- Email: [email protected]
Synthesis and biological evaluation of naphthoquinone-coumarin conjugates as topoisomerase II inhibitors
Accepted Manuscript Synthesis and Biological Evaluation of Naphthoquinone-Coumarin Conjugates as Topoisomerase II Inhibitors Idaira Hueso-Falcón, Ángel Amesty, Laura Anaissi-Afonso, Isabel LorenzoCastrillejo, Félix Machín, Ana Estévez-Braun PII: DOI: Reference:
S0960-894X(16)31307-5 http://dx.doi.org/10.1016/j.bmcl.2016.12.040 BMCL 24529
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
17 May 2016 13 December 2016 14 December 2016
Please cite this article as: Hueso-Falcón, I., Amesty, A., Anaissi-Afonso, L., Lorenzo-Castrillejo, I., Machín, F., Estévez-Braun, A., Synthesis and Biological Evaluation of Naphthoquinone-Coumarin Conjugates as Topoisomerase II Inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/ j.bmcl.2016.12.040
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and Biological Evaluation of Naphthoquinone-Coumarin Conjugates as Topoisomerase II Inhibitors
Idaira Hueso-Falcóna, Ángel Amesty†,a, Laura Anaissi-Afonso†,b, Isabel LorenzoCastrillejo b, Félix Machín*,b, Ana Estévez-Braun*,a
a
Instituto Universitario de Bio-Orgánica (CIBICAN), Departamento de Química Orgánica, Universidad de
La Laguna, 38206, Spain. E-mail: [email protected]; Tel: +34 922 318576 b
Unidad de Investigación Hospital Universitario Nuestra Señora de La Candelaria, 38010, Tenerife, Spain.
E-mail: [email protected]; Tel: +34 922 602951
† These authors equally contributed to this work.
* Co-corresponding authors
1
ABSTRACT
Based on previous Topoisomerase II docking studies of naphthoquinone derivatives, a series
of
naphthoquinone-coumarin
conjugates
was
synthesized
through
a
multicomponent reaction from aromatic aldehydes, 4-hydroxycoumarin and 2hydroxynaphthoquinone. The hybrid structures were evaluated against the isoform of human topoisomerase II (hTopoII), Escherichia coli DNA Gyrase and E. coli Topoisomerase I. All tested compounds inhibited the hTopoII-mediated relaxation of negatively supercoiled circular DNA in the low micromolar range. This inhibition was specific since neither DNA Gyrase nor Topoisomerase I were affected. Cleavage assays pointed out that naphthoquinone-coumarins act by catalytically inhibiting hTopoII. ATPase assays and molecular docking studies further pointed out that the mode of action is related to the hTopoII ATP-binding site.
Keywords: 2-hydroxynaphthoquinone, 4-hydroxycoumarin, privileged structures, topoisomerase II, ATPase, docking 2
Quinone derivatives may be toxic to cell by a number of mechanisms, including redox cycling, generation of free radicals, Michael-type arylation of thiol groups in biomolecules, DNA intercalation and bioreductive alkylation via quinone methide formation.1-3 As a consequence, the molecular framework of a great number of pharmaceuticals and biologically important compounds contains a quinone moiety. Nowadays, their important pharmacological activity is also attributed to the inhibition of enzymes,
such as
bacterial topoisomerase
II-DNA gyrase,4 mammalian
topoisomerase I and II,5-8 HIV-1 integrase,9-10 etc. Representative examples of this class of compounds are the well known anticancer drugs of the anthracycline series, doxorubicin and mitoxanthrone, the action of which is believed to occur via topoisomerase II inhibition.11-12 In addition, a number of naphthoquinone analogues from plumbagin,13 shikonin,14-15 naphthazarin116-17 and -lapachone,18-21 have also been found to inhibit similar enzymes. On the other hand, coumarins (2H-chromen-2-one) are a large family of compounds of both natural and synthetic origin22-24 that are also important because of the pharmacological activities they display, such as antimicrobial, anti-HIV, anti-inflammatory and antitumoral.25-29 There are many examples of coumarin derivatives that inhibit DNA-associated enzymes, such as topoisomerases or gyrase. 30-32 Due to their potential applications in cancer therapy, extensive studies have been carried out on the design and synthesis of coumarin derivatives with improved anticancer activity.33-34 Therefore, coumarin and quinone represent promising scaffolds in medicinal chemistry. Taking into account the above mentioned features and based on previous docking studies of naphthoquinone derivatives into the ATP domain of topoisomerase II, we have combined both privileged structures in triarylmethane-like compounds. Thus, in this paper we present the synthesis of novel coumarin-naphthoquinone 3
conjugates as potential agents that target human topoisomerase II through their binding to the ATP pocket. We have previously published the synthesis of a set of pyranonaphthoquinones as catalytic inhibitors of the enzyme topoisomerase II in vitro.35 Docking studies revealed the existence of a key -cation interaction between the aromatic ring and the magnesium present into the ATPase domain of hTopoII. One of the carbonyl of the quinonic nucleus also showed an effective hydrogen bonding interaction with the residue Asn122. Continuing with our interest in synthesizing antitumoral naphthoquinone derivatives,35 we aim at achieving new compounds with good interactions into the ATP pocket. Thus, we decided the preparation of naphthoquinone-coumarin conjugates, since this
type
of
compounds
showed
better
interactions
than
our
previous
pyranonaphthoquinones in docking models. With respect to the pyranonaphthoquinone, the hybrid compound, besides the -cation interaction, shows two additional hydrogen bonding interactions through the carbonyls of the coumarin and naphthoquinone moieties. Furthermore, more hydrophobic interactions are also observed. The naphthoquinone-coumarin conjugates (4) were synthesized through a onepot three-component reaction from 2-hydroxy-1,4-naphthoquinone (1), aromatic aldehydes (2), and 4-hydroxycoumarin (3) in EtOH under reflux conditions (Scheme 1).
4
Scheme 1. Multicomponent synthesis of coumarin-naphthoquinone conjugates (4).
Since 2-hydroxy-1,4-naphthoquinone (1) and 4-hydroxy-coumarin (3) are adequate synthetic equivalents to a 1,3-dicarbonyl compound, both compounds, in the presence of an aldehyde can react to afford the corresponding quinone methide intermediate. In order to explore, which one shows the highest nucleophilicity we calculated the Fukui function, which is one of the widely used local density functional descriptor to model chemical reactivity and site-selectivity.36,37 The calculated values of the Fukui function (f-) indicated that the highest value for 4-hydroxycoumarin (3) was located at C-3 (fk-=0.21), while for 2-hydroxy-naphthoquinone the highest value was located at C-3 (fk-=0.32). From these results the reactive intermediate methylene quinone is presumably formed from 2-hydroxy-1,4-naphthoquinone (1). Thus, the synthesis of these hybrid compounds can be rationalized by the initial formation of a conjugated electron-deficient enone (A) through a Knoevenagel condensation of the 2-hydroxy-1,4-naphthoquinone (1) and an aldehyde (2). The next step of this mechanism could involve a Michael addition of the 4-hydroxy-coumarin (3) to the reactive quinone methide intermediate to yield the intermediate (B) which
5
experiments various intramolecular proton transfers to produce the final product (Scheme 1). When the reaction was carried out in the presence of different catalysts to favors the Knoevenagel condensation such as EDDA (ethylendiaminediacetate), piperidine, Et3N, N-acetylpiperidin or L-proline in all cases we obtained 1,4-naphthoquinone dimers instead of the desired naphthoquinone-coumarin conjugates. These dimers are obtained from the nucleophilic attack of 2-hydroxy-1,4-naphthoquinone instead of 4hydroxy-coumarin. Only in the absence of catalyst, under reflux in EtOH we obtained the corresponding hybrids in moderate yields. The results obtained using this reaction conditions and several aromatic aldehydes are summarized in Table 1.
Table 1. Synthesis of coumarin-naphthoquinone conjugates. Entry
R
Compound
Yielda
1
4-Br-C6H5
4a
61
2
4-Cl-C6H5
4b
60
3
4-F-C6H5
4c
42
4
2-F-C6H5
4d
35
5
4-NO2-C6H5
4e
31
6
C6H5
4f
31
7
3,4-methylenedioxyphenyl
4g
33
a
Isolated yield
The compounds were tested in vitro against human Topoisomerase II (hTopoII) using the purified enzyme and a set of small molecules of circular DNA which carries a preferred sequence for Topoisomerase II (pRYG plasmid).5-6, 38 All 6
tested compounds inhibited the hTopoII-mediated relaxation of negatively supercoiled pRYG in the low micromolar range (Fig. 1). The degree of inhibition at 100 M was comparable to the known hTopoII inhibitor merbarone and superior to the clinically used hTopoII inhibitors etoposide and ICRF-187 (Fig 1A). Since the quinone moiety has been proposed to oxidize dithiothreitol (DTT),39 and this is the reducing agent employed in most commercial buffers to keep hTopoII active throughout the reaction, we decided to repeat the relaxation assay in a home-made buffer based on 2mercaptoethanol (Fig 1B). In this buffer, inhibition with etoposide, ICRF-187 and merbarone was somewhat weaker; however, full inhibition was maintained for all coumarin-naphthoquinones. Relaxation assays performed at lower concentrations of the tested coumarin-naphthoquinones showed that they all have an IC50 in the low submicromolar range, between 10 and 30 M (Fig 1C).
7
A
SC
Compound (100 M)
1% DMSO
pRYG Lin
+T2
-T2
Etop ICRF Merb
4a
4b
4c
4d
4e
4f
4g
4e
4f
4g
SC[d] Nck + Rel PRTs
SC
B
pRYG SC
Lin
Compound (100 M)
1% DMSO +T2
-T2
Etop ICRF Merb
4a
4b
4c
4d
SC[d] Nck + Rel PRTs SC
C % Inhibition of hTopoII
120 100 80 60 40 20 0 4a
4b
4c
4d
4e
4f
4g
Compound
Fig. 1. Topo II-mediated relaxation assays of supercoiled circular DNA upon incubation with coumarin-naphthoquinone hybrids 4a-g. A. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (SC forms) and products (relaxed topoisomers) of the hTopoII relaxation reaction. Covalently closed negatively supercoiled pRYG plasmid (SC forms) was treated with 2U hTopo II in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. The assay was performed with the commercially supplied reaction buffer that contains dithiothreitol (DTT) as reducing agent. B. Like in panel A but using a home-made reaction buffer 8
with 2-mercaptoethanol as reducing agent. Etoposide (Etop), ICRF-187 (ICRF) and Merbarone (Merb) were included as controls for inhibition. They act on the Top2 catalytic cycle at different levels (see text for details) C. Quantitation of percentage of hTopo IIα inhibition of three independent relaxation assays (mean + SEM) for each coumarin-naphthoquinone hybrid at 10, 30 and 100 M (from lightest to darkest grey boxes). These assays were carried out with the commercial reaction buffer.
Next, we performed two experiments aimed to get mechanistic insights into the way these novel compounds were inhibiting hTopo IIα. This enzyme has a complex catalytic cycle and one of the most critical steps is the formation of the short-lived cleavage complex intermediate, whose stabilization for longer than usual is the mechanism of action of clinically relevant hTopo IIα inhibitors (so-called “poisons”) such as etoposide. Compounds that inhibit hTopo IIα without stabilizing the cleavage complex are all referred as “catalytic inhibitors” and they can act either before or after the formation of the cleavage complex (e.g., merbarone and ICRF-187, respectively).40 Thus, in order to address whether any of the coumarin-naphthoquinones were “poisoning” the enzyme we performed a cleavage assay using etoposide as a positive control (Fig 2A). Indeed, we found that etoposide gave a triad of equivalent upper bands, being the lowest in that triad the consequence of the stabilization of a single cleavage product (i.e., linearized plasmid, Lin). Nevertheless, we found no enrichment for that intermediate in any of the tested coumarin-naphthoquinones. This points out that these compounds are novel catalytic inhibitors of hTopo IIα. The second mechanistically-related experiment we carried out was an ATPase inhibition assay for the hTopo IIα. ATP is needed at different steps of the catalytic cycle. Any compound that interferes with ATP binding or hydrolysis fit well with the catalytic inhibition mode of action.41 As stated above, and show below in detail (see docking studies), we already predicted an interaction between the compounds and the hTopo IIα 9
ATP pocket during the design of the coumarin-naphthoquinones hybrids. Indeed, hTopoIIα-mediated ATPase was inhibited with all compounds, although they showed different strengths (Fig 2B). In general, the order of ATPase inhibition was 4e ≥ 4d > 4a ≥ 4g > 4b ≥ 4c = 4f. This order is slightly different that the one observed for the relaxation activity, where inhibition was more homogeneous and somewhat stronger. This difference in strength and order suggests that these compounds could also be acting upon other steps in the catalytic cycle aside from the ATP pocket.
A SC
Nck
Compound (100 M)
1% DMSO
pRYG Lin
-Top2 +Top2 Etop
4a
4b
4c
4d
4e
4f
SC[d] + Nck Rel[d] Lin
SC Rel
B
Fig 2. Topo II-mediated cleavage and ATPase assays of supercoiled circular DNA upon incubation with coumarin-naphthoquinone hybrids 4a-g. A. Agarose electrophoresis run under the presence of ethidium bromide to separate substrates (SC forms) and intermediate products (linearized plasmid) of the hTopo II cleavage assay. Covalently 10
4g
closed negative supercoiled pRYG plasmid (SC forms) was treated with 10U hTopo II in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. Etoposide (Etop) was included as a positive control. Nck (nicked plasmid), Rel (fully relaxed plasmid), PTRs (partially relaxed plasmid topoisomers), SC (supercoiled plasmid), SC[d] (SC form of the plasmid catenated dimer), Rel[d] (fully relaxed dimer). B. Time course plots of hTopo II-mediated ATPase activity in the presence of 10, 30 and 100 μM of the seven coumarin-naphthoquinone conjugates. Activity was measure in an ATPase/Pyruvate kinase/Lactate dehydrogenase linked assay as decline of NADH measured at 340 nm. The percentages of activity relative to the 1% DMSO control are indicated on the right of each declining curve.
Next, we addressed whether this human Topoisomerase II inhibition was specific by testing the activity of the prokaryotic Topoisomerase II-like DNA gyrase under the presence of the new synthesized coumarin-naphthoquinones. Noteworthy, Gyrase also utilizes ATP for the reaction in a sort of similar catalytic cycle.42 We performed the Gyrase assay and found that no a single coumarin-naphthoquinones inhibited DNA gyrase at 100 μM; however, ciprofloxaxin, a known Gyrase inhibitor, 42 fully did (Fig 3A). Finally, we also checked if any compound could interfere with the other major enzyme that changes DNA topology, topoisomerase I (TopoI). We further used this enzyme in a secondary unwinding assay that questions whether any of the coumarinnaphthoquinones could intercalate within the DNA.43 For these assays, we used chloroquine, a known DNA intercalator and TopoI inhibitor, 44 as a control. TopoI greatly relaxed the SC form without any added compound, whereas chloroquine delayed complete relaxation (Fig 3B). Contrary to hTopoIIα, we did not observe signs TopoI inhibition with any of the coumarin-naphthoquinones. Likewise, no inhibition whatsoever was seen when the relaxed substrate (instead of the SC forms) was employed (Fig 3C). We thus concluded that the tested coumarin-naphthoquinones are 11
not DNA intercalators and that the hTopoIIα inhibition is specific since mechanistically related enzymes such as Gyrase and TopoI were not inhibited.
A
pBSKS Rel
SC
Compound (100 M)
DMSO -Gy
+Gy Cipro Etop ICRF Merb
4a
4b
4c
4d
4e
4f
Nck + Rel PRTs SC
B
pBSKS Rel
Compound (100 M)
DMSO
SC
-T1 +T1
pBSKS
DMSO
Chlor Etop
4a
4b
4c
4d
4e
4f
4g
4f
4g
Nck + Rel
PRTs SC
C
Rel
SC
-T1 +T1
Compound (100 M) Chlor Etop
4a
4b
4c
4d
4e
Nck + Rel
PRTs
SC
Fig. 3. Gyrase-mediated supercoiling and Topoisomerase I-mediated relaxation of circular DNA upon incubation with coumarin-naphthoquinone hybrids 4a-g. A. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (a ladder of partially relaxed forms) and products (monomeric SC) of the E. coli DNA gyrase reaction. The substrate was treated with 2.5U DNA gyrase in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. Etoposide (Etop), ICRF-187 (ICRF) and Merbarone (Merb) were also included. B. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (monomeric SC) and products (relaxed forms) of the Topoisomerase I reaction. The 12
4g
substrate used, SC form, directly questions whether any compound inhibits TopoI. The substrate was treated with 2.5U of E. coli TopoI in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. Chloroquine (Chlor), a reported TopoI catalytic inhibitor, and Etoposide (Etop) were included as controls. C. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (a ladder of partially relaxed forms) and products (relaxed forms) of the Topoisomerase I reaction. The substrate used, partially relaxed forms, questions whether any compound that could have appeared as an inhibitor in the previous panel was actually a DNA intercalator. Except for the substrate, the reaction was set up as indicated in panel B.
The naphthoquinone-coumarin conjugates, possessing a chiral carbon, were obtained as racemic mixtures. Thus, we analyzed the binding into the ATP pocket of Topoisomerase II of both enantiomers in the obtained series (4a-4g) using the Glide software.45 An analysis of the docking results shows that the compounds fit well into the ATP-binding site, being observed the existence of a key -cation interaction between the aromatic ring of naphthoquinone moiety and the magnesium present into the ATPase domain for the S-enantiomers, while for the R-enantiomers this type of interaction was observed with the aromatic ring of the coumarin moiety. The glide score values for the R- and S-enantiomers were similar [i.e (R)-4e; -7.93 Kcalmol-1 (S)-4e; -8.00 Kcalmol-1]. For the S-enantiomer two hydrogen bonds were detected between the residue Lys 168 and one of the carbonyl groups of the naphthoquinonic nucleus, and Ser 149 with the carbonyl group of the coumarin moiety (Fig. 4A). For the R-enantiomer similar H-bond interactions were found with the residues Asn 150 and Ser 149 (Fig. 4B). Hydrophobic interactions between the aromatic ring of the coumarin (S-enantiomer) or naphthoquinone (R-enantiomer) and Ile 125, Val 137, Ile 141 and Pro 126 were also observed. Most of the mentioned interactions were also detected in the rest of the compounds (see Supplementary material). Additional interactions were observed in 13
specific cases, for example the nitro group of compound 4e showed a hydrogen bond interaction with the residue Asn 120.
A)
B)
Fig. 4. Docking model and key interactions of compounds (S)-4e and (R)-4e.
Only in the case of compound 4b a different posse is adopted. Thus, for the S enantiomer two hydrogen bonds with the residue Asn 150 were detected but the cation interaction was not observed since the aromatic ring does not overlap with the magnesium cation, while for the R-enantiomer the -cation interaction exists but no hydrogen bonds were detected (See Supplementary Material). 14
In conclusion, a novel series of naphthoquinone-coumarin conjugates as specific eukaryotic topoisomease II catalytic inhibitors has been synthesized based on Docking studies into the ATP pocket of Topoisomerase II. The hybrid compounds, besides the cation interaction which is present in the earlier pyranonaphthoquinone series, shows additional hydrogen bonding and hydrophobic interactions which increase their effectiveness as topoisomerase II catalytic inhibitors. The physicochemical descriptors were calculated for compounds 4a-4g (see Supplementary material), and they do not violate the optimal requirements for druggability, which suggests that these hybrids are promising lead compounds for further research.
15
References and Notes 1. Bolton, J.L.; Trush, M.A.; Penning, T.M.; Dryhurst, G.; Monks, T.J. Chem. Res. Toxicol. 2000, 13, 135. 2. O’Brien, P. J. Chem Biol. Interact. 1991, 80, 1. 3. Powis, G. Pharmacol. Ther. 1987, 35, 57. 4. Barret, J.F.; Gootz, T.D.; McGuirk, P.R.; Farrell, C.A. Solowski, S.A. Antimicrob. Agents Chemother. 1989, 33, 1697. 5. Jiménez-Alonso, A.; Chávez, H.; Estévez-Braun, A; Ravelo, A.G. Pérez-Sacau, E.; Machín, F. J. Med. Chem. 2008, 51, 6761. 6. Sperry, J.; Lorenzo-Castrillejo, I.; Brimble, M.A. Machín, F. Bioorg. Med. Chem. 2009, 17, 7131. 7. Hu, C.X.; Zuo, Z.L.; Xiong, B.; Ma, J.G.; Geng, M.Y.; Lin, L.P.; Jiang, H.L.; Ding, J. Mol. Pharmacol. 2006, 70, 1593. 8. Gurbani, D.; Kukshal, V.; Laubental, J.; Kumar, A.; Pandey, A.; Tripathi, S.; Arora, A.; Jain, S.K.; Ramachandran, R.; Anderson, D.; Dhawan, A. Toxicol. Sciences 2012, 126, 372. 9. Fensen, M.R.; Kohn, K.W.; Leteurtre, F.; Pommier, Y. Proc. Natl. Acad. Sci. USA 1993, 90, 2399. 10. Brinkworth, R.I.; Fairlie, D.P. Biochim. Biophys. Acta 1995, 1253, 5. 11. Ramachandran, C.; Samy, T.S.A.; Huang, X.L.; Yuan, Z.K.; Krishan, A. Biochem. Pharmacol. 1993, 45, 1367. 12. Smart, D.J. Halicka, H.D. Schmuck, G.; Traganos, F.; Darzynkiewicz, Z.; Williams, G.M.. Mutant. Res. 2008, 641, 43. 13. Yamashita, F.N.; Nagashima, A.Y.; Nakano, M. Antimicrob. Agents and Chemother. 1992, 36, 2589. 16
14. Yang, F.; Chen, Y.; Duan, W.; Zhang, C.; Zhu, H.; Ding, J. Int. J. Cancer, 2006, 119, 1184. 15. Ahn, B.Z.; Baik, K.U.; Kweon, G.R. J. Med. Chem. 1995, 38, 1044. 16. Song, G.Y.; Kim, Y.; You, Y.J.; Cho, H.; Kim, S.H.; Sok, D.E.; Ahn, B.Z. Arch. der Pharmazie 2000, 333, 87. 17. Song, G.Y.; Kim, Y.; Zheng, X.G.; You, Y.J.; Cho, H.; Sok, D.E.; Ahn, B.Z. Eur. J. Med. Chem. 2000, 35, 291. 18. Quevedo, O.; García-Luis, J.; Lorenzo-Castrillejo, I.; Machin, F. Chem. Res. Toxicol. 2011, 24, 2106. 19. Estevez-Souza, A.; Figuereido, D.V.; Esteves, A.; Cámara, C.A.; Vargas, M.D.; Pinto, A.C. Echevarria, A.C. Braz. J. Med. Biol. Res. 2007, 40, 1399. 20. Ravelo, A.G.; Estévez-Braun, A.; Pérez-Sacau, E. Studies in Natural Products Chemistry, 2003, 29, 719. 21. Ramos-Perez, C.; Lorenzo-Castrillejo, I.; Quevedo, O.; Garcia-Luis, J.; MatosPerdomo, E.; Medina-Coello, C.; Estevez-Braun, A.; Machin, F. Biochem. Pharmacol. 2014, 92, 206. 22. Estévez-Braun, A.; González, A.G. Coumarins. Nat. Prod. Rep. 1997, 14, 465. 23. Venugopala, K.N.; Rashmi, V.; Odhav, B. Biomed. Res. Int. 2013, ID 963248. Doi:10.1155/2013/963248. 24. Farook, M.; Ashish, P.; Nida, K. J. Med. Pharm. Allied Sci., 2013, 4, 43. 25. Zhao, H.; Donnelly, A.C.; Kusuma, B.R.; Brandt, G.E.; Brown, D.; Rajewski, R.A.; Vielhauer, G.; Holzbeierlein, J. Cohen, M.S.; Blagg, B.S., J Med Chem. 2011, 54, 3839. 26. Stefanachi, A.; Favia, A.D.; Nicolotti, O.; Leonetti, F.; Pisani, L.; Catto, M.; Zimmer, C.; Hartmann, R.W., Carotti A. J. Med Chem. 2011, 54, 1613. 17
27. Dayam, R.; Gunla, R.; Al-Mawsawi, L.Q.; Neamati, N. Med. Res. Reviews, 2008, 28, 118. 28. Basanagouda, M.; Shivashankar, K.; Kulkarni, M.V.; Rasal, V.P.; Patel, H.; Mutha, S.S.; Mohite, A.A. Eur J Med Chem. 2010, 45, 1151. 29. Hadjipavlou-Litina, D. J.; Litinas, K. E.; Kontogiorgis, C. Anti-Inflam & AntiAllergy Agents Med Chem. 2007, 4, 293. 30. Bailly, C. Chem. Rev. 2012, 112, 3611. 31. Facompré, M.; Tardy, C.; Bal-Mahieu, C.; Colson, P.; Perez, C.; Manzanares, I.; Cuevas, C.; Bailly, C. Cancer Res. 2003, 63, 7392. 32. Zhao, L; Yao, Y.; Li, S.; Lv, M.; Chen, H.; Li, X. Bioorg. Med. Chem.2014, 24, 900. 33. Sashidhara, K.V.; Kumar, A.; Kumar, M.; Sarkar, J.; Sinha, S. Biorg. Med. Chem. Lett. 2010, 20, 7205. 34. Medina, F.G.; Marrero, J.G.; Macías-Alonso, M.; González, M.C.; CórdovaGuerrero, I.; Teisseier-García, A.; Osegueda-Robles, S. Nat. Prod. Report. 2015, 32, 1472. 35. McNaughton-Smith, G.; Estévez-Braun, A.; Jiménez Alonso, S.; Gutiérrez-Ravelo, A.; Fernández-Pérez, L.; Díaz-Chico, N.PCT Int. Appl.(2014),WO 2014016314 A1201401. Eur. Pat. Appl.(2014), EP 2690094 A1 20140129. 36. Parr, R. G.; Yang, W. J. Am. Chem. Soc., 1985, 106, 4049. 37. Yang, W.; Mortier, W. J. J. Am. Chem. Soc., 1986, 108, 5708. 38. Quintana-Espinoza, P.; García-Luis, J.; Amesty, A.; Martín-Rodríguez, P.; LorenzoCastrillejo, I.; Ravelo, A. G.; Fernández-Pérez, L.; Machín, F.; Estévez-Braun, A. Bioorg. Med. Chem. 2013, 21, 6484. 39. Krishnan, P.; Bastow, K. F. Biochem. Pharmacol. 2000, 60, 1367–79. 18
40. Nitiss, J. L. Nat. Rev. Cancer 2009, 9, 338–50. 41. Schmidt, B. H.; Osheroff, N.; Berger, J. M. Nat. Struct. Mol. Biol. 2012, 19, 1147– 1154. 42. Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. Chem. Biol. 2010, 17, 421-433. 43. Shen, L. L. Methods Mol. Biol. 2001, 95, 149–60. 44. Sorensen, M.; Sehested, M.; Jensen, P. B. Biochem. Pharmacol. 1997, 54, 373-80. 45. Glide software, Glide, version 6.2; Schrodinger, LLC: New York, NY, 2014.
19
Supplementary material Supplementary data (procedure for the preparation of compounds 4a-4g, in vitro human TopoII-mediated DNA relaxation, cleavage and ATPase assays, in vitro E. coli Gyrase and Topoisomerase I assays, Molecular modeling studies, physicochemical descriptors, 1H NMR and
13
C NMR spectra) associated with this
article can be found at http://dx.doi.org /
Acknowledgments Financial support was provided by Spanish MINECO (SAF 2012-37344-C03-01, SAF 2015-65113-C2-1-R to AEB, BFU2015-63902-R to FM), Instituto de Salud Carlos III (PS12/00280 to FM), and the EU Research Potential (FP7-REGPOT-2012-CT201231637-IMBRAIN). AA thanks CDCH-Venezuela for a predoctoral fellowship. IHF thanks MINECO for a predoctoral contract. FM thanks Clara Volz for technical help.
20
O
O
OH OH O
+R 1 O
H
+
2
OH HO
EtOH O 3
O O
R
O
O 4
Multicomponent reaction
Topoisomerase II inhibitors
21
S0960-894X(16)31307-5 http://dx.doi.org/10.1016/j.bmcl.2016.12.040 BMCL 24529
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
17 May 2016 13 December 2016 14 December 2016
Please cite this article as: Hueso-Falcón, I., Amesty, A., Anaissi-Afonso, L., Lorenzo-Castrillejo, I., Machín, F., Estévez-Braun, A., Synthesis and Biological Evaluation of Naphthoquinone-Coumarin Conjugates as Topoisomerase II Inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/ j.bmcl.2016.12.040
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and Biological Evaluation of Naphthoquinone-Coumarin Conjugates as Topoisomerase II Inhibitors
Idaira Hueso-Falcóna, Ángel Amesty†,a, Laura Anaissi-Afonso†,b, Isabel LorenzoCastrillejo b, Félix Machín*,b, Ana Estévez-Braun*,a
a
Instituto Universitario de Bio-Orgánica (CIBICAN), Departamento de Química Orgánica, Universidad de
La Laguna, 38206, Spain. E-mail: [email protected]; Tel: +34 922 318576 b
Unidad de Investigación Hospital Universitario Nuestra Señora de La Candelaria, 38010, Tenerife, Spain.
E-mail: [email protected]; Tel: +34 922 602951
† These authors equally contributed to this work.
* Co-corresponding authors
1
ABSTRACT
Based on previous Topoisomerase II docking studies of naphthoquinone derivatives, a series
of
naphthoquinone-coumarin
conjugates
was
synthesized
through
a
multicomponent reaction from aromatic aldehydes, 4-hydroxycoumarin and 2hydroxynaphthoquinone. The hybrid structures were evaluated against the isoform of human topoisomerase II (hTopoII), Escherichia coli DNA Gyrase and E. coli Topoisomerase I. All tested compounds inhibited the hTopoII-mediated relaxation of negatively supercoiled circular DNA in the low micromolar range. This inhibition was specific since neither DNA Gyrase nor Topoisomerase I were affected. Cleavage assays pointed out that naphthoquinone-coumarins act by catalytically inhibiting hTopoII. ATPase assays and molecular docking studies further pointed out that the mode of action is related to the hTopoII ATP-binding site.
Keywords: 2-hydroxynaphthoquinone, 4-hydroxycoumarin, privileged structures, topoisomerase II, ATPase, docking 2
Quinone derivatives may be toxic to cell by a number of mechanisms, including redox cycling, generation of free radicals, Michael-type arylation of thiol groups in biomolecules, DNA intercalation and bioreductive alkylation via quinone methide formation.1-3 As a consequence, the molecular framework of a great number of pharmaceuticals and biologically important compounds contains a quinone moiety. Nowadays, their important pharmacological activity is also attributed to the inhibition of enzymes,
such as
bacterial topoisomerase
II-DNA gyrase,4 mammalian
topoisomerase I and II,5-8 HIV-1 integrase,9-10 etc. Representative examples of this class of compounds are the well known anticancer drugs of the anthracycline series, doxorubicin and mitoxanthrone, the action of which is believed to occur via topoisomerase II inhibition.11-12 In addition, a number of naphthoquinone analogues from plumbagin,13 shikonin,14-15 naphthazarin116-17 and -lapachone,18-21 have also been found to inhibit similar enzymes. On the other hand, coumarins (2H-chromen-2-one) are a large family of compounds of both natural and synthetic origin22-24 that are also important because of the pharmacological activities they display, such as antimicrobial, anti-HIV, anti-inflammatory and antitumoral.25-29 There are many examples of coumarin derivatives that inhibit DNA-associated enzymes, such as topoisomerases or gyrase. 30-32 Due to their potential applications in cancer therapy, extensive studies have been carried out on the design and synthesis of coumarin derivatives with improved anticancer activity.33-34 Therefore, coumarin and quinone represent promising scaffolds in medicinal chemistry. Taking into account the above mentioned features and based on previous docking studies of naphthoquinone derivatives into the ATP domain of topoisomerase II, we have combined both privileged structures in triarylmethane-like compounds. Thus, in this paper we present the synthesis of novel coumarin-naphthoquinone 3
conjugates as potential agents that target human topoisomerase II through their binding to the ATP pocket. We have previously published the synthesis of a set of pyranonaphthoquinones as catalytic inhibitors of the enzyme topoisomerase II in vitro.35 Docking studies revealed the existence of a key -cation interaction between the aromatic ring and the magnesium present into the ATPase domain of hTopoII. One of the carbonyl of the quinonic nucleus also showed an effective hydrogen bonding interaction with the residue Asn122. Continuing with our interest in synthesizing antitumoral naphthoquinone derivatives,35 we aim at achieving new compounds with good interactions into the ATP pocket. Thus, we decided the preparation of naphthoquinone-coumarin conjugates, since this
type
of
compounds
showed
better
interactions
than
our
previous
pyranonaphthoquinones in docking models. With respect to the pyranonaphthoquinone, the hybrid compound, besides the -cation interaction, shows two additional hydrogen bonding interactions through the carbonyls of the coumarin and naphthoquinone moieties. Furthermore, more hydrophobic interactions are also observed. The naphthoquinone-coumarin conjugates (4) were synthesized through a onepot three-component reaction from 2-hydroxy-1,4-naphthoquinone (1), aromatic aldehydes (2), and 4-hydroxycoumarin (3) in EtOH under reflux conditions (Scheme 1).
4
Scheme 1. Multicomponent synthesis of coumarin-naphthoquinone conjugates (4).
Since 2-hydroxy-1,4-naphthoquinone (1) and 4-hydroxy-coumarin (3) are adequate synthetic equivalents to a 1,3-dicarbonyl compound, both compounds, in the presence of an aldehyde can react to afford the corresponding quinone methide intermediate. In order to explore, which one shows the highest nucleophilicity we calculated the Fukui function, which is one of the widely used local density functional descriptor to model chemical reactivity and site-selectivity.36,37 The calculated values of the Fukui function (f-) indicated that the highest value for 4-hydroxycoumarin (3) was located at C-3 (fk-=0.21), while for 2-hydroxy-naphthoquinone the highest value was located at C-3 (fk-=0.32). From these results the reactive intermediate methylene quinone is presumably formed from 2-hydroxy-1,4-naphthoquinone (1). Thus, the synthesis of these hybrid compounds can be rationalized by the initial formation of a conjugated electron-deficient enone (A) through a Knoevenagel condensation of the 2-hydroxy-1,4-naphthoquinone (1) and an aldehyde (2). The next step of this mechanism could involve a Michael addition of the 4-hydroxy-coumarin (3) to the reactive quinone methide intermediate to yield the intermediate (B) which
5
experiments various intramolecular proton transfers to produce the final product (Scheme 1). When the reaction was carried out in the presence of different catalysts to favors the Knoevenagel condensation such as EDDA (ethylendiaminediacetate), piperidine, Et3N, N-acetylpiperidin or L-proline in all cases we obtained 1,4-naphthoquinone dimers instead of the desired naphthoquinone-coumarin conjugates. These dimers are obtained from the nucleophilic attack of 2-hydroxy-1,4-naphthoquinone instead of 4hydroxy-coumarin. Only in the absence of catalyst, under reflux in EtOH we obtained the corresponding hybrids in moderate yields. The results obtained using this reaction conditions and several aromatic aldehydes are summarized in Table 1.
Table 1. Synthesis of coumarin-naphthoquinone conjugates. Entry
R
Compound
Yielda
1
4-Br-C6H5
4a
61
2
4-Cl-C6H5
4b
60
3
4-F-C6H5
4c
42
4
2-F-C6H5
4d
35
5
4-NO2-C6H5
4e
31
6
C6H5
4f
31
7
3,4-methylenedioxyphenyl
4g
33
a
Isolated yield
The compounds were tested in vitro against human Topoisomerase II (hTopoII) using the purified enzyme and a set of small molecules of circular DNA which carries a preferred sequence for Topoisomerase II (pRYG plasmid).5-6, 38 All 6
tested compounds inhibited the hTopoII-mediated relaxation of negatively supercoiled pRYG in the low micromolar range (Fig. 1). The degree of inhibition at 100 M was comparable to the known hTopoII inhibitor merbarone and superior to the clinically used hTopoII inhibitors etoposide and ICRF-187 (Fig 1A). Since the quinone moiety has been proposed to oxidize dithiothreitol (DTT),39 and this is the reducing agent employed in most commercial buffers to keep hTopoII active throughout the reaction, we decided to repeat the relaxation assay in a home-made buffer based on 2mercaptoethanol (Fig 1B). In this buffer, inhibition with etoposide, ICRF-187 and merbarone was somewhat weaker; however, full inhibition was maintained for all coumarin-naphthoquinones. Relaxation assays performed at lower concentrations of the tested coumarin-naphthoquinones showed that they all have an IC50 in the low submicromolar range, between 10 and 30 M (Fig 1C).
7
A
SC
Compound (100 M)
1% DMSO
pRYG Lin
+T2
-T2
Etop ICRF Merb
4a
4b
4c
4d
4e
4f
4g
4e
4f
4g
SC[d] Nck + Rel PRTs
SC
B
pRYG SC
Lin
Compound (100 M)
1% DMSO +T2
-T2
Etop ICRF Merb
4a
4b
4c
4d
SC[d] Nck + Rel PRTs SC
C % Inhibition of hTopoII
120 100 80 60 40 20 0 4a
4b
4c
4d
4e
4f
4g
Compound
Fig. 1. Topo II-mediated relaxation assays of supercoiled circular DNA upon incubation with coumarin-naphthoquinone hybrids 4a-g. A. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (SC forms) and products (relaxed topoisomers) of the hTopoII relaxation reaction. Covalently closed negatively supercoiled pRYG plasmid (SC forms) was treated with 2U hTopo II in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. The assay was performed with the commercially supplied reaction buffer that contains dithiothreitol (DTT) as reducing agent. B. Like in panel A but using a home-made reaction buffer 8
with 2-mercaptoethanol as reducing agent. Etoposide (Etop), ICRF-187 (ICRF) and Merbarone (Merb) were included as controls for inhibition. They act on the Top2 catalytic cycle at different levels (see text for details) C. Quantitation of percentage of hTopo IIα inhibition of three independent relaxation assays (mean + SEM) for each coumarin-naphthoquinone hybrid at 10, 30 and 100 M (from lightest to darkest grey boxes). These assays were carried out with the commercial reaction buffer.
Next, we performed two experiments aimed to get mechanistic insights into the way these novel compounds were inhibiting hTopo IIα. This enzyme has a complex catalytic cycle and one of the most critical steps is the formation of the short-lived cleavage complex intermediate, whose stabilization for longer than usual is the mechanism of action of clinically relevant hTopo IIα inhibitors (so-called “poisons”) such as etoposide. Compounds that inhibit hTopo IIα without stabilizing the cleavage complex are all referred as “catalytic inhibitors” and they can act either before or after the formation of the cleavage complex (e.g., merbarone and ICRF-187, respectively).40 Thus, in order to address whether any of the coumarin-naphthoquinones were “poisoning” the enzyme we performed a cleavage assay using etoposide as a positive control (Fig 2A). Indeed, we found that etoposide gave a triad of equivalent upper bands, being the lowest in that triad the consequence of the stabilization of a single cleavage product (i.e., linearized plasmid, Lin). Nevertheless, we found no enrichment for that intermediate in any of the tested coumarin-naphthoquinones. This points out that these compounds are novel catalytic inhibitors of hTopo IIα. The second mechanistically-related experiment we carried out was an ATPase inhibition assay for the hTopo IIα. ATP is needed at different steps of the catalytic cycle. Any compound that interferes with ATP binding or hydrolysis fit well with the catalytic inhibition mode of action.41 As stated above, and show below in detail (see docking studies), we already predicted an interaction between the compounds and the hTopo IIα 9
ATP pocket during the design of the coumarin-naphthoquinones hybrids. Indeed, hTopoIIα-mediated ATPase was inhibited with all compounds, although they showed different strengths (Fig 2B). In general, the order of ATPase inhibition was 4e ≥ 4d > 4a ≥ 4g > 4b ≥ 4c = 4f. This order is slightly different that the one observed for the relaxation activity, where inhibition was more homogeneous and somewhat stronger. This difference in strength and order suggests that these compounds could also be acting upon other steps in the catalytic cycle aside from the ATP pocket.
A SC
Nck
Compound (100 M)
1% DMSO
pRYG Lin
-Top2 +Top2 Etop
4a
4b
4c
4d
4e
4f
SC[d] + Nck Rel[d] Lin
SC Rel
B
Fig 2. Topo II-mediated cleavage and ATPase assays of supercoiled circular DNA upon incubation with coumarin-naphthoquinone hybrids 4a-g. A. Agarose electrophoresis run under the presence of ethidium bromide to separate substrates (SC forms) and intermediate products (linearized plasmid) of the hTopo II cleavage assay. Covalently 10
4g
closed negative supercoiled pRYG plasmid (SC forms) was treated with 10U hTopo II in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. Etoposide (Etop) was included as a positive control. Nck (nicked plasmid), Rel (fully relaxed plasmid), PTRs (partially relaxed plasmid topoisomers), SC (supercoiled plasmid), SC[d] (SC form of the plasmid catenated dimer), Rel[d] (fully relaxed dimer). B. Time course plots of hTopo II-mediated ATPase activity in the presence of 10, 30 and 100 μM of the seven coumarin-naphthoquinone conjugates. Activity was measure in an ATPase/Pyruvate kinase/Lactate dehydrogenase linked assay as decline of NADH measured at 340 nm. The percentages of activity relative to the 1% DMSO control are indicated on the right of each declining curve.
Next, we addressed whether this human Topoisomerase II inhibition was specific by testing the activity of the prokaryotic Topoisomerase II-like DNA gyrase under the presence of the new synthesized coumarin-naphthoquinones. Noteworthy, Gyrase also utilizes ATP for the reaction in a sort of similar catalytic cycle.42 We performed the Gyrase assay and found that no a single coumarin-naphthoquinones inhibited DNA gyrase at 100 μM; however, ciprofloxaxin, a known Gyrase inhibitor, 42 fully did (Fig 3A). Finally, we also checked if any compound could interfere with the other major enzyme that changes DNA topology, topoisomerase I (TopoI). We further used this enzyme in a secondary unwinding assay that questions whether any of the coumarinnaphthoquinones could intercalate within the DNA.43 For these assays, we used chloroquine, a known DNA intercalator and TopoI inhibitor, 44 as a control. TopoI greatly relaxed the SC form without any added compound, whereas chloroquine delayed complete relaxation (Fig 3B). Contrary to hTopoIIα, we did not observe signs TopoI inhibition with any of the coumarin-naphthoquinones. Likewise, no inhibition whatsoever was seen when the relaxed substrate (instead of the SC forms) was employed (Fig 3C). We thus concluded that the tested coumarin-naphthoquinones are 11
not DNA intercalators and that the hTopoIIα inhibition is specific since mechanistically related enzymes such as Gyrase and TopoI were not inhibited.
A
pBSKS Rel
SC
Compound (100 M)
DMSO -Gy
+Gy Cipro Etop ICRF Merb
4a
4b
4c
4d
4e
4f
Nck + Rel PRTs SC
B
pBSKS Rel
Compound (100 M)
DMSO
SC
-T1 +T1
pBSKS
DMSO
Chlor Etop
4a
4b
4c
4d
4e
4f
4g
4f
4g
Nck + Rel
PRTs SC
C
Rel
SC
-T1 +T1
Compound (100 M) Chlor Etop
4a
4b
4c
4d
4e
Nck + Rel
PRTs
SC
Fig. 3. Gyrase-mediated supercoiling and Topoisomerase I-mediated relaxation of circular DNA upon incubation with coumarin-naphthoquinone hybrids 4a-g. A. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (a ladder of partially relaxed forms) and products (monomeric SC) of the E. coli DNA gyrase reaction. The substrate was treated with 2.5U DNA gyrase in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. Etoposide (Etop), ICRF-187 (ICRF) and Merbarone (Merb) were also included. B. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (monomeric SC) and products (relaxed forms) of the Topoisomerase I reaction. The 12
4g
substrate used, SC form, directly questions whether any compound inhibits TopoI. The substrate was treated with 2.5U of E. coli TopoI in the presence of 100 μM of each coumarin-naphthoquinone hybrid in 1% (v/v) DMSO. Chloroquine (Chlor), a reported TopoI catalytic inhibitor, and Etoposide (Etop) were included as controls. C. Agarose electrophoresis run in the absence of ethidium bromide to separate substrates (a ladder of partially relaxed forms) and products (relaxed forms) of the Topoisomerase I reaction. The substrate used, partially relaxed forms, questions whether any compound that could have appeared as an inhibitor in the previous panel was actually a DNA intercalator. Except for the substrate, the reaction was set up as indicated in panel B.
The naphthoquinone-coumarin conjugates, possessing a chiral carbon, were obtained as racemic mixtures. Thus, we analyzed the binding into the ATP pocket of Topoisomerase II of both enantiomers in the obtained series (4a-4g) using the Glide software.45 An analysis of the docking results shows that the compounds fit well into the ATP-binding site, being observed the existence of a key -cation interaction between the aromatic ring of naphthoquinone moiety and the magnesium present into the ATPase domain for the S-enantiomers, while for the R-enantiomers this type of interaction was observed with the aromatic ring of the coumarin moiety. The glide score values for the R- and S-enantiomers were similar [i.e (R)-4e; -7.93 Kcalmol-1 (S)-4e; -8.00 Kcalmol-1]. For the S-enantiomer two hydrogen bonds were detected between the residue Lys 168 and one of the carbonyl groups of the naphthoquinonic nucleus, and Ser 149 with the carbonyl group of the coumarin moiety (Fig. 4A). For the R-enantiomer similar H-bond interactions were found with the residues Asn 150 and Ser 149 (Fig. 4B). Hydrophobic interactions between the aromatic ring of the coumarin (S-enantiomer) or naphthoquinone (R-enantiomer) and Ile 125, Val 137, Ile 141 and Pro 126 were also observed. Most of the mentioned interactions were also detected in the rest of the compounds (see Supplementary material). Additional interactions were observed in 13
specific cases, for example the nitro group of compound 4e showed a hydrogen bond interaction with the residue Asn 120.
A)
B)
Fig. 4. Docking model and key interactions of compounds (S)-4e and (R)-4e.
Only in the case of compound 4b a different posse is adopted. Thus, for the S enantiomer two hydrogen bonds with the residue Asn 150 were detected but the cation interaction was not observed since the aromatic ring does not overlap with the magnesium cation, while for the R-enantiomer the -cation interaction exists but no hydrogen bonds were detected (See Supplementary Material). 14
In conclusion, a novel series of naphthoquinone-coumarin conjugates as specific eukaryotic topoisomease II catalytic inhibitors has been synthesized based on Docking studies into the ATP pocket of Topoisomerase II. The hybrid compounds, besides the cation interaction which is present in the earlier pyranonaphthoquinone series, shows additional hydrogen bonding and hydrophobic interactions which increase their effectiveness as topoisomerase II catalytic inhibitors. The physicochemical descriptors were calculated for compounds 4a-4g (see Supplementary material), and they do not violate the optimal requirements for druggability, which suggests that these hybrids are promising lead compounds for further research.
15
References and Notes 1. Bolton, J.L.; Trush, M.A.; Penning, T.M.; Dryhurst, G.; Monks, T.J. Chem. Res. Toxicol. 2000, 13, 135. 2. O’Brien, P. J. Chem Biol. Interact. 1991, 80, 1. 3. Powis, G. Pharmacol. Ther. 1987, 35, 57. 4. Barret, J.F.; Gootz, T.D.; McGuirk, P.R.; Farrell, C.A. Solowski, S.A. Antimicrob. Agents Chemother. 1989, 33, 1697. 5. Jiménez-Alonso, A.; Chávez, H.; Estévez-Braun, A; Ravelo, A.G. Pérez-Sacau, E.; Machín, F. J. Med. Chem. 2008, 51, 6761. 6. Sperry, J.; Lorenzo-Castrillejo, I.; Brimble, M.A. Machín, F. Bioorg. Med. Chem. 2009, 17, 7131. 7. Hu, C.X.; Zuo, Z.L.; Xiong, B.; Ma, J.G.; Geng, M.Y.; Lin, L.P.; Jiang, H.L.; Ding, J. Mol. Pharmacol. 2006, 70, 1593. 8. Gurbani, D.; Kukshal, V.; Laubental, J.; Kumar, A.; Pandey, A.; Tripathi, S.; Arora, A.; Jain, S.K.; Ramachandran, R.; Anderson, D.; Dhawan, A. Toxicol. Sciences 2012, 126, 372. 9. Fensen, M.R.; Kohn, K.W.; Leteurtre, F.; Pommier, Y. Proc. Natl. Acad. Sci. USA 1993, 90, 2399. 10. Brinkworth, R.I.; Fairlie, D.P. Biochim. Biophys. Acta 1995, 1253, 5. 11. Ramachandran, C.; Samy, T.S.A.; Huang, X.L.; Yuan, Z.K.; Krishan, A. Biochem. Pharmacol. 1993, 45, 1367. 12. Smart, D.J. Halicka, H.D. Schmuck, G.; Traganos, F.; Darzynkiewicz, Z.; Williams, G.M.. Mutant. Res. 2008, 641, 43. 13. Yamashita, F.N.; Nagashima, A.Y.; Nakano, M. Antimicrob. Agents and Chemother. 1992, 36, 2589. 16
14. Yang, F.; Chen, Y.; Duan, W.; Zhang, C.; Zhu, H.; Ding, J. Int. J. Cancer, 2006, 119, 1184. 15. Ahn, B.Z.; Baik, K.U.; Kweon, G.R. J. Med. Chem. 1995, 38, 1044. 16. Song, G.Y.; Kim, Y.; You, Y.J.; Cho, H.; Kim, S.H.; Sok, D.E.; Ahn, B.Z. Arch. der Pharmazie 2000, 333, 87. 17. Song, G.Y.; Kim, Y.; Zheng, X.G.; You, Y.J.; Cho, H.; Sok, D.E.; Ahn, B.Z. Eur. J. Med. Chem. 2000, 35, 291. 18. Quevedo, O.; García-Luis, J.; Lorenzo-Castrillejo, I.; Machin, F. Chem. Res. Toxicol. 2011, 24, 2106. 19. Estevez-Souza, A.; Figuereido, D.V.; Esteves, A.; Cámara, C.A.; Vargas, M.D.; Pinto, A.C. Echevarria, A.C. Braz. J. Med. Biol. Res. 2007, 40, 1399. 20. Ravelo, A.G.; Estévez-Braun, A.; Pérez-Sacau, E. Studies in Natural Products Chemistry, 2003, 29, 719. 21. Ramos-Perez, C.; Lorenzo-Castrillejo, I.; Quevedo, O.; Garcia-Luis, J.; MatosPerdomo, E.; Medina-Coello, C.; Estevez-Braun, A.; Machin, F. Biochem. Pharmacol. 2014, 92, 206. 22. Estévez-Braun, A.; González, A.G. Coumarins. Nat. Prod. Rep. 1997, 14, 465. 23. Venugopala, K.N.; Rashmi, V.; Odhav, B. Biomed. Res. Int. 2013, ID 963248. Doi:10.1155/2013/963248. 24. Farook, M.; Ashish, P.; Nida, K. J. Med. Pharm. Allied Sci., 2013, 4, 43. 25. Zhao, H.; Donnelly, A.C.; Kusuma, B.R.; Brandt, G.E.; Brown, D.; Rajewski, R.A.; Vielhauer, G.; Holzbeierlein, J. Cohen, M.S.; Blagg, B.S., J Med Chem. 2011, 54, 3839. 26. Stefanachi, A.; Favia, A.D.; Nicolotti, O.; Leonetti, F.; Pisani, L.; Catto, M.; Zimmer, C.; Hartmann, R.W., Carotti A. J. Med Chem. 2011, 54, 1613. 17
27. Dayam, R.; Gunla, R.; Al-Mawsawi, L.Q.; Neamati, N. Med. Res. Reviews, 2008, 28, 118. 28. Basanagouda, M.; Shivashankar, K.; Kulkarni, M.V.; Rasal, V.P.; Patel, H.; Mutha, S.S.; Mohite, A.A. Eur J Med Chem. 2010, 45, 1151. 29. Hadjipavlou-Litina, D. J.; Litinas, K. E.; Kontogiorgis, C. Anti-Inflam & AntiAllergy Agents Med Chem. 2007, 4, 293. 30. Bailly, C. Chem. Rev. 2012, 112, 3611. 31. Facompré, M.; Tardy, C.; Bal-Mahieu, C.; Colson, P.; Perez, C.; Manzanares, I.; Cuevas, C.; Bailly, C. Cancer Res. 2003, 63, 7392. 32. Zhao, L; Yao, Y.; Li, S.; Lv, M.; Chen, H.; Li, X. Bioorg. Med. Chem.2014, 24, 900. 33. Sashidhara, K.V.; Kumar, A.; Kumar, M.; Sarkar, J.; Sinha, S. Biorg. Med. Chem. Lett. 2010, 20, 7205. 34. Medina, F.G.; Marrero, J.G.; Macías-Alonso, M.; González, M.C.; CórdovaGuerrero, I.; Teisseier-García, A.; Osegueda-Robles, S. Nat. Prod. Report. 2015, 32, 1472. 35. McNaughton-Smith, G.; Estévez-Braun, A.; Jiménez Alonso, S.; Gutiérrez-Ravelo, A.; Fernández-Pérez, L.; Díaz-Chico, N.PCT Int. Appl.(2014),WO 2014016314 A1201401. Eur. Pat. Appl.(2014), EP 2690094 A1 20140129. 36. Parr, R. G.; Yang, W. J. Am. Chem. Soc., 1985, 106, 4049. 37. Yang, W.; Mortier, W. J. J. Am. Chem. Soc., 1986, 108, 5708. 38. Quintana-Espinoza, P.; García-Luis, J.; Amesty, A.; Martín-Rodríguez, P.; LorenzoCastrillejo, I.; Ravelo, A. G.; Fernández-Pérez, L.; Machín, F.; Estévez-Braun, A. Bioorg. Med. Chem. 2013, 21, 6484. 39. Krishnan, P.; Bastow, K. F. Biochem. Pharmacol. 2000, 60, 1367–79. 18
40. Nitiss, J. L. Nat. Rev. Cancer 2009, 9, 338–50. 41. Schmidt, B. H.; Osheroff, N.; Berger, J. M. Nat. Struct. Mol. Biol. 2012, 19, 1147– 1154. 42. Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. Chem. Biol. 2010, 17, 421-433. 43. Shen, L. L. Methods Mol. Biol. 2001, 95, 149–60. 44. Sorensen, M.; Sehested, M.; Jensen, P. B. Biochem. Pharmacol. 1997, 54, 373-80. 45. Glide software, Glide, version 6.2; Schrodinger, LLC: New York, NY, 2014.
19
Supplementary material Supplementary data (procedure for the preparation of compounds 4a-4g, in vitro human TopoII-mediated DNA relaxation, cleavage and ATPase assays, in vitro E. coli Gyrase and Topoisomerase I assays, Molecular modeling studies, physicochemical descriptors, 1H NMR and
13
C NMR spectra) associated with this
article can be found at http://dx.doi.org /
Acknowledgments Financial support was provided by Spanish MINECO (SAF 2012-37344-C03-01, SAF 2015-65113-C2-1-R to AEB, BFU2015-63902-R to FM), Instituto de Salud Carlos III (PS12/00280 to FM), and the EU Research Potential (FP7-REGPOT-2012-CT201231637-IMBRAIN). AA thanks CDCH-Venezuela for a predoctoral fellowship. IHF thanks MINECO for a predoctoral contract. FM thanks Clara Volz for technical help.
20
O
O
OH OH O
+R 1 O
H
+
2
OH HO
EtOH O 3
O O
R
O
O 4
Multicomponent reaction
Topoisomerase II inhibitors
21