDCl3): d = ss-84.20(s, 3 F, CF3) ppm; IR (KBr): 3386, 1738, 1692, 1631, 1371, 1198, 759cm 1; ESI-MS: m/z 457 [M+Na]+; HRMS (ESI) Anal. Calcd for C22H17F3NaO6 m/z 457.0874 [M+Na]+, found 457.0858.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxicity evaluation of highly functionalized pyranochromenes and pyranopyrans Narender Reddy Emmadi a, Krishnaiah Atmakur a,⇑, Ganesh Kumar Chityal b,⇑, Sujitha Pombala b, Jagadeesh Babu Nanubolu c a
Crop Protection Chemicals Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India Chemical Biology Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India c Laboratory of X-ray crystallography, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India b
a r t i c l e
i n f o
Article history: Received 15 May 2012 Revised 21 August 2012 Accepted 5 September 2012 Available online 23 September 2012 Keywords: 4-Hydroxy chromenes Ethyltrifluoro acetoacetate Aromatic aldehydes Tetrahydropyrano[3,2-c]chromenes Dihydropyrano[3,2-b]pyrans
a b s t r a c t A series of fluorinated tetrahydropyrano[3,2-c]chromenes and dihydropyrano[3,2-b]pyran derivatives have been synthesized and their in vitro cytotoxic activities have been determined in cervical cancer cell line (HeLa), human breast adenocarcinoma cell line (MDA-MB-231 and MCF-7) and human alveolar adenocarcinoma cell line (A549). Compounds 4g, 4k, 4p showed a very potent activity against MDA-MB-231, and 4c, 4p showed promising activity against MCF-7, while compounds 4c, 4g, 4p showed moderate activity against HeLa. Ó 2012 Elsevier Ltd. All rights reserved.
Pyranochromenes also known as pyranobenzopyrans are an important class of oxygen containing heterocycles that have attracted significant importance in the field of organic and natural product chemistry.1,2 Chromene, the basic skeleton of pyranochromenes is the isomer of coumarins and also noted to be present as an essential core moiety in numerous natural and biologically active compounds.3–7 Further, pyranochromenes and pyrano pyran scaffolds are one of the most commonly encountered heterocycles, which forms the important component of pharmacophores for a number of compounds having medicinal significance8 including anticancer, antiviral, antibacterial, antioxidant, anti-HIV,9–11 antihyperglycemic and anti-dyslipidemic activities.12 Meanwhile the presence of fluorine, especially trifluoromethyl group at a specified position in a molecule alters the reactivity because of its strong electron withdrawing character, lipophilicity and metabolic stability. The special character of fluorine has been thoroughly exploited in the design of novel targets for pharmaceutical, agrochemical and material science industries.13–16 In the recent past, a number of methods have been reported for the synthesis of dihydropyranochromenes17–22 and benzo[h]chromenes.23–29 However, few reports have appeared in the literature on the synthesis of tetrahydropyrano[3,2-c]chromenes30–32 and no re-
ports are available on trifluoromethyl substituted pyrano[3,2-c] chromenes and dihydropyrano[3,2-b]pyrans. With this background and also as a part of our continuous interest on the synthesis of trifluoromethyl substituted heterocycles of biological interest,33,34 herein we report the synthesis and cytotoxicity evaluation of hitherto unreported fluorinated tetrahydropyrano [3,2-c]chromenes and dihydropyrano[3,2-b]pyran derivatives. Initially, a model reaction was tried to prepare the fluorinated tetrahydropyrano[3,2-c]chromenes starting with simple benzaldehyde (1) ethyltrifluoro acetoacetate (2) and 4-hydroxy-2H-chromene-2-one (3a) (Scheme 1), in water at room temperature followed by at reflux temperature using a series of catalysts. Though the water mediated reaction method for the synthesis of pyranochromenes using various catalysts, that is, diammonium hydrogen phosphate17 TBAB,20 DBU21 and basic alumina24 are well documented in the literature, it did not work for our substrates. In a second attempt, the same reaction was conducted in ethanol
CHO
E-mail addresses: [email protected] (K. Atmakur), [email protected] (G.K. Chityal). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.09.018
O
F3C 1
⇑ Corresponding authors. Tel.: +91 40 27191436; fax: +91 40 27193382.
O
OH OEt
2
O 3a
Catalyst
O
Ethanol, reflux
Scheme 1.
F3C O O
OH COOEt O 4a
7262
N. R. Emmadi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7261–7264
As the optimum parameters were established, the generality of the reaction has been studied with varying substituent’s on (1), (3) (Scheme 2) and observed that the methodology was compatible to all the substituent’s. However, high yields were observed when electron withdrawing groups were present in the Para position (4d, 4e, 4m and 4o) to aldehyde functional group in 1 (Table 2). The same reaction (Scheme 1) when refluxed in ethanol without any catalyst, very poor yields were obtained (15% in 24 h, Table 1). The methodology was extended to the preparation of dihydro pyrano[3,2-b]pyrans (4s–w) by reacting compounds (1), (2) with 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (3b) and the desired products 4s-w were obtained in high yields. Structure of the products 4 were well characterized by their proton 1H NMR spectroscopy, where C-3 and C-4 protons were found to be trans to each other based on their chemical shifts (doublet) and coupling constants (J = 11.52 and 11.33). This data is in agreement with the earlier report35. In order to find out the absolute configuration of 4, (as there are three stereogenic centers in the molecule and possible for isomers) X-ray crystallography studies36 have been conducted on 4a. Based on the crystallography data (Fig. 1), it was observed that the
Table 1
S.No
Catalyst (mol %)
Time (h)
Yield (%)
1 2 3 4 5 6 7
TEA(30) DBU(10) DBU(20) DBU(30) DABCO(30) Piperdine(30) NH4OAc(30) N-Methy Imidazole(30) —
12 12 12 6 10 12 12 12 12 24
25 52 63 70 56 64 35 40 40 15
8 9
with various catalysts. Though the solvent role is not clear, interesting results were obtained in ethanol at reflux temperature with DBU as the catalyst. Encouraged by the results, the reaction was carried out with different mole ratios (Table 1) with different catalysts. However 30 mol % DBU proved to be an effective and high yielding parameter within 6 h.
OH
R2
O 3a
CHO
O 1
R
R
O
F3C
1
OEt
2
R
F3C O O
O
OH COOEt R1 O 4 a-r
R
DBU(30%) Ethanol, reflux 6h
O
2 HO
O OH
HO
O 3b
O
O
CF3 OH COOEt
R1 R 4 s-w
Scheme 2.
Table 2
S. No
(4)
R
R1
R2
Time (h)
Yield (%)
Melting point (°C)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q
H Cl F NO2 CN Me OMe OH OH Iso propyl OMe H NO2 F CN OH H
H H H H H H H H OMe H OMe H H H H H H
H H H H H H H H H H H F F F F F Me
6 6 7 6 6 7 8 7 8 6 7 6 6 6 7 8 6
70 65 67 72 74 64 68 65 58 67 61 68 75 66 70 62 67
115–116 138–140 107–108 112–114 114–115 144–146 134–136 163–165 110–112 145–147 158–160 145–146 118–120 142–144 109–111 170–171 172–174
18
4r
—
H
8
60
186–188
H H H H OMe
— — — — —
6 6 8 7 8
68 65 67 58 64
150–152 112–114 173–174 170–171 159–160
CHO
N
19 20 21 22 23
4s 4t 4u 4v 4w
H Me OMe OH OMe
N. R. Emmadi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7261–7264 Table 3 Cytotoxicity data, IC50 (lM) Entry
Compound
HeLa
MDA-MB-231
MCF-7
A549
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
4c 4e 4g 4h 4i 4j 4k 4m 4n 4o 4p 4r 4t 4u 4v Doxorubicin
11.01 — 11.23 — — — 87.01 — — — 11.45 — — — — <1
11.27 — 2.20 — — — 7.71 — — — 6.61 — — — — <1
7.67 — 71.7 — — — 53.89 — — — 6.54 — — — — 1
— — 82.37 — — — 34.28 — — — 33.86 — — — — 1
-No activity. HeLa- Cervical cancer cell line. MDA-MB-231- Human breast adenocarcinoma cell line. MCF-7- Human breast adenocarcinoma cell line. A549- Human alveolar adenocarcinoma cell line.
Figure 1. (a) The molecular structure of 4a. (b) The molecular structure of 4a with the atom-numbering scheme. The compound crystallized in the monoclinic space group P21/c with two molecules in the asymmetric unit. Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radius.
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Further, compounds in 4 series were taken-up to evaluate the cytotoxicity, since they are lipophilic in nature due to the presence of trifluoromethyl group and expected to penetrate into the membrane in considerable concentration and suppose to exhibit potent activity. Therefore, the cytotoxicity was determined against a panel of human tumor cell lines such as cervical cancer cell line (HeLa), human breast adenocarcinoma cell lines (MDA-MB-231 and MCF-7), human alveolar adenocarcinoma cell line (A549) and the results are tabulated in (Table 3) and human alveolar adenocarcinoma cell line (A549). In the series of pyranochromene derivatives, compounds 4c, 4g, 4k, 4p having fluorine, methoxy, di-methoxy and hydroxy functional groups on C-4 phenyl ring had the distinct tendency to increase the in vitro antitumor potency and exhibited an excellent cytotoxicity against MDA-MB-231, MCF-7 and HeLa. Compounds 4g, 4k, and 4p showed potent activity against MDA-MB-231 with IC50 value of 2.20, 7.71 and 6.61 lM (Table 2, entries 3, 7 and 11). Compounds 4c, 4p showed very promising activity against MCF-7 with IC50 value of 7.67 and 6.54 lM (Table 2, entries 1, 11) respectively and compounds 4c, 4g, 4p showed moderate activity with IC50 value of 11.01, 11.23 and 11.45 lM against HeLa (Table 2, entries 1,3,11). Cytotoxicity data was assessed on the basis of measurement of in vitro growth of tumor cell lines in 96 well plates by cellmediated reduction of tetrazolium salt to water insoluble formazan crystals using doxorubicin as a standard. The compounds were tested for cytotoxicity against a panel of four different tumor cell lines: A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), HeLa derived from human cervical cancer cells (ATCC No. CCL-2), MDA-MB-231 derived from human breast adenocarcinoma cells (ATCC No. HTB-26) and MCF7 derived from human breast adenocarcinoma cells (ATCC No. HTB-22) using the MTT assay.38 The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the doseresponse curves. IC50 values (in lM) are expressed as the average of two independent experiments. In conclusion, we prepared hitherto unreported fluorinated tetrahydro pyrano[3,2-c]chromenes,39 dihydropyrano[3,2-b]pyran derivatives using simple reaction conditions in a single pot and evaluated their in vitro cytotoxicity against a panel of human tumor cell lines. In the series of pyranochromene derivatives, four compounds 4c, 4g, 4k and 4p showed very potent activity and the data is helpful to further exploit these compounds for anticancer activity mechanistic studies and also helpful to design and synthesize more such derivatives to be taken-up for further studies. Acknowledgments Authors are thankful to Director IICT for constant support and ENR is thankful to CSIR New Delhi for SRF.
compound exists as a racemic mixture (enantiomers and are non separable) comprising the SSR and RRS configuration at C-2, C-3 and C-4 asymmetric centers. These studies support the 1H NMR data of 4a. Attempts to prepare the dehydrated product of 4 by reported protocols especially for the trifluoromethyl substituted heterocycles were unsuccessful in spite of refluxing them in toluene in presence of several reagents such as concentrated H2SO4, P2O5, PTSA, TFA and also PPTS and TPP independently. This may be attributed to the fact that the weaker electron donating character of oxygen in the pyran ring, as the dehydration is an elimination reaction where the activated complex stability is more dependent on the involvement of lone pair electrons on the heteroatom that is present in the heterocyclic ring.37
Supplementary data Supplementary data (X-ray crystallographic data, experimental procedure and spectroscopic data of all compounds) associated with this article can be found, in the online version, at http:// dx.doi.org/10.1016/j.bmcl.2012.09.018. References and notes 1. Hepworth, J. In Comprehensive Heterocyclic Chemistry; Katrizky, A. R., Rees, C. W., Eds.; Pergamon: Oxford, UK, 1984; Vol. 3, p 737. 2. Ellis, G. P. In Chromenes, Chromanones, and Chromones in the Chemistry of Heterocyclic Compounds; Wiley: New York, 1977; Vol. 31, p 11. 3. Mandal, T. K.; Kuznetsov, V. V.; Soldatenkov, A. T. Chem. Heterocycl. Compd. 1994, 30, 867.
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29. Elagamey, A. G. A.; El-Taweel, F. M. A. A.; Khodeir, M. N. M.; Elnagdi, M. H. Bull. Chem. Soc. Jpn. 1993, 66, 464. 30. Cravotto, G.; Nano, G. M.; Palmisano, G.; Tagliapietra, S. Synthesis 2003, 8, 1286. 31. Dong, Z.; Feng, J.; Cao, W.; Liu, X.; Lin, L.; Feng, X. Tetrahedron Lett. 2011, 52, 3433. 32. Cravotto, G.; Nano, G. M.; Palmisano, G.; Pilati, T.; Tagliapietra, S. J. Heterocycl. Chem. 2001, 38, 965. 33. Krishnaiah, A.; Narsaiah, B. J. Fluorine Chem. 2001, 109, 183. 34. Krishnaiah, A.; Reddy, E. N.; Sridhar, B. J. Hetercycl. Chem. Accepted and is in press. 35. Li, D.; Song, L.; Song, S.; Zhu, S. Z. J. Fluorine Chem. 2007, 128, 952. 36. Crystal data for compound 4a: C22H17F3O6, M = 434.36, colorless block, 0.40 0.28 0.13 mm3, monoclinic, space group P21/c (No. 14), a = 13.8545(12), b = 12.5537(11), c = 24.054(2) Å, b = 93.980(2)°, V = 4173.5(6) Å3, Z = 8, Dc = 1.383 g/cm3, F0 0 0 = 1792, CCD area detector, MoKa radiation, k = 0.71073 Å, T = 294(2)K, 2hmax = 50.0°, 39308 reflections collected, 7349 unique (Rint = 0.0254). Final GooF = 1.021, R1 = 0.0436, wR2 = 0.1170, R indices based on 5622 reflections with I > 2r(I) (refinement on F2), 607 parameters. l = 0.118 mm 1. CCDC 892543 contains supplementary crystallographic data for the structure. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 (0)1223 336 033; email: [email protected]]. 37. Li, D.; Song, L.; Li, X. F.; Xing, C. H.; Peng, W. M.; Zhu, S. Z. Eur. J. Org. Chem. 2007, 3520. 38. Mosmann, T. J. Immunol. Methods 1983, 65, 55. 39. General procedure for preparation of compounds 4: A mixture of 4-hydroxy coumarine (3a) or kojicacid (3b) (1.2 mmol), ethyl trifluoacetoacetate (2) (1.3 mmol), appropriate aldehyde (1) (1.3 mmol) in ethanol (10 ml) in presence of 30 mol % DBU was refluxed for a stipulated period by monitoring the reaction progress by TLC. After completion of the reaction, ethanol was evaporated under reduced pressure and the residue was purified by flash column chromatography using hexane and ethyl acetate as the eluent to get compound 4. Ethyl 2-hydroxy-5-oxo-4-phenyl-2-(trifluoro methyl)-2,3,4,5-tetra hydro pyrano chromene-3-carboxylate[3,2-c] 4a: mp = 115–116 °C; 1H NMR (CDCl3, 300 MHz): d 7.85 (d, J = 7.55 Hz, 1H), 7.55 (t, J = 7.55 Hz, 1H), 7.35–7.20 (m, 5H), 7.16–7.12 (m, 2H), 4.20 (d, J = 11.33 Hz, 1H), 4.08 (q, J = 7.17 Hz, 2H), 3.15 (d, J = 11.52 Hz, 1H), 1.04 (t, J = 7.17 Hz, 3H) ppm; 19F NMR (500 MHz, CDCl3): d = ss-84.20(s, 3 F, CF3) ppm; IR (KBr): 3386, 1738, 1692, 1631, 1371, 1198, 759cm 1; ESI-MS: m/z 457 [M+Na]+; HRMS (ESI) Anal. Calcd for C22H17F3NaO6 m/z 457.0874 [M+Na]+, found 457.0858.