- Email: [email protected]
Novel anthraquinone based chalcone analogues containing an imine fragment: Synthesis, cytotoxicity and anti-angiogenic activity
Bioorganic & Medicinal Chemistry Letters 24 (2014) 65–71
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Novel anthraquinone based chalcone analogues containing an imine fragment: Synthesis, cytotoxicity and anti-angiogenic activity Branka Kolundzˇija a, Violeta Markovic´ b, Tatjana Stanojkovic´ a, Ljubinka Joksovic´ b, Ivana Matic´ a, Nina Todorovic´ c, Marijana Nikolic´ d, Milan D. Joksovic´ b,⇑ a
Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia Faculty of Science, Department of Chemistry, University of Kragujevac, R. Domanovic´a 12, 34000 Kragujevac, Serbia Institute for Chemistry, Technology and Metallurgy, Njegoševa 12, 11000 Belgrade, Serbia d Faculty of Medicinal Sciences, University of Kragujevac, S. Markovic´a 69, 34000 Kragujevac, Serbia b c
a r t i c l e
i n f o
Article history: Received 2 October 2013 Revised 25 November 2013 Accepted 29 November 2013 Available online 4 December 2013 Keywords: Chalcone Anthraquinone Cytotoxicity Angiogenesis
a b s t r a c t A new class of imine derivatives of hybrid chalcone analogues containing anthraquinone scaffold was synthesized and evaluated for their in vitro cytotoxic activity against HeLa, LS174, and A549 cancer cells. The compound 5n with furan ring linked to imino group showed potent activity against all target cells with IC50 values ranging from 1.76 to 6.11 lM. A mode of action study suggested that compounds induced changes typical for apoptosis in HeLa cells. The most active compounds inhibited tubulogenesis and 5h was found to exhibit a strong anti-angiogenic effect. Ó 2013 Elsevier Ltd. All rights reserved.
Natural and synthetic chalcones are known cytotoxic pharmacophores that act by disruption of the cell cycle, inhibition of angiogenesis, binding MDM2 human oncoprotein and induction of apoptosis.1 During the last years, a number of pharmacologically interesting hybrid chalcone analogues were synthesized involving structural modification to improve their anticancer potential and chemoprotective properties. The examples of these transformations are introduction of different substituents on aromatic rings, replacement of phenyl rings A or B with heterocyclic, polyaromatic or organometallic structures and substitution on enone part of chalcone. Some new classes of hybrid chalcone compounds with pronounced activity against various types of cancer cell lines contain Mannich bases of heterocyclic chalcones,2 dihydrobenzofuran,3 imidazopyridine/pyrimidine,4 3-arylquinoline,5 quinoline-2-one,6 naphthalene,7 indole,8,9 pyrazole,10 quinoxaline,11 amidobenzothiazole,12 and ferrocene.13 Tumor angiogenesis is a physiological process that is involved in the formation of a network of blood capillaries supplying cancerous growths with oxygen and nutritients.14 Chalcone analogues exhibit antitumor activities through various mechanisms and inhibition of angiogenesis is one of them.15 The natural chalcone, xanthohumol and its synthetic derivatives inhibit angiogenesis
⇑ Corresponding author. Tel.: +381 34 336 223. E-mail address: [email protected] (M.D. Joksovic´). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.075
by suppressing vascular endothelial growth factor (VEGF),16 while the flavonoid precursor 4-hydroxychalcone inhibits this process by affecting both VEGF and basic fibroblast growth factor (bFGF) intracellular signaling pathways.17 The chalcone derivatives bearing furan and thiophene heterocycles demonstrated strong antiangiogenic activity18 as well as quinolil-thienyl chalcones in the role of VEGFR-2 kinase inhibitors.19 On the other hand, some natural anthraquinones like aloe-emodin, emodin and rhein displayed anti-angiogenic effects in the zebrafish model. Contrary, chrysophanol with no substitution and physcion with the substituted methoxy group did not show any anti-angiogenic activity. Emodin was the most potent anti-angiogenic compound inhibiting endothelial cell proliferation, migration and tube formation in a dose-dependent manner.20,21 Some representative chalcone and anthraquinone-based structures are presented in Figure 1. In continuation with our current interest toward synthesis of bioactive anthraquinone compounds,22 in this work we explored the possibility to combine the potential synergic anticarcinogenic effects of chalcone moiety and anthraquinone scaffold. To the best of our knowledge, there is no literature data on preparation and biological activity of anthraquinone based chalcone analogues. Thus, we designed and synthesized a new class of chalcones with anthraquinone as A-ring and different arylimino or alkylimino substituents in para-position of B-ring, together with an evaluation of their antiproliferative and anti-angiogenic potential.
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B. Kolundzˇija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71
Figure 1. Basic structure of chalcone (1) and some representative chalcone and anthraquinone-based derivatives with antitumor properties (2–6).
Starting aromatic amines were selected to obtain the compounds with a good coverage of polar, steric, electron-donating and electron-withdrawing properties. Our further strategy was
based on replacement of the phenyl group by the benzyl one as well as more flexible alkyl group bonded to heterocyclic furan and thiophene rings.
Scheme 1. Reagents and conditions: (a) CH3COCl, AlCl3, CH2Cl2, 2 h, 0 °C, 5 M HCl; (b) CrO3, CH3COOH, 5 min reflux, H2O; (c) terephthalaldehyde, NaOH, MeOH, 5 h, reflux; (d) primary amines, AcOH, dioxane, 12–48 h, reflux.
B. Kolundzˇija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71 Table 1 Concentrations of 5a–q that induced a 50% decrease in HeLa (2000 cells per well), LS174 (7000 cells per well), A549 (5000 cells per well), and MRC5 (5000 cells per well), cell survival expressed as IC50 (lM). The compounds were incubated with cells for 72 h and IC50 values are presented as the mean ± SD, determined from the results of MTT assay in three independent experiment IC50 (lM)
Compounds
5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q Cisplatin
HeLa
LS174
A549
MRC5
4.46 ± 1.51 6.58 ± 0.85 4.17 ± 1.14 8.82 ± 0.53 5.02 ± 0.54 5.94 ± 1.35 5.01 ± 0.88 1.93 ± 0.89 9.78 ± 0.01 6.57 ± 0.22 4.62 ± 0.81 3.09 ± 0.86 1.45 ± 0.34 1.82 ± 0.68 4.19 ± 0.79 5.97 ± 0.04 5.34 ± 1.13 2.10 ± 0.20
10.56 ± 0.09 10.18 ± 0.32 9.54 ± 0.68 8.99 ± 3.23 17.22 ± 2.12 5.98 ± 0.84 8.70 ± 0.85 17.18 ± 2.24 11.30 ± 0.82 7.51 ± 0.10 8.55 ± 2.07 7.60 ± 1.06 6.61 ± 0.49 1.76 ± 0.78 11.03 ± 0.37 10.66 ± 0.38 5.30 ± 0.45 5.54 ± 1.03
11.04 ± 0.37 11.06 ± 0.33 9.38 ± 0.54 13.01 ± 0.51 14.51 ± 1.31 7.63 ± 0.91 8.17 ± 1.06 9.99 ± 0.46 15.73 ± 0.75 7.80 ± 1.51 10.18 ± 0.46 7.98 ± 0.67 7.78 ± 0.26 6.11 ± 0.45 9.79 ± 0.74 11.44 ± 1.92 6.01 ± 0.09 11.92 ± 2.19
16.14 ± 0.03 17.08 ± 1.24 13.52 ± 2.40 16.68 ± 0.35 11.75 ± 1.30 11.66 ± 0.81 17.16 ± 1.74 16.69 ± 0.85 12.74 ± 2.65 16.15 ± 0.52 12.40 ± 1.64 11.00 ± 1.31 14.55 ± 0.72 15.11 ± 3.56 11.93 ± 0.33 15.96 ± 1.36 11.87 ± 0.65 14.21 ± 1.54
The synthesis of new hybrid chalcone analogues 5a–q is presented in Scheme 1 while experimental protocols are given in Supplementary content. The commercially available anthracene 1 was acylated with acetyl chloride to 1-acetylanthracene 223 which was then oxidized by chromium (VI) oxide in glacial acetic acid yielding 1-acetylanthraquinone 3.24 The ketone 3 was recrystallized from ethanol and condensed with terephthalaldehyde in presence of sodium hydroxide affording anthraquinone aldehyde 4. In the next step, the final chalcone conjugates containing imine fragment were prepared by condensation of aldehyde 4 and a series of aromatic and aliphatic primary amines in dioxane. The presence of
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anthraquinone unit significantly decreases reactivity of aldehyde group and some reactions required an excess of amine reagent and prolonged reaction time. Importantly, anthraquinone also make imine functional group extremely resistant to hydrolysis under neutral conditions. A suspension of 5g in water was stirred for 24 h and no detectable quantity of aldehyde was observed by NMR. In the beginning of our investigations, we first synthesized a chalcone product by reaction of acetylanthraquinone 3 and benzaldehyde, but its cytotoxic activity was very low. However, derivatization of aldehyde 4 in the para-position of the B-ring through imine formation by condensation with different primary amines dramatically enhanced cytotoxicity. The obtained imines were particularly active against A549 cell lines, making these compounds comparable to cisplatin. The cytotoxic properties of all synthesized compounds were evaluated in vitro against human cervix adenocarcinoma (HeLa), human colon carcinoma (LS174), nonsmall cell lung carcinoma (A549) and normal human fetal lung fibroblast cell line (MRC-5) by MTT assay. Cisplatin was used as a reference drug. The results are summarized in Table 1 with IC50 values defined as the concentrations corresponding to 50% growth inhibition. Evidently, the substituents of imine part of molecule strongly influence on cytotoxic effect of the synthesized compounds. The introduction of electron-donating methyl group at all three positions of phenyl moiety did not improve activity in comparison with unsubstituted phenyl group. However, the presence of the voluminous electron-withdrawing substituents such as –Cl and –CF3, especially in meta-position, caused a significant increase of cytotoxic activity in inhibition of HeLa cells. The replacement of phenyl group with cyclohexyl one led to slight decrease of antiproliferative activity. Although alkyl groups had no effect on cytotoxicity, aromatic and heterocyclic rings linked to methylene group showed potent activity toward HeLa cell lines with IC50 values from 1.82 to 5.34 lM. The compounds 5n and 5q containing furan and phenyl rings were also efficient in inhibition of LS174 and A549 cells.
Figure 2. Representative histograms of cell-cycle distribution after 24 h treatment of A549 cells with IC50 of compounds 5h, 5m, 5n and 5q. Cell-cycle distribution was measured by flow cytometric analysis of DNA content after treatment with these compounds.
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The significant cytotoxic activity of 5h, 5m, 5n and 5q led us to investigate the mechanism of action of these compounds by cytofluorimetric analysis, using propidium iodide to label DNA. Flow
cytometry was employed to examine the changes in DNA content. A representative cell cycle analysis of A549 cell lines treated with 5h, 5m, 5n and 5q for 24 h is given in Figure 2. The effect of com-
Figure 3. Photomicrographs of AO/EB stained control HeLa cells, and HeLa cells treated with IC50 and 2IC50 concentrations of 5h, 5m, 5n and 5q within 24 h.
B. Kolundzˇija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71
Figure 4. DNA fragmentation detection by 2% agarose gel electrophoresis. C1 and C2 represent control samples while 5h, 5m, 5n and 5q lanes represent treatment with IC50 of respective compounds.
pounds on cell cycle distribution of malignant cells is shown in Table S1 (Supplementary material). There were no significant changes of sub-G1 populations on all tested cell lines after 24 h treatment with IC50 dose of investigated compounds. The percentage change in sub-G1 in A549 cells treated with 5q for 24 h was marked with increase to 15.29%. In addition, any relevant change in the basal level in G1, S and G2-M population was not observed in the investigated cells after 24 h of treatment. Moreover, the A549 and LS174 cells showed significant changes in G2/M arrest (29.31% and 68.89%, respectively) after 24 h of treatment with 5h (see Fig. 2 and Table S1). The G2/M arrest is associated with evident reduction in S phase. Figure 3 shows the results of fluorescence microscopy of HeLa cells coupled with acridine orange/ethidium bromide (AO/EB) double staining for the occurrence of morphological changes and DNA condensation after treatment with 5h, 5m, 5n and 5q. HeLa cells treated with IC50 and 2IC50 concentrations of these compounds exhibited nuclear shrinkage and chromatin condensation, compared to the untreated control cells. The presence of a ladder DNA fragmentation pattern typical for apoptotic cells was evaluated after 24 h of exposure of HeLa cells to IC50 of 5h, 5m, 5n, and 5q. However, alterations in nuclear morphology may not necessarily involve DNA fragmentation.25 Despite the significant cytotoxicity of compounds observed towards HeLa cells, no DNA ladder formation was detected upon their treatment (Fig. 4). In this regard, it should be noted that 5h, 5m, 5n, and 5q do
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not show significant accumulation in sub-G1 phase of the cell cycle on HeLa cells. The activities of caspase-3 and -8, in 5h, 5m, 5n, and 5q-treated HeLa cells were detected by specific caspase inhibitors using flow cytometry (Fig. 5). Bearing in mind the absence of DNA fragmentation and the fact that these compounds do not lead to significant accumulation of sub-G1 phase of the cell cycle, one could conclude that the cell death induced by these compounds should be independent of caspase-3. Unexpectedly, the analysis revealed the presence of active caspase-3 in HeLa cells treated with 5h, 5n, and 5q while 5m showed that inhibitors of caspase-3, had almost no effect on the activity of this compound. Thus, only specific caspase-8 inhibitor significantly suppressed the caspase activity and we can conclude that 5m shows activity mainly via mitochondrial pathway through activation of caspase-8 in HeLa cells. In general, these caspase activities, coupled with the absence of DNA fragmentation, may indicate a possible inhibition of the endonuclease activity DFF40/CAD by these compounds.26–29 Further studies are needed for more detailed understanding of the relationship between DFF40/CAD endonuclease and caspase-3 activity. Blocking angiogenesis could be a strategy to arrest tumor growth and anti-angiogenic drug development has attracted a lot of research interest. Without blood vessels, tumors cannot grow beyond a critical size or metastasize to another organ.30 The tube formation activity of EA.hy926s is important in vitro endpoint for angiogenesis. Examined compounds 5h, 5m, 5n, and 5q inhibited tubulogenesis as it is shown in Figure 6A where untreated EA.hy926s formed elongated tube-like structures. The treatment with IC20 sub-toxic dose of investigated compounds resulted in a significant decrease in capillary tube formation and this effect was very strong for 5h. The IC20 values of these compounds on tube formation inhibition of EA.hy926 ranged from 4.9 to 5.5 lM (sub-toxic effect of these compounds is illustrated in Figure 6B. Our observations were consistent with previously cited reports that many natural and synthetic chalcones possess anti-angiogenic activity. Next, we examined the effect of the investigated compounds on the activity of matrix metalloproteinases. HeLa cells were treated with sub-cytotoxic concentrations (IC20) of 5h, 5m, 5n and 5q for 24 h in serum-free RPMI-1640. The supernatant was collected and tested for matrix metalloproteinase activity by gelatin zymography. As it is shown in Figure 7, activity of MMP-s was significantly reduced compared to the controls C1 and C2. Our results showed that the investigated compounds inhibited the activity of these enzymes. MMP-2 has been strongly implicated in angiogenesis and promotion of the tumor metastatic potential playing crucial role not only in invasion, but also in determination of cancer cell transformation, growth, apoptosis and signal transduction.31– 33 The inhibitory effects confirmed by 5h, 5m, 5n and 5q against MMP-2 secretion could be a starting point for further development
Figure 5. Effects of 5h, 5m, 5n and 5q on caspase-3 and -8 inhibitors. Data are shown as mean ± SD. HeLa cells were exposed to IC90 concentrations of investigated compounds for 24 h in the presence of specific caspase inhibitors (final concentration—40 lM) as described.
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Figure 6. (A) EA.hy926 cell tube formation on Matrigel with and without an added compound. (B) Sub-toxic effect of the investigated compounds on EA.hy926 after 24 h incubation, without Matrigel.
Figure 7. Treatment of HeLa cells with sub-toxic concentrations of 5h, 5m, 5n and 5q within 24 h reduces MMP-2 secretion as demonstrated by gelatin zymography. C1 and C2 represent control.
of these compounds as anti-angiogenic agents for metastatic malignancies. In summary, we designed and prepared a series of new hybrid chalcones with an anthraquinone unit. Generally, introduction of electron withdrawing substituents in meta-position of the phenyl ring was beneficial for good activity toward all cell lines. The compound 5n containing furan ring was found to be especially active against the growth of all tested malignant cell lines. It was noted that these compounds did not have tendency to significant accumulation of cells in sub-G1 phase. Only 5h led to an increase in sub-G1 phase in A549 cells as well as changes in G2/M arrest of A549 and LS174 cells. In addition, 5h, 5m, 5n and 5q induced the activities of caspase-3 and -8 in treated Hela cells, and exhibited strong anti-angiogenic activity. Finally, these compounds showed potent inhibition against MMP-2 secretion. Acknowledgement The authors are grateful to the Ministry of Science and Technological Development of the Republic of Serbia for financial support (Grant Nos. 172016 and 175011).
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11.075. References and notes 1. Go, M. L.; Wu, X.; Liu, X. L. Curr. Med. Chem. 2005, 12, 483. 2. Reddy, M. V. B.; Su, C. R.; Chiou, W. F.; Liu, Y. N.; Chen, R. Y. H.; Bastow, K. F.; Lee, K. H.; Wu, T. S. Bioorg. Med. Chem. 2008, 16, 7358. 3. Nagaraju, M.; Deepthi, E. G.; Ashwini, C.; Vishnuvardhan, M. V. P. S.; Nayak, V. L.; Chandra, R.; Ramakrishna, S.; Gawali, B. B. Bioorg. Med. Chem. Lett. 2012, 22, 4314. 4. Kamal, A.; Reddy, J. S.; Ramaiah, M. J.; Dastagiri, D.; Bharathi, E. V.; Sagar, M. V. P.; Pushpavalli, S. N. C. V. L.; Ray, P.; Bhadra, M. P. Chem. Commun. 2010, 355. 5. Tseng, C. H.; Chen, Y. L.; Hsu, C. Y.; Chen, T. C.; Cheng, C. M.; Tso, H. C.; Lu, Y. J.; Tseng, C. C. Eur. J. Med. Chem. 2013, 59, 274. 6. Abonia, R.; Insuasty, D.; Castillo, J.; Insuasty, B.; Quiroga, J.; Nogueras, M.; Cobo, J. Eur. J. Med. Chem. 2012, 57, 29. 7. Winter, E.; Chiaradia, L. D.; de Cordova, C. A. S.; Nunes, R. J.; Yunes, R. A.; Creczynski-Pasa, T. B. Bioorg. Med. Chem. 2010, 18, 8026. 8. Robinson, M. W.; Overmeyer, J. H.; Young, A. M.; Erhardt, P. W.; Maltese, W. A. J. Med. Chem. 2012, 55, 1940. 9. Kumar, D.; Kumar, N. M.; Akamatsu, K.; Kusaka, E.; Harada, H.; Ito, T. Bioorg. Med. Chem. Lett. 2010, 20, 3916.
B. Kolundzˇija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71 10. Insuasty, B.; Tigreros, A.; Orozco, F.; Quiroga, J.; Abonia, R.; Nogueras, M.; Sanchez, A.; Cobo, J. Bioorg. Med. Chem. 2010, 18, 4965. 11. Mielcke, T. R.; Mascarello, A.; Chiela, E. F.; Zanin, R. F.; Lenz, G.; Leal, P. C.; Chirardia, L. D.; Yunes, R. A.; Nunes, R. J.; Battastini, A. M. O.; Morone, F. B.; Campos, M. M. Eur. J. Med. Chem. 2012, 48, 255. 12. Kamal, A.; Mallareddy, A.; Suresh, P.; Shaik, T. B.; Nayak, V. L.; Kishor, C.; Shetti, R. V. C. R. N. C.; Rao, N. S.; Tamboli, J. R.; Ramakrishna, S.; Addlagatta, A. Bioorg. Med. Chem. 2012, 20, 3480. 13. Zsoldos-Mády, V.; Chámpai, A.; Szabó, R.; Mészáros-Alapi, E.; Pásztor, J.; Hudecz, F.; Sohár, P. ChemMedChem 2006, 1, 1119. 14. Bergers, G.; Benjamin, L. E. Nat. Rev. Cancer 2003, 3, 401. 15. Mojzis, J.; Varinska, L.; Mojzisova, G.; Kostova, I.; Mirossay, L. Pharmacol. Res. 2008, 57, 259. 16. Kim, J. A.; Kang, Y.; Thapa, D.; Lee, J. S.; Park, M. A.; Lee, K. H.; Lyoo, W. S.; Lee, Y. R. Biomol. Ther. 2009, 17, 422. 17. Varinska, L.; van Wijhe, M.; Belleri, M.; Mitola, S.; Perjesi, P.; Presta, M.; Koolwijk, P.; Ivanova, L.; Mojzis, J. Eur. J. Pharmacol. 2012, 691, 125. 18. Lee, J. S.; Kang, Y.; Kim, J. T.; Thapa, D.; Lee, E. S.; Kim, J. A. Eur. J. Pharmacol. 2012, 677, 22. 19. Rizvi, S. U. F.; Siddiqui, H. L.; Nisar, M.; Khan, N.; Khan, I. Bioorg. Med. Chem. Lett. 2012, 22, 942. 20. He, Z. H.; He, M. F.; Ma, S. C.; But, P. P. H. J. Ethnopharmacol. 2009, 121, 313. 21. Kaneshiro, T.; Morioka, T.; Inamine, M.; Kinjo, T.; Arakaki, J.; Chiba, I.; Sunagawa, N.; Suzui, M.; Yoshimi, N. Eur. J. Pharmacol. 2006, 553, 46.
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22. Markovic´, V.; Janic´ijevic´, A.; Stanojkovic´, T.; Kolundzˇija, B.; Sladic´, D.; Vujcˇic´, M.; Janovic´, B.; Joksovic´, L.; Djurdjevic´, P. T.; Todorovic´, N.; Trifunovic´, S.; Joksovic´, M. D. Eur. J. Med. Chem. 2013, 64, 228. 23. Stolka, M.; Yanus, J. F.; Pearson, J. M. Macromolecules 1976, 9, 715. 24. Bassilios, H. F.; Shawky, M.; Salem, A. Y. Recl. Trav. Chim. Pays-Bas 1963, 82, 298. 25. Shrivastava, A.; Tiwari, M.; Sinha, R. A.; Kumar, A.; Balapure, A. K.; Bajpai, V. K.; Sharma, R.; Mitra, K.; Tandon, A.; Godbole, M. M. J. Biol. Chem. 2006, 281, 19762. 26. Iglesias-Guimarais, V.; Gil-Guiñon, E.; Gabernet, G.; García-Belinchón, M.; Sánchez-Osuna, M.; Elisenda Casanelles, E.; Comella, J. X.; Yuste, V. J. J. Biol. Chem. 2012, 287, 7766. 27. Falcieri, E.; Martelli, A. M.; Bareggi, R.; Cataldi, A.; Cocco, L. Biochem. Biophys. Res. Commun. 1993, 193, 19. 28. Sikora, E.; Bielak-Zmijewska, A.; Magalska, A.; Piwocka, K.; Mosieniak, G.; Kalinowska, M.; Widlak, P.; Cymerman, I. A.; Bujnicki, J. M. Mol. Cancer Ther. 2006, 5, 927. 29. Widak, P. Acta Biochim. Pol. 2000, 47, 1037. 30. Folkman, J.; Shing, Y. J. Biol. Chem. 1992, 267, 10931. 31. Kessenbrock, K.; Plaks, V.; Werb, Z. Cell 2010, 141, 52. 32. Fang, J. M.; Shing, Y.; Wiederschain, D.; Yan, L.; Butterfield, C.; Jackson, G.; Harper, J.; Tamvakopoulos, G.; Moses, M. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3884. 33. Stearns, M.; Stearns, M. E. Oncol. Res. 1996, 8, 69.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Novel anthraquinone based chalcone analogues containing an imine fragment: Synthesis, cytotoxicity and anti-angiogenic activity Branka Kolundzˇija a, Violeta Markovic´ b, Tatjana Stanojkovic´ a, Ljubinka Joksovic´ b, Ivana Matic´ a, Nina Todorovic´ c, Marijana Nikolic´ d, Milan D. Joksovic´ b,⇑ a
Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia Faculty of Science, Department of Chemistry, University of Kragujevac, R. Domanovic´a 12, 34000 Kragujevac, Serbia Institute for Chemistry, Technology and Metallurgy, Njegoševa 12, 11000 Belgrade, Serbia d Faculty of Medicinal Sciences, University of Kragujevac, S. Markovic´a 69, 34000 Kragujevac, Serbia b c
a r t i c l e
i n f o
Article history: Received 2 October 2013 Revised 25 November 2013 Accepted 29 November 2013 Available online 4 December 2013 Keywords: Chalcone Anthraquinone Cytotoxicity Angiogenesis
a b s t r a c t A new class of imine derivatives of hybrid chalcone analogues containing anthraquinone scaffold was synthesized and evaluated for their in vitro cytotoxic activity against HeLa, LS174, and A549 cancer cells. The compound 5n with furan ring linked to imino group showed potent activity against all target cells with IC50 values ranging from 1.76 to 6.11 lM. A mode of action study suggested that compounds induced changes typical for apoptosis in HeLa cells. The most active compounds inhibited tubulogenesis and 5h was found to exhibit a strong anti-angiogenic effect. Ó 2013 Elsevier Ltd. All rights reserved.
Natural and synthetic chalcones are known cytotoxic pharmacophores that act by disruption of the cell cycle, inhibition of angiogenesis, binding MDM2 human oncoprotein and induction of apoptosis.1 During the last years, a number of pharmacologically interesting hybrid chalcone analogues were synthesized involving structural modification to improve their anticancer potential and chemoprotective properties. The examples of these transformations are introduction of different substituents on aromatic rings, replacement of phenyl rings A or B with heterocyclic, polyaromatic or organometallic structures and substitution on enone part of chalcone. Some new classes of hybrid chalcone compounds with pronounced activity against various types of cancer cell lines contain Mannich bases of heterocyclic chalcones,2 dihydrobenzofuran,3 imidazopyridine/pyrimidine,4 3-arylquinoline,5 quinoline-2-one,6 naphthalene,7 indole,8,9 pyrazole,10 quinoxaline,11 amidobenzothiazole,12 and ferrocene.13 Tumor angiogenesis is a physiological process that is involved in the formation of a network of blood capillaries supplying cancerous growths with oxygen and nutritients.14 Chalcone analogues exhibit antitumor activities through various mechanisms and inhibition of angiogenesis is one of them.15 The natural chalcone, xanthohumol and its synthetic derivatives inhibit angiogenesis
⇑ Corresponding author. Tel.: +381 34 336 223. E-mail address: [email protected] (M.D. Joksovic´). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.075
by suppressing vascular endothelial growth factor (VEGF),16 while the flavonoid precursor 4-hydroxychalcone inhibits this process by affecting both VEGF and basic fibroblast growth factor (bFGF) intracellular signaling pathways.17 The chalcone derivatives bearing furan and thiophene heterocycles demonstrated strong antiangiogenic activity18 as well as quinolil-thienyl chalcones in the role of VEGFR-2 kinase inhibitors.19 On the other hand, some natural anthraquinones like aloe-emodin, emodin and rhein displayed anti-angiogenic effects in the zebrafish model. Contrary, chrysophanol with no substitution and physcion with the substituted methoxy group did not show any anti-angiogenic activity. Emodin was the most potent anti-angiogenic compound inhibiting endothelial cell proliferation, migration and tube formation in a dose-dependent manner.20,21 Some representative chalcone and anthraquinone-based structures are presented in Figure 1. In continuation with our current interest toward synthesis of bioactive anthraquinone compounds,22 in this work we explored the possibility to combine the potential synergic anticarcinogenic effects of chalcone moiety and anthraquinone scaffold. To the best of our knowledge, there is no literature data on preparation and biological activity of anthraquinone based chalcone analogues. Thus, we designed and synthesized a new class of chalcones with anthraquinone as A-ring and different arylimino or alkylimino substituents in para-position of B-ring, together with an evaluation of their antiproliferative and anti-angiogenic potential.
66
B. Kolundzˇija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71
Figure 1. Basic structure of chalcone (1) and some representative chalcone and anthraquinone-based derivatives with antitumor properties (2–6).
Starting aromatic amines were selected to obtain the compounds with a good coverage of polar, steric, electron-donating and electron-withdrawing properties. Our further strategy was
based on replacement of the phenyl group by the benzyl one as well as more flexible alkyl group bonded to heterocyclic furan and thiophene rings.
Scheme 1. Reagents and conditions: (a) CH3COCl, AlCl3, CH2Cl2, 2 h, 0 °C, 5 M HCl; (b) CrO3, CH3COOH, 5 min reflux, H2O; (c) terephthalaldehyde, NaOH, MeOH, 5 h, reflux; (d) primary amines, AcOH, dioxane, 12–48 h, reflux.
B. Kolundzˇija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71 Table 1 Concentrations of 5a–q that induced a 50% decrease in HeLa (2000 cells per well), LS174 (7000 cells per well), A549 (5000 cells per well), and MRC5 (5000 cells per well), cell survival expressed as IC50 (lM). The compounds were incubated with cells for 72 h and IC50 values are presented as the mean ± SD, determined from the results of MTT assay in three independent experiment IC50 (lM)
Compounds
5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q Cisplatin
HeLa
LS174
A549
MRC5
4.46 ± 1.51 6.58 ± 0.85 4.17 ± 1.14 8.82 ± 0.53 5.02 ± 0.54 5.94 ± 1.35 5.01 ± 0.88 1.93 ± 0.89 9.78 ± 0.01 6.57 ± 0.22 4.62 ± 0.81 3.09 ± 0.86 1.45 ± 0.34 1.82 ± 0.68 4.19 ± 0.79 5.97 ± 0.04 5.34 ± 1.13 2.10 ± 0.20
10.56 ± 0.09 10.18 ± 0.32 9.54 ± 0.68 8.99 ± 3.23 17.22 ± 2.12 5.98 ± 0.84 8.70 ± 0.85 17.18 ± 2.24 11.30 ± 0.82 7.51 ± 0.10 8.55 ± 2.07 7.60 ± 1.06 6.61 ± 0.49 1.76 ± 0.78 11.03 ± 0.37 10.66 ± 0.38 5.30 ± 0.45 5.54 ± 1.03
11.04 ± 0.37 11.06 ± 0.33 9.38 ± 0.54 13.01 ± 0.51 14.51 ± 1.31 7.63 ± 0.91 8.17 ± 1.06 9.99 ± 0.46 15.73 ± 0.75 7.80 ± 1.51 10.18 ± 0.46 7.98 ± 0.67 7.78 ± 0.26 6.11 ± 0.45 9.79 ± 0.74 11.44 ± 1.92 6.01 ± 0.09 11.92 ± 2.19
16.14 ± 0.03 17.08 ± 1.24 13.52 ± 2.40 16.68 ± 0.35 11.75 ± 1.30 11.66 ± 0.81 17.16 ± 1.74 16.69 ± 0.85 12.74 ± 2.65 16.15 ± 0.52 12.40 ± 1.64 11.00 ± 1.31 14.55 ± 0.72 15.11 ± 3.56 11.93 ± 0.33 15.96 ± 1.36 11.87 ± 0.65 14.21 ± 1.54
The synthesis of new hybrid chalcone analogues 5a–q is presented in Scheme 1 while experimental protocols are given in Supplementary content. The commercially available anthracene 1 was acylated with acetyl chloride to 1-acetylanthracene 223 which was then oxidized by chromium (VI) oxide in glacial acetic acid yielding 1-acetylanthraquinone 3.24 The ketone 3 was recrystallized from ethanol and condensed with terephthalaldehyde in presence of sodium hydroxide affording anthraquinone aldehyde 4. In the next step, the final chalcone conjugates containing imine fragment were prepared by condensation of aldehyde 4 and a series of aromatic and aliphatic primary amines in dioxane. The presence of
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anthraquinone unit significantly decreases reactivity of aldehyde group and some reactions required an excess of amine reagent and prolonged reaction time. Importantly, anthraquinone also make imine functional group extremely resistant to hydrolysis under neutral conditions. A suspension of 5g in water was stirred for 24 h and no detectable quantity of aldehyde was observed by NMR. In the beginning of our investigations, we first synthesized a chalcone product by reaction of acetylanthraquinone 3 and benzaldehyde, but its cytotoxic activity was very low. However, derivatization of aldehyde 4 in the para-position of the B-ring through imine formation by condensation with different primary amines dramatically enhanced cytotoxicity. The obtained imines were particularly active against A549 cell lines, making these compounds comparable to cisplatin. The cytotoxic properties of all synthesized compounds were evaluated in vitro against human cervix adenocarcinoma (HeLa), human colon carcinoma (LS174), nonsmall cell lung carcinoma (A549) and normal human fetal lung fibroblast cell line (MRC-5) by MTT assay. Cisplatin was used as a reference drug. The results are summarized in Table 1 with IC50 values defined as the concentrations corresponding to 50% growth inhibition. Evidently, the substituents of imine part of molecule strongly influence on cytotoxic effect of the synthesized compounds. The introduction of electron-donating methyl group at all three positions of phenyl moiety did not improve activity in comparison with unsubstituted phenyl group. However, the presence of the voluminous electron-withdrawing substituents such as –Cl and –CF3, especially in meta-position, caused a significant increase of cytotoxic activity in inhibition of HeLa cells. The replacement of phenyl group with cyclohexyl one led to slight decrease of antiproliferative activity. Although alkyl groups had no effect on cytotoxicity, aromatic and heterocyclic rings linked to methylene group showed potent activity toward HeLa cell lines with IC50 values from 1.82 to 5.34 lM. The compounds 5n and 5q containing furan and phenyl rings were also efficient in inhibition of LS174 and A549 cells.
Figure 2. Representative histograms of cell-cycle distribution after 24 h treatment of A549 cells with IC50 of compounds 5h, 5m, 5n and 5q. Cell-cycle distribution was measured by flow cytometric analysis of DNA content after treatment with these compounds.
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The significant cytotoxic activity of 5h, 5m, 5n and 5q led us to investigate the mechanism of action of these compounds by cytofluorimetric analysis, using propidium iodide to label DNA. Flow
cytometry was employed to examine the changes in DNA content. A representative cell cycle analysis of A549 cell lines treated with 5h, 5m, 5n and 5q for 24 h is given in Figure 2. The effect of com-
Figure 3. Photomicrographs of AO/EB stained control HeLa cells, and HeLa cells treated with IC50 and 2IC50 concentrations of 5h, 5m, 5n and 5q within 24 h.
B. Kolundzˇija et al. / Bioorg. Med. Chem. Lett. 24 (2014) 65–71
Figure 4. DNA fragmentation detection by 2% agarose gel electrophoresis. C1 and C2 represent control samples while 5h, 5m, 5n and 5q lanes represent treatment with IC50 of respective compounds.
pounds on cell cycle distribution of malignant cells is shown in Table S1 (Supplementary material). There were no significant changes of sub-G1 populations on all tested cell lines after 24 h treatment with IC50 dose of investigated compounds. The percentage change in sub-G1 in A549 cells treated with 5q for 24 h was marked with increase to 15.29%. In addition, any relevant change in the basal level in G1, S and G2-M population was not observed in the investigated cells after 24 h of treatment. Moreover, the A549 and LS174 cells showed significant changes in G2/M arrest (29.31% and 68.89%, respectively) after 24 h of treatment with 5h (see Fig. 2 and Table S1). The G2/M arrest is associated with evident reduction in S phase. Figure 3 shows the results of fluorescence microscopy of HeLa cells coupled with acridine orange/ethidium bromide (AO/EB) double staining for the occurrence of morphological changes and DNA condensation after treatment with 5h, 5m, 5n and 5q. HeLa cells treated with IC50 and 2IC50 concentrations of these compounds exhibited nuclear shrinkage and chromatin condensation, compared to the untreated control cells. The presence of a ladder DNA fragmentation pattern typical for apoptotic cells was evaluated after 24 h of exposure of HeLa cells to IC50 of 5h, 5m, 5n, and 5q. However, alterations in nuclear morphology may not necessarily involve DNA fragmentation.25 Despite the significant cytotoxicity of compounds observed towards HeLa cells, no DNA ladder formation was detected upon their treatment (Fig. 4). In this regard, it should be noted that 5h, 5m, 5n, and 5q do
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not show significant accumulation in sub-G1 phase of the cell cycle on HeLa cells. The activities of caspase-3 and -8, in 5h, 5m, 5n, and 5q-treated HeLa cells were detected by specific caspase inhibitors using flow cytometry (Fig. 5). Bearing in mind the absence of DNA fragmentation and the fact that these compounds do not lead to significant accumulation of sub-G1 phase of the cell cycle, one could conclude that the cell death induced by these compounds should be independent of caspase-3. Unexpectedly, the analysis revealed the presence of active caspase-3 in HeLa cells treated with 5h, 5n, and 5q while 5m showed that inhibitors of caspase-3, had almost no effect on the activity of this compound. Thus, only specific caspase-8 inhibitor significantly suppressed the caspase activity and we can conclude that 5m shows activity mainly via mitochondrial pathway through activation of caspase-8 in HeLa cells. In general, these caspase activities, coupled with the absence of DNA fragmentation, may indicate a possible inhibition of the endonuclease activity DFF40/CAD by these compounds.26–29 Further studies are needed for more detailed understanding of the relationship between DFF40/CAD endonuclease and caspase-3 activity. Blocking angiogenesis could be a strategy to arrest tumor growth and anti-angiogenic drug development has attracted a lot of research interest. Without blood vessels, tumors cannot grow beyond a critical size or metastasize to another organ.30 The tube formation activity of EA.hy926s is important in vitro endpoint for angiogenesis. Examined compounds 5h, 5m, 5n, and 5q inhibited tubulogenesis as it is shown in Figure 6A where untreated EA.hy926s formed elongated tube-like structures. The treatment with IC20 sub-toxic dose of investigated compounds resulted in a significant decrease in capillary tube formation and this effect was very strong for 5h. The IC20 values of these compounds on tube formation inhibition of EA.hy926 ranged from 4.9 to 5.5 lM (sub-toxic effect of these compounds is illustrated in Figure 6B. Our observations were consistent with previously cited reports that many natural and synthetic chalcones possess anti-angiogenic activity. Next, we examined the effect of the investigated compounds on the activity of matrix metalloproteinases. HeLa cells were treated with sub-cytotoxic concentrations (IC20) of 5h, 5m, 5n and 5q for 24 h in serum-free RPMI-1640. The supernatant was collected and tested for matrix metalloproteinase activity by gelatin zymography. As it is shown in Figure 7, activity of MMP-s was significantly reduced compared to the controls C1 and C2. Our results showed that the investigated compounds inhibited the activity of these enzymes. MMP-2 has been strongly implicated in angiogenesis and promotion of the tumor metastatic potential playing crucial role not only in invasion, but also in determination of cancer cell transformation, growth, apoptosis and signal transduction.31– 33 The inhibitory effects confirmed by 5h, 5m, 5n and 5q against MMP-2 secretion could be a starting point for further development
Figure 5. Effects of 5h, 5m, 5n and 5q on caspase-3 and -8 inhibitors. Data are shown as mean ± SD. HeLa cells were exposed to IC90 concentrations of investigated compounds for 24 h in the presence of specific caspase inhibitors (final concentration—40 lM) as described.
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Figure 6. (A) EA.hy926 cell tube formation on Matrigel with and without an added compound. (B) Sub-toxic effect of the investigated compounds on EA.hy926 after 24 h incubation, without Matrigel.
Figure 7. Treatment of HeLa cells with sub-toxic concentrations of 5h, 5m, 5n and 5q within 24 h reduces MMP-2 secretion as demonstrated by gelatin zymography. C1 and C2 represent control.
of these compounds as anti-angiogenic agents for metastatic malignancies. In summary, we designed and prepared a series of new hybrid chalcones with an anthraquinone unit. Generally, introduction of electron withdrawing substituents in meta-position of the phenyl ring was beneficial for good activity toward all cell lines. The compound 5n containing furan ring was found to be especially active against the growth of all tested malignant cell lines. It was noted that these compounds did not have tendency to significant accumulation of cells in sub-G1 phase. Only 5h led to an increase in sub-G1 phase in A549 cells as well as changes in G2/M arrest of A549 and LS174 cells. In addition, 5h, 5m, 5n and 5q induced the activities of caspase-3 and -8 in treated Hela cells, and exhibited strong anti-angiogenic activity. Finally, these compounds showed potent inhibition against MMP-2 secretion. Acknowledgement The authors are grateful to the Ministry of Science and Technological Development of the Republic of Serbia for financial support (Grant Nos. 172016 and 175011).
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