lable at ScienceDirect

European Journal of Medicinal Chemistry 86 (2014) 797e805

Contents lists avai

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis and evaluation of new antitumor 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones

Andrey E. Shchekotikhin a, b, *, Valeria A. Glazunova c, d, Lyubov G. Dezhenkova a,Yuri N. Luzikov a, Vladimir N. Buyanov b, Helena M. Treshalina c, Nina A. Lesnaya c,Vladimir I. Romanenko c, Dmitry N. Kaluzhny e, Jan Balzarini f, Keli Agama g,Yves Pommier g, Alexander A. Shtil c, d, Maria N. Preobrazhenskaya a

a Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11 B. Pirogovskaya Street, Moscow 119021, Russiab Mendeleyev University of Chemical Technology, 9 Miusskaya Square, Moscow 125190, Russiac Blokhin Cancer Center, 24 Kashirskoye Shosse, Moscow 115478, Russiad Moscow Engineering and Physics Institute, 31 Kashirskoye Shosse, Moscow 115409, Russiae Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow 119991, Russiaf Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgiumg Developmental Therapeutics Branch, National Cancer Institute, NIH, 37 Convent Drive, 37-5068, Bethesda, MD 20892, USA

a r t i c l e i n f o

Article history:Received 12 June 2014Received in revised form2 September 2014Accepted 6 September 2014Available online 8 September 2014

Keywords:Naphtho[2,3-f]indole-5,10-dionesDNA ligandsTopoisomerase 1/2 inhibitorsCircumvention of multidrug resistanceAntitumor activity

* Corresponding author. Gause Institute of New AnMedical Sciences, 11 B. Pirogovskaya Street, Moscow

E-mail address: shchekotikhin@mail.ru (A.E. Shche

http://dx.doi.org/10.1016/j.ejmech.2014.09.0210223-5234/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

A series of new 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones 6e13 bearing the cyclicdiamine in the position 3 of the indole ring was synthesized. The majority of new compounds demon-strated a superior cytotoxicity than doxorubicin against a panel of mammalian tumor cells with de-terminants of altered drug response, that is, Pgp expression or p53 inactivation. For naphtho[2,3-f]indole-5,10-diones 6e9 bearing 3-aminopyrrolidine in the side chains, the ability to bind double-stranded DNA and inhibit topoisomerases 1 and 2 mediated relaxation of supercoiled DNA weredemonstrated. Only one isomer, (R)-4,11-dihydroxy-3-((pyrrolidin-3-ylamino)methyl)-1H-naphtho[2,3-f]indole-5,10-dione (7) induced the formation of specific DNA cleavage products similar to the knowntopoisomerase 1 inhibitors camptothecin and indenoisoquinoline MJeIIIe65, suggesting a role of thestructure of the side chain of 3-aminomethylnaphtho[2,3-f]indole-5,10-diones in interaction with thetarget. Compound 7 demonstrated an antitumor activity in mice with P388 leukemia transplantswhereas its enantiomer 6was inactive. Thus, 3-aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione emerge as a new prospective chemotype for the search of antitumor agents.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

The ability to interact with a variety of intracellular targets,including nucleic acids [1e3], topoisomerases (Top1 and Top2)[4e6], telomerase [7,8], protein kinases [9e11], receptors [12,13]etc., makes the anthraquinone moiety an important pharmaco-phore for rational drug design. Indeed, natural and synthetic de-rivatives of hydroxyanthraquinones (e.g., anthracyclines,mitoxantrone and emodin) are widely used as prototypes for thedesign of anticancer drug candidates [14e19]. In particular, thealtered drug response, a major challenge for chemotherapy, stim-ulates the search for agents whose cytotoxicity is retained forpleotropically resistant tumor cells [20e25].

tibiotics, Russian Academy of119021, Russia.kotikhin).

served.

Previously we have identified the linear pyrroloquinizarine(4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione) as a promisingscaffold for the search of agents active against resistant tumor cells[26]. Indeed, 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones were able to circumvent two clinically validatedmechanisms of drug resistance; that is, P-glycoprotein (Pgp)expression (an MDR phenotype) and loss of function of pro-apoptotic p53. Furthermore, aminomethylation of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione with cyclic diaminessuch as piperazine or quinuclidine, significantly increased thecytotoxicity. One of themost potent in this series was the derivative2 (Fig. 1) carrying piperazine in the side chain. This compound wasmore active than doxorubicin (DOX; 1) against MDR K562/4leukemic cells selected for survival in the presence of 1 and thecolon carcinoma HCT116p53KO variant with deleted p53. The

O

O

OH

OH

OH

O

O

O

OH

NH2

O

OH

Doxorubicin (1)

2

O

O

OH

OH

NH

N NH

Fig. 1. Structures of DOX (1) and naphtho[2,3-f]indole-5,10-dione 2.

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805798

potency of 2 has been attributed to the formation of stable com-plexes with double stranded DNA and attenuation of Top1 activity[27].

Clinical efficacy of Top1 and Top2 inhibitors (e.g., DOX, etopo-side, irinotecan) can explain the continuous interest in the searchfor novel topoisomerase blockers as anticancer agents withimproved therapeutic properties [28e31]. In this study weexplored new Top1 and Top2 antagonists based on 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione scaffold bearing thecyclic diamine in the side chain (position 3 of the heterocycle). Theoptimised synthetic procedure yielded a series of new 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones. Ahigh affinity to double stranded DNA and potent attenuation ofTop1 and Top2 by new naphthoindolediones were associated withcytotoxicity (at submicromolar or low micromolar concentrations)against wild type and drug resistant cell lines. The selected 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione 7 with (S)-3-aminopyrrolidine in the side chain demonstrated therapeutic effi-cacy in vivo against a transplanted murine tumor.

2. Results and discussion

2.1. Chemistry

To prepare a new series of 3-aminomethylnaphtho[2,3-f]indole-5,10-diones we modified the previously reported scheme that wasbased on transamination reaction of gramine analogs [26]. The keystep in the synthesis of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones is demethylation of starting 4,11-dimethoxynaphtho[2,3-f]indole-5,10-diones. For demethylation procedure we used mildLewis acids such as BСl3, BBr3, B(CF3CO2)3 or CeCl3 [26,32e34].However, the efficiency of these reactions is moderate and dependson the structure of functional groups in the heterocyclic nucleus ofnaphtho[2,3-f]indole-5,10-diones. Treatment of methoxynaph-thoindoledione 3 with common demethylation agents is prob-lematic due to reactivity of the aminomethyl residue. Interestingly,we observed that the hydrochloride salt of the gramine analogue 3was unstable during storage. Major products of decomposition of 3were identified by TLC and mass-spectrometry as demethoxy de-rivatives. We assumed that HCl can be useful for demethylation ofmethoxynaphtho[2,3-f]indole-5,10-diones. We found that, despitesome stabilization in aqueous or methanol HCl, 3 can be easilydemethylated by the solution of HCl in anhydrous acetic acid toproduce the hydrochloride of gramine 4 in 92% yield (Scheme 1).The subsequent treatment of this salt with anion exchange resingave the free base of 4 (Scheme 1). Thus, we developed a novelconvenient method of naphtho[2,3-f]indole-5,10-dione 3 deme-thylation that produced the gramine analogue 4 in twice bigger

yield than the procedure with Lewis acid [34]. This new methodfacilitates the access to other bioactive 4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones [32] since gramine 4 is a key intermediate fortheir multistep synthesis. Additionally, this method can be usefulfor dealkylation of other methoxynaphthoindolediones and relatedcompounds.

The interaction of gramine 4 with methyl iodide produced thequaternary salt 5. The latter intermediate was used to synthesizethe aminomethyl derivatives in the reaction of transaminationwithBoc-protected amino derivatives of pyrrolidine, piperidine or dia-zabicyclo[2.2.1]heptane (Scheme 2). At the final step the unblock-ing of intermediate Boc derivatives with a solution of HCl in ethergave the corresponding 3-aminomethylnaphtho[2,3-f]indole-5,10-diones 6e13 in 55e64% yields.

The resulting compounds 6e13 were purified by re-precipitation. The analytical and spectroscopic data were in fullaccordance with the assigned structures. The purified compoundswere used for biophysical, cell culture and in vivo studies.

2.2. Biology

2.2.1. Antiproliferative activity of naphtho[2,3-f]indole-5,10-dionesThe antiproliferative activity of hydrochlorides of 4,11-

dihydroxynaphtho[2,3-f]indole-5,10-diones 2, 6e13 was studiedusing a panel of wild type tumor cell lines and isogenic drugresistant sublines: murine leukemia L1210/0, Т-lymphocyte leu-kemia Molt4/C8, K562 myeloid leukemia and its MDR (Pgpexpressing) K562/4 variant, as well as colon carcinoma НСТ116 andHCT116p53KO variant with inactivation of pro-apoptotic p53.

All new derivatives of naphtho[2,3-f]indole-5,10-diones inhibi-ted tumor cell proliferation at submicromolar to low micromolarconcentrations. The most potent were stereoisomers 6 and 7 withthe 3-aminopyrrolidine residue attached to the chromophoremoiety via the exocyclic nitrogen (Fig. 2 and Table S1). Theregioisomers 8, 9 with diamine conjugated to naphtho[2,3-f]indole-5,10-diones through the cyclic nitrogenwere less potent. Anincreased size of the cyclic amine ring in the side chain (10e12) aswell as the fused diamine (diazabicyclo[2.2.1]heptane) in 13 alsodiminished the antiproliferative potency (Fig. 2).

Importantly, all new compounds were cytotoxic for wild typecell lines and their isogenic drug resistant counterparts. The K562/4 cells express functional Pgp and are resistant to Pgp transportedagents such as DOX (1, resistance indices RI z 8.8, seeSupplementary Material, Table S1). In striking contrast, for naphtho[2,3-f]indole-5,10-diones 2, 6e13 the resistance indices were closeto 1; for compounds 8, 11 and 13 the activity against Pgp-positivecells was even higher than for the parental K562 counterparts(RI ¼ 0.8; 0.4; and 0.3, respectively; Table S1). In contrast to DOX,

O

O

OH

OH

NH

NMe2

4 (78%)3

1.HCl/AcOH

O

O

OMe

OMe

NH

NMe2

2. Dowex (OH-form)

Scheme 1. Demethylation of dimethoxynaphtho[2,3-f]indole-5,10-dione 3.

O

O

NH

HO

HO

NH

NH

6 7 8 9 10 11 12 13

NH

NH

NMe3 I

1. HXBoc

O

O

NH

HO

HO

XH

2. HCl

5 6-13

N

NH2

N

NH2

*2HCl

N NH2

NH

NH

NH

NHXH= N

NH

4

Scheme 2. Synthesis of 3-aminomethylnaphtho[2,3-f]indole-5,10-diones 6e13.

Fig. 2. Relationship between chemical structure and antiproliferative activity (�logIC50) for 3-aminomethylnaphtho[2,3-f]indole-5,10-diones 2, 6e13. DOX (1) was used as areference drug. Values of IC50 (mean of 3 experiments) are shown in Supplementary Material, Table S1.

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805 799

almost all new naphtho[2,3-f]indole-5,10-diones (except 9 and 10)were capable of inducing p53-independent cell death. Moreover,the derivatives 7 and 12 demonstrated a more pronounced toxicityfor HCT116p53KO (p53�/�) cells than for parental HCT116 cells(RI ¼ 0.75 and 0.5, respectively) as opposed to the sensitivity ofthese cell lines to 1 (RI ¼ 3.1; Table S1). Thus, the designed naphtho[2,3-f]indole-5,10-diones demonstrated the improved anti-proliferative activity against tumor cells with the determinants ofaltered drug response, that is, Pgp expression and p53 inactivation.Optimization of the structure of the side chain in 3-

aminomethylnaphtho[2,3-f]indole-5,10-diones allowed us toselect compounds 6e9 for further studies.

2.2.2. Interaction of naphtho[2,3-f]indole-5,10-diones with duplexDNA

Like structurally close anthraquinone containing compounds[3,35], 3-aminomethyl derivatives of naphtho[2,3-f]indole-5,10-dione can bind DNA and interfere with enzymes involved in thefunctionality of nucleic acids. We tested DNA binding of newisomeric naphtho[2,3-f]indole-5,10-diones 6e9 with different

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140

6789

[L]x

[DN

A]/(

F 0-F),

x1012

DNA concentration, μM(bp)

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805800

substituents at the side chains. Titration of 6e9 (1.2 mM each) withdouble stranded DNA (up to 65 mM (bp)) resulted in a drop offluorescence intensity with minimal changes of fluorescencespectra (Fig. 3). The observed quench of fluorescence of naphtho[2,3-f]indole-5,10-dione chromophores was characteristic of drug-DNA complex formation [36]. The binding constants determinedby Scott's method (Fig. 4) were similar for the pairs of stereoiso-mers 6, 7 and 8, 9 (Table 1). This similarity means that stereo-chemistry of 3-aminopyrrolidine residue in the side chain ofnaphtho[2,3-f]indole-5,10-diones insignificantly affected the affin-ity to DNA. In striking contrast, the mode of linking of naphtho[2,3-f]indole-5,10-dione to diamine was important. Indeed, the DNAbinding constants of 6 and 7 (with diamine conjugated throughpyrrolidine nitrogen) were 3-fold bigger than the respective valuesfor regioisomers 8 and 9 with the side chain conjugated via theexocyclic amino group (Table 1). These results demonstrated thehigh affinity of new compounds to DNA and highlighted the role ofthe structure of the side chain of 3-aminomethylnaphtho[2,3-f]indole-5,10-diones in the drug-DNA complex formation.

Fig. 4. Binding of naphtho[2,3-f]indole-5,10-diones 6e9 to double stranded DNA.

Table 1Binding constants (Kb) of complex formation of naph-tho[2,3-f]indole-5,10-diones 6e9 with double strandedDNA determined by fluorescence titration.

Compound Kb, 105 М�1

6 11 ± 17 10 ± 18 3.5 ± 0.49 3.4 ± 0.3

Fig. 5. Top1 inhibition by naphtho[2,3-f]indole-5,10-diones 6e9.

2.2.3. Top1 and Top2 inhibitionPreviously we have identified Top1 as a target for the cytotox-

icity of naphthoindolediones [27,32]. In this study we testedwhether isomeric compounds 6e9 bearing 3-aminopyrrolidineresidue in the side chains can affect Top1 and Top2. These com-pounds were selected because of their high potency againstparental and drug resistant tumor cells (Fig. 2 and Table S1).Relaxation of supercoiled plasmid DNA by Top1 was attenuatedwith 6e9 in a dose dependent manner; the increase of concen-trations from 0.5 mM to 5 mM yielded slowly migrating DNA top-oisomers (Fig. 5). Interestingly, stereochemistry of the side chainwas important for inhibition of Top1-mediated relaxation; at 2 mMof 9 (R-isomer) the plasmid relaxation was inhibited whereas S-isomer 8 caused a smaller effect even at 5 mМ. In striking contrast,the reference drug camptothecin (CPT) attenuated Top1-mediatedDNA relaxation only at >> 10 mM. Thus, among 3-aminomethylderivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione,compound 9 was the most potent Top1 inhibitor. In control ex-periments we observed no influence of tested compounds (atconcentration as high as 20 mM) on DNA mobility in the absence ofTop1 (data not shown).

Next, we addressed the question of specificity of Top1 inhibitionby 6e9 using a Top1 cleavage assay [37]. Among this tested seriesonly compound 7 showed a specific DNA cleavage similar forreference Top1 inhibitors CPT and indenoisoquinoline MJeIIIe65(NSC 706744 [38]) (Fig. 6). The induction of Top1-DNA complexesby 7 at submicromolar concentrations was dose-dependent, asevidenced by the increased intensity of DNA cleavage bands.However, the highest concentration of 7 blocked the formation ofDNA cleavage products and evoked an increased band of the full-

0

50

100

150

200

500 550 600 650 700

Fluo

resc

ence

, au

Wavelength, nm

A

0

50

100

150

200

250

500 550 600 650 700

Fluo

resc

ence

, au

Wavelength, nm

B

Fig. 3. Changes of fluorescence spectra of 6e9 upon titration with double stranded DNA. Opthe compound in complexes with DNA. (A) 6; (B) 7; (C) 8; (D) 9.

length DNA substrate, a common characteristic of strong DNAintercalators [39]. Notably, 7 has unique cleavage pattern as itinduced DNA cleavage complexes at several sites similar to CPT(sites 92 and 119) and indenoisoquinoline MJeIIIe65 (sites 48, 97and 108). Probably, the observed differences for 7 and its isomers 6,8 and 9 in Top1 cleavage assay could ultimately lead to differentpharmacological effects.

0

100

200

300

400

500

600

500 550 600 650 700

Fluo

resc

ence

, au

Wavelength, nm

C

0

100

200

300

400

500

600

700

500 550 600 650 700

Fluo

resc

ence

, au

Wavelength, nm

D

en markers, fluorescence of free compound in the buffer; filled markers, fluorescence of

Fig. 6. Top1 mediated DNA cleavage by 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione 7. Lane 1: DNA alone; lane 2: þ Top1; lane 3: þ camptothecin 1 mM; lane 4: þindenoisoquinoline MJeIIIe65, 1 mM; lanes 5e8: þ compound 7 at 0.1, 1, 10 and100 mM, respectively. The numbers on the left and arrows indicate cleavage site po-sitions. A 117 bp DNA substrate was used in this assay but, to facilitate comparison, thecleavage sites are numbered to be consistent with the commonly used 161 bp DNAsubstrate [40].

Fig. 8. Mean survival (MS) of P388 tumor bearing mice treated with 6, 7 and 1.

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805 801

We next tested whether naphthoindolediones 6e9 affect Top2-mediated relaxation of supercoiled DNA. Fig. 7 shows DNA top-oisomers generated by Top2 in the absence (lane Top2) or presenceof 6e9. Compound 8 almost completely inhibited plasmid relaxa-tion at 2 mМ whereas other tested compounds were less potent.Notably, stereoisomers 8, 9 with terminal primary amino groups,despite a lower affinity to DNA, were more potent against Top2 andTop1, respectively, than their isomers 6, 7.

Fig. 7. Top2 inhibition by naphtho[2,3-f]indole-5,10-diones 6e9.

Thus, we showed that the potency of inhibition of Top1 andTop2 depends on the structure and stereochemistry of the sidechains of naphthoindolediones. These results also indicated thatthe structure of the side chain can influence the mode of enzymeinhibition. As known for other intercalating agents, naph-thoindolediones can inhibit topoisomerases by altering local DNAconformation [41,42]. However, the lack of direct correlation be-tween DNA affinity of 6e9 and Top1 or Top2 inhibitory potency, aswell as specific DNA cleavage by Top1 in the presence of 7 suggestanother mechanism of enzyme inhibition by these compounds. Thespecific Top-mediated DNA cleavage is a hallmark of Top1 poisonsthat stabilize drug-DNA-enzyme complexes [4, 43] . Supposedly,the carbonyl or hydroxyl groups of the anthraquinone moiety, aswell as 3-aminomethyl substituent in 6e13 (similarly to the aminosugar in anthracyclines [44]), could be involved in the stabilizationof above mentioned complexes. On the other hand, the diaminefragment of the side chain may be involved in stabilization of ter-tiary complexes by hydrogen or electrostatic interactions withfunctional groups of the enzyme. Also, 3-aminomethyl group in6e13 can participate in the formation of a covalent link betweenthe ligand and the protein. This hypothesis comes from the fact that3-aminomethylnaphthoindolediones retain the reactive propertiesof gramine [26,32,45] that is capable of alkylating a wide range ofO-, N-, C- and S-nucleophiles [46].

2.2.4. In vivo testingGiven that compound 7 demonstrated a high potency against

drug resistant cells and specificity of Top1 inhibition, we tested itsantitumor efficacy in mice bearing the transplanted leukemia P388.The i.p. injections of 7 increased the life span of mice in a dose-depending manner. Mean survival (MS) of mice in the controlgroup was 10.2 days (Fig. 8). After injection of 7 in a single dose of80 mg/kg MS reached 14.3 days (T/C ¼ 140 ± 15%, p � 0.05); at25 mg/kg or 30 mg/kg daily for 5 days the MS values were 14.0 and15.8 days (T/C ¼ 137 ± 11% and 155 ± 17%, p � 0.05), respectively(Fig. 8). Treatment with 7 was well tolerated up to daily dose of45 mg/kg (5 days).

The efficacy of 7was comparable with that for 1 administered atmaximum tolerated single dose of 8 mg/kg (MS ¼ 14.3 days; T/C ¼ 140 ± 22%, p � 0.05%). In striking contrast, the stereoisomer 6used in the same doses as 7 did not increase the life span of P388tumor bearing mice (Fig. 8).

3. Conclusion

A series of new 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones 6e13 bearing the cyclic diamine in the sidechain was prepared. In doing so we optimised the scheme of

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805802

synthesis and developed a method of demethylation of their keyprecursor, 3-dimethylamino-4,11-dimethoxynaphtho[2,3-f]indole-5,10-dione 3. The new procedure of O-demethylation by treatmentof 3 with HCl in acetic acid gave the hydrochloride of 3-dimethylamino-4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione 4in a high yield (92%). This improvement provides excellent oppor-tunity for increasing the yields of target compounds. Also, thenewly developed method of mild dealkylation may be useful fordeprotection of alkoxy groups of other alkoxynaphthoindoledionesand related compounds.

A potent (in submicromolar to low micromolar concentrations)cytotoxicity against a panel of mammalian tumor cells wasobserved for all novel 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones 6e13. The majority of new compoundsdemonstrated a superior potency than the reference drug DOXagainst cells with Pgp expression or p53 inactivation. Naphtho[2,3-f]indole-5,10-diones 6e9 bearing 3-aminopyrrolidine in the sidechains were found to bind double stranded DNA and affect Top1 orTop2 mediated DNA relaxation. These parameters strongly depen-ded on the mode of attachment of the diamine residue to thenaphtho[2,3-f]indole-5,10-dione moiety. Importantly, only theisomer 7 induced the formation of specific DNA cleavage productssimilar for classical Top1 inhibitors camptothecin and inden-oisoquinoline MJeIIIe65. Moreover, the derivative of (R)-amino-pyrrolidine 7 increased the life span of mice bearing P388 leukemiawhile its enantiomer 6was inactive. Altogether, this study providesevidence that naphtho[2,3-f]indole-5,10-diones bearing the cyclicdiamine in the side chain in the position 3 can be a perspectivechemotype for exploration of antitumor drug candidates withimproved properties.

4. Experimental section

4.1. General methods

The NMR spectra of all newly synthesized compounds wererecorded on a Varian VXR-400 instrument operating at 400 MHz(1H NMR) and 100 MHz (13C NMR). Chemical shifts were measuredin D2O using sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4acid (1H NMR) or methanol (13C NMR) as internal standards. Thesignals in the 13C NMR spectra were assigned by the APT (attachedproton test) method. Analytical TLC was performed on silica gelF254 plates (Merck) and column chromatography on Silica GelMerck 60. Melting points were determined on a Buchi SMP-20apparatus and are uncorrected. High resolution mass spectrawere recorded by electron spray ionization on a Bruker DaltonicsmicroOTOF-QII instrument. UV spectra were recorded on Hitachi-U2000 spectrophotometer. HPLC was performed using ShimadzuClass-VP V6.12SP1 system (GraseSmart RP-18, 6 � 250 mm). Elu-ents: A, H3PO4 (0.01M); B, MeCN. All solutionswere evaporated at areduced pressure on a Buchi-R200 rotary evaporator at the tem-perature below 50 �C. All products were vacuum dried at roomtemperature. All solvents, chemicals, and reagents were obtainedcommercially and used without purification. Naphtho[2,3-f]indole-5,10-diones 2 and 3 were prepared as described [26]. The purity ofcompounds 4e13 was >95% as determined by HPLC analysis. Allreagents were from SigmaeAldrich unless specified otherwise.

4.1.1. 3-((Dimethylamino)methyl)-4,11-dihydroxy-1H-naphtho[2,3-f]indole-5,10-dione (4)

To a solution of dimethylaminomethyl derivative 3 (500 mg,1.4 mmol [26]) in glacial acetic acid (20 mL) the solution (9%) ofhydrogen chloride in glacial acetic acid (20 mL) was added. Themixturewas stirred for 24 h and evaporated in vacuum. The residuewas dissolved in hot water (3e4 mL), filtered, then product was

precipitated with acetone-ether mixture (1:1), collected by filtra-tion and dried. The yield of hydrochloride 4 was 465 mg (92%) as ared solid, mp 238-239 �С. HPLC (LW ¼ 260 nm, gradient B20/ 60% (30 min)) tR ¼ 18.3 min, purity 95.7%. 1H NMR (400 MHz,D2O) d 7.46 (1H, d, H9), 7.30 (3H, m, H6,7,8), 7.09 (1H, s, H2), 4.13 (2H,s, СН2), 2.85 (6Н, s, NMe2). HRMS (ESI) calculated for C19H17N2O4[MþH]þ 337.1170, found 337.1178.

For preparation of the free base of 4, an aqueous solution ofhydrochloride 4 was eluted through column with hydroxy form ofDowex 1x2 resin. The eluant was evaporated under vacuum and thesolid residue was used at the next steps without purification. Foranalytical purposes the crude product was recrystallized frommethanol to afford 85% free base of 4 as a dark red powder with mp151e153 �C (mp 151e152 �C [34]).

4.1.2. 4,11-Dihydroxy-3-[(dimethylamino)methyl]-1H-naphtho[2,3-f]indole-5,10-dione iodomethylate (5)

This compound was prepared from 4 as described earlier [26].HPLC (LW ¼ 260 nm, gradient B 20 / 60% (30 min)) tR ¼ 19.0 min,purity 95.5%. HRMS (ESI) calculated for C20H19N2O4 [M]þ 351.1339,found 351.1342.

4.1.3. (S)-4,11-Dihydroxy-3-((pyrrolidin-3-ylamino)methyl)-1H-naphtho[2,3-f]indole-5,10-dione dihydrochloride (6)

A stirring solution of iodomethylate 5 (100 mg, 0.2 mmol) and(S)-1-Boc-3-aminopyrrolidine (100 mg, 2.5 mmol) in chloroform(10 mL) was refluxed for 1 h. The mixture was diluted with chlo-roform (25 mL), washed with water and aqueous solution ofNaHCO3 (1%), dried, and evaporated. The residue was purified bycolumn chromatography on silica gel in chloroformemethanol(2:0/ 2:1). The red solid obtained after evaporationwas dissolvedin hot chloroform (1 mL), and a solution of HCl in methanol (1.25 N,1 mL) was added. The mixture was stirred overnight and evapo-rated. The residue was dissolved in hot water (0.5 mL), filtered andthen product was precipitated with acetone e ether mixture (1:1),collected by filtration and dried. The yield of dihydrochloride 6was58 mg (61%) as a red solid, mp 212e213 �С (dec.). HPLC(LW ¼ 260 nm, gradient B 10/ 40% (30 min)) tR ¼ 21.3 min, purity98.0%. 1H NMR (400 MHz, D2O) d 7.54 (1H, d, J ¼ 7.5 Hz, H9), 7.38(1H, d, J¼ 7.5 Hz, H6), 7.35 (2H, m, H7,8), 7.09 (1H, s, H2), 4.21 (1Н, m,NСH2СH), 4.13 (1Н, m, СН2N), 3.92 (1Н, m, NСH2СH), 3.68 (1Н, m,NСH2СH2), 3.58 (2Н, m, NСH2СH2СН), 2.70 (1Н, m, J ¼ 7.5 Hz,СH2СH2СH), 2.34 (1Н, m, J ¼ 7.0 Hz, СH2СH2СH). HRMS (ESI)calculated for C21H20N3O4 [MþH]þ 378.1448, found 378.1441.Analysis calculated for C21H19N3O4$2HCl$H2O: C 53.86, H 4.95, N8.97. Found: C 53.30, H 5.17, N 8.81.

4.1.4. (R)-4,11-Dihydroxy-3-((pyrrolidin-3-ylamino)methyl)-1H-naphtho[2,3-f]indole-5,10-dione dihydrochloride (7)

This compound was prepared from 5 and (R)-1-Boc-3-aminopyrrolidine as described for 6. A red powder, yield 58%, mp212e213 �С (dec.). HPLC (LW ¼ 260 nm, gradient B 10 / 40%(30 min)) tR ¼ 21.3 min, purity 99.8%. 1H NMR (400 MHz, D2O) d 1HNMR (400 MHz, D2O) d 7.54 (1H, d, J ¼ 7.5 Hz, H9), 7.38 (1H, d,J ¼ 7.5 Hz, H6), 7.34 (2H, m, H7,8), 7.09 (1H, s, H2), 4.21 (1Н, m,NСH2СH), 4.13 (1Н, m, СН2N), 3.92 (1Н, m, NСH2СH), 3.68 (1Н, m,NСH2СH2), 3.58 (2Н, m, NСH2СH2СН), 2.70 (1Н, m, J ¼ 7.5 Hz,СH2СH2СH), 2.34 (1Н, m, J ¼ 7.0 Hz, СH2СH2СH); 13C NMR(100MHz, D2O, 70 �C) d 172.58 (C), 168.47 (C), 168.34 (C),165.00 (C),131.59 (C), 129.74 (2C), 123.34 (C), 112.16 (C), 105.88 (C), 105.44 (C),132.57 (CH), 132.44 (CH), 129.98 (CH), 124.58 (2CH), 55.61 (CH),47.08 (CH2), 44.93 (CH2), 42.17 (CH2), 27.68 (CH2); HRMS (ESI)calculated for C21H20N3O4 [MþH]þ 378.1448, found 378.1445.Analysis calculated for C21H19N3O4$2HCl$H2O: C 53.86, H 4.95, N8.97. Found: C 53.75, H 5.31, N 9.18.

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805 803

4.1.5. (S)-3-((3-Aminopyrrolidin-1-yl)methyl)-4,11-dihydroxy-1H-naphtho[2,3-f]indole-5,10-dione dihydrochloride (8)

This compound was prepared from 5 and (S)-3-(Boc-amino)pyrrolidine as described for 6. A red powder, yield 64%, mp218e219 �C (dec.). HPLC (LW ¼ 260 nm, gradient B 10 / 40%(30min)) tR¼ 21.4 min, purity 99.1%. 1H NMR (400MHz, D2O) d 7.63(1H, d, J ¼ 7.5 Hz, H9), 7.54 (1H, d, J ¼ 7.5 Hz, H6), 7.40 (1H, t,J¼ 7.5 Hz, H8), 7.35 (1H, t, J¼ 7.5 Hz, H7), 7.13 (1H, s, H2), 4.37 (2Н, s,СН2N), 4.18 (1Н, dd, J ¼ 7.4 Hz, NСH2СH), 3.87 (1Н, m, NСH2СH),3.62 (1Н, m, NСH2СH2), 3.52 (2Н, m, NСH2СH2СН), 2.61 (1Н, m,J ¼ 7.4 Hz, СH2СH2СH), 2.19 (1Н, m, J ¼ 7.0 Hz, СH2СH2СH). HRMS(ESI) calculated for C21H20N3O4 [MþH]þ 378.1448, found 378.1438.Analysis calculated for C21H19N3O4$2HCl$H2O: C 53.86, H 4.95, N8.97. Found: C 54.08, H 4.78, N 9.25.

4.1.6. (R)-3-((3-Aminopyrrolidin-1-yl)methyl)-4,11-dihydroxy-1H-naphtho[2,3-f]indole-5,10-dione dihydrochloride (9)

This compound was prepared from 5 and (R)-3-(Boc-amino)pyrrolidine as described for 6. A red powder, yield 62%, mp218e219 �C (dec.). HPLC (LW ¼ 260 nm, gradient B 10 / 40%(30min)) tR¼ 21.4min, purity 98.4%. 1H NMR (400MHz, D2O) d 7.64(1H, d, J ¼ 7.5 Hz, H9), 7.53 (1H, d, J ¼ 7.5 Hz, H6), 7.42 (1H, t,J¼ 7.5 Hz, H8), 7.38 (1H, t, J¼ 7.5 Hz, H7), 7.13 (1H, s, H2), 4.32 (2Н, s,СН2N), 4.20 (1Н, dd, J ¼ 7.4 Hz, NСH2СH), 3.82 (1Н, m, NСH2СH),3.62 (1Н, m, NСH2СH2), 3.48 (2Н, m, NСH2СH2, СН), 2.64 (1Н, m,J ¼ 7.4 Hz, СH2СH2СH), 2.22 (1Н, m, J ¼ 7.0 Hz, СH2СH2СH); 13CNMR (100 MHz, D2O) d 170.70 (C), 169.83 (2C), 163.02 (C), 130.99(C), 129.74 (C), 129.65 (C), 123.10 (C), 110.89 (C), 105.54 (C), 105.11(C), 132.69 (CH), 132.52 (CH), 131.13 (CH), 124.49 (2CH), 48.27 (CH),55.68 (CH2), 52.38 (CH2), 49.68 (CH2), 28.27 (CH2); HRMS (ESI)calculated for C21H20N3O4 [MþH]þ 378.1448, found 378.1451.Analysis calculated for C21H19N3O4$2HCl$H2O: C 53.86, H 4.95, N8.97. Found: C 53.72, H 5.14, N 8.82.

4.1.7. (R, S)-4,11-Dihydroxy-3-((piperidin-3-ylamino)methyl)-1H-naphtho[2,3-f]indole-5,10-dione dihydrochloride (10)

This compound was prepared from 5 and (R,S)-1-Boc-3-aminopiperidine as described for 6. A red powder, yield 68%, mp254e256 �C (dec.). HPLC (LW ¼ 260 nm, gradient B 10 / 40%(30min)) tR¼ 21.6 min, purity 97.7%. 1H NMR (400MHz, D2O) d 7.60(1H, d, J ¼ 7.5 Hz, H9), 7.41 (2H, m, H7,8), 7.36 (1H, d, J ¼ 7.5 Hz, H6),7.06 (1H, s, H2), 4.12 (2Н, s, СН2N), 3.85 (1Н, d, J¼ 12.6 Hz,СHСH2N),3.65 (1Н, m, СH), 3.54 (1Н, d, J ¼ 12.5 Hz, NСH2СH), 3.22 (1Н, d,J¼ 12.6 Hz, СHСH2N), 3.11 (1Н, d, J¼ 12.7 Hz, NСH2СH2), 2.40 (1Н, d,J¼ 12.5 Hz, NСH2СH2), 2.23 (1Н, d, J¼ 12.6 Hz, СHСH2), 1.86 (2Н,М,СH2СH2). HRMS (ESI) calculated for C22H22N3O4 [MþH]þ 392.1605,found 392.1602. Analysis calculated for C22H21N3O4$2HCl$H2O: C54.78, H 5.22, N 8.71. Found: C 54.65, H 5.10, N 8.82.

4.1.8. 3-((4-Аminopiperidin-1-yl)methyl)-4,11-dihydroxy-1H-naphtho[2,3-f]indole-5,10-dione dihydrochloride (11)

This compound was prepared from 5 and 4-(Boc-amino)piper-idine as described for 6. A red powder, yield 73%, mp 253e255 �C(dec.). HPLC (LW ¼ 260 nm, gradient B 10 / 40% (30 min))tR ¼ 22.0 min, purity 97.3%. 1H NMR (400 MHz, D2O) d 7.72 (1H, d,J ¼ 7.5 Hz, H9), 7.50 (23H, m, H6,7,8), 7.25 (1H, s, H2), 4.29 (2Н, s,СН2N), 3.66 (3Н, m, СH, 2СH2N), 3.20 (2Н, t, J ¼ 12.0 Hz, 2СH2N),2.41 (2Н, d, J ¼ 12.8 Hz, СH2СHСH2), 2.03 (2Н, m, СH2СHСH2).HRMS (ESI) calculated for C22H22N3O4 [MþH]þ 392.1605, found392.1603. Analysis calculated for C22H21N3O4$2HCl$H2O: C 54.78, H5.22, N 8.71. Found: C 54.56, H 5.34, N 8.80.

4.1.9. 4,11-Dihydroxy-3-((piperidin-4-ylamino)methyl)-1H-naphtho[2,3-f]indole-5,10-dione dihydrochloride (12)

This compound was prepared from 5 and 1-Boc-4-aminopiperidine as described for 6. A red powder, yield 64%, mp>260 �C (dec.). HPLC (LW¼ 260 nm, gradient B 10/ 40% (30 min))tR ¼ 21.6 min, purity 96.6%. 1H NMR (400 MHz, D2O) d 7.55 (1H, d,J¼ 7.5 Hz, H9), 7.40 (1H, d, J¼ 7.5 Hz, H6), 7.35 (2H,m, H7,8), 7.08 (1H,s, H2), 4.15 (2Н, s, СН2N), 3.74 (2Н, d, J¼ 12.8 Hz, 2СH2N), 3.63 (1Н, t,J ¼ 12.1 Hz, СH), 3.22 (2Н, t, J ¼ 12.7 Hz, 2СH2N), 2.50 (2Н, d,J ¼ 12.8 Hz, СH2СHСH2), 2.04 (2Н, m, СH2СHСH2); 13C NMR(100 MHz, D2O, 70 �C) d 173.14 (C), 168.04 (C), 167.86 (C), 165.53 (C),131.69 (C), 129.71 (2C), 123.41 (C), 112.75 (C), 105.89 (C), 105.45 (C),132.57 (CH), 132.46 (CH), 129.76 (CH), 124.58 (2CH), 52.35 (CH),42.61 (2CH2), 40.16 (CH2), 25.52 (2CH2); HRMS (ESI) calculated forC22H22N3O4 [MþH]þ 392.1605, found 392.1594. Analysis calculatedfor C22H21N3O4$2HCl$H2O: C 54.78, H 5.22, N 8.71. Found: C 54.60,H 5.10, N 8.40.

4.1.10. 3-(((1S,4S)-2,5-Diazabicyclo[2.2.1]heptan-2-yl)methyl)-4,11-dihydroxy-1H-naphtho[2,3-f]indole-5,10-dionedihydrochloride (13)

This compound was prepared from 5 and (1S,4S)-(�)-2-Boc-2,5-diazabicyclo[2.2.1]heptane. A red powder, yield 61%, mp226e227 �C. HPLC (LW ¼ 260 nm, gradient B 10 / 40% (30 min))tR ¼ 21.6 min, purity 97.7%. 1H NMR (400 MHz, D2O) d 7.48 (1H, br s,H9), 7.38e7.24 (3H, br m, H6,7,8), 7.12 (1H, s, H2), 4.68 (1H, s, СH),4.57 (1H, s, СH), 4.39 (1Н, d, J ¼ 15.2 Hz, СН2N), 4.30 (1Н, d,J ¼ 14.1 Hz, СН2N), 3.75 (2Н, t, J ¼ 12.6 Hz, NСН2СН), 3.61 (2Н, d,J¼ 12.6 Hz, NСН2СН), 2.63 (1Н, d, J ¼ 12.3 Hz, СНСН2СН), 2.32 (1Н,d, J ¼ 12.3 Hz, СНСН2СН). HRMS (ESI) calculated for C22H20N3O4[MþH]þ 390.1448, found 390.1446. Analysis calculated forC22H21N3O4$2HCl$H2O: C 55.01, H 4.83, N 8.75. Found: C 55.22, H5.09, N 9.01.

4.2. Cell culture and cytotoxicity assays

The K562 human leukemia cell line (American Type CultureCollection; ATCC, Manassas, VA) and its Pgp-positive subline K562/4 selected for survival in the continuous presence of DOX (gift of A.Saprin), the HCT116 colon carcinoma cell line (ATCC) with wild typep53 and the HCT116p53KO subline (both p53 alleles deleted [47])(generated in B. Vogelstein lab, Johns Hopkins University, Balti-more, MD; gift of B. Kopnin) were cultured in Dulbecco modifiedEagle's medium supplemented with 5% fetal calf serum (HyClone,Logan, UT), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mLstreptomycin. The murine leukemia L1210 and human T-lympho-cyte Molt4/C8 cell line (ATCC) was propagated in RPMI-1640 me-dium supplemented with 10% fetal calf serum, 0.075% NaHCO3 and2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL strepto-mycin at 37 �C, 5% CO2 in humidified atmosphere. Cells in loga-rithmic phase of growth were used in the experiments. Novelcompounds were dissolved in 10% aqueous DMSO as 10 mM stocksolutions followed by serial dilutions in water immediately beforeexperiments. The assays were performed in 96-well microtiterplates. To eachwell (5e7.5)� 104 tumor cells and a given amount ofthe test compound were added. The cells were allowed to prolif-erate for 48 h (L1210) or 72 h (all other cell lines) at 37 �C in ahumidified CO2-controlled atmosphere. Cell viability was assessedbyMTT-assay [48] or by counting cells in a Coulter counter. The IC50was defined as the concentration of the compound that inhibitedcell viability by 50%.

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805804

4.3. Drug-DNA complex formation

The binding of compounds 6e9 to DNA was determined in100 mM KCl, 10 mM Na phosphate buffer, pH 7.8 at 20 �C. Fluo-rescence spectra were recorded with Cary Eclipse fluorescencespectrophotometer (Varian Inc, USA; excitation 490 nm, emissionat 500e700 nm). The concentration of calf thymus DNA (ctDNA,double stranded; moles of bp) was determined in a sodium phos-phate buffered solution at 20 �C using the molar extinction coeffi-cient ε[ctDNA] ¼ 13,200 M(bp)�1 cm�1. Binding constants weredetermined by linear approximation of the ratio [L]$[DNA]/(F0�F)where [L] is the concentration of the compound, F0 and F arefluorescence intensities of the compound in the absence or pres-ence of DNA, respectively.

4.4. Top1 and Top2 relaxation assays

One or two units of purified Top1 (Promega, USA) were incu-bated with 250 ng of supercoiled pBR322 plasmid DNA (Fermentas,Lithuania) in the buffer (35mM TriseHCl, pH 8.0; 72 mMKCl, 5 mMMgCl2, 5 mM dithiothreitol, 2 mM spermidine, 100 mg/mL bovineserum albumin) in the presence of 0.1% DMSO (vehicle control) orcompounds 6e9 at 37 �C for 30min. For Top2 activity assays, 250 ngof supercoiled pBR322 plasmid and 4 units of purified enzyme(TopoGen, USA) were incubated in the buffer (50 mМ TriseHCl, рН8.0; 150 mМ NaCl, 10 mМ MgCl2, 0.5 mМ dithiothreitol, 30 mg/mLbovine serum albumin, 2 mМ АТP) in the presence of 0.1% DMSO(vehicle control) or compounds 6e9 at 37 �C for 30 min. The re-actions were terminated by the addition of sarcosyl (up to 1%). Thenproteinase K was added (final concentration 50 mg/mL), and thereaction mixtures were incubated for 30 min (Top1) and 15 min(Top2) at 37 �C. DNA topoisomers were resolved by electrophoresisin 1% agarose gel (3 h, 70 V) in the buffer containing 40 mМ Tris-base, 1 mМ EDTA, 30 mМ glacial acetic acid. Gels were stainedwith ethidium bromide after electrophoresis. The vehicle had noeffect on Top1 or Top2 mediated DNA relaxation (not shown).

4.5. Top1 mediated DNA cleavage reactions

Human recombinant Top1 was purified from baculovirus [40].DNA cleavage reactions were performed as described [37] with aDNA substrate consisting of a 117-bp oligonucleotide (IntegratedDNA Tech.) encompassing previously identified Top1 cleavage sitesin the 161-bp fragment from pBluescript SK(�) phagemid DNA. The117-bp oligonucleotide contains a single 50-cytosine overhang,which was 30end-labeled by fill-in reaction with [32P]dGTP in React2 buffer (50 mM TriseHCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl) inthe presence of 0.5 units of DNA polymerase I (Klenow fragment,New England BioLabs). Unincorporated [32P] dGTP was removedusing mini Quick Spin DNA columns (Roche). Approximately 2 nMof radiolabeled DNA substrate was incubated with recombinantTop1 in 20 mL of the reaction buffer (10mM TriseHCl pH 7.5, 50mMKCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 mg/mL bovine serum al-bumin) at 25 �C for 20 min in the presence of the indicated con-centrations of compounds. Reactions were terminated by addingsodium dodecyl sulfate (0.5% final concentration) followed by theaddition of two volumes of the loading dye (80% formamide,10mMsodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and0.1% bromophenol blue). Aliquots of reaction mixtures were sub-jected to 20% denaturing polyacrylamide gel electrophoresis. Gelswere dried and visualized with a phosphorimager and ImageQuantsoftware (Molecular Dynamics). For simplicity, cleavage sites werenumbered as previously described in the 161-bp fragment [40].

4.6. Animals and tumor models

The DBA2 or BDF1 [DBA2 � C57Bl6] (female, 19e21 g) mice werekept in the animal facility at Blokhin Cancer Center. Animals weregiven food and water ad libitum. The P388 leukemia cells (106 permouse) were transplanted i.p. according to the protocol [49]. Nextday after tumor cell inoculation mice were divided into groups(n ¼ 4e6) and treated with compounds 6, 7, 1 (reference drug) orsaline (control). The doses are indicated in the Section 2.2.4. Thetherapeutic effect was expressed as the increase of life span (ILS)calculated as MS (days) in drug treated (T) group divided by MS(days) in control (C) cohort (T/C � 100%). Animals were monitoreddaily for behavioral and nutritional habits, and weight loss. Theminimal criterion of therapeutic efficacy for screening was T/C�125%. Statistical difference between groups was calculated usingStudent's t-test. Values p � 0.05 were considered significant.

Acknowledgments and funding

The authors thank A. Korolev and N. Maliutina (Gause Instituteof New Antibiotics) for HRMS and HPLS analyses; S. Sitdikova and F.Donenko (Blokhin Cancer Center) for assistance in animal testing,and L. van Berckelaer (KU Leuven) for help in cell proliferation as-says. Y. Pommier and K. Agama are supported by the Center forCancer Research, Intramural Program of the US National CancerInstitute (Z01 BC006161).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.09.021. These data include MOLfiles and InChiKeys of the most important compounds described inthis article.

References

[1] A. Rescifina, C. Zagni, M.G. Varrica, V. Pistar�a, A. Corsaro, Recent advances insmall organic molecules as DNA intercalating agents: synthesis, activity, andmodeling, Eur. J. Med. Chem. 74 (2014) 95e115.

[2] Y. Liu, E. Peacey, J. Dickson, C.P. Donahue, S. Zheng, G. Varani, M.S. Wolfe,Mitoxantrone analogues as ligands for a stem-loop structure of tau pre-mRNA,J. Med. Chem. 52 (2009) 6523e6526.

[3] S. Dogra, P. Awasthi, S. Tripathi, T.P. Pradeep, M.S. Nair, R. Barthwal, NMR-based structure of anticancer drug mitoxantrone stacked with terminal basepair of DNA hexamer sequence d-(ATCGAT)2, J. Biomol. Struct. Dyn. (2013).http://dx.doi.org/10.1080/07391102.2013.809021.

[4] Y. Pommier, DNA topoisomerase I inhibitors: chemistry, biology, and inter-facial inhibition, Chem. Rev. 109 (2009) 2894e2902.

[5] A. Romero, T. Cald�es, E. Díaz-Rubio, M. Martín, Topoisomerase 2 alpha: a realpredictor of anthracycline efficacy? Clin. Transl. Oncol. 14 (2012) 163e168.

[6] C. Sheng, Z. Miao, W. Zhang, New strategies in the discovery of novel non-camptothecin topoisomerase I inhibitors, Curr. Med. Chem. 18 (2011)4389e4409.

[7] T.V.T. Le, S. Han, J. Chae, H.-J. Park, G-Quadruplex binding ligands: fromnaturally occurring to rationally designed molecules, Curr. Pharm. Des. 18(2012) 1948e1972.

[8] Y.R. Lee, D.S. Yu, Y.C. Liang, K.F. Huang, S.J. Chou, T.C. Chen, C.C. Lee, C.L. Chen,S.H. Chiou, H.S. Huang, New approaches of PARP-1 inhibitors in human lungcancer cells and cancer stem-like cells by some selected anthraquinone-derived small molecules, PLoS One 8 (2013) e56284.

[9] X. Wan, W. Zhang, L. Li, Y. Xie, W. Li, N. Huang, A new target for an old drug:identifying mitoxantrone as a nanomolar inhibitor of PIM1 kinase via kinome-wide selectivity modeling, J. Med. Chem. 56 (2013) 2619e2629.

[10] Z. Liang, J. Ai, X. Ding, X. Peng, D. Zhang, R. Zhang, Y. Wang, F. Liu, M. Zheng,H. Jiang, H. Liu, M. Geng, C. Luo, Anthraquinone derivatives as potent in-hibitors of c-Met kinase and the extracellular signaling pathway, ACS Med.Chem. Lett. 4 (2013) 408e413.

[11] G. Cozza, A. Gianoncelli, M. Montopoli, L. Caparrotta, A. Venerando, F. Meggio,L.A. Pinna, G. Zagotto, S. Moro, Identification of novel protein kinase CK1 deltainhibitors through structure-based virtual screening, Bioorg. Med. Chem. Lett.18 (2008) 5672e5675.

[12] T.S. Feng, Z.Y. Yuan, R.Q. Yang, S. Zhao, F. Lei, X.Y. Xiao, D.M. Xing, W.H. Wang,Y. Ding, L.J. Du, Purgative components in rhubarbs: adrenergic receptor in-hibitors linked with glucose carriers, Fitoterapia (2013) 236e246.

A.E. Shchekotikhin et al. / European Journal of Medicinal Chemistry 86 (2014) 797e805 805

[13] D. Shrimali, M.K. Shanmugam, A.P. Kumar, J. Zhang, B.K. Tan, K.S. Ahn, G. Sethi,Targeted abrogation of diverse signal transduction cascades by emodin for thetreatment of inflammatory disorders and cancer, Cancer Lett. 341 (2013)139e149.

[14] J.-H. Chiang, J.-S. Yang, C.-Y. Ma, M.-D. Yang, H.-Y. Huang, T.-C. Hsia, H.-M. Kuo, P.-P. Wu, T.-H. Lee, J.-G. Chung, Danthron, an anthraquinone deriva-tive, induces DNA damage and caspase cascades-mediated apoptosis in SNU-1human gastric cancer cells through mitochondrial permeability transitionpores and Bax-triggered pathways, Chem. Res. Toxicol. 24 (2011) 20e29.

[15] G.-Z. Zeng, J.-T. Fan, J.-J. Xu, Y. Li, N.-H. Tan, Apoptosis induction and G2/Marrest of 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone from Rubia yunna-nensis in human cervical cancer Hela cells, Die Pharm. 68 (2013) 293e299.

[16] H.-Y. Tu, A.-M. Huang, C.-H. Teng, T.-C. Hour, S.-C. Yang, Y.-S. Pu, C.-N. Lin,Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis inNTUB1 cells, Bioorg. Med. Chem. 19 (2011) 5670e5678.

[17] D.-H. Shi, W. Huang, C. Li, Y.-W. Liu, S.-F. Wang, Design, synthesis and mo-lecular modeling of aloe-emodin derivatives as potent xanthine oxidase in-hibitors, Eur. J. Med. Chem. 75 (2014) 289e296.

[18] D. Bhasin, J.P. Etter, S.N. Chettiar, M. Mok, P.-K. Li, Antiproliferative activitiesand SAR studies of substituted anthraquinones and 1,4-naphthoquinones,Bioorg. Med. Chem. Lett. 23 (2013) 6864e6867.

[19] A. Di Salvo, P. Dugois, D. Tandeo, M. Peltekian, P.K.T. Lin, Synthesis, cytotox-icity and DNA binding of oxoazabenzo[de]anthracenes derivatives in coloncancer Caco-2 cells, Eur. J. Med. Chem. 69 (2013) 754e761.

[20] M. Rebucci, C. Michiels, Molecular aspects of cancer cell resistance tochemotherapy, Biochem. Pharmacol. 85 (2013) 1219e1226.

[21] H.J. Broxterman, K.J. Gotink, H.M.W. Verheul, Understanding the causes ofmultidrug resistance in cancer: a comparison of doxorubicin and sunitinib,Drug. Resist. Updat. 12 (2009) 114e126.

[22] M. Piekarski, A. Jelinska, Anthracyclines still prove effective in anticancertherapy, Mini Rev. Med. Chem. 13 (2013) 627e634.

[23] M.-T. Ma, M. He, Y. Wang, X.-Y. Jiao, L. Zhao, X.-F. Bai, Z.-J. Yu, H.-Z. Wu, M.-L. Sun, Z.-G. Song, M.-J. Wei, MiR-487 a resensitizes mitoxantrone (MX)-resistant breast cancer cells (MCF-7/MX) to MX by targeting breast cancerresistance protein (BCRP/ABCG2), Cancer Lett. 339 (2013) 107e115.

[24] S. Sangthong, H. Ha, T. Teerawattananon, N. Ngamrojanavanich, N. Neamati,N. Muangsin, Overcoming doxorubicin-resistance in the NCI/ADR-RES modelcancer cell line by novel anthracene-9,10-dione derivatives, Bioorg. Med.Chem. Lett. 23 (2013) 6156e6160.

[25] K. Pors, Z. Paniwnyk, P. Teesdale-Spittle, J.A. Plumb, E. Willmore, C.A. Austin,L.H. Patterson, Alchemix: a novel alkylating anthraquinone with potent ac-tivity against anthracycline- and cisplatin-resistant ovarian Cancer, Mol.Cancer Ther. 2 (2003) 607e610.

[26] A.E. Shchekotikhin, A.A. Shtil, Y.N. Luzikov, T.V. Bobrysheva, V.N. Buyanov,M.N. Preobrazhenskaya, 3-Aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione for circumvention of anticancerdrug resistance, Bioorg. Med. Chem. 13 (2005) 2285e2291.

[27] L.G. Dezhenkova, O.Y. Susova, A.E. Shchekotikhin, M.N. Preobrazhenskaya,A.A. Shtil, Naphtho[2,3-f]indole-5,10-dione aminoalkyl derivatives: a newclass of topoisomerase I inhibitors, Bull. Exp. Biol. Med. 145 (2008) 334e337.

[28] X. Wang, Z. Chen, L. Tong, S. Tan, W. Zhou, T. Peng, K. Han, J. Ding, H. Xie, Y. Xu,Naphthalimides exhibit in vitro antiproliferative and antiangiogenic activitiesby inhibiting both topoisomerase II (topo II) and receptor tyrosine kinases(RTKs), Eur. J. Med. Chem. 65 (2013) 477e486.

[29] J. Qiu, B. Zhao, Y. Shen, W. Chen, Y. Ma, Y. Shen, A novel p-terphenyl derivativeinducing cell-cycle arrest and apoptosis in MDA-MB-435 cells through topo-isomerase inhibition, Eur. J. Med. Chem. 68 (2013) 192e202.

[30] S.-E. Park, I.-H. Chang, K.-Y. Jun, E. Lee, E.-S. Lee, Y. Na, Y. Kwon, 3-(3-Butylamino-2-hydroxy-propoxy)-1-hydroxy-xanthen-9-one acts as a topo-isomerase IIa catalytic inhibitor with low DNA damage, Eur. J. Med. Chem. 69(2013) 139e145.

[31] P.-C. Lv, K. Agama, C. Marchand, Y. Pommier, M. Cushman, Design, Synthesis,and biological evaluation of O-2-modified indenoisoquinolines as dual

topoisomerase I-tyrosyl-DNA posphodiesterase I inhibitors, J. Med. Chem. 57(2014) 4324e4336.

[32] A.E. Shchekotikhin, L.G. Dezhenkova, O.Y. Susova, V.A. Glazunova,Y.N. Luzikov, Y.B. Sinkevich, V.N. Buyanov, A.A. Shtil, M.N. Preobrazhenskaya,Naphthoindole-based analogues of tryptophan and tryptamine: synthesis andcytotoxic properties, Bioorg. Med. Chem. 15 (2007) 2651e2659.

[33] A.E. Shchekotikhin, V.N. Buyanov, M.N. Preobrazhenskaya, Synthesis of 1-(omega-aminoalkyl)naphthoindolediones with antiproliferative properties,Bioorg. Med. Chem. 12 (2004) 3923e3930.

[34] A.E. Shchekotikhin, E.P. Baberkina, K.F. Turchin, V.N. Buyanov, N.N. Suvorov,Naphtoindoles. 10. Synthesis of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione and some of its derivatives, Chem. Heterocycl. Compd. 37 (2001)944e948.

[35] V. Verebov�a, J. Adamcik, P. Danko, D. Podhradský, P. Mi�skovský, J. Stani�cov�a,Anthraquinones quinizarin and danthron unwind negatively supercoiled DNAand lengthen linear DNA, Biochem. Biophys. Res. Commun. 444 (2014) 50e55.

[36] R. Scott, Some comments on the BenesieHildebrand equation, Recl. Des. Trav.Chim. Des. Pays-Bas 75 (1956) 787e789.

[37] T.S. Dexheimer, Y. Pommier, DNA cleavage assay for the identification oftopoisomerase I inhibitors, Nat. Protoc. 3 (2008) 1736e1750.

[38] S. Antony, M. Jayaraman, G. Laco, G. Kohlhagen, K.W. Kohn, M. Cushman,Y. Pommier, Differential induction of topoisomerase I-DNA cleavage com-plexes by the indenoisoquinoline MJ-III-65 (NSC 706744) and camptothecin:base sequence analysis and activity against camptothecin-resistant top-oisomerases I, Cancer Res. 63 (2003) 7428e7435.

[39] K. Wassermann, J. Markovits, C. Jaxel, G. Capranico, K.W. Kohn, Y. Pommier,Effects of morpholinyl doxorubicins, doxorubicin, and actinomycin D onmammalian DNA topoisomerases I and II, Biochem. Pharmacol. 38 (1990)38e45.

[40] P. Pourquier, L.-M. Ueng, J. Fertala, D. Wang, H.-K. Park, J.M. Essigmann, M.-A. Bjornsti, Y. Pommier, Induction of reversible complexes between eukary-otic DNA topoisomerase I and DNA-containing oxidative base damages 7,8-dihydro-8-oxoguanine and 5-hydroxycytosine, J. Biol. Chem. 274 (1999)8516e8523.

[41] Y. Pommier, J. Covey, D. Kerrigan, W. Mattes, J. Markovits, K.W. Kohn, Role ofDNA intercalation in the inhibition of purified mouse leukemia (L1210) DNAtopoisomerase II by 9-aminoacridines, Biochem. Pharmacol. 36 (1987)3477e3486.

[42] M.F. Brana, M. Cacho, A. Gradillas, B. de Pascual-Teresa, A. Ramos, Intercalatorsas anticancer drugs, Curr. Pharm. Des. 7 (2001) 1745e1780.

[43] Y. Pommier, P. Pourquier, Y. Fan, D. Strumberg, Mechanism of action ofeukaryotic DNA topoisomerase I and drugs targeted to the enzyme, Biochem.Biophys. Acta 1400 (1998) 83e105.

[44] S.B. Howerton, A. Nagpal, L.D. Williams, Surprising roles of electrostatic in-teractions in DNA-ligand complexes, Biopolymers 69 (2003) 87e99.

[45] A.E. Shchekotikhin, V.A. Glazunova, Y.N. Luzikov, V.N. Buyanov, O.Y. Susova,A.A. Shtil, M.N. Preobrazhenskaya, Synthesis and structureeactivity relation-ship studies of 4,11ediaminonaphtho[2,3ef]indolee5,10ediones, Bioorg.Med. Chem. 14 (2006) 5241e5251.

[46] B.B. Semenov, V.G. Granik, Chemistry of N-(1H-indol-3-ylmethyl)-N,N-dime-thylamine (gramine): a review, Pharm. Chem. J. 38 (2004) 287e310.

[47] F. Bunz, A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J.P. Brown, J.M. Sedivy,K.W. Kinzler, B. Vogelstein, Requirement for p53 and p21 to sustain G2 arrestafter DNA damage, Science 282 (1998) 1497e1501.

[48] A.E. Shchekotikhin, V.A. Glazunova, L.G. Dezhenkova, E.K. Shevtsova,V.F. Traven, J. Balzarini, H.S. Huang, A.A. Shtil, M.N. Preobrazhenskaya, Thefirst series of 4,11ebis[(2eaminoethyl)amino]anthra[2,3eb]fur-ane5,10ediones: synthesis and antiproliferative characteristics, Eur. J. Med.Chem. 46 (2011) 423e428.

[49] T. Balandin, E. Edelweis, N. Andronova, H. Treshalina, A. Sapozhnikov, S. Deev,Antitumor activity and toxicity of anti-HER2 immunoRNase scFv 4D5-dibar-nase in mice bearing human breast cancer xenograft, Investig. New. Drugs 29(2011) 22e32.