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

Accepted Manuscript

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

Andrey E. Shchekotikhin, Valeria A. Glazunova, Lyubov G. Dezhenkova, Yuri N.Luzikov, Vladimir N. Buyanov, Helena M. Treshalina, Nina A. Lesnaya, VladimirI. Romanenko, Dmitry N. Kaluzhny, Jan Balzarini, Keli Agama, Yves Pommier,Alexander A. Shtil, Maria N. Preobrazhenskaya

PII: S0223-5234(14)00839-3

DOI: 10.1016/j.ejmech.2014.09.021

Reference: EJMECH 7327

To appear in: European Journal of Medicinal Chemistry

Received Date: 12 June 2014

Revised Date: 2 September 2014

Accepted Date: 6 September 2014

Please cite this article as: A.E. Shchekotikhin, V.A. Glazunova, L.G. Dezhenkova, Y.N. Luzikov, V.N.Buyanov, H.M. Treshalina, N.A. Lesnaya, V.I. Romanenko, D.N. Kaluzhny, J. Balzarini, K. Agama,Y. Pommier, A.A. Shtil, M.N. Preobrazhenskaya, Synthesis and evaluation of new antitumor 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones, European Journal of Medicinal Chemistry(2014), doi: 10.1016/j.ejmech.2014.09.021.

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http://dx.doi.org/10.1016/j.ejmech.2014.09.021

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ACCEPTED MANUSCRIPTSynthesis and evaluation of new antitumor 3-aminomethyl-4,11-

dihydroxynaphtho[2,3-f]indole-5,10-diones

Andrey E. Shchekotikhin, Valeria A. Glazunova, Lyubov G. Dezhenkova, Dmitry N. Kaluzhny,

Yuri N. Luzikov, Vladimir N. Buyanov, Helena M. Treshalina, Nina A. Lesnaya, Vladimir I.

Romanenko, Jan Balzarini, Keli Agama, Yves Pommier, Alexander A. Shtil and Maria N.

Preobrazhenskaya

Graphical abstract

O

O

OH

OH

NH

O

O

OMe

OMe

NH

HNNMe2

NH

- antitumor DNA ligand;- active against drug resistant tumor cells;- potent Top1/2 blocker.

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Revised manuscript Synthesis and evaluation of new antitumor 3-aminomethyl-4,11-


Andrey E. Shchekotikhin, Valeria A. Glazunova, Lyubov G. Dezhenkova, Dmitry N. Kaluzhny, Yuri N.

Luzikov, Vladimir N. Buyanov, Helena M. Treshalina, Nina A. Lesnaya, Vladimir I. Romanenko, Jan

Balzarini, Keli Agama, Yves Pommier, Alexander A. Shtil and Maria N. Preobrazhenskaya

Graphical abstract

O

O

OH

OH

NH

O

O

OMe

OMe

NH

HNNMe2

NH

- antitumor DNA ligand;- active against drug resistant tumor cells;- potent Top1/2 blocker.

Keywords:

naphtho[2,3-f]indole-5,10-diones; DNA ligands; topoisomerase 1/2 inhibitors; circumvention of

multidrug resistance; antitumor activity

Highlights: • A series of novel naphtho[2,3-f]indole-5,10-diones was designed and synthesized

• The designed derivatives showed an improved antiproliferative activity

• A high affinity of designed derivatives to double stranded DNA was demonstrated

• The inhibition of Top1 and Top2 by naphthoindolediones was demonstrated

• One compound showed an induction of Top1 mediated DNA cleavage and antitumor activity

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Synthesis and evaluation of new antitumor 3-aminomethyl-4,11-


Andrey E. Shchekotikhin a,b,* ,Valeria A. Glazunova с, 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 d, Jan Balzarini e, Keli Agama f, Yves Pommier f, Alexander A. Shtil с and Maria N.

Preobrazhenskaya a

a Gause Institute of New Antibiotics, Russian Academy of Medical Sciences,

11 B. Pirogovskaya Street, Moscow 119021, Russia b.Mendeleyev University of Chemical Technology, 9 Miusskaya Square, Moscow 125190, Russia c Blokhin Cancer Center, 24 Kashirskoye shosse, Moscow 115478, Russia, & Moscow Engineering

and Physics Institute, 31 Kashirskoye shosse, Moscow 115409, Russia

d Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street,

Moscow 119991, Russia

e Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium f Developmental Therapeutics Branch, National Cancer Institute, NIH, 37 Convent Drive, 37-5068,

Bethesda, MD 20892, USA

Abstract

A series of new 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones 6-13 bearing

the cyclic diamine in the position 3 of the indole ring was synthesized. The majority of new

compounds demonstrated a superior cytotoxicity than doxorubicin against a panel of mammalian

tumor cells with determinants of altered drug response, that is, Pgp expression or p53 inactivation. For

naphtho[2,3-f]indole-5,10-diones 6-9 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

were demonstrated. 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 known topoisomerase 1 inhibitors camptothecin and indenoisoquinoline MJ-III-65, suggesting a

role of the structure of the side chain of 3-aminomethylnaphtho[2,3-f]indole-5,10-diones in interaction

with the target. Compound 7 demonstrated an antitumor activity in mice with P388 leukemia

transplants whereas its enantiomer 6 was 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.

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1. Introduction

The ability to interact with a variety of intracellular targets, including nucleic acids [1-3],

topoisomerases (Top1 and Top2) [4-6], telomerase [7, 8], protein kinases [9-11], receptors [12, 13]

etc., makes the anthraquinone moiety an important pharmacophore for rational drug design. Indeed,

natural and synthetic derivatives of hydroxyanthraquinones (e.g., anthracyclines, mitoxantrone and

emodin) are widely used as prototypes for the design of anticancer drug candidates [14-19]. In

particular, the altered drug response, a major challenge for chemotherapy, stimulates the search for

agents whose cytotoxicity is retained for pleotropically resistant tumor cells [20-25].

Previously we have identified the linear pyrroloquinizarine (4,11-dihydroxynaphtho[2,3-

f]indole-5,10-dione) as a promising scaffold 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 validated mechanisms 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 diamines such as

piperazine or quinuclidine, significantly increased the cytotoxicity. One of the most potent in this

series was the derivative 2 (Fig. 1) carrying piperazine in the side chain. This compound was more

active than doxorubicin (DOX; 1) against MDR K562/4 leukemic cells selected for survival in the

presence of 1 and the colon carcinoma HCT116p53KO variant with deleted p53. The potency of 2 has

been attributed to the formation of stable complexes with double stranded DNA and attenuation of

Top1 activity [27].

O

O

OH

OH

OH

O

O

O

OH

NH2

O

OH

Doxorubicin (1)

2

O

O

OH

OH

NH

N NH

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

Clinical efficacy of Top 1 and Top 2 inhibitors (e.g., DOX, etoposide, irinotecan) can explain

the continuous interest in the search for novel topoisomerase inhibitors as anticancer agents with

improved therapeutic properties [28-31]. In this study we explored new Top1 and Top2 antagonists

based on 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione scaffold bearing the cyclic diamine in the

* Corresponding author. Fax 7 499 2450295; e-mail: [email protected]

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side chain (position 3 of the heterocycle). The optimised synthetic procedure yielded a series of new

3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones. A high affinity to double stranded

DNA and potent attenuation of Top1 and Top2 by new naphthoindolediones were associated with

cytotoxicity (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 efficacy 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 was based on transamination reaction of gramine analogs [26]. The

key step 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 mild

Lewis acids such as BСl3, BBr3, B(CF3CO2)3 or CeCl3 [26, 32-34]. However, the efficiency of these

reactions is moderate and depends on the structure of functional groups in the heterocyclic nucleus of

naphtho[2,3-f]indole-5,10-diones. Treatment of methoxynaphthoindoledione 3 with common

demethylation agents is problematic due to reactivity of the aminomethyl residue. Interestingly, we

observed that the hydrochloride salt of the gramine analogue 3 was unstable during storage. Major

products of decomposition of 3 were identified by TLC and mass-spectrometry as demethoxy

derivatives. We assumed that HCl can be useful for demethylation of methoxynaphtho[2,3-f]indole-

5,10-diones. We found that, despite some stabilization in aqueous or methanol HCl, 3 can be easily

demethylated by the solution of HCl in anhydrous acetic acid to produce the hydrochloride of gramine

4 in 92% yield (Scheme 1). The subsequent treatment of this salt with anion exchange resin gave the

free base of 4 (Scheme 1). Thus, we developed a novel convenient method of naphtho[2,3-f]indole-

5,10-dione 3 demethylation that produced the gramine analogue 4 in twice bigger yield than the

procedure with Lewis acid [34]. This new method facilitates the access to other bioactive 4,11-

dihydroxynaphtho[2,3-f]indole-5,10-diones [32] since gramine 4 is a key intermediate for their

multistep synthesis. Additionally, this method can be useful for dealkylation of other

methoxynaphthoindolediones and related compounds.

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.

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The interaction of gramine 4 with methyl iodide produced the quaternary salt 5. The latter

intermediate was used to synthesize the aminomethyl derivatives in the reaction of transamination

with Boc-protected amino derivatives of pyrrolidine, piperidine or diazabicyclo[2.2.1]heptane

(Scheme 2). At the final step the unblocking of intermediate Boc derivatives with a solution of HCl in

ether gave the corresponding 3-aminomethylnaphtho[2,3-f]indole-5,10-diones 6-13 in 55-64% yields.

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 6-13.

The resulting compounds 6-13 were purified by re-precipitation. The analytical and

spectroscopic data were in full accordance with the assigned structures. The purified compounds were

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-diones

The antiproliferative activity of hydrochlorides of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-

diones 2, 6-13 were studied using a panel of wild type tumor cell lines and isogenic drug resistant

sublines: murine leukemia L1210/0, Т-lymphocyte leukemia Molt4/C8, К562 myeloid leukemia and

its MDR (Pgp expressing) K562/4 variant, as well as colon carcinoma НСТ116 and HCT116p53KO

variant with inactivation of pro-apoptotic p53.

All new derivatives of naphtho[2,3-f]indole-5,10-diones inhibited tumor cell proliferation at

submicromolar to low micromolar concentrations. The most potent were stereoisomers 6 and 7 with

the 3-aminopyrrolidine residue attached to the chromophore moiety via the exocyclic nitrogen (Fig. 2

and Table S1). The regioisomers 8, 9 with diamine conjugated to naphtho[2,3-f]indole-5,10-diones

through the cyclic nitrogen were less potent. An increased size of the cyclic amine ring in the side

chain (10-12) as well as the fused diamine (diazabicyclo[2.2.1]heptane) in 13 also diminished the

antiproliferative potency (Fig. 2).

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Importantly, all new compounds were cytotoxic for wild type cell lines and their isogenic drug

resistant counterparts. The K562/4 cells express functional Pgp and are resistant to Pgp transported

agents such as DOX (1, resistance indices RI≈8.8, see Supplementary Material, Table S1). In striking

contrast, for naphtho[2,3-f]indole-5,10-diones 2, 6-13 the resistance indices were close to 1; for

compounds 8, 11 and 13 the activity against Pgp-positive cells was even higher than for the parental

K562 counterparts (RI=0.8; 0.4; and 0.3, respectively; Table S1). In contrast to DOX, 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 toxicity for

HCT116p53KO (p53-/-) cells than for parental HCT116 cells (RI=0.75 and 0.5, respectively) as

opposed to the sensitivity of these cell lines to 1 (RI=3.1; Table S1). Thus, the designed naphtho[2,3-

f]indole-5,10-diones demonstrated the improved antiproliferative activity against tumor cells with the

determinants of altered 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 to select

compounds 6-9 for further studies.

Figure 2. Relationship between chemical structure and antiproliferative activity (–logIC50) for 3-

aminomethylnaphtho[2,3-f]indole-5,10-diones 2, 6-13. DOX (1) was used as a reference drug. Values

of IC50 (mean of 3 experiments) are shown in Supplementary Material, Table S1.

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

Like structurally close anthraquinone containing compounds [3, 35], 3-aminomethyl

derivatives of naphtho[2,3-f]indol-5,10-dione can bind DNA and interfere with enzymes involved in

the functionality of nucleic acids. We tested DNA binding of new isomeric naphtho[2,3-f]indole-5,10-

diones 6-9 with different substituents at the side chains. Titration of 6-9 (1.2 µM each) with double

stranded DNA (up to 65 µM (bp)) resulted in a drop of fluorescence intensity with minimal changes of

fluorescence spectra (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

determined by Scott’s method (Fig. 4) were similar for the pairs of stereoisomers 6, 7 and 8, 9 (Table

1). This similarity means that stereochemistry of 3-aminopyrrolidine residue in the side chain of

naphtho[2,3-f]indol-5,10-diones insignificantly affected the affinity to DNA. In striking contrast, the

mode of linking of naphtho[2,3-f]indole-5,10-dione to diamine was important. Indeed, the DNA

binding constants of 6 and 7 (with diamine conjugated through pyrrolidine nitrogen) were 3-fold

bigger than the respective values for regioisomers 8 and 9 with the side chain conjugated via the

exocyclic amino group (Table 1). These results demonstrated the high affinity of new compounds to

DNA and highlight the role of the structure of the side chain of 3-aminomethylnaphtho[2,3-f]indol-

5,10-diones in the drug-DNA complex formation.

0

50

100

150

200

500 550 600 650 700

Flu

ores

cenc

e, a

u

Wavelength, nm

A

0

50

100

150

200

250

500 550 600 650 700

Flu

ores

cenc

e, a

u

Wavelength, nm

B

0

100

200

300

400

500

600

500 550 600 650 700

Flu

ores

cenc

e, a

u

Wavelength, nm

C

0

100

200

300

400

500

600

700

500 550 600 650 700

Flu

ores

cenc

e, a

u

Wavelength, nm

D

Figure 3. Changes of fluorescence spectra of 6-9 upon titration with double stranded DNA.

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

compound in complexes with DNA. (A) 6; (B) 7; (C) 8; (D) 9.

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140

6789

[L]x

[DN

A]/(

F0-F

), x

1012

DNA concentration, µM(bp)

Figure 4. Binding of naphtho[2,3-f]indole-5,10-diones 6-9 to double stranded DNA.

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Table 1. Binding constants (Kb) of complex formation of naphtho[2,3-f]indole-5,10-diones 6-9 with

double stranded DNA determined by fluorescence titration.

Compound Kb, 105 М

-1

6 11±1

7 10±1

8 3.5±0.4

9 3.4±0.3

2.2.3. Top1 and Top2 inhibition

Previously we have identified Top1 as a target for the cytotoxicity of naphthoindolediones [27,

32]. In this study we tested whether isomeric compounds 6-9 bearing a 3-aminopyrrolidine residue in

the side chains can affect Top1 and Top2. These compounds were selected because of their high

potency against parental and drug resistant tumor cells (Fig. 2 and Table S1). Relaxation of

supercoiled plasmid DNA by Top1 was attenuated with 6-9 in a dose dependent manner; the increase

of concentrations from 0.5 µM to 5 µM yielded slowly migrating DNA topoisomers (Fig. 5).

Interestingly, stereochemistry of the side chain was important for inhibition of Top1-mediated

relaxation; at 2 µM of 9 (R-isomer) the plasmid relaxation was inhibited whereas S-isomer 8 caused a

smaller effect even at 5 µМ. In striking contrast, the reference drug camptothecin (CPT) attenuated

Top1-mediated DNA relaxation only at >> 10 µM. Thus, among 3-aminomethyl derivatives of 4,11-

dihydroxynaphtho[2,3-f]indole-5,10-dione, compound 9 was the most potent Top1 inhibitor. In

control experiments we observed no influence of tested compounds (at concentration as high as 20

µM) on DNA mobility in the absence of Top1 (data not shown).

Figure 5. Top1 inhibition by naphtho[2,3-f]indole-5,10-diones 6-9.

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Next, we addressed the question of specificity of Top1 inhibition by 6-9 using a Top1 cleavage

assay [37]. Among this tested series only compound 7 showed a specific DNA cleavage similar for

reference Top1 inhibitors CPT and indenoisoquinoline MJ-III-65 (NSC 706744 [38]) (Fig. 6). The

induction of Top1-DNA complexes by 7 at submicromolar concentrations was dose-dependent, as

evidenced by the increased intensity of DNA cleavage bands. However, the highest concentration of 7

blocked the formation of DNA cleavage products and evoked an increased band of the full-length

DNA substrate, a common characteristic of strong DNA intercalators [39]. Notably, 7 has unique

cleavage pattern as it induced DNA cleavage complexes at several sites similar to CPT (sites 92 and

119) and indenoisoquinoline MJ-III-65 (sites 48, 97 and 108). Probably, the observed differences for 7

and its isomers 6, 8 and 9 in Top1 cleavage assay could ultimately lead to different pharmacological

effects. D

NA a

lone

7Top1 n

o d

rug

CPT

MJ-I

II-6

5

0.1

µM

1 µ

M

10 µ

M

100 µ

M

44

62

92

97

106

119

Figure 6. Top1 mediated DNA cleavage by 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione 7.

Lane 1: DNA alone; lane 2: + Top1; lane 3: + camptothecin 1 µM; lane 4: + indenoisoquinoline MJ-

III-65, 1 µM; lanes 5-8: + compound 7 at 0.1, 1, 10 and 100 µM, respectively. The numbers on the left

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and arrows indicate cleavage site positions. A 117 bp DNA substrate was used in this assay but, to

facilitate comparison, the cleavage sites are numbered to be consistent with the commonly used 161

bp DNA substrate [40].

We next tested whether naphthoindolediones 6-9 affect Top2-mediated relaxation of supercoiled

DNA. Fig. 7 shows DNA topoisomers generated by Top2 in the absence (lane Top2) or presence of 6-

9. Compound 8 almost completely inhibited plasmid relaxation at 2 µМ 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 Top 2 and Top 1, respectively, than their isomers 6,

7.

Figure 7. Top2 inhibition by naphtho[2,3-f]indole-5,10-diones 6-9.

Thus, we showed that the potency of inhibition of Top1 and Top2 depends on the structure and

stereochemistry of the side chains of naphthoindolediones. These results also indicated that the

structure of the side chain can influence the mode of enzyme inhibition. As known for other

intercalating agents, naphthoindolediones can inhibit topoisomerases by altering local DNA

conformation [41, 42]. However, the lack of direct correlation between DNA affinity of 6-9 and Top1

or Top2 inhibitory potency, as well as specific DNA cleavage by Top 1 in the presence of 7 suggest

another mechanism of enzyme inhibition by these compounds. The specific Top-mediated DNA

cleavage is a hallmark of Top 1 poisons that stabilize drug-DNA-enzyme complexes [43, 44].

Supposedly, the carbonyl or hydroxyl groups of the anthraquinone moiety, as well as 3-aminomethyl

substituent in 6-13 (similarly to the amino sugar in anthracyclines [45]), could be involved in the

stabilization of above mentioned complexes. On the one hand, the diamine fragment of the side chain

may be involved in stabilization of tertiary complexes by hydrogen or electrostatic interactions with

functional groups of the enzyme. Also, 3-aminomethyl group in 6-13 can participate in the formation

of a covalent link between the ligand and the protein. This hypothesis comes from the fact that 3-

aminomethylnaphtoindoldiones retain the reactive properties of gramine [26, 32, 46] that is capable of

alkylating a wide range of O-, N-, C- and S-nucleophiles [47].

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2.2.4. In vivo testing

Given that compound 7 demonstrated a high potency against drug resistant cells and specificity

of Top1 inhibition, we tested its antitumor 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 the mice in the control group was 10.2 days (Fig. 8). After injection of 7 in a single dose of 80

mg/kg MS reached 14.3 days (T/C=140±15%, p ≤ 0.05); at 25 mg/kg or 30 mg/kg daily for 5 days the

MS values were 14.0 and 15.8 days (T/C=137±11% and 155±17%, p ≤ 0.05), respectively (Fig. 8).

Treatment with 7 was well tolerated up to daily dose of 45 mg/kg (5 days).

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

The efficacy of 7 was comparable with that for 1 administered at maximum tolerated single dose

of 8 mg/kg (MS = 14.3 days; T/C=140±22%, p ≤ 0.05%). In striking contrast, the stereoisomer 6 used

in the same doses as 7 did not increase the life span of P388 tumor bearing mice (Fig. 8).

3. Conclusion

A series of new 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones 6-13 bearing

the cyclic diamine in the side chain was prepared. In doing so we optimised the scheme of synthesis

and developed a method of demethylation of their key precursor, 3-dimethylamino-4,11-

dimethoxynaphtho[2,3-f]indole-5,10-dione 3. The new procedure of O-demethylation by treatment of

3 with HCl in acetic acid gave the hydrochloride of 3-dimethylamino-4,11-dihydxynaphtho[2,3-

f]indole-5,10-dione 4 in a high yield (92%). This improvement provides excellent opportunity for

increasing the yields of target compounds. Also, the newly developed method of mild dealkylation

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may be useful for deprotection of alkoxy groups of other alkoxynaphthoindolediones and related

compounds.

A potent (in submicromolar to low micromolar concentrations) cytotoxicity against a panel of

mammalian tumor cells was observed for all novel 3-aminomethyl-4,11-dihydroxynaphtho[2,3-

f]indole-5,10-diones 6-13. The majority of new compounds demonstrated a superior potency than the

reference drug DOX against cells with Pgp expression or p53 inactivation. Naphtho[2,3-f]indole-5,10-

diones 6-9 bearing 3-aminopyrrolidine in the side chains were found to bind double stranded DNA

and affect Top1 or Top2 mediated DNA relaxation. These parameters strongly depended on the mode

of attachment of the diamine residue to the naphtho[2,3-f]indole-5,10-dione moiety. Importantly, only

the isomer 7 induced the formation of specific DNA cleavage products similar for classical Top1

inhibitors camptothecin and indenoisoquinoline MJ-III-65. Moreover, the derivative of (R)-

aminopyrrolidine 7 increased the life span of mice bearing P388 leukemia while its enantiomer 6 was

inactive. Altogether, this study provides evidence that naphtho[2,3-f]indole-5,10-diones bearing the

cyclic diamine in the side chain in the position 3 can be a perspective chemotype for exploration of

antitumor drug candidates with improved properties.

4. Experimental section

4.1. General methods

The NMR spectra of all newly synthesized compounds were recorded on a Varian VXR-400

instrument operating at 400 MHz (1H NMR) and 100 MHz (13C NMR). Chemical shifts were

measured in D2O using sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (1H NMR) or

methanol (13C NMR) as internal standards. The signals in the 13C NMR spectra were assigned by the

APT (attached proton test) method. Analytical TLC was performed on silica gel F254 plates (Merck)

and column chromatography on Silica Gel Merck 60. Melting points were determined on a Buchi

SMP-20 apparatus and are uncorrected. High resolution mass spectra were recorded by electron spray

ionization on a Bruker Daltonics microOTOF-QII instrument. UV spectra were recorded on Hitachi-

U2000 spectrophotometer. HPLC was performed using Shimadzu Class-VP V6.12SP1 system

(GraseSmart RP-18, 6×250 mm). Eluents: A, H3PO4 (0.01 M); B, MeCN. All solutions were

evaporated at a reduced pressure on a Buchi-R200 rotary evaporator at the temperature below 50 °C.

All products were vacuum dried at room temperature. All solvents, chemicals, and reagents were

obtained commercially and used without purification. Naphtho[2,3-f]indole-5,10-diones 2 and 3 were

prepared as described [26]. The purity of compounds 4-13 was >95% as determined by HPLC

analysis. All reagents were from Sigma-Aldrich unless specified otherwise.

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

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To a solution of dimethylaminomethyl derivative 3 (500 mg, 1.4 mmol [26]) in glacial acetic

acid (20 ml) the solution (9%) of hydrogen chloride in glacial acetic acid (20 ml) was added. The

mixture was stirred for 24 h and evaporated in vacuum. The residue was dissolved in hot water (3-4

mL), filtered, then product was precipitated with acetone-ether mixture (1:1), collected by filtration

and dried. The yield of hydrochloride 4 was 465 mg (92 %) as a red solid, mp 238-239 ºС. HPLC

(LW=260 nm, gradient B 20 → 60% (30 min)) tR=18.3 min, purity 95.7%. 1H NMR (400 MHz, D2O)

δ 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 [M+H]+ 337.1170, found 337.1178.

For preparation of the free base of 4, an aqueous solution of hydrochloride 4 was eluted

through column with hydroxy form of Dowex 1x2 resin. The eluent was evaporated under vacuum and

the solid residue was used at the next steps without purification. For analytical purposes the crude

product was recrystallized from methanol to afford 85% free base of 4 as a dark red powder with mp

151-153°C (mp 151-152°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=260 nm, gradient B

20 → 60% (30 min)) tR=19.0 min, purity 95.5%. HRMS (ESI) calculated for C20H19N2O4 [M] +

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

chloroform (25 mL), washed with water and aqueous solution of NaHCO3 (1%), dried, and

evaporated. The residue was purified by column chromatography on silica gel in chloroform–

methanol (2:0→2:1). The red solid obtained after evaporation was dissolved in hot chloroform (1 mL),

and a solution of HCl in methanol (1.25 N, 1 mL) was added. The mixture was stirred overnight and

evaporated. The residue was dissolved in hot water (0.5 mL), filtered and then product was

precipitated with acetone - ether mixture (1:1), collected by filtration and dried. The yield of

dihydrochloride 6 was 58 mg (61%) as a red solid, mp 212-213 ºС (dec.). HPLC (LW=260 nm,

gradient B 10 → 40% (30 min)) tR=21.3 min, purity 98.0%. 1H NMR (400 MHz, D2O) δ 7.54 (1H, d,

J=7.5 Hz, H9), 7.38 (1H, d, J=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=7.5 Hz, СH2СH2СH), 2.34 (1Н, m, J=7.0 Hz, СH2СH2СH). HRMS

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(ESI) calculated for C21H20N3O4 [M+H] + 378.1448, found 378.1441. Analysis calculated for

C21H19N3O4.2HCl.H2O: C 53.86, H 4.95, N 8.97. 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


This compound was prepared from 5 and (R)-1-Boc-3-aminopyrrolidine as described for 6. A

red powder, yield 58%, mp 212- 213 ºС (dec.). HPLC (LW=260 nm, gradient B 10 → 40% (30 min))

tR=21.3 min, purity 99.8%. 1H NMR (400 MHz, D2O) δ 1H NMR (400 MHz, D2O) δ 7.54 (1H, d,

J=7.5 Hz, H9), 7.38 (1H, d, J=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=7.5 Hz, СH2СH2СH), 2.34 (1Н, m, J=7.0 Hz, СH2СH2СH); 13C NMR

(100 MHz, D2O, 70 ºC) δ 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 [M+H]+ 378.1448, found 378.1445. Analysis calculated for C21H19N3O4.2HCl.H2O: C

53.86, H 4.95, N 8.97. Found: C 53.75, H 5.31, N 9.18.

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


This compound was prepared from 5 and (S)-3-(Boc-amino)pyrrolidine as described for 6. A

red powder, yield 64%, mp 218-219 ºC (dec.). HPLC (LW=260 nm, gradient B 10 → 40% (30 min))

tR=21.4 min, purity 99.1%. 1H NMR (400 MHz, D2O) δ 7.63 (1H, d, J=7.5 Hz, H9), 7.54 (1H, d,

J=7.5 Hz, H6), 7.40 (1H, t, J=7.5 Hz, H8), 7.35 (1H, t, J=7.5 Hz, H7), 7.13 (1H, s, H2), 4.37 (2Н, s,

СН2N), 4.18 (1Н, dd, J=7.4 Hz, NСH2СH), 3.87 (1Н, m, NСH2СH), 3.62 (1Н, m, NСH2СH2), 3.52

(2Н, m, NСH2СH2СН), 2.61 (1Н, m, J=7.4 Hz, СH2СH2СH), 2.19 (1Н, m, J=7.0 Hz, СH2СH2СH).

HRMS (ESI) calculated for C21H20N3O4 [M+H]+ 378.1448, found 378.1438. Analysis calculated for


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


This compound was prepared from 5 and (R)-3-(Boc-amino)pyrrolidine as described for 6. A


tR=21.4 min, purity 98.4%. 1H NMR (400 MHz, D2O) δ 7.64 (1H, d, J=7.5 Hz, H9), 7.53 (1H, d,

J=7.5 Hz, H6), 7.42 (1H, t, J=7.5 Hz, H8), 7.38 (1H, t, J=7.5 Hz, H7), 7.13 (1H, s, H2), 4.32 (2Н, s,

СН2N), 4.20 (1Н, dd, J=7.4 Hz, NСH2СH), 3.82 (1Н, m, NСH2СH), 3.62 (1Н, m, NСH2СH2), 3.48

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(2Н, m, NСH2СH2, СН), 2.64 (1Н, m, J=7.4 Hz, СH2СH2СH), 2.22 (1Н, m, J=7.0 Hz, СH2СH2СH); 13C NMR (100 MHz, D2O) δ 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


53.86, H 4.95, N 8.97. 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


This compound was prepared from 5 and (R,S)-1-Boc-3-aminopiperidine as described for 6. A


tR=21.6 min, purity 97.7%. 1H NMR (400 MHz, D2O) δ 7.60 (1H, d, J=7.5 Hz, H9), 7.41 (2H, m,

H7,8), 7.36 (1H, d, J=7.5 Hz, H6), 7.06 (1H, s, H2), 4.12 (2Н, s, СН2N), 3.85 (1Н, d, J=12.6 Hz,

СHСH2N), 3.65 (1Н, m, СH), 3.54 (1Н, d, J=12.5 Hz, NСH2СH), 3.22 (1Н, d, J=12.6 Hz, СHСH2N),

3.11 (1Н, d, J=12.7 Hz, NСH2СH2), 2.40 (1Н, d, J=12.5 Hz, NСH2СH2), 2.23 (1Н, d, J=12.6 Hz,

СHСH2), 1.86 (2Н, м, СH2СH2). HRMS (ESI) calculated for C22H22N3O4 [M+H] + 392.1605, found

392.1602. Analysis calculated for C22H21N3O4.2HCl.H2O: C 54.78, 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


This compound was prepared from 5 and 4-(Boc-amino)piperidine as described for 6. A red

powder, yield 73%, mp 253-255 ºC (dec.). HPLC (LW=260 nm, gradient B 10 → 40% (30 min))

tR=22.0 min, purity 97.3%. 1H NMR (400 MHz, D2O) δ 7.72 (1H, d, J=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=12.0 Hz,

2СH2N), 2.41 (2Н, d, J=12.8 Hz, СH2СHСH2), 2.03 (2Н, m, СH2СHСH2). HRMS (ESI) calculated for


54.78, H 5.22, 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


This compound was prepared from 5 and 1-Boc-4-aminopiperidine as described for 6. A red

powder, yield 64%, mp >260 ºC (dec.). HPLC (LW=260 nm, gradient B 10 → 40% (30 min)) tR=21.6

min, purity 96.6%. 1H NMR (400 MHz, D2O) δ 7.55 (1H, d, J=7.5 Hz, H9), 7.40 (1H, d, J=7.5 Hz,

H6), 7.35 (2H, m, H7,8), 7.08 (1H, s, H2), 4.15 (2Н, s, СН2N), 3.74 (2Н, d, J=12.8 Hz, 2СH2N), 3.63

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(1Н, t, J=12.1 Hz, СH,), 3.22 (2Н, t, J=12.7 Hz, 2СH2N), 2.50 (2Н, d, J=12.8 Hz, СH2СHСH2), 2.04

(2Н, m, СH2СHСH2); 13C NMR (100 MHz, D2O, 70 ºC) δ 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 for C22H22N3O4 [M+H] + 392.1605, found 392.1594. Analysis calculated for


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-dione dihydrochloride (13).

This compound was prepared from 5 and (1S,4S)-(−)-2-Boc-2,5-diazabicyclo[2.2.1]heptane. A

red powder, yield 61%, mp 226-227 ºC. HPLC (LW=260 nm, gradient B 10 → 40% (30 min)) tR=21.6

min, purity 97.7%. 1H NMR (400 MHz, D2O) δ 7.48 (1H, br s, H9), 7.38-7.24 (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=15.2 Hz, СН2N), 4.30 (1Н, d, J=14.1

Hz, СН2N), 3.75 (2Н, t, J=12.6 Hz, NСН2СН), 3.61 (2Н, d, J=12.6 Hz, NСН2СН), 2.63 (1Н, d,

J=12.3 Hz, СНСН2СН), 2.32 (1Н, d, J=12.3 Hz, СНСН2СН). HRMS (ESI) calculated for C22H20N3O4

[M+H] + 390.1448, found 390.1446. Analysis calculated for C22H21N3O4.2HCl.H2O: C 55.01, H 4.83,

N 8.75. Found: C 55.22, H 5.09, N 9.01.

4.2. Cell culture and cytotoxicity assays

The K562 human leukemia cell line (American Type Culture Collection; 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 type p53 (p53-/-) and the

HCT116p53KO subline (both p53 alleles deleted [48]) (generated in B.Vogelstein lab, Johns Hopkins

University, Baltimore, MD; gift of B.Kopnin) were cultured in Dulbecco modified Eagle’s medium

supplemented with 5% fetal calf serum (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/mL

penicillin, and 100 µg/mL streptomycin. The murine leukemia L1210 and human T-lymphocyte

Molt4/C8 cell line (ATCC) was propagated in RPMI-1640 medium supplemented with 10% fetal calf

serum, 0.075% NaHCO3 and 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin

at 37°C, 5% CO2 in humidified atmosphere. Cells in logarithmic phase of growth were used in the

experiments. Novel compounds were dissolved in 10% aqueous DMSO as 10 mM stock solutions

followed by serial dilutions in water immediately before experiments.The assays were performed in

96-well microtiter plates. To each well (5-7.5)×104 tumor cells and a given amount of the test

compound were added. The cells were allowed to proliferate for 48 h (L1210) or 72 h (all other cell

lines) at 37 °C in a humidified CO2-controlled atmosphere. Cell viability was assessed by MTT-assay

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[49] or by counting cells in a Coulter counter. The IC50 was defined as the concentration of the

compound that inhibited cell viability by 50%.

4.3. Drug-DNA complex formation

The binding of compounds 6-9 to DNA was determined in 100 mM KCl, 10 mM Na phosphate

buffer, pH 7.8 at 20°C. Fluorescence spectra were recorded with Cary Eclipse fluorescence

spectrophotometer (Varian Inc, USA; excitation 490 nm, emission at 500-700 nm). The concentration

of calf thymus DNA (ctDNA, double stranded; moles of bp) was determined in a sodium phosphate

buffered solution at 20°C using the molar extinction coefficient ε[ctDNA]=13200 M(bp)-1·cm-1.

Binding constants were determined by linear approximation of the ratio [L]·[DNA]/(F0-F) where [L] is

the concentration of the compound, F0 and F are fluorescence intensities of the compound in the

absence or presence of DNA, respectively.

4.4. Top1 and Top2 relaxation assays

One or two units of purified Top1 (Promega, USA) were incubated with 250 ng of supercoiled

pBR322 plasmid DNA (Fermentas, Lithuania) in the buffer (35 mM Tris-HCl, pH 8.0; 72 mM KCl, 5

mM MgCl2, 5 mM dithiotreitol, 2 mM spermidine, 100 µg/mL bovine serum albumin) in the presence

of 0.1% DMSO (vehicle control) or compounds 6-9 at 37 °C for 30 min. For Top2 activity assays, 250

ng of supercoiled plasmid DNA pBR322 and 4 units of purified enzyme (TopoGen, USA) were

incubated in the buffer (50 mМ Tris-HCl, рН 8.0; 150 mМ NaCl, 10 mМ MgCl2, 0.5 mМ

dithiothreitol, 30 µg/mL bovine serum albumin, 2 mМ АТP) in the presence of 0.1% DMSO (vehicle

control) or compounds 6-9 at 37 °C for 30 min. The reactions were terminated by the addition of

sarcosyl (up to 1%). Then proteinase K was added (final concentration 50 µg/mL), and the reaction

mixtures were incubated for 30 min (Top1) and 15 min (Top2) at 37 °C. DNA topoisomers were

resolved by electrophoresis in 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 stained with ethidium bromide after electrophoresis.

4.5.Top1 mediated DNA cleavage reactions

Human recombinant Top1 was purified from baculovirus [40]. DNA cleavage reactions were

performed as described [37] with a DNA substrate consisting of a 117-bp oligonucleotide (Integrated

DNA Tech.,) encompassing previously identified Top1 cleavage sites in the 161-bp fragment from

pBluescript SK(-) phagemid DNA. The 117-bp oligonucleotide contains a single 5’-cytosine

overhang, which was 3’end-labeled by fill-in reaction with [32P]dGTP in React 2 buffer (50 mM Tris-

HCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl) in the presence of 0.5 units of DNA polymerase I

(Klenow fragment, New England BioLabs). Unincorporated [32P] dGTP was removed using mini

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Quick Spin DNA columns (Roche, Indianapolis, IN). Approximately 2 nM of radiolabeled DNA

substrate was incubated with recombinant Top1 in 20 µL of the reaction buffer (10 mM Tris-HCl pH

7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 µg/mL bovine serum albumin) at 25 °C for 20

min in the presence of the indicated concentrations of compounds. Reactions were terminated by

adding sodium dodecyl sulphate (0.5% final concentration) followed by the addition of two volumes

of the loading dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene

cyanol, and 0.1% bromphenol blue). Aliquots of reaction mixtures were subjected to 20% denaturing

polyacrylamide gel electrophoresis. Gels were dried and visualized with a phosphoimager and

ImageQuant software (Molecular Dynamics). For simplicity, cleavage sites were numbered as

previously described in the 161-bp fragment [40].

4.6. Animals and tumor models

The DBA2 or BDF1 [DBA2 x C57Bl6] (female, 19-21 g) mice were kept in the animal facility at

Blokhin Cancer Center. Animals were given food and water ad libitum. The P388 leukemia cells (106

per mouse) were transplanted i.p. according to the protocol [50]. Next day after tumor cell inoculation

mice were divided into groups (n=4-6) and treated with compounds 6, 7, 1 (reference drug) or saline

(control). The doses are indicated in the section 2.2.4. The therapeutic 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 x 100%). Animals were monitored daily for behavioral and nutritional habits,

and weight loss. The minimal criterion of therapeutic efficacy for screening was T/C>125%.

Statistical difference between groups was calculated using Student’s t-test. Values p ≤0.05 were

considered significant.

Acknowledgements and funding

The authors thank A. Korolev and N. Maliutina (Gause Institute of New Antibiotics) for

HRMS and HPLS analyses; N. Lesnaya, S. Sitdikova and F. Donenko (Blokhin Cancer Center) for

assistance in animal testing, and L. van Berckelaer (KU Leuven) for help in cell proliferation assays.

Y. Pommier and K. Agama are supported by the Center for Cancer Research, Intramural Program of

the US National Cancer Institute (Z01 BC006161).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://XXXXXXXXX.

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М-1

6 11±17 10±18 3.5±0.49 3.4±0.3

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ACCEPTED MANUSCRIPTHighlights: • A series of novel naphtho[2,3-f]indole-5,10-diones was designed and synthesized

• The designed derivatives showed an improved antiproliferative activity

• A high affinity of designed derivatives to double stranded DNA was demonstrated

• The inhibition of Top1 and Top2 by naphthoindolediones was demonstrated

• One compound showed an induction of Top1 mediated DNA cleavage and antitumor activity

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Supplementary Material

Synthesis and evaluation of new antitumor 3-aminomethyl-4,11-


Andrey E. Shchekotikhina,b,1

,Valeria A. Glazunovaс, 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. Kaluzhnyd, Jan Balzarini

e, Keli Agama

f, Yves Pommier

f, Alexander A. Shtil

с

and Maria N. Preobrazhenskayaa

aGause Institute of New Antibiotics, Russian Academy of Medical Sciences,

11 B. Pirogovskaya Street, Moscow 119021, Russia b.

Mendeleyev University of Chemical Technology, 9 Miusskaya Square, Moscow 125190, Russia cBlokhin Cancer Center, Russian Academy of Medical Sciences, 24 Kashirskoyeshosse, Moscow

115478, Russia

dEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street,

Moscow 119991, Russia

eRega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium

f Developmental Therapeutics Branch, National Cancer Institute, NIH, 37 Convent Drive, 37-5068,

Bethesda, MD 20892, USA

Table S1. Antiproliferative activity (IC50,

aμM) and resistance indices (RI) of naphtho[2,3-f]indole-

5,10-diones 6-13 and reference drugs 1, 2.

compd L1210 Molt4/C8 K562 K562/4 RIb HCT116 HCT116p53KO RI

c

6 0.080.04 0.420.01 0.50.1 0.90.1 1.8 0.80.2 1.20.2 1.5

7 0.070.02 0.360.01 0.40.1 0.40.06 1.0 1.60.4 1.20.1 0.75

8 0.70.11 1.20.1 0.60.1 0.50.1 0.8 1.40.4 1.50.1 1.1

9 0.70.12 1.50.1 0.50.1 0.60.2 1.2 1.60.3 2.00.2 1.25

10 0.900.07 2.60.21 0.70.1 0.60.1 0.9 1.90.6 3.90.1 2.1

11 0.360.06 1.00.23 1.20.1 0.50.1 0.4 1.60.1 2.90.1 1.8

12 1.40.14 3.01.8 0.20.1 0.20.1 1.0 1.40.1 0.70.15 0.5

13 1.50.1 1.70.1 1.50.1 0.40.1 0.3 6.40.4 6.91.1 1.1

1 0.370.07 0.200.02 0.80.2 7.00.3 8.8 1.40.1 4.40.4 3.1

2 1.60.1 5.00.2 1.20.2 1.20.2 1.0 2.10.2 2.60.3 1.2 aIC50, μM, mean S.D. of 3 experiments.

b RI, resistance index: IC50(K562/4)/IC50(K562).

c RI, resistance index: IC50(HCT116p53KO)/IC50(HCT116).

1Corresponding author. Fax 7 499 2450295; e-mail: [email protected]

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1H,

13C NMRSpectra

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

dihydrochloride (6)

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

dihydrochloride (7)

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

dihydrochloride (8)

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

dihydrochloride (9)

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

dihydrochloride (10)

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


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


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3-(((1S,4S)-2,5-Diazabicyclo[2.2.1]heptan-2-yl)methyl)-4,11-dihydroxy-1H-naphtho[2,3-

f]indole-5,10-dione dihydrochloride (13)

Documents

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