Bioorganic & Medicinal Chemistry 24 (2016) 651–660
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of ferrocenyl indeno[1,2-c] isoquinolines as topoisomerase II inhibitors Nathalie Wambang a,b, Nadège Schifano-Faux c, Alexandre Aillerie a, Brigitte Baldeyrou d, Camille Jacquet a, Christine Bal-Mahieu d, Till Bousquet a,⇑, Sylvain Pellegrini a, Peter T. Ndifon b, Samuel Meignan d, Jean-François Goossens c, Amélie Lansiaux e, Lydie Pélinski a,⇑ a
Univ. Lille, CNRS, Centrale Lille, ENSCL, Univ. Artois, UMR 8181—UCCS—Unité de Catalyse et Chimie du Solide, F-59000 Lille, France Univ. Yaoundé 1, Laboratoire de Chimie de Coordination, BP 812, Yaoundé, Cameroon Univ. Lille, CHU Lille, EA 7365—GRITA—Groupe de Recherche sur les formes Injectables et les Technologies Associées, F-59000 Lille, France d Univ. Lille, Inserm, U908—CPAC—Cell Plasticity and Cancer, F-59000 Lille, France e Département de Recherches Médicales, Groupement hospitalier de l’Institut Catholique de Lille, 59000 Lille, France b c
a r t i c l e
i n f o
Article history: Received 14 October 2015 Revised 15 December 2015 Accepted 17 December 2015 Available online 18 December 2015 Keywords: Indenoisoquinoline Ferrocene Topoisomerase II inhibitor Drug binding Cytotoxicity
a b s t r a c t Three series of indeno[1,2-c]isoquinolines bearing a ferrocenyl entity were synthesized and evaluated for DNA interaction, topoisomerase I and II inhibition, and cytotoxicity against breast human cancer cell lines. In the first and second series, the ferrocenyl scaffold was inserted as a linker between the two nitrogen atoms. In the last series, it was introduced at the end of the carbon chain. The present study showed that the ferrocenyl entity enhanced the topoisomerase II inhibition. Most compounds showed a potent growth inhibitory effect on MDA-MB-231 cell line with the IC50 in lM range. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Topoisomerases I and II (Topo I and II), essential enzymes for replication, transcription and recombination of DNA, are attractive targets for a number of successful chemotherapeutic agents. The main classification of topoisomerase is based on the enzyme’s ability to generate a single-stranded (type I) or double-stranded (type II) breaks in the DNA double helix.1–3 In the both cases, a religation step leads to the final relaxation of DNA. Camptothecin 1 and its derivatives such as topotecan and irinotecan interact with the DNA–enzyme complex and inhibit the religation mechanism in the presence of topoisomerase I (Fig. 1). However, the low water solubility, the reversibility of the camptothecin–DNA–Topo I cleavage complex and the hydrolytically unstable lactone ring limited its clinical utility.4–6 Moreover, the enzyme–DNA complexes stabilized by indotecan 2 and indimitecan 3 are more persistent than those induced by camptothecin (Fig. 1). Recently, a number of indenoisoquinolines have been identified as novel Topo I inhibitors ⇑ Corresponding authors. Tel.: +33 (0) 3 20 43 48 93; fax: +33 (0) 3 20 43 65 85 (T.B.); tel.: +33 (0) 3 20 43 65 01; fax: +33 (0) 3 20 43 65 85 (L.P.). E-mail addresses: [email protected] (T. Bousquet), lydie.pelinski@ univ-lille1.fr (L. Pélinski). http://dx.doi.org/10.1016/j.bmc.2015.12.033 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
exhibiting better pharmacokinetic features and greater chemical stability than camptothecin.7–10 Interestingly, structural modifications of the indenoisoquinolines provided good topoisomerase II inhibitors.11–13 Among them, the most potent compound on Topo II was an indenoisoquinoline-5,11-dione with a basic tertiary amino group linked to the indenoisoquinoline through an ethoxy spacer group. Topoisomerase II poisons such as etoposide 4 (ETO), mitoxantrone 5 and doxorubicin 6 represent some of the most important and widely prescribed anticancer drugs, particularly for treatment of leukemias, lymphomas, breast and lung cancers (Fig. 1).14–16 Clinical uses of these drugs are limited by their toxicities and development of drug resistance.17,18 New generation of other synthetic and natural products has been developed as topoisomerase II inhibitors.19 Particularly, Top2-targeted drugs such as F14512,20 C-131121 and (R)-XK46922 are currently subject to clinical development. Organometallic compounds have been found to be promising anticancer-drug candidates.23–26 During the last years, halfsandwich Ru(II) arene complexes such as RAPTA-C 7 and [(g6-C6H4Ph)Ru(N,N-en)Cl)]+ (en = 1,2-ethylenediamine) 8, came into the focus of interest due to their biological activity and pharmacological properties.27–29 Among the organometallic
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N. Wambang et al. / Bioorg. Med. Chem. 24 (2016) 651–660
O O
O O
N
O
H 3 CO N
H 3 CO
O
O O
N
N
H 3 CO
N N
H 3 CO
O
Camptothecin 1 Me
O
O
OH
O O HO
N
O
Indotecan (NSC 743400) 2
Indimitecan (NSC 725776) 3
O HO
O
OH
O
O
HN
H N
O
O
OH
OH
OH OH
O O OMe O
O OH
O
HN
OCH 3
H3 CO
N H
OH
H 3C HO
OH Etoposide 4
Mitoxantrone 5
OH
O
O NH2
Doxorubicin 6
Figure 1. Examples of representative Topo I and Topo II inhibitors.
compounds, ferrocenyl phenols 9 developed by Jaouen’s group constitute a new class of molecules possessing both a ferrocene and a tamoxifen diphenylmethylene moiety (Fig. 2). These compounds demonstrate a high in vitro antiproliferative activity on both hormono-dependent (MCF-7) and -independent (MDA-MB231) breast cancer cell lines. Furthermore, they are kinetically stable, relatively lipophilic and have good redox properties.30–34 Since this pioneering work, the use of ferrocene in bioorganometallic chemistry has been growing in the recent years, not only for their antitumor properties but also for their antimalarial, antifungal and DNA-cleaving activities.35–40 Particularly ferrocenyl derivatives, such as azalactone ferrocene and thiomorpholide amido methyl ferrocene, are found to be potent inhibitors of topoisomerase II with preferential action against the b isoform.41,42 As a part of our ongoing effort to develop ferrocenyl drugs,43–45 we report herein the synthesis of novel indenoisoquinolines bearing a ferrocenyl moiety on the side chain (Fig. 3). The enhancing effects of the ferrocene have been evaluated on the cytotoxicity, DNA interaction and topoisomerase I and II inhibition.
monoprotected diamines followed by deprotection of carbamates by hydrochloric acid.48,49 Alkylation of the primary amines with (ferrocenylmethyl)trimethylammonium iodide 21 in the presence of potassium carbonate has afforded ferrocenyl indenoisoquinolines 22–25 in 33–56% yields (Scheme 1). A disubstitution of the amine group occurred during this reaction providing the disubstituted ferrocenyl compounds 26–29 in 19–44% yields.50 The reductive amination in the presence of 2-(N,N-dimethylaminomethyl)ferrocene carboxaldehyde 30 and sodium triacetoxyborohydride on amines 18 and 19 led to ferrocenyl indenoisoquinolines 31 and 32 in 65% and 60% yields respectively. The methylation or ethylation of secondary ferrocenyl amines 23 and 24 were realized by reductive amination in the presence of methanal or ethanal and sodium triacetoxyborohydride leading to compounds 33–35 (Scheme 1). Finally, the condensation of ferrocene carboxylic acid on amines 18 and 19 in the presence of EDCI/HOBt afforded ferrocenyl amides 36 and 37 in 59% and 35% yields, respectively (Scheme 2). 2.2. DNA thermal denaturation
2. Results and discussion 2.1. Chemistry The synthesis of ferrocenyl indenoisoquinolines 14–16 and 22–35 are described in Scheme 1. The condensation of ferrocenyl diamines 11–13, obtained by a described procedure,46 with benzo[d]indeno[1,2-b]pyran-5,11-dione 1047 provided the targeted ferrocenyl compounds 14–16 in 43–67% yields. Indenoisoquinolines 17–20 were obtained by condensation of the benzo[d]indeno[1,2-b]pyran-5,11-dione 10 with primary
OH +
Ph Ru Cl
Cl
Ru
N
P
Cl N N
NH 2
PF6-
NH2 Fe O(CH 2) nN(CH3) 2
RAPTA-C 7
8
Ferrocenyl phenols 9
Figure 2. Organometallic anticancer agents.
The ability of the drugs to protect calf thymus DNA (CT DNA, 42% GC bp) against thermal denaturation was used as an indicator of the relative capacity of indenoisoquinoline derivatives to bind and to stabilize the DNA double helix. DTm values (DTm = Tmdrug–DNA complex TmDNA alone) ranging from 0 to 27.5 °C are reported in Table 1.51–54 Ferrocene-free indenoisoquinoline derivatives bearing an alkylamino side chain from two to five methylene groups (17–20) were considered as reference compounds in our study. N-Substituted compounds with an alkyl (22–25) or acyl (36–37) ferrocenyl moiety were synthesized to amplify the structure affinity relationships in indenoisoquinoline derivatives. In the alkyl series, as showed in Table 1, the amines 17, 19 and 20 displayed better DTm values than their ferrocenyl counterparts (22, 24, 25). However, the best value is obtained for the ferrocenyl derivative 23 possessing a three methylene spacer (DTm = 21.6 °C) in comparison to its amino analog 18 (DTm = 12.9 °C). Moreover, the presence of an additional protonable site on the ferrocenyl moiety in 31 and 32 resulted in an increase of DTm values at 25 °C and 27.5 °C, respectively. It can be assumed that this additional basic nitrogen interacts with anionic phosphate groups of the DNA backbone.
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N. Wambang et al. / Bioorg. Med. Chem. 24 (2016) 651–660
O
O
N
N
NR 2
Fe
O
O R = CH3 R, R = (CH 2) 5 R, R = (CH 2CH2 )2 NCH 3
Fe
R' N ( )n
N
O n = 2, 3, 4, 5 R' = H, Me, Et
( )n
H N
Fe
NMe 2
O n = 3, 4
Figure 3. Design of ferrocenyl indenoisoquinolines.
O
O
O
H2N
+
a
NR2
Fe
N
O
NR2
Fe
O
10
11: R = CH3 12: R, R = -(CH 2 )5 13: R, R = -(CH 2 CH2 )2 NCH3
14: R = CH3 15: R, R = -(CH 2 )5 16: R, R = -(CH 2 CH2 )2 NCH3
b, c O
O
O
d N O 17: n = 18 : n = 19: n = 20: n =
NH2
+
N(CH3 )3 I
Fe
N
( )n
e
Fe
Fe +
N
( )n
O 22: n = 23: n = 24: n = 25: n =
21
2 3 4 5
H N
O 26: 27 : 28: 29:
2 3 4 5
N
( )n
Fe n=2 n=3 n=4 n=5
f
O
O
N
( )n
H N
Fe
N
NMe 2
O
R' N
Fe
( )n
O
31 : n = 3 32: n = 4
33 : n = 3, R' = Me 34: n = 4, R' = Me 35: n = 4, R' = Et
Scheme 1. Reagents and conditions: (a) CHCl3, reflux, 12 h, 43–67%; (b) H2N(CH2)nNHBoc, CHCl3, reflux, 12 h; (c) HCl, iPrOH, 12 h; (d) K2CO3, CH3CN, reflux, 12 h, 33–65% for 22–25 and 19–44% for 26–29; (e) 2-(N,N-dimethylaminomethyl)ferrocene carboxaldehyde 30, NaBH(OAc)3, MeOH, 2 h, 60–65%; (f) HCHO or CH3CHO, NaBH(OAc)3, MeOH, 1 h or 30 h, 26–88%.
O
O
N
NH ( )n 2
O 18: n = 3 19: n = 4
N
Fe
H N ( )n
O
O
36: n = 3 37: n = 4
Scheme 2. Reagents and conditions: Ferrocene carboxylic acid, EDCI, HOBt, CH2Cl2, 12 h, 35–59%.
Additionally, N-alkylation of monoferrocenyl derivatives by a methyl or an ethyl group (33 and 35) led to a decrease of DTm values (33 and 35 vs 23 and 24). However, compound 34 displayed a higher DTm than 24 (DTm = 18.5 °C and 13.70 °C, respectively) suggesting an effect of the methylene units number from the indenoisoquinoline ring to the nitrogen atom bearing a methyl group, on DNA interaction.
The position of the ferrocenyl moiety with regard to the indenoisoquinoline ring was investigated with the three compounds 14–16 and led to lower DTm values than 23. However, it is interesting to note, that the replacement of the dimethylamino group in 14 by a bulkier cyclic amine such as piperidine in 15 is not favorable for DNA interaction with DTm values 16.8 °C and 6.8 °C, respectively. Similarly, the diferrocenyl compounds (26–29) displayed low binding affinity to DNA compared to the monoferrocenyl derivatives (22–25). It is noteworthy that in the both series, the best results were observed for compounds 23 and 27, bearing a N6-lactam side chain of three methylene units (DTm = 21.6 °C and 12.8 °C, respectively). For the acyl series, the acylation of the amines 18 and 19 altered drastically the capacity of the drug to bind DNA as indicated with DTm values calculated for compounds 36 and 37 (0 and 1 °C, respectively). 2.3. Fluorescence measurements DNA binding affinities for some of ferrocenyl compounds were quantified by means of fluorescence Kapp (Table 1). As a weak
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Table 1 Interaction of indenoisoquinolines and ferrocenyl indenoisoquinolines with DNA Compd
DTma (°C)
Kapp (107 M1)b
14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 31 32 33 34 35 36 37 Eto 4
16.8 6.8 Ndc 9.9 12.9 19.5 17.5 5.6 21.6 13.7 11.7 4.9 12.8 4.6 Ndc 25 27.5 3.4 18.5 7.5 0 1 0e
0.83 ± 0.01 Nd Nd Nd 1.38 ± 0.25 1.53 ± 0.13 1.14 ± 0.38 Nd 1.41 ± 0.26 0.45 ± 0.03 0.42 ± 0.11 Nd 11.5%d Nd Nd 3.52 ± 0.52 6.49 ± 0.23 Nd 0.82 ± 0.14 Nd Nd Nd 28%f
Variations in melting temperature (DTm = Tmdrug–DNA complex TmDNA alone) at ratio of 1. b Apparent binding constant measured by fluorescence using [BET]/[DNA] = 1.26. c Nd: not determined (low aqueous solubility). d Displacement of BET with 11.9 lM of compound 27. e Ref. 11. f Displacement of BET with 10 lM of etoposide. a
fluorescence was observed upon DNA titration, an indirect method was privileged.55,56 We used the conventional fluorescence quenching assay based on DNA binding competition between the intercalating drug ethidium bromide (BET) and the tested molecules.57–59 We decided to focus this study on the compounds with a DTm value superior to 11 °C. As reported in Table 1, Kapp values ranged from 0.42 107 (compound 25) to 6.49 107 M1 (compound 32). The introduction of a ferrocenyl moiety on the primary amine of the N6 lactam side chain decreased DNA affinity (24, 25 vs 19, 20) while an equivalent Kapp value was observed for, compounds 18 and 23. However, in accordance with DTm values, compound 23, possessing three methylene units, exhibited a higher Kapp value than 24 and 25. In addition, the introduction of a ferrocenyl moiety as linker between the two nitrogen atoms led to a lower DNA affinity (14 vs 23). Increasing the length of the N6 lactam side chain from three to five methylene units did not bring significant variations in Kapp values. Furthermore, the diferrocenyl compound 27 displayed lower DNA affinity than its monoferrocenyl analog 23. In agreement with DTm values, the N-alkylation of monoferrocenyl derivative 24 by a methyl group (compound 34) provided an increase of Kapp value. However, the N-methylation of compound 23 led to a weaker DNA ligand (23 vs 33). Finally, the addition of a second protonable site on the side chain resulted in an increase of Kapp values (31 and 32 vs 23 and 24). These compounds displayed the highest DTm and Kapp values, indicating both good affinity for DNA and duplex stabilization. This could be attributed to the additional stabilizing interaction, potentially between anionic phosphate groups of the DNA backbone and the additional protonable site. 2.4. Drug binding mechanism Different binding modes (intercalation and/or groove binding) can account for the melting temperature elevation. Therefore, to
understand the drug binding mechanism, a DNA unwinding assay based on the relaxation of native supercoiled plasmid DNA in the presence of both topoisomerase I and ethidium bromide for compounds 14, 22–24, 26–28 was used (Fig. 4A). The presence of supercoiled DNA (Sc) in Figure 4A could reveal that 14 and 23 were the best DNA interacting agents with highest supercoiled bands. From these results, a DNA relaxation in the presence of increasing concentrations of these two selected compound was performed (Fig. 4B). As shown in Figure 4B, the DNA relaxed by topoisomerase I (lane Topo I) generated a family of DNA topoisomers with a slow electrophoretic mobility. The drug was then added while topoisomerase I was maintained in the reaction mixture. When the drug concentration was increased, the closed circular duplex DNA was progressively supercoiled, indicating that the drug intercalated into DNA at low concentration for 23. These results were consistent with the Kapp value obtained with ethidium bromide competition and suggest an intercalation mode for the two selected compounds 14 and 23. 2.5. Topoisomerase inhibition A conventional DNA relaxation assay was performed to assess the effects of the compounds on the catalytic activity of human topoisomerases I and II. Supercoiled plasmid DNA was treated with either topoisomerase I or II in the presence of tested drugs and the DNA relaxation products were then resolved by gel electrophoresis on agarose gels containing ethidium bromide.59 From this study, none of the tested compounds was found to behave as a topoisomerase I poison (Fig. 4A). Indeed, unlike camptothecin (reference drug), no topoisomerase I-induced strand breaks visualized in nicked bands was observed with any of the compounds. The same results were obtained for the other compounds (see Supporting information). In Topo I inhibition experiments, we have also confirmed results obtained with DNA thermal denaturation studies for 14, 19, 23, 24, 31–32 (highest Kapp values observed in Table 1). Following this study, the influence of ferrocenyl moiety on topoisomerase II inhibition was then examined. The linear DNA results from the dissociation of the Topo II homodimer from cleaved DNA by SDS. In this case, the reference drug was etoposide, which produced a marked level of DNA double-stranded breaks corresponding to linear DNA (Lin). The formation of Topo II– DNA–drug cleavage complex could be evidenced by the appearance of linear DNA for Topo II. Interestingly, from this study, inhibition of topoisomerase II was clearly detected with these ferrocenyl compounds (Fig. 5). The presence of a ferrocenyl moiety on the primary amine of the N6 lactam side chain seems to play a major role in topoisomerase II inhibition as the compound 24 displayed high poisoning activity (Fig. 5A) whereas 19 had no effect (Fig. 5B). However, the conversion of the ferrocenyl methylene amino group to a ferrocenamide group suppressed the activity (Fig. 5B, 24 vs 37). On the other hand, the introduction of a ferrocenyl moiety as linker between the amine and the lactam nitrogen, led to compounds exhibiting weak Topo II inhibition (Fig. 5A, compound 14). The replacement of the dimethylamino group in compound 14 by a piperidine or a N-methylpiperazine (compounds 15 and 16), abrogated the activity (data not shown). The position of the ferrocenyl moiety at the end of the N6 lactam chain seems to be critical for topoisomerase II inhibition since the compound 24 was more potent than 14 (Fig. 5A). The length of the N6 lactam side chain (from two to five methylene units) had an impact on the topoisomerase inhibition abilities. Indeed, for monoferrocenyl derivatives 22–25, especially the compound 24 with a four methylene spacer, displayed the highest
N. Wambang et al. / Bioorg. Med. Chem. 24 (2016) 651–660
655
Figure 4. Topoisomerase I inhibition. (A) Effects of compounds 22–24, 26–28 and 14 on the relaxation of plasmid DNA by human topoisomerase I. Native supercoiled pUC19 plasmid DNA (130 ng, lane DNA) was incubated with 8 units topoisomerase I in the absence (lane Topo I) or the presence of tested compounds at 20 lM. Camptothecin (CPT) was used as reference at 20 lM. DNA samples were separated by electrophoresis on a 1% agarose gel containing 1 lg/mL ethidium bromide. (B) Effects of compounds 23 and 14 on the relaxation of plasmid DNA by human topoisomerase I. Native supercoiled pUC19 plasmid DNA (130 ng, lane DNA) was incubated with 4 units topoisomerase I in the absence (lane Topo I) or the presence of tested compounds at indicated concentration (1–20 lM). DNA samples were separated by electrophoresis on a 1% agarose gel which was stained with ethidium bromide after DNA migration. Gels were photographed under UV light. Nck: nicked, Sc: supercoiled, Rel: relaxed, Topo: topoisomers products. Gels were photographed under UV light.
Figure 5. Topoisomerase II inhibition (A) Effects of compounds 22–24, 26–28 and 14 on the relaxation of plasmid DNA by human topoisomerase II at 50 lM. (B) Effects of compounds 31, 32, 25, 29, 34, 37, 19 and 24 on the relaxation of plasmid DNA by human topoisomerase II at 50 lM. (C) Effects of compounds 24, 34 and 35 on the relaxation of plasmid DNA by human topoisomerase II at 25 and 50 lM. Native supercoiled pUC19 plasmid DNA (350 ng, lane DNA) was incubated with 8 units of topoisomerase II in the absence (lane Topo II) or the presence of tested compounds at 50 lM. Etoposide was used as reference at 25 and 50 lM. DNA samples were separated by electrophoresis on a 1% agarose gel containing 1 lg/mL ethidium bromide. Gels were photographed under UV light. Nck: nicked, Sc: supercoiled, Lin: linear, Rel: relaxed.
activity whereas compound 27 with three methylene chain displayed the best activity among the diferrocenyl counterparts 26–29 (Fig. 5A and B). As an example, if we compare these two compounds, we can observe that the band corresponding to linear DNA (double-stranded breaks) is more marked for the compound 24 which appeared to be more potent than 27 (Fig. 5A). It is worth noting that the compound 24 and its alkylated analogs 34 and 35 exhibited a similar inhibition activity (Fig. 5C). Finally, the introduction of a second protonable site on the ferrocenyl moiety into compounds 31 and 32 suppressed the topoisomerase II inhibition (Fig. 5B, 31 and 32 vs 24). As these compounds displayed the highest DTm and Kapp values, it can be assumed that a strong DNA interaction is detrimental to topoisomerase II inhibition. Structure–activity relationships of ferrocenyl derivatives have been summarized in Figure 6. As can be observed in previous studies,41,42 the positive electrostatic potential induced by iron through cyclopentadienyl ring may provide weak interaction of
Figure 6. Structure–activity relationships of ferrocenyl derivatives.
the ferrocene with the negative charged phosphate backbone. However, topoisomerase II interaction requires the presence of ferrocenyl moiety in indenoisoquinoline derivatives.
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2.6. In vitro antiproliferative activity The antiproliferative activity of the synthesized ferrocenyl compounds was tested with etoposide 4, as reference compound, on breast cell line (MDA-MB-231) using a colorimetric cell proliferation assay. Only cytotoxicities of compounds exhibiting topoisomerase II inhibition and best DNA interacting agents were evaluated. The ability of the new analogs to inhibit the growth of cancer cells is summarized in Table 2. The results show that most ferrocenyl indenoisoquinolines exhibited a significant antiproliferative activity against MDA-MB231 breast cancer cells with IC50 values ranging from 0.95 to 10.41 lM. In particular, four derivatives 23, 24, 31, and 32 displayed good cytotoxicities (
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of ferrocenyl indeno[1,2-c] isoquinolines as topoisomerase II inhibitors Nathalie Wambang a,b, Nadège Schifano-Faux c, Alexandre Aillerie a, Brigitte Baldeyrou d, Camille Jacquet a, Christine Bal-Mahieu d, Till Bousquet a,⇑, Sylvain Pellegrini a, Peter T. Ndifon b, Samuel Meignan d, Jean-François Goossens c, Amélie Lansiaux e, Lydie Pélinski a,⇑ a
Univ. Lille, CNRS, Centrale Lille, ENSCL, Univ. Artois, UMR 8181—UCCS—Unité de Catalyse et Chimie du Solide, F-59000 Lille, France Univ. Yaoundé 1, Laboratoire de Chimie de Coordination, BP 812, Yaoundé, Cameroon Univ. Lille, CHU Lille, EA 7365—GRITA—Groupe de Recherche sur les formes Injectables et les Technologies Associées, F-59000 Lille, France d Univ. Lille, Inserm, U908—CPAC—Cell Plasticity and Cancer, F-59000 Lille, France e Département de Recherches Médicales, Groupement hospitalier de l’Institut Catholique de Lille, 59000 Lille, France b c
a r t i c l e
i n f o
Article history: Received 14 October 2015 Revised 15 December 2015 Accepted 17 December 2015 Available online 18 December 2015 Keywords: Indenoisoquinoline Ferrocene Topoisomerase II inhibitor Drug binding Cytotoxicity
a b s t r a c t Three series of indeno[1,2-c]isoquinolines bearing a ferrocenyl entity were synthesized and evaluated for DNA interaction, topoisomerase I and II inhibition, and cytotoxicity against breast human cancer cell lines. In the first and second series, the ferrocenyl scaffold was inserted as a linker between the two nitrogen atoms. In the last series, it was introduced at the end of the carbon chain. The present study showed that the ferrocenyl entity enhanced the topoisomerase II inhibition. Most compounds showed a potent growth inhibitory effect on MDA-MB-231 cell line with the IC50 in lM range. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Topoisomerases I and II (Topo I and II), essential enzymes for replication, transcription and recombination of DNA, are attractive targets for a number of successful chemotherapeutic agents. The main classification of topoisomerase is based on the enzyme’s ability to generate a single-stranded (type I) or double-stranded (type II) breaks in the DNA double helix.1–3 In the both cases, a religation step leads to the final relaxation of DNA. Camptothecin 1 and its derivatives such as topotecan and irinotecan interact with the DNA–enzyme complex and inhibit the religation mechanism in the presence of topoisomerase I (Fig. 1). However, the low water solubility, the reversibility of the camptothecin–DNA–Topo I cleavage complex and the hydrolytically unstable lactone ring limited its clinical utility.4–6 Moreover, the enzyme–DNA complexes stabilized by indotecan 2 and indimitecan 3 are more persistent than those induced by camptothecin (Fig. 1). Recently, a number of indenoisoquinolines have been identified as novel Topo I inhibitors ⇑ Corresponding authors. Tel.: +33 (0) 3 20 43 48 93; fax: +33 (0) 3 20 43 65 85 (T.B.); tel.: +33 (0) 3 20 43 65 01; fax: +33 (0) 3 20 43 65 85 (L.P.). E-mail addresses: [email protected] (T. Bousquet), lydie.pelinski@ univ-lille1.fr (L. Pélinski). http://dx.doi.org/10.1016/j.bmc.2015.12.033 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
exhibiting better pharmacokinetic features and greater chemical stability than camptothecin.7–10 Interestingly, structural modifications of the indenoisoquinolines provided good topoisomerase II inhibitors.11–13 Among them, the most potent compound on Topo II was an indenoisoquinoline-5,11-dione with a basic tertiary amino group linked to the indenoisoquinoline through an ethoxy spacer group. Topoisomerase II poisons such as etoposide 4 (ETO), mitoxantrone 5 and doxorubicin 6 represent some of the most important and widely prescribed anticancer drugs, particularly for treatment of leukemias, lymphomas, breast and lung cancers (Fig. 1).14–16 Clinical uses of these drugs are limited by their toxicities and development of drug resistance.17,18 New generation of other synthetic and natural products has been developed as topoisomerase II inhibitors.19 Particularly, Top2-targeted drugs such as F14512,20 C-131121 and (R)-XK46922 are currently subject to clinical development. Organometallic compounds have been found to be promising anticancer-drug candidates.23–26 During the last years, halfsandwich Ru(II) arene complexes such as RAPTA-C 7 and [(g6-C6H4Ph)Ru(N,N-en)Cl)]+ (en = 1,2-ethylenediamine) 8, came into the focus of interest due to their biological activity and pharmacological properties.27–29 Among the organometallic
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N. Wambang et al. / Bioorg. Med. Chem. 24 (2016) 651–660
O O
O O
N
O
H 3 CO N
H 3 CO
O
O O
N
N
H 3 CO
N N
H 3 CO
O
Camptothecin 1 Me
O
O
OH
O O HO
N
O
Indotecan (NSC 743400) 2
Indimitecan (NSC 725776) 3
O HO
O
OH
O
O
HN
H N
O
O
OH
OH
OH OH
O O OMe O
O OH
O
HN
OCH 3
H3 CO
N H
OH
H 3C HO
OH Etoposide 4
Mitoxantrone 5
OH
O
O NH2
Doxorubicin 6
Figure 1. Examples of representative Topo I and Topo II inhibitors.
compounds, ferrocenyl phenols 9 developed by Jaouen’s group constitute a new class of molecules possessing both a ferrocene and a tamoxifen diphenylmethylene moiety (Fig. 2). These compounds demonstrate a high in vitro antiproliferative activity on both hormono-dependent (MCF-7) and -independent (MDA-MB231) breast cancer cell lines. Furthermore, they are kinetically stable, relatively lipophilic and have good redox properties.30–34 Since this pioneering work, the use of ferrocene in bioorganometallic chemistry has been growing in the recent years, not only for their antitumor properties but also for their antimalarial, antifungal and DNA-cleaving activities.35–40 Particularly ferrocenyl derivatives, such as azalactone ferrocene and thiomorpholide amido methyl ferrocene, are found to be potent inhibitors of topoisomerase II with preferential action against the b isoform.41,42 As a part of our ongoing effort to develop ferrocenyl drugs,43–45 we report herein the synthesis of novel indenoisoquinolines bearing a ferrocenyl moiety on the side chain (Fig. 3). The enhancing effects of the ferrocene have been evaluated on the cytotoxicity, DNA interaction and topoisomerase I and II inhibition.
monoprotected diamines followed by deprotection of carbamates by hydrochloric acid.48,49 Alkylation of the primary amines with (ferrocenylmethyl)trimethylammonium iodide 21 in the presence of potassium carbonate has afforded ferrocenyl indenoisoquinolines 22–25 in 33–56% yields (Scheme 1). A disubstitution of the amine group occurred during this reaction providing the disubstituted ferrocenyl compounds 26–29 in 19–44% yields.50 The reductive amination in the presence of 2-(N,N-dimethylaminomethyl)ferrocene carboxaldehyde 30 and sodium triacetoxyborohydride on amines 18 and 19 led to ferrocenyl indenoisoquinolines 31 and 32 in 65% and 60% yields respectively. The methylation or ethylation of secondary ferrocenyl amines 23 and 24 were realized by reductive amination in the presence of methanal or ethanal and sodium triacetoxyborohydride leading to compounds 33–35 (Scheme 1). Finally, the condensation of ferrocene carboxylic acid on amines 18 and 19 in the presence of EDCI/HOBt afforded ferrocenyl amides 36 and 37 in 59% and 35% yields, respectively (Scheme 2). 2.2. DNA thermal denaturation
2. Results and discussion 2.1. Chemistry The synthesis of ferrocenyl indenoisoquinolines 14–16 and 22–35 are described in Scheme 1. The condensation of ferrocenyl diamines 11–13, obtained by a described procedure,46 with benzo[d]indeno[1,2-b]pyran-5,11-dione 1047 provided the targeted ferrocenyl compounds 14–16 in 43–67% yields. Indenoisoquinolines 17–20 were obtained by condensation of the benzo[d]indeno[1,2-b]pyran-5,11-dione 10 with primary
OH +
Ph Ru Cl
Cl
Ru
N
P
Cl N N
NH 2
PF6-
NH2 Fe O(CH 2) nN(CH3) 2
RAPTA-C 7
8
Ferrocenyl phenols 9
Figure 2. Organometallic anticancer agents.
The ability of the drugs to protect calf thymus DNA (CT DNA, 42% GC bp) against thermal denaturation was used as an indicator of the relative capacity of indenoisoquinoline derivatives to bind and to stabilize the DNA double helix. DTm values (DTm = Tmdrug–DNA complex TmDNA alone) ranging from 0 to 27.5 °C are reported in Table 1.51–54 Ferrocene-free indenoisoquinoline derivatives bearing an alkylamino side chain from two to five methylene groups (17–20) were considered as reference compounds in our study. N-Substituted compounds with an alkyl (22–25) or acyl (36–37) ferrocenyl moiety were synthesized to amplify the structure affinity relationships in indenoisoquinoline derivatives. In the alkyl series, as showed in Table 1, the amines 17, 19 and 20 displayed better DTm values than their ferrocenyl counterparts (22, 24, 25). However, the best value is obtained for the ferrocenyl derivative 23 possessing a three methylene spacer (DTm = 21.6 °C) in comparison to its amino analog 18 (DTm = 12.9 °C). Moreover, the presence of an additional protonable site on the ferrocenyl moiety in 31 and 32 resulted in an increase of DTm values at 25 °C and 27.5 °C, respectively. It can be assumed that this additional basic nitrogen interacts with anionic phosphate groups of the DNA backbone.
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O
O
N
N
NR 2
Fe
O
O R = CH3 R, R = (CH 2) 5 R, R = (CH 2CH2 )2 NCH 3
Fe
R' N ( )n
N
O n = 2, 3, 4, 5 R' = H, Me, Et
( )n
H N
Fe
NMe 2
O n = 3, 4
Figure 3. Design of ferrocenyl indenoisoquinolines.
O
O
O
H2N
+
a
NR2
Fe
N
O
NR2
Fe
O
10
11: R = CH3 12: R, R = -(CH 2 )5 13: R, R = -(CH 2 CH2 )2 NCH3
14: R = CH3 15: R, R = -(CH 2 )5 16: R, R = -(CH 2 CH2 )2 NCH3
b, c O
O
O
d N O 17: n = 18 : n = 19: n = 20: n =
NH2
+
N(CH3 )3 I
Fe
N
( )n
e
Fe
Fe +
N
( )n
O 22: n = 23: n = 24: n = 25: n =
21
2 3 4 5
H N
O 26: 27 : 28: 29:
2 3 4 5
N
( )n
Fe n=2 n=3 n=4 n=5
f
O
O
N
( )n
H N
Fe
N
NMe 2
O
R' N
Fe
( )n
O
31 : n = 3 32: n = 4
33 : n = 3, R' = Me 34: n = 4, R' = Me 35: n = 4, R' = Et
Scheme 1. Reagents and conditions: (a) CHCl3, reflux, 12 h, 43–67%; (b) H2N(CH2)nNHBoc, CHCl3, reflux, 12 h; (c) HCl, iPrOH, 12 h; (d) K2CO3, CH3CN, reflux, 12 h, 33–65% for 22–25 and 19–44% for 26–29; (e) 2-(N,N-dimethylaminomethyl)ferrocene carboxaldehyde 30, NaBH(OAc)3, MeOH, 2 h, 60–65%; (f) HCHO or CH3CHO, NaBH(OAc)3, MeOH, 1 h or 30 h, 26–88%.
O
O
N
NH ( )n 2
O 18: n = 3 19: n = 4
N
Fe
H N ( )n
O
O
36: n = 3 37: n = 4
Scheme 2. Reagents and conditions: Ferrocene carboxylic acid, EDCI, HOBt, CH2Cl2, 12 h, 35–59%.
Additionally, N-alkylation of monoferrocenyl derivatives by a methyl or an ethyl group (33 and 35) led to a decrease of DTm values (33 and 35 vs 23 and 24). However, compound 34 displayed a higher DTm than 24 (DTm = 18.5 °C and 13.70 °C, respectively) suggesting an effect of the methylene units number from the indenoisoquinoline ring to the nitrogen atom bearing a methyl group, on DNA interaction.
The position of the ferrocenyl moiety with regard to the indenoisoquinoline ring was investigated with the three compounds 14–16 and led to lower DTm values than 23. However, it is interesting to note, that the replacement of the dimethylamino group in 14 by a bulkier cyclic amine such as piperidine in 15 is not favorable for DNA interaction with DTm values 16.8 °C and 6.8 °C, respectively. Similarly, the diferrocenyl compounds (26–29) displayed low binding affinity to DNA compared to the monoferrocenyl derivatives (22–25). It is noteworthy that in the both series, the best results were observed for compounds 23 and 27, bearing a N6-lactam side chain of three methylene units (DTm = 21.6 °C and 12.8 °C, respectively). For the acyl series, the acylation of the amines 18 and 19 altered drastically the capacity of the drug to bind DNA as indicated with DTm values calculated for compounds 36 and 37 (0 and 1 °C, respectively). 2.3. Fluorescence measurements DNA binding affinities for some of ferrocenyl compounds were quantified by means of fluorescence Kapp (Table 1). As a weak
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Table 1 Interaction of indenoisoquinolines and ferrocenyl indenoisoquinolines with DNA Compd
DTma (°C)
Kapp (107 M1)b
14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 31 32 33 34 35 36 37 Eto 4
16.8 6.8 Ndc 9.9 12.9 19.5 17.5 5.6 21.6 13.7 11.7 4.9 12.8 4.6 Ndc 25 27.5 3.4 18.5 7.5 0 1 0e
0.83 ± 0.01 Nd Nd Nd 1.38 ± 0.25 1.53 ± 0.13 1.14 ± 0.38 Nd 1.41 ± 0.26 0.45 ± 0.03 0.42 ± 0.11 Nd 11.5%d Nd Nd 3.52 ± 0.52 6.49 ± 0.23 Nd 0.82 ± 0.14 Nd Nd Nd 28%f
Variations in melting temperature (DTm = Tmdrug–DNA complex TmDNA alone) at ratio of 1. b Apparent binding constant measured by fluorescence using [BET]/[DNA] = 1.26. c Nd: not determined (low aqueous solubility). d Displacement of BET with 11.9 lM of compound 27. e Ref. 11. f Displacement of BET with 10 lM of etoposide. a
fluorescence was observed upon DNA titration, an indirect method was privileged.55,56 We used the conventional fluorescence quenching assay based on DNA binding competition between the intercalating drug ethidium bromide (BET) and the tested molecules.57–59 We decided to focus this study on the compounds with a DTm value superior to 11 °C. As reported in Table 1, Kapp values ranged from 0.42 107 (compound 25) to 6.49 107 M1 (compound 32). The introduction of a ferrocenyl moiety on the primary amine of the N6 lactam side chain decreased DNA affinity (24, 25 vs 19, 20) while an equivalent Kapp value was observed for, compounds 18 and 23. However, in accordance with DTm values, compound 23, possessing three methylene units, exhibited a higher Kapp value than 24 and 25. In addition, the introduction of a ferrocenyl moiety as linker between the two nitrogen atoms led to a lower DNA affinity (14 vs 23). Increasing the length of the N6 lactam side chain from three to five methylene units did not bring significant variations in Kapp values. Furthermore, the diferrocenyl compound 27 displayed lower DNA affinity than its monoferrocenyl analog 23. In agreement with DTm values, the N-alkylation of monoferrocenyl derivative 24 by a methyl group (compound 34) provided an increase of Kapp value. However, the N-methylation of compound 23 led to a weaker DNA ligand (23 vs 33). Finally, the addition of a second protonable site on the side chain resulted in an increase of Kapp values (31 and 32 vs 23 and 24). These compounds displayed the highest DTm and Kapp values, indicating both good affinity for DNA and duplex stabilization. This could be attributed to the additional stabilizing interaction, potentially between anionic phosphate groups of the DNA backbone and the additional protonable site. 2.4. Drug binding mechanism Different binding modes (intercalation and/or groove binding) can account for the melting temperature elevation. Therefore, to
understand the drug binding mechanism, a DNA unwinding assay based on the relaxation of native supercoiled plasmid DNA in the presence of both topoisomerase I and ethidium bromide for compounds 14, 22–24, 26–28 was used (Fig. 4A). The presence of supercoiled DNA (Sc) in Figure 4A could reveal that 14 and 23 were the best DNA interacting agents with highest supercoiled bands. From these results, a DNA relaxation in the presence of increasing concentrations of these two selected compound was performed (Fig. 4B). As shown in Figure 4B, the DNA relaxed by topoisomerase I (lane Topo I) generated a family of DNA topoisomers with a slow electrophoretic mobility. The drug was then added while topoisomerase I was maintained in the reaction mixture. When the drug concentration was increased, the closed circular duplex DNA was progressively supercoiled, indicating that the drug intercalated into DNA at low concentration for 23. These results were consistent with the Kapp value obtained with ethidium bromide competition and suggest an intercalation mode for the two selected compounds 14 and 23. 2.5. Topoisomerase inhibition A conventional DNA relaxation assay was performed to assess the effects of the compounds on the catalytic activity of human topoisomerases I and II. Supercoiled plasmid DNA was treated with either topoisomerase I or II in the presence of tested drugs and the DNA relaxation products were then resolved by gel electrophoresis on agarose gels containing ethidium bromide.59 From this study, none of the tested compounds was found to behave as a topoisomerase I poison (Fig. 4A). Indeed, unlike camptothecin (reference drug), no topoisomerase I-induced strand breaks visualized in nicked bands was observed with any of the compounds. The same results were obtained for the other compounds (see Supporting information). In Topo I inhibition experiments, we have also confirmed results obtained with DNA thermal denaturation studies for 14, 19, 23, 24, 31–32 (highest Kapp values observed in Table 1). Following this study, the influence of ferrocenyl moiety on topoisomerase II inhibition was then examined. The linear DNA results from the dissociation of the Topo II homodimer from cleaved DNA by SDS. In this case, the reference drug was etoposide, which produced a marked level of DNA double-stranded breaks corresponding to linear DNA (Lin). The formation of Topo II– DNA–drug cleavage complex could be evidenced by the appearance of linear DNA for Topo II. Interestingly, from this study, inhibition of topoisomerase II was clearly detected with these ferrocenyl compounds (Fig. 5). The presence of a ferrocenyl moiety on the primary amine of the N6 lactam side chain seems to play a major role in topoisomerase II inhibition as the compound 24 displayed high poisoning activity (Fig. 5A) whereas 19 had no effect (Fig. 5B). However, the conversion of the ferrocenyl methylene amino group to a ferrocenamide group suppressed the activity (Fig. 5B, 24 vs 37). On the other hand, the introduction of a ferrocenyl moiety as linker between the amine and the lactam nitrogen, led to compounds exhibiting weak Topo II inhibition (Fig. 5A, compound 14). The replacement of the dimethylamino group in compound 14 by a piperidine or a N-methylpiperazine (compounds 15 and 16), abrogated the activity (data not shown). The position of the ferrocenyl moiety at the end of the N6 lactam chain seems to be critical for topoisomerase II inhibition since the compound 24 was more potent than 14 (Fig. 5A). The length of the N6 lactam side chain (from two to five methylene units) had an impact on the topoisomerase inhibition abilities. Indeed, for monoferrocenyl derivatives 22–25, especially the compound 24 with a four methylene spacer, displayed the highest
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Figure 4. Topoisomerase I inhibition. (A) Effects of compounds 22–24, 26–28 and 14 on the relaxation of plasmid DNA by human topoisomerase I. Native supercoiled pUC19 plasmid DNA (130 ng, lane DNA) was incubated with 8 units topoisomerase I in the absence (lane Topo I) or the presence of tested compounds at 20 lM. Camptothecin (CPT) was used as reference at 20 lM. DNA samples were separated by electrophoresis on a 1% agarose gel containing 1 lg/mL ethidium bromide. (B) Effects of compounds 23 and 14 on the relaxation of plasmid DNA by human topoisomerase I. Native supercoiled pUC19 plasmid DNA (130 ng, lane DNA) was incubated with 4 units topoisomerase I in the absence (lane Topo I) or the presence of tested compounds at indicated concentration (1–20 lM). DNA samples were separated by electrophoresis on a 1% agarose gel which was stained with ethidium bromide after DNA migration. Gels were photographed under UV light. Nck: nicked, Sc: supercoiled, Rel: relaxed, Topo: topoisomers products. Gels were photographed under UV light.
Figure 5. Topoisomerase II inhibition (A) Effects of compounds 22–24, 26–28 and 14 on the relaxation of plasmid DNA by human topoisomerase II at 50 lM. (B) Effects of compounds 31, 32, 25, 29, 34, 37, 19 and 24 on the relaxation of plasmid DNA by human topoisomerase II at 50 lM. (C) Effects of compounds 24, 34 and 35 on the relaxation of plasmid DNA by human topoisomerase II at 25 and 50 lM. Native supercoiled pUC19 plasmid DNA (350 ng, lane DNA) was incubated with 8 units of topoisomerase II in the absence (lane Topo II) or the presence of tested compounds at 50 lM. Etoposide was used as reference at 25 and 50 lM. DNA samples were separated by electrophoresis on a 1% agarose gel containing 1 lg/mL ethidium bromide. Gels were photographed under UV light. Nck: nicked, Sc: supercoiled, Lin: linear, Rel: relaxed.
activity whereas compound 27 with three methylene chain displayed the best activity among the diferrocenyl counterparts 26–29 (Fig. 5A and B). As an example, if we compare these two compounds, we can observe that the band corresponding to linear DNA (double-stranded breaks) is more marked for the compound 24 which appeared to be more potent than 27 (Fig. 5A). It is worth noting that the compound 24 and its alkylated analogs 34 and 35 exhibited a similar inhibition activity (Fig. 5C). Finally, the introduction of a second protonable site on the ferrocenyl moiety into compounds 31 and 32 suppressed the topoisomerase II inhibition (Fig. 5B, 31 and 32 vs 24). As these compounds displayed the highest DTm and Kapp values, it can be assumed that a strong DNA interaction is detrimental to topoisomerase II inhibition. Structure–activity relationships of ferrocenyl derivatives have been summarized in Figure 6. As can be observed in previous studies,41,42 the positive electrostatic potential induced by iron through cyclopentadienyl ring may provide weak interaction of
Figure 6. Structure–activity relationships of ferrocenyl derivatives.
the ferrocene with the negative charged phosphate backbone. However, topoisomerase II interaction requires the presence of ferrocenyl moiety in indenoisoquinoline derivatives.
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2.6. In vitro antiproliferative activity The antiproliferative activity of the synthesized ferrocenyl compounds was tested with etoposide 4, as reference compound, on breast cell line (MDA-MB-231) using a colorimetric cell proliferation assay. Only cytotoxicities of compounds exhibiting topoisomerase II inhibition and best DNA interacting agents were evaluated. The ability of the new analogs to inhibit the growth of cancer cells is summarized in Table 2. The results show that most ferrocenyl indenoisoquinolines exhibited a significant antiproliferative activity against MDA-MB231 breast cancer cells with IC50 values ranging from 0.95 to 10.41 lM. In particular, four derivatives 23, 24, 31, and 32 displayed good cytotoxicities (
