Structure-Activity Relationship Study of Dexrazoxane Analogues Reveals ICRF-193 as the Most Potent Bisdioxopiperazine against Anthracycline Toxicity to Cardiomyocytes Due to Its Strong Topoisomerase IIβ Interactions

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Structure−Activity Relationship Study of Dexrazoxane Analogues

Reveals ICRF-193 as the Most Potent Bisdioxopiperazine against

Anthracycline Toxicity to Cardiomyocytes Due to Its Strong

Topoisomerase IIβ Interactions

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Anna Jirkovsk á ,* Galina Karabanovich, Jan Kubes, Veronika Skalick á , Iuliia Melnikova, Jan Kor á becny,

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Tom á s Kucera, Eduard Jirkovsky, Lucie Nov á kov á , Hana Bavlovic Pisk á ckov á , Josef Skoda,

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Martin Sterba, Caroline A. Austin, Tom á s Simunek, and Jaroslav Roh *

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ABSTRACT: Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced

cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested

that the inhibition and/or depletion of topoisomerase IIβ (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated

a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with

TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure−activity relationships were

demonstrated on stereoisomeric forms of 4,4′-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-

derivative 11 (ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in

cardiomyocytes more eﬃciently than dexrazoxane, and showed the highest cardioprotective eﬃciency. Importantly, the observed

ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identiﬁes ICRF-

193 as the new lead compound in the development of eﬃcient cardioprotective agents.

their anticancer eﬀect and established catalytic inhibition of

topoisomerase II (TOP2) as the key mechanism.^6,7In-depth

mechanistic studies generally supported the catalytic mode of

TOP2 inhibition,⁸⁻¹¹despite some studies that oﬀered an

alternative perspective.^12,13DEX and other bisdioxopiperazines

do not bind directly to the adenosine 5′-triphosphate (ATP)-

binding pocket of TOP2 but instead they bind to the so-called

DEX binding site where they bridge and stabilize a transient

INTRODUCTION

■

The bisdioxopiperazines have been originally discovered as

drug candidates accidentally while attempting to develop less

polar and thus cell-permeable derivatives of the metal chelator

ethylenediaminetetraacetic acid (EDTA) as antineoplastic

agents.¹Notable antiproliferative eﬀects were observed in

several members of this class²and further early preclinical

studies performed with ICRF-159 (razoxane, a racemic mixture

of dexrazoxane and levrazoxane, Figure 1) demonstrated high

antitumor activity and antimetastatic eﬀects when combined

with established anticancer drugs, including anthracyclines

(ANT).^3,4The same eﬀect was found in its more soluble

enantiomer dexrazoxane (ICRF-187, DEX) or another

bisdioxopiperazine ICRF-154.⁵Further investigations dis-

proved the validity of the metal chelation hypothesis for

Received: December 14, 2020

Published: March 22, 2021

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suggested other binding sites, mainly in the core domain of the

protein.^15,16Nevertheless, the crystal structure of the core part

of TOP2 with bound DEX or other bisdioxopiperazine has not

been described yet.

Over decades, the structure−activity relationships (SAR) of

bisdioxopiperazines have been well characterized with respect

to both topoisomerase II (TOP2) inhibition and anticancer

eﬀects.¹⁷⁻²⁰However, with the exception of sobuzoxane,²¹

none of these agents has progressed to advanced clinical

evaluation or routine clinical use. Even the most potent

analogue of this class, ICRF-193, has not progressed beyond its

use as an experimental tool and model TOP2 catalytic

inhibitor.

Interestingly, the cardioprotective eﬀects of bisdioxopiper-

azine compounds were recognized later, and in contrast to the

anticancer eﬀects, it has been successfully translated into

clinical use. In cardioprotective settings, DEX has dominated

as its markedly increased solubility allowed intravenous

administration.²²So far, DEX has been the only drug that

unambiguously protected the heart against chronic type of

ANT cardiotoxicity in diﬀerent animal models²³and has been

approved for this indication in clinical practice.²⁴Despite some

concerns, meta-analyses of all randomized controlled trials

conﬁrmed that signiﬁcant cardioprotection is achieved without

interference with anticancer eﬀects of ANTs.^24,25

The mechanism(s) of cardioprotective activity of DEX

remains controversial. DEX has been found to undergo

metabolization to EDTA-like ring-opened chelating agent

ADR-925 (Figure 1).^26,27ANTs and particularly their iron

complexes are redox-active and can form highly toxic reactive

oxygen species (ROS). It has been widely accepted that DEX

Figure 1. Bisdioxopiperazine ICRF-187 (DEX), its metabolite ADR-

925 and several DEX analogues previously studied for protection

against anthracycline cardiotoxicity.

dimer interface between the two ATPase protomers of

TOP2.¹⁴This site was conﬁrmed by studies using mutated

enzymes and later by the crystallization experiments. Based on

the mutations of diﬀerent amino acids, some studies also

Figure 2. Structures of DEX analogues 1−12 that were prepared and studied in this work.

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Scheme 1. Synthesis of 4,4′-(2-Hydroxypropane-1,3-diyl)bis(piperazine-2,6-dione) (1), 4-(2-(2-Oxomorpholin-4-

a

yl)ethyl)piperazine-2,6-dione (2), and 4-(2-(3-Oxopiperazin-1-yl)ethyl)piperazine-2,6-dione (3)

a

Reagents and conditions: (a) 1. HCONH₂, vacuum (30 mbar), 110 °C, 1.5 h; 2. 155 °C, 5 h, Ar; (b) 1. BrCH₂COOtBu, K₂CO₃, tetrahydrofuran

(THF), H₂O, reﬂux, 4 h, 91%; 2. H₂, Pd/C, MeOH, room temperature (rt), 24 h, 94%; (c) Br(CH₂)₂NHCOOtBu, K₂CO₃, CH₃CN, reﬂux, 48 h,

55%; (d) 1. 36% aq. HCl, 90 °C, 2 h; 2. HCONH₂, vacuum (30 mbar), 110 °C, 1 h; 3. HCONH₂, 160 °C, 5 h, Ar, 24%.

prevents ANT-induced oxidative injury via iron chelation. In

this regard, the lipophilicity of DEX ensures its penetration

into the cardiac cells, where, upon the hydrolytic opening, the

metabolite chelates iron ions and displaces them from the

ANT complexes. However, several studies showed that other

eﬀective iron chelators^23,28or DEX analogues with preserved

iron-chelating properties^29,30were not able to protect the heart

from ANT cardiotoxicity. Furthermore, an accumulating body

of evidence implies that an alternative mechanism may be

important for cardioprotection.³¹⁻³³In particular, interaction

with topoisomerase IIβ (TOP2B), which is the predominant

TOP2 isoform in cardiomyocytes,^34,35has been attributed to

the cardioprotective eﬀects of DEX against ANT cardiotox-

icity. The latter hypothesis has been supported by a

nonpharmacological approach where cardiac-speciﬁc TOP2B

knock-out eﬀectively protected mice from ANT cardiotox-

icity.³⁶

In sharp contrast to anticancer eﬀects, SAR studies of

bisdioxopiperazines with respect to cardioprotection have been

scarce and this matter remains poorly understood. Besides

DEX, only demethylated DEX analogue ICRF-154,³⁷its

prodrug sobuzoxane (MST-16), and morpholine prodrug of

ICRF-159 probimane^21,31,38showed a clear cardioprotective

potential. Other bisdioxopiperazine derivatives tested so far,

including close DEX homologues ICRF-161²⁹and ICRF-

192,³⁷amide analogue MK-15,³⁰or N,N′-dimethyl analogue

ICRF-239,³⁹showed negative results.

DEX analogues with respect to cardioprotection. We focused

on their interaction with both TOP2 isoforms and the iron-

chelating activities of their hydrolysis products to provide

mechanistic explanations of the observed eﬀects. The ﬁrst two

DEX analogues 1 and 2 were designed to produce iron-

chelating metabolites, as they are the closed-ring derivatives of

known iron chelators 1,3-diamino-2-hydroxypropane-

N,N,N′,N′-tetraacetic acid and N-(2-hydroxyethyl)-

ethylenediamine-N,N′,N′-triacetic acid, respectively (Figure

2). DEX analogue 1 is a close analogue of ICRF-161, while

2 and particularly 3 are analogues of ICRF-154. DEX analogue

3 diﬀers from ICRF-154 only in one oxo group; it carries one

amide group instead of an imide group. Five-membered ring

analogues 4−6 were designed to mimic the original 3,5-

dioxopiperazine cycles in DEX. Especially in analogues 5 and

6, both imides and two basic amines as in the original DEX

molecule are preserved and in the latter case also the distance

of the two imide end groups is close to that of DEX. The

analogues 7−12 were designed to closely mimic the structure

of DEX; the changes were made merely in the linker between

two 3,5-dioxopiperazine moieties. Analogue 7 carries one more

3,5-dioxopiperazine connected to the linker of the original

DEX. Analogue 8 was derived from a known chelator trans-1,2-

cyclohexanediamine-N,N,N′,N′-tetraacetic acid; thus, its linker

between both 3,5-dioxopiperazines is a part of cyclohexyl ring.

Analogues 9−12 are the simplest homologues of DEX, with

merely one inserted methyl group. Since both enantiomers,

DEX (ICRF-187) and levrazoxane (LEV, ICRF-186), are

similarly eﬀective TOP2 inhibitors⁷and also similarly eﬀective

Hence, the aim of this work was to design, synthesize, and

examine the SAR in the series of several structural types of

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Scheme 2. Synthesis of 1,1′-(Propane-1,2-diyl)bis(imidazolidine-2,4-dione) (4), 3,3′-(Hydrazine-1,2-diyl)bis(pyrrolidine-2,5-

a

dione) (5), and N,N′-bis(2,5-Dioxopyrrolidin-3-yl)-N,N′-dimethylethylenediamine (6)

a

Reagents and conditions: (a) HOCH₂CN, H₂O, rt, 12 h, 77%; (b) 1. NaOH, H₂O, 80 °C, 3 h; 2. KOCN, H₂O, reﬂux, 1 h, 5%; (c) N₂H₄.H₂O,

EtOH, rt, 48 h, 69%; (d) N,N′-dimethylethylenediamine (DMEDA), THF, rt, 10 h, 38%.

Scheme 3. Unwanted Rearrangements Observed during the Addition of Diamines to Maleimide

against ANT-induced cardiotoxicity in vivo,⁴⁰most analogues

were prepared and studied as the racemates (4, 8, 9 and 12) or

the mixtures of all diastereoisomers (5 and 6, Figure 2).

Although 4-(2-(3-oxopiperazin-1-yl)ethyl)piperazine-2,6-

dione (3) was the lactam analogue of compound 2, a

completely diﬀerent procedure had to be used to successfully

prepare it. Several attempts to use chelator 13b as the starting

material failed. Thus, the synthesis started from N-

benzylethylenediamine (14) that was converted in two steps

to tri(tert-butyl) 1,2-diaminoethane-N,N′,N′-triacetate (15).³⁹

The reaction of substrate 15 with tert-butyl (2-bromoethyl)-

carbamate, followed by complete deprotection and cyclization

in formamide, providing DEX analogue 3 in 11% overall yield

(Scheme 1).

1,1′-(Propane-1,2-diyl)bis(imidazolidine-2,4-dione) (4) was

prepared using a three-step procedure from 1,2-diaminopro-

pane 17. The ﬁrst step consisted of the preparation of

(propane-1,2-diyldiimino)diacetonitrile (18) using Strecker

synthesis. The resulting dinitrile 18 was hydrolyzed to the

corresponding diacetic acid, which upon the reaction with

KOCN⁴²gave DEX analogue 4 in 4% overall yield (Scheme

2).

RESULTS

■

Chemistry. 4,4′-(2-Hydroxypropane-1,3-diyl)bis-

(piperazine-2,6-dione) (1) and 4-(2-(2-oxomorpholin-4-yl)-

ethyl)piperazine-2,6-dione (2) were prepared from known

chelators 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic

acid (13a) and N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-

triacetic acid (13b), respectively, using standard DEX synthesis

consisted of heating in formamide under reduced pressure at

100−110 °C for 1−2 h, followed by heating under an argon

atmosphere at 150−160 °C for 5 h.⁴¹However, the free

hydroxy group in substrate 13a facilitated the unwanted

formation of lactone type product 2-(2-((3,5-dioxopiperazin-1-

yl)methyl)-6-oxomorpholino)acetamide (1b) that further

complicated the isolation and puriﬁcation of target product

1. Thus, the yield of DEX analogue 1 was only 14% (Scheme

1). The yield of DEX analogue 2 was 39% and was isolated

using a standard workup; it crystallized upon evaporation of

the excess formamide and addition of cold methanol.

The syntheses of succinimide-type DEX analogues 5 and 6

consisted of the addition of appropriate diamine to two

molecules of maleimide (Scheme 2). There were two main

problems with these reactions. The intermediates, which were

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a

Scheme 4. Six-Step Synthesis of 4,4′,4′′-(Propane-1,2,3-triyl)tris(piperazine-2,6-dione) (7)

a

Reagents and conditions: (a) MsCl, Et₃N, CH₃CN, rt, 48 h, 99%; (b) NaN₃, N,N-dimethylformamide (DMF), 85 °C, 5 h, 91%; (c) 1. N₂H₄.H₂O,

Pd/C, EtOH, reﬂux, 12 h; 2. 36% aq. HCl, EtOH, 70%; (d) BrCH₂COOtBu, K₂CO₃, THF, H₂O, reﬂux, 25 h, 44%; (e) 1. 36% aq. HCl, H₂O, 90

°C, 4.5 h; 2. HCONH₂, vacuum (30 mbar), 115 °C, 1.5 h; 3. HCONH₂, 160 °C, 5 h, Ar, 40%.

a

Scheme 5. Syntheses of DEX Analogues 8−12

a

Reagents and conditions: (a) 1. HCONH₂, vacuum (30 mbar), 110 °C, 1.5 h; 2. HCONH₂, 155 °C, 5 h, Ar, 57%; (b) MsCl, Et₃N, CH₂Cl₂, rt, 48

h, 76%; (c) NaN₃, DMF, 85 °C, 5 h, 60%; (d) 1. H₂, Pd/C, MeOH, 1 month; 2. BrCH₂COOtBu, K₂CO₃, THF, H₂O, reﬂux, 12 h, 29%; (e) 36%

aq. HCl, 90 °C, 4 h, 97%; (f) 1. HCONH₂, vacuum (3 mbar), 110 °C, 1.5 h; 2. HCONH₂, 155 °C, 5 h, Ar, 44%; (g) CH₂O, H₂SO₄, NaCN, H₂O,

35 °C, 40 h, 19%; (h) Ba(OH)_2.8H₂O, H₂O, reﬂux, 24 h, 58%; (i) 1. HCONH₂, vacuum (30 mbar), 110 °C, 1.5 h; 2. HCONH₂, 155 °C, 5 h, Ar,

18%; (j) MsCl, Et₃N, CH₂Cl₂, rt, 2 h, 99%; (k) NaN₃, DMF, 85 °C, 6 h; (l) 1. H₂, Pd/C, MeOH, 1 month; 2. HCl (g), 38% over two steps; (m)

BrCH₂COOtBu, K₂CO₃, THF, H₂O, reﬂux, 24 h, 76% (37a), 4% (37b); (n) 36% aq. HCl, H₂O, 85 °C, 3 h, 99%; (o) SOCl₂, MeOH, rt, 24 h,

91%; (p) HCONH₂, NaH, dioxane, Ar, rt, 24 h, 55%; (q) 1. HCONH₂, vacuum (30 mbar), 110 °C, 1.5 h; 2. HCONH₂, 150−160 °C, 5 h, Ar, 20%.

formed after the addition of a single amine group to one

maleimide, tended to rearrange into lactam products (Scheme

3A). Moreover, when ethylenediamine or 1,2-diaminopropane

were used, the ﬁnal products quickly rearranged to the

corresponding lactams (Scheme 3B). The former unwanted

rearrangement can be reduced by the slow addition of diamine

to the excess of maleimide. To prevent the latter type of

rearrangement, we were forced to use N,N′-dimethylethylene-

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Figure 3. Toxicities of DEX and compounds 1, 2, 7−12, and their protective eﬀects on the DAU-induced cardiotoxicity in primary isolated rat

cardiomyocytes. The data from four experiments are expressed as mean standard deviation (SD). Statistical signiﬁcance was determined in

GraphPad Prism 8 using the one-way analysis of variance (ANOVA) with the Holm−Sidak post hoc test and was accepted at P ≤ 0.05: c, signiﬁcant

diﬀerence to control and d, signiﬁcant diﬀerence to DAU.

diamine to obtain a stable tertiary amine-containing DEX

analogue 6.

hexane-1,2-diyl)bis(piperazine-2,6-dione) (8) was commer-

cially available. 1,2-Diaminobutane-N,N,N′,N′-tetraacetic acid

dihydrochloride (29) used for the synthesis of 4,4′-(butane-

1,2-diyl)bis(piperazine-2,6-dione) (9) was prepared from 1,2-

butanediol (25) using the same protocol as for the preparation

of DEX analogue 7 (Scheme 5). In the synthesis of 4,4′-(2-

methylpropane-1,2-diyl)bis(piperazine-2,6-dione) (10), the

corresponding tetraacetic acid 32 was prepared from 2-

methyl-1,2-diaminopropane (30). Due to the sterically

hindered amine in position 2, Strecker synthesis was used to

prepare tetranitrile 31, which was hydrolyzed to acid 32 as

described previously.⁴⁴Final cyclization in formamide gave

only a low yield of product and several products of elimination

were detected. Elimination proceeded, especially at the

quaternary carbon in the linker. In the synthesis of meso-4,4′-

(butane-2,3-diyl)bis(piperazine-2,6-dione) (11), known as

ICRF-193, we started from the commercially available 2,3-

butanediol 33, while the ratio between meso form and

4,4′,4′′-(Propane-1,2,3-triyl)tris(piperazine-2,6-dione) (7)

was prepared using a six-step procedure starting from glycerol

19 (Scheme 4). First, glycerol was mesylated and then

converted to 1,2,3-triazidopropane 21 by the reaction with

sodium azide. 1,2,3-Triazidopropane 21 was reduced on Pd/C

to propane-1,2,3-triamine trihydrochloride (22) according to

the known method.⁴³Its alkylation with tert-butyl bromoace-

tate yielded hexa(tert-butyl) 1,2,3-triaminopropane-

N,N,N′,N′,N′′,N′′-hexaacetate (23). Hydrolysis of 23 followed

by the ﬁnal cyclization using a standard procedure in

formamide resulted in the formation of ﬁnal DEX analogue

7 in 11% overall yield.

The last step of the preparation of DEX analogues 8−11 was

the cyclization of corresponding tetraacetic acids. Chelator

trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid

monohydrate 24 used for the synthesis of trans-4,4′-(cyclo-

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Figure 4. Antiproliferative activity of DAU, DEX, and compound 11 (ICRF-193). HL-60 cells were incubated for 72 h. The toxicity was assessed

by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Absorbances of the treatment wells were expressed

as a percent of control wells. Data are presented as mean

SD of four independent experiments. Statistical signiﬁcance was determined in

GraphPad Prism 8 using the one-way ANOVA with the Holm−Sidak post hoc test and was accepted at P ≤ 0.05: c, signiﬁcant to control.

Figure 5. Antiproliferative activity of DEX and compounds 1, 2, and 7−12 and their inﬂuence on the antiproliferative eﬀect of daunorubicin

(DAU). HL-60 cells were incubated for 72 h with DEX and its analogues 1, 2, and 7−12 either alone (10 and 100 μM, light gray columns) or in

combination with 15 nM DAU (dark gray columns for combinations, black columns for DAU alone). The toxicity was assessed by the XTT assay.

Absorbances of the treatment wells were expressed as a percent of dimethyl sulfoxide (DMSO) control wells (white columns). Data are presented

as mean SD of four independent experiments. Statistical signiﬁcance was determined in GraphPad Prism 8 using the one-way ANOVA with the

Holm−Sidak post hoc test and was accepted at P ≤ 0.05: c, signiﬁcant diﬀerence to untreated control and d, signiﬁcant diﬀerence to DAU.

racemate in it was 2.5:1 according to NMR. Surprisingly, after

the reduction of azide 35 to diamine and its conversion to

dihydrochloride 36, predominantly meso form crystallized

from the reaction mixture and was isolated as 92% pure (8%

was racemic form). Upon alkylation of diamine 36 with tert-

butyl bromoacetate, both meso-tetraester 37a and rac-tetraester

37b were isolated using column chromatography. Then, in the

cyclization step of tetraacetic acids 38a and 38b, a standard

procedure in formamide under elevated temperatures was

used. However, only rac-tetraacetic acid 38b was successfully

converted to rac-4,4′-(butane-2,3-diyl)bis(piperazine-2,6-

dione) (12) using this method. For the cyclization of meso-

tetraacetic acid 38a, we used the cyclization procedure of its

tetramethylester 39 in dioxane at room temperature as

described previously.²⁹Finally, meso-4,4′-(butane-2,3-diyl)bis-

(piperazine-2,6-dione) (11) was prepared using a seven-step

procedure in 14% overall yield (Scheme 5). As the meso-

derivative 11 was later found to be a very potent TOP2B

inhibitor and protectant of cardiomyocytes against ANT-

induced toxicity, its presence in racemate 12 as an impurity

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Figure 6. Concentration dependence of inhibition of recombinant human TOP2A (A) and TOP2B (B) by DEX and its analogues 11 and 12 (1−

1000 μM). Isolated kDNA was incubated with either enzyme isoform in a reaction buﬀer and 1/10 volume of each compound diluted in 10%

DMSO (ﬁnal DMSO concentration 1%) for 30 min at 37 °C. In reactions performed in the presence of DEX, analogue 11 or 12 (1−1000 μM) are

compared to the reaction with no TOP2 activity and with complete TOP2 activity. DMSO was added to the reactions where no other inhibitor was

included in the same ﬁnal concentration (1%). The signal of the treated samples was normalized to the respective control on the same gel

(untreated sample, 100%), and the data are expressed as mean SD.

might have an impact on the results of the following in vitro

studies. Therefore, we thoroughly analyzed the purity of

racemate 12 using high-performance liquid chromatography-

tandem mass spectrometry (HPLC-MS/MS) and found that it

contained only a very low amount (0.035%) of its meso-form

11 (see Figure S6, Supporting Information).

In Vitro Assay of Cardioprotective Activity. DEX and

all its prepared analogues 1−12 were evaluated for their ability

to protect primary cultures of rat neonatal ventricular

cardiomyocytes (NVCMs) against daunorubicin (DAU)-

induced toxicity. DAU (1.2 μM) treatment caused approx-

imately 40−50% cytotoxicity (measured as the leakage of total

intracellular lactate dehydrogenase (LDH) to the culture

media; Figures 3 and S1, black columns). The protective

activities of DEX and its analogues were assessed by the

pretreatment of the culture for 3 h with subsequent

cotreatment with DAU for 3 h (Figures 3 and S1, dark gray

columns). This schedule was based on our previous ﬁndings

and previously published literature.^30,45The own toxicities of

DEX and its analogues to NVCMs were also examined

(Figures 3 and S1, light-gray columns).

All of the assessed agents were generally nontoxic on their

own in the tested concentration range (10−100 μM). Apart

from DEX, only four analogues displayed signiﬁcant protective

activity against DAU toxicity. Three of them, 8, 9, and 12, were

less eﬀective than DEX. However, analogue 11 (ICRF-193)

oﬀered markedly better protection of NVCMs against DAU

toxicity than DEX (signiﬁcant protection was observed from 1

μM). Noteworthily, stereoisomers 11 and 12, which diﬀer just

in the spatial orientation of methyl groups in the linker,

showed signiﬁcantly diﬀerent potential to protect NVCMs,

although stereoisomers DEX and LEV and their racemate were

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Figure 7. Depletion of TOP2B from neonatal rat ventricular cardiomyocytes. The cells were incubated with DEX or its analogues 1, 2, and 7−12

for 24 h, and TOP2B protein abundance was determined by immunoblotting with normalization to total protein using stain-free technology. The

signal of the treated samples was normalized to the respective control on the same blot (untreated sample, 100%). The data of four independent

experiments are expressed as mean SD. Statistical signiﬁcance was determined using the one-way ANOVA with the Holm−Sidak post hoc test

and was accepted at P ≤ 0.05: c, signiﬁcant diﬀerence to untreated control.

equally eﬀective.⁴⁰The EC₅₀value, i.e., the concentration that

reduced the toxicity of DAU toward cardiomyocytes to 50%,

was 4 μM for meso-derivative 11, while >100 μM for both

DEX and rac-form 12. Surprisingly, analogue 10 with high

structural similarity to DEX and to both analogues 9 and 11

showed no signiﬁcant protection or any nonsigniﬁcant trend

toward this eﬀect under the same conditions.

Inhibition of Proliferation of Leukemic Cells and the

Eﬀect on the Antiproliferative Activity of Daunorubicin.

The antiproliferative eﬀects of DEX and its analogues 1−12

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Figure 8. Crystal structure of DEX ((A) yeast, PDB ID: 1QZR)¹⁴and top-scored docking poses for 10 (B), meso-11 (C), (R,R)-12 (D), and (S,S)-

12 (E) in the DEX binding pocket of TOP2. The ligands are displayed in orange, brown, dark blue, purple, and yellow, respectively; important

amino acid residues responsible for ligand anchoring are shown in green and light blue (the same color among residues determines a speciﬁc

receptor protomer labeled either as “A” or “B”). Hydrogen bond contacts are rendered by black dashed lines; distances are measured in angstroms

(Å). The rest of the receptor is displayed in the light-gray cartoon.

alone or in combination with DAU were studied in human

leukemia HL-60 cell line. First, IC₅₀values of both DAU and

DEX alone were assessed (15 nM and 18 μM, respectively;

Figure 4). Then, the antiproliferative activity of DEX analogues

was assessed in the same concentration ranges as in DEX

(Figures 5 and S2; light gray columns) as limited solubility did

not allow testing of higher concentrations in many analogues.

The greatest inhibition of proliferation was seen in analogue 11

(IC₅₀= 0.037 μM, Figures 4C and 5H), and a signiﬁcant eﬀect

was also noted in analogues 9 and 12, although only in the

highest concentration tested (100 μM, Figure 5F,I, respec-

tively). However, it should be noted that the eﬀects observed

in the latter compound (12) could be partially attributed to the

presence of a very low amount (0.035%) of highly potent

meso-form 11 as an impurity (Figure S6). Analogue 8 was

without any eﬀect even in the highest concentration.

Interestingly, analogues 1 and 6, which had no activity in the

cardioprotective assay (Figures 3B and S1D, respectively), also

signiﬁcantly inhibited proliferation of cancer cells at the highest

assayed concentration (Figures 5B and S2D, respectively).

Then, the eﬀects of DEX and its analogues on the

antiproliferative activity of DAU were assessed. Most of the

analogues had insigniﬁcant or weak eﬀects on the proliferation

of HL-60 cells, and IC₅₀values were often not reached and the

classical Chou−Talalay analysis⁴⁶could not be performed.

Hence, we used low and high concentrations (10 and 100 μM,

respectively) of the DEX and its analogues in combination

with DAU in the calculated IC₅₀value (15 nM). The most

important ﬁnding is that none of the tested compounds had

any negative eﬀect on the antiproliferative eﬀect of DAU under

the tested conditions, which is a clear prerequisite for their

potential use as cardioprotective agents in the clinics. In

contrast, signiﬁcant enhancement of the antiproliferative eﬀect

of DAU was noted in DEX and analogues 6, 9, and 12 at 100

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μM, while compound 11 showed the enhancement eﬀect at

both 10 and 100 μM. To understand the mechanistic details of

this observation, there will be a need to investigate the

contribution of G2/M checkpoints known to be activated by

TOP2 catalytic inhibitors, including ICRF-193 (11), because

many cancer cells lack these cell cycle responses.^47,48

we redocked DEX into the TOP2 DEX binding site, which

showed a high degree of homology with an excellent root-

mean-square deviation (RMSD) value of 0.4 Å, indicating high

validity of the applied docking protocol (RMSD calculated

with LigRMSD 1.0⁵³). The superimposed structure of DEX

from the crystal structure and docked DEX revealed a very

close arrangement within the TOP2 DEX binding site. The

only small diﬀerence between the docked DEX and the “bat-

wing” conformation of DEX seen in the original crystal

structure^14,54can be found in the ﬂipped conformation of one

of the dioxopiperazine rings (Figure S5).

Our docking studies revealed that DEX interacts with the

primary dimer interface formed by 14 amino acid residues

(Figure 8A). DEX exerts only two hydrogen bonds between

terminal imide hydrogens and Gln365 residues at distances of

1.7 and 1.8 Å. We have also found one additional hydrogen

bond between the carbonyl oxygen of DEX and the hydroxyl

group from Thr27 (2.3 Å). From the crystallography analysis,

it is also evident that the attached methyl group at the ethylene

linker is somewhat disordered, which generated two possible

conformations (for the sake of clarity, we displayed only one

conformer).¹⁴The most active meso-derivative 11 (Figure 8C)

demonstrated an almost identical arrangement to that of DEX

in the TOP2 DEX binding site, conveying two essential

hydrogen bonds between carbonyl oxygens of Gln365 residues

and terminal imide hydrogens (with distances of 1.9 and 1.7

Å). Most importantly, both methyl groups at the linker can

contact their neighboring Tyr28 residues from each protomer

via methyl−π interactions. In the case of DEX binding, this

type of interaction is represented for only one Tyr28 residue.

Stronger interaction of meso-11 than DEX could also be

explained by higher ﬂexibility and related “entropic penalty” of

DEX compared to meso-11 after accommodation into the DEX

binding site of TOP2. The conformation of meso-11 is more

rigid, and its dioxopiperazine units are naturally ﬁxed in very

beneﬁcial positions.

Two protruding geminal methyl groups of analogue 10

(Figure 8B) placed one hydroxyl group from dioxopiperazine

unit comparable to DEX or meso-11 within the TOP2 binding

pocket; however, the second dioxopiperazine stands aside from

the Gln365 residue of the second protomer, providing

hydrogen bonds with two water molecules only. Thus, complex

10-TOP2 can be regarded as the least suitable for contacting

both protomers simultaneously. Although other two stereo-

isomers, namely, (R,R)-12 (Figure 8D) and (S,S)-12 (Figure

8E), have been previously reported to be equally potent TOP2

inhibitors,⁴⁴the docking results showed that in contrast to

stereoisomer (R,R)-12, stereoisomer (S,S)-12 unexpectedly

adopted beneﬁcial conformation very similar to that of DEX.

Speciﬁcally, (S,S)-12 established contact with both Gln365

residues, while (R,R)-12 is linked to a single Gln365 residue

only, since its linker imposes the second dioxopiperazine

moiety to a less energetically favorable position. To further

verify the results of the docking experiments, especially the

unexpectedly positive result of compound (S,S)-12, we

extended our docking analysis by molecular dynamics (MD)

simulations (Figure 9). Similar to the docking results, MD

indicated that DEX and meso-derivative 11 adopted nearly the

same spatial arrangement in the TOP2 DEX binding site with

apparent hydrogen bonds formed between carbonyl oxygens of

Gln365 residues and terminal imide hydrogens. In addition,

higher TOP2B inhibition activity of meso-11 can be attributed

to hydrophobic interactions of the protruding methyl in the

Interactions with Topoisomerase II Isoforms. First, the

inhibitory eﬀects of DEX and its analogues 1−11 on the

relaxation of supercoiled DNA by puriﬁed human recombinant

TOP2 isoforms were studied in 1 mM ﬁnal concentration

(Figure S3A (TOP2A),B (TOP2B)). The ﬁnal concentration

was limited by the solubility of the compounds and the

maximal amount of DMSO tolerable in the assay. Besides

DEX, only the analogues 9 and 11 inhibited the recombinant

enzymes, with a greater eﬀect noticeable with analogue 11.

Detailed analysis of TOP2 inhibition by the analogues 11 and

12 showed roughly 1 order of magnitude higher potency of

meso-derivative 11 compared to DEX, while rac-form 12 was

roughly 1 order of magnitude less eﬀective than DEX (Figure

S3C (TOP2A),D (TOP2B)). These results closely correlated

with the results obtained in another assay, where the inhibitory

eﬀects of DEX and its analogues 11 and 12 on the

decatenation of kDNA by puriﬁed human recombinant

TOP2 isoforms were studied and the respective IC₅₀values

were calculated (Figure 6A (TOP2A),B (TOP2B)). Meso-

derivative 11 was again found to be the strongest inhibitor of

both TOP2A and TOP2B among the studied compounds with

IC₅₀values of 2 and 3 μM, respectively. DEX showed

approximately 10-times and racemic derivative 12 approx-

imately 100-times lower potency as compared to meso-

derivative 11. All of the observed eﬀects of DEX and its

analogues 11 and 12 were the same for both TOP2 isoforms in

both assays.

Previously, DEX and ICRF-193 were documented to induce

depletion of TOP2 in various models in vitro and also in

vivo.^{30,36,49−51}Based on these results, we assessed the

depletion of TOP2 in NVCMs after 24 h of incubation

(Figures 7 and S4). The results of this assay correlated well

with the results of the cardioprotective assay (Figures 3 and

S1) and with the exception of compound 8 also with inhibition

of puriﬁed recombinant TOP2A and TOP2B isoforms (Figures

6 and S3). The latter discrepancy may be explained by only a

small eﬀect of analogue 8 on the cardioprotective assay (Figure

3E) and a much longer time allowed for the depletion as

compared to the TOP2 inhibition assay. This suggests that the

TOP2 depletion assay may be more sensitive and may better

correlate with the cardioprotection.

In accordance with cardioprotection and TOP2 inhibition,

analogue 11 showed the strongest depletion of TOP2B (strong

eﬀect already at 1 μM), while its rac-form 12 or analogue 9

were only eﬀective at concentrations ≥30 μM.

Molecular Modeling and Dynamic Simulation Stud-

ies. To explain the marked diﬀerences between DEX and its

close analogues 10, meso-11, and rac-12 ((R,R)-12 and (S,S)-

12) in their ability to inhibit and deplete TOP2B in NVCMs,

we have applied molecular modeling (Figure 8). As a receptor

structure, we used yeast TOP2 (PDB ID: 1QZR).¹⁴The

rationale for the choice of the receptor was dictated by (i) high

resolution (1.90 Å) and (ii) absolute similarity of the

respective ATP-binding sites and related DEX binding site

between human TOP2B and yeast TOP2. The latter accounts

for all of the critical residues involved in the anchoring of DEX

in the TOP2 binding site in yeast.^14,52To validate our model,

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was driven by their diﬀerent pharmacological proﬁles in the

above-mentioned bioassayscompound 1 showed slight

antiproliferative eﬀects (Figure 5B) and no cardioprotection

(Figure 3B) and compound 2 showed no eﬀect in either assay

(Figures 3C and 5C). Compound 11 (ICRF-193) was more

eﬀective than DEX in both assays (Figures 3H and 5H), while

its stereoisomeric form 12 was less eﬀective than DEX in both

assays (Figures 3I and 5I). The iron chelation activity of the

parent compounds and their hydrolysis products was assessed

by the displacement of the Fe³⁺ions from the complex with

DAU (Figure 10), which was used previously in similar

studies.⁵⁵As a positive control, a model experimental iron

chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was

used, which displaced iron eﬃciently and almost completely.

As published before, the hydrolysis product of DEX, ADR-

925, displaced Fe³⁺ions from the complex with DAU

eﬀectively, contrary to DEX itself. The hydrolysis products of

analogues 1 and 2, primarily designed for this purpose,

displaced Fe³⁺even more eﬃciently than ADR-925. The

analogue 2 displaced iron from the complex even after the

addition of the parent compound (apparently due to its

prompt hydrolysis). On the other hand, the hydrolysis product

of analogue 11 was the least eﬀective iron chelator despite

being apparently the most eﬀective in both cardioprotective

and antiproliferative bioassays. The chelation eﬃciency of the

hydrolysis product of its stereoisomeric counterpartcom-

pound 12was comparable, although their cardioprotective

eﬃciency was signiﬁcantly diﬀerent.

DISCUSSION

■

Initially, the iron chelation was the main hypothesis used to

explain both antiproliferative and cardioprotective eﬀects of

bisdioxopiperazine agents. This was driven by the original

design of (dex)razoxane as a cell-permeable prodrug of an

EDTA-like chelator. In the case of cardioprotection, this

notion was further strengthened by a coincidental description

of iron-catalyzed prooxidative eﬀects of ANTs in the

heart.⁵⁵⁻⁵⁸Iron chelation was early questioned and ﬁnally

disproved as the main mechanism of the antiproliferative

eﬀects of bisdioxopiperazines,⁵⁹and the eﬀects have been soon

ascribed to the catalytic inhibition of TOP2.^6,7ICRF-193 has

been identiﬁed as the most potent TOP2 inhibitor and

antiproliferative agents of this class.^17,18,60However, the iron

chelation remained an often cited theory of cardioprotective

eﬀects of DEX also used in documents issued by regulatory

authorities.^28,61

Analogue 1 (Figure 3), which diﬀers from the previously

described compound ICRF-161 just by the presence of the

hydroxyl group in the aliphatic linker, completely lacked the

activity in our cardioprotective bioassay. The hydrolysis

product of 1 is a known commercial metal chelator; therefore,

it was not a surprise that it eﬀectively displaced Fe³⁺ions from

the complex with DAU in our present study. Analogue 1 is

thus similar to ICRF-161 in the iron-chelating activity of its

hydrolysis product and also in the lack of TOP2 inhibitory

activity of the parent compound.²⁹Noteworthily, both

compound 1 studied herein and ICRF-161 tested previously

show a complete lack of cardioprotective potential, which

seems to be attributable to the impaired TOP2B interaction.²⁹

Hence, the prolongation of the linker is not a perspective

modiﬁcation of the DEX molecule regarding cardioprotective

eﬀects despite retained or even enhanced iron-chelating

activity. The weak antiproliferative activity of bisdioxopiper-

Figure 9. Molecular dynamic simulations of enantiomers (R,R)-12

(panel (A); purple) and (S,S)-12 (panel (A); yellow) and meso-

derivative 11 (panel (B); blue) and DEX (panel (B); orange) in the

DEX binding pocket of TOP2 (PDB ID: 1QZR).¹⁴Important amino

acid residues responsible for ligand anchoring are shown in green and

light blue lines (the same color among residues determines speciﬁc

receptor protomer labeled either as “A” or “B”). Hydrogen bond

contacts are rendered by black dashed lines; distances are measured in

angstroms (Å).

linker (Figure 9B). In contrast to the docking results, the MD

simulation revealed that both (S,S)-12 and (R,R)-12

enantiomers adopted signiﬁcantly diﬀerent and less beneﬁcial

arrangements compared to that of DEX or meso-derivative 11

(Figure 9A). Thus, the results of MD simulation of DEX, 11,

(S,S)-12, and (R,R)-12 seem to correspond better with the

experimental data than the results of docking analysis.

From the standpoint of in silico predictions, the activity of

each ligand can be anticipated upon the linker stereochemistry

along with the bulkiness of the attached moieties (methyl,

ethyl, etc.). For all of the compounds selected for in silico

studies (i.e., DEX, 10, meso-11, (R,R)-12, and (S,S)-12), their

respective methyl groups at the ethylene linkers impact the

conformation of the linker and the orientation of dioxopiper-

azine moieties mainly due to steric reasons, playing a critical

role for the ligand binding to TOP2.

Displacement of Iron from the Complex with DAU.

To assess whether the iron chelation activity of the tested

compounds is linked to their pharmacological activity in

cardiomyocytes and/or cancer cells, iron chelation was

compared for DEX and analogues 1, 2, 11, and 12 and their

hydrolysis products ADR-925, 1-hp, 2-hp, 11-hp, and 12-hp

(Figure 10C), respectively. The selection of these compounds

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Figure 10. Displacement of Fe³⁺from the DAU−Fe³⁺complex measured as the absorbance change at λ = 600 nm. (A) Time proﬁles of the

displacementthe data are presented as mean values, standard deviations were omitted for clarity. After 3 min of equilibration, the tested

compounds (100 μM) or DMSO (solvent control) were added and the absorbance was monitored for 10 min. (B) The endpoint absorbances

presented as means SD; statistical signiﬁcance (one-way ANOVA, Holm−Sidak post hoc test P ≤ 0.05; f, signiﬁcantly diﬀerent from DAU−Fe³⁺

complex). (C) Structure of the racemic form of ADR-925 and expected hydrolysis products 1-hp, 2-hp, 11-hp, and 12-hp.

Figure 11. Structure−activity relationships of bisdioxopiperazines with respect to TOP2B inhibition/depletion and their cardioprotective activity.

azine 1 can be related to its iron chelation ability, as iron

chelators are known for their antiproliferative eﬀects.⁶²

NVCM cells against DAU toxicity. Importantly, the most

potent cardioprotective agents in this study (11, ICRF-193)

and its hydrolysis product 11-hp were the least eﬀective in the

iron chelation assay, and their eﬃciency was roughly the same

as those of their stereoisomeric counterparts 12 and 12-hp,

respectively. However, rac-form 12 showed markedly weaker

cardioprotective activity than its meso-form 11. All of the

above-mentioned ﬁndings suggest that metal-chelating activ-

ities of bisdioxopiperazines and their metabolites do not

Compound 2 also produced a well-known metal-chelating

metabolite, which displaced iron complexed with DAU even

more eﬀectively than ADR-925. Several other structurally

related analogues evaluated in this study, i.e., compounds 3 and

7−12, are also very likely to be hydrolytically opened to

EDTA-like iron-chelating metabolites. However, only com-

pounds 8, 9, 11, and 12 had the signiﬁcant ability to protect

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Figure 12. In vitro cardioprotective eﬃciency of DEX and stereoisomers 11 and 12 correlates with their inhibitory/depletory activity toward TOP2,

not with their chelation potential.

correspond with cardioprotective eﬀects observed in this and

previous studies,^30,39,45and thus metal chelation is unlikely to

determine eﬀective cardioprotection by bisdioxopiperazine

derivatives.

carbon linker between both dioxopiperazine rings is

detrimental for TOP2B activity and corresponding pharmaco-

logical eﬀects. Regarding other modiﬁcations of the DEX

molecule, it is apparent that the removal of just one oxo group

from ICRF-154 in analogue 3, i.e., replacement of one imide by

amide, completely deteriorates both the TOP2 interaction and

correspondingly also the cardioprotective activity. This ﬁnding

further supported our previous results that both imide groups

were necessary for eﬀective protection.³⁰The replacement of

dioxopiperazine cycles by a ﬁve-membered imidazolidine-2,4-

dione or a succinimide ring in analogues 4−6 resulted in

complete loss of activity in all assays employed in this study,

although the distance of both imides in the analogue 6 is

similar to DEX. Unfortunately, due to the chemical instability

of succinimide-type analogues, we were not able to examine

analogues with 1,2-diaminopropane or ethylenediamine linker,

i.e., the structures closer to parent DEX. Taken all together, the

present data strongly indicate very narrow structure−activity

relationships of bisdioxopiperazines with respect to both

TOP2B inhibition and cardioprotection. The same is true for

their TOP2A inhibition, which is in line with the well-

documented mechanisms of antiproliferative eﬀects. We have

conﬁrmed previously that TOP2A is not expressed in

signiﬁcant amounts in NVCMs used in this study;⁶³therefore,

its role in cardioprotective eﬀects observed in the present study

is highly unlikely.

The present study shows that ICRF-193 (i.e., derivative in

the meso-conﬁguration) shows the highest cardioprotective

potential. Previously, ICRF-193 (11) was not considered for

further cardioprotective studies because it had been reported

to be too toxic¹and in an initial single-dose model the authors

had reported no cardioprotection.⁶⁴Hence, with respect to the

former ﬁndings, a thorough analysis of cardioprotective eﬀects

and systemic toxicity in the in vivo model of chronic ANT

toxicity is warranted. Given the increased potency of analogue

11 over DEX in our in vitro assays, optimal in vivo dose will

have to be determined. However, the structural symmetry of

the analogue 11 led to a sharp decrease of its solubility, which

is the issue that must be resolved prior to future in vivo

pharmacological evaluation as a potential cardioprotective drug

candidate.

Until the discovery of TOP2B, which is the predominant

TOP2 isoform in cardiomyocytes,^34,35,63TOP2 inhibition was

not considered as a plausible mechanism for DEX

cardioprotection. However, our present results obtained with

DEX and its analogues 8−12 point to the key role of the

inhibition/depletion of TOP2B in their cardioprotective

activity. Cardioprotective potency observed in these experi-

ments very closely mirrors their ability to inhibit and/or

deplete TOP2, which is in accordance with the results of a

recent study of Hasinoﬀ.⁵²From the SAR point of view, the

substitution and conﬁguration of the two-carbon linker

between both dioxopiperazine rings were found to be the

determining structural factors responsible for eﬀective

inhibition/depletion of TOP2B and related cardioprotective

activity of DEX and its analogues 8−12 (Figure 11).

Achiral DEX analogue 10 with just one additional methyl on

the original chiral carbon in the linker was unable to inhibit or

deplete TOP2B, which was accompanied by the lack of the

corresponding cardioprotective eﬀects. Low cardioprotective

potential of cyclohexyl analogue 8 and ethyl analogue 9

(ICRF-192), seen only at the highest tested concentration

(Figure 3E,F, respectively), corresponded with the moderate

depletion of TOP2B in cardiomyocytes (Figure 7) and in the

case of slightly more potent analogue 9 also with the positive

ﬁnding in the TOP2B inhibitory assay (Figure S3). Compound

11 (ICRF-193) has been found to be both the most potent

TOP2 inhibitor and cardioprotectant against ANT toxicity,

signiﬁcantly surpassing clinically used DEX, as well as all

previously tested bisdioxopiperazine derivatives. Markedly

decreased ability of analogue 12, i.e., the stereoisomeric

counterpart of the most active meso-derivative 11, to inhibit

TOP2B and to protect cardiomyocytes against ANT toxicity is

among the strongest evidence that the cardioprotective

eﬃciency of bisdioxopiperazines is TOP2B-dependent and

not related to the chelation eﬃciency (Figure 12).

Furthermore, the docking experiment provided a mechanistic

explanation to this end. The substituents on the linker and

their conﬁguration were recognized to be essential for the

eﬀective interaction of bisdioxopiperazines with the DEX

binding site of TOP2.

Considering that both ANTs and bisdioxopiperazines are

TOP2 targeting compounds, there could be possible concerns

about unwanted “prevention” of ANT toxicity also toward the

cancer cells. This question becomes even more relevant given

the indiscriminate inhibitory activity of DEX as well as other

bisdioxopiperazines on both TOP2 isoforms seen in this study.

Most importantly, in clinical settings, no reduction of ANT

The remaining analogues 3−7, which were inactive against

TOP2B, as shown in Figures 7, S3, and S4, were also

completely inactive as cardioprotectants. The example of

analogue 7 conﬁrms that the bulkier substitution on the two-

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N,N,N′,N′-tetraacetic acid (13a) (5 g, 15.5 mmol) was mixed with

formamide (32 mL), and the reaction mixture was heated under

reduced pressure (30 mbar) at 110 °C for 1.5 h. Then, the reaction

ﬂask was ﬁlled with argon and the reaction mixture was heated to

150−160 °C for 5 h. Upon completion, formamide was distilled oﬀ

under reduced pressure (30 mbar). The residue was separated using

column chromatography (mobile phase: EtOAc/acetone, gradient

25−100% acetone) and two crystalline products, 4,4′-(2-hydrox-

ypropane-1,3-diyl)bis(piperazine-2,6-dione) (1) and 2-(2-((3,5-diox-

opiperazin-1-yl)methyl)-6-oxomorpholino)acetamide (1b), were ob-

tained. Both products were washed with methanol and dried.

4,4′-(2-Hydroxypropane-1,3-diyl)bis(piperazine-2,6-dione) (1).

Yield: 14% as a white solid (R_f= 0.75, mobile phase: acetone); mp

173−175 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 11.05 (s, 2H), 4.74

(d, J = 4.6 Hz, 1H), 3.82−3.75 (m, 1H), 3.36 (s, 8H), 2.45 (dd, J =

12.9, 4.3 Hz, 2H), 2.36 (dd, J = 12.9, 7.0 Hz, 2H). ¹³C NMR (126

MHz, DMSO-d₆) δ 171.71, 66.35, 59.74, 56.01. Anal. calcd for

C₁₁H₁₆N₄O₅: C, 46.48; H, 5.67; N, 19.71. Found: C, 46.69; H, 5.35;

N, 19.62. HRMS (ESI⁺) calcd for (C₁₁H₁₆N₄O₅+ H)⁺m/z:

285.11935; found: 285.1201.

antineoplastic activity by DEX was found in multiple

randomized clinical trials and their meta-analyses. Also, in

our study, neither DEX, nor any of the analogues, including the

most potent TOP2 inhibitor ICRF-193 (11), diminished the

antiproliferative activity of DAU on leukemic cells.

CONCLUSIONS

■

In vitro evaluation of a series of DEX analogues showed that

their cardioprotective activity tightly correlated with their

ability to inhibit and deplete TOP2B in the NVCM cells.

Furthermore, it revealed very narrow SAR between DEX and

its cardioprotective activity, where the addition of even one

methyl group substantially decreased or completely deterio-

rated the TOP2B inhibition and cardioprotective activity, as

documented by the analogues 9 and 10, respectively. The most

eﬃcient TOP2B inhibitor 11 (ICRF-193) also displayed the

strongest depletory activity toward TOP2B and showed the

most eﬃcient protection of cardiomyocytes against ANT-

induced toxicity. In contrast, its racemic stereoisomer 12

showed markedly decreased ability to inhibit TOP2B, which

was accompanied by the decreased cardioprotective eﬃciency.

Such tight SAR was further conﬁrmed by docking analysis that

showed the important role of the substituents on the linker and

its conﬁguration in the interaction with the DEX binding site

of TOP2. However, the very low solubility of analogue 11

precludes rational in vivo evaluation; thus, a suitable

formulation or a prodrug must be developed prior to the

advanced in vivo studies. Our study also supported the recent

ﬁndings that the iron-chelating eﬃciency of the studied

compounds or their metabolites/hydrolysis products is not

essential for their cardioprotective action and further high-

lighted the role of TOP2B in the pathogenesis of ANT

cardiotoxicity.

2-(2-((3,5-Dioxopiperazin-1-yl)methyl)-6-oxomorpholino)-

acetamide (1b). Yield: 34% as a white solid; (R_f= 0.3, mobile phase:

acetone); mp 187−189 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 11.14

(s, 1H), 7.42−7.28 (m, 1H), 7.25−7.06 (m, 1H), 4.80−4.60 (m, 1H),

3.48−3.36 (m, 5H), 3.25 (d, J = 17.3 Hz, 1H), 3.05−2.94 (m, 2H),

2.87 (dd, J = 12.3, 3.6 Hz, 1H), 2.79−2.69 (m, 2H), 2.55 (dd, J =

12.4, 7.9 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 171.44, 171.07,

167.27, 76.58, 58.94, 57.12, 55.68, 54.60, 51.23.

4-(2-(2-Oxomorpholin-4-yl)ethyl)piperazine-2,6-dione (2). N-(2-

Hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid (13b) (16.5 g,

0.06 mol) was mixed with formamide (66 mL), and the reaction

mixture was heated under reduced pressure (30 mbar) at 110 °C for

1.5 h. Then, the reaction ﬂask was ﬁlled with argon and the reaction

mixture was heated to 150−160 °C for 5 h. Upon completion,

formamide was distilled oﬀ under reduced pressure (30 mbar), the

residue was cooled to rt, and MeOH (30 mL) was added. The

resulting suspension was stirred at rt overnight. The crystalline

product was ﬁltered, washed with another MeOH (50 mL), and dried

1

EXPERIMENTAL SECTION

in air. Yield: 39% (5.52 g) as a yellowish solid; mp 151−153 °C. H

■

NMR (500 MHz, DMSO-d₆) δ 11.05 (s, 1H), 4.29 (t, J = 5.1 Hz,

2H), 3.36 (s, 4H), 3.29 (s, 2H, overlapped with water), 2.69 (t, J = 5.1

Hz, 2H), 2.61 (t, J = 6.3 Hz, 2H), 2.52 (m, J = 6.2 Hz, 2H, overlapped

with DMSO). ¹³C NMR (126 MHz, DMSO-d₆) δ 171.62, 167.45,

68.59, 55.30, 55.24, 53.52, 51.90, 48.45. Anal. calcd for C₁₀H₁₅N₃O₄:

C, 49.79; H, 6.27; N, 17.42. Found: C, 49.91; H, 6.48; N, 17.62.

HRMS (ESI⁺) calcd for (C₁₀H₁₅N₃O₄+ H₂O + H)⁺m/z: 260.12410;

found: 260.1250.

General. The structural identities of the prepared compounds

were conﬁrmed by ¹H NMR and ¹³C NMR spectroscopy and by high-

resolution mass spectrometry (HRMS). Each of the tested

compounds had ≥95% purity, as determined using elemental analysis.

All chemicals used for synthesis were obtained from Sigma-Aldrich

(Schnelldorf, Germany) and were used as received. Thin-layer

chromatography (TLC) was performed on Merck aluminum plates

with silica gel 60 F₂₅₄. Merck Kieselgel 60 (0.040−0.063 mm) was

used for column chromatography. Melting points were recorded with

Synthesis of 4-(2-(3-Oxopiperazin-1-yl)ethyl)piperazine-

2,6-dione (3). Tri(tert-butyl) N-(2-(tert-Butoxycarbonylamino)-

ethyl)-1,2-diaminoethane-N,N′,N′-triacetate (16). The mixture of

tri(tert-butyl) 1,2-diaminoethane-N,N′,N′-triacetate (15) (2 g, 5

mmol), tert-butyl (2-bromoethyl)carbamate (1.34 g, 6 mmol), and

potassium carbonate (0.89 g, 6.45 mmol) in CH₃CN (35 mL) was

reﬂuxed for 48 h. The reaction mixture was cooled down to rt and

ﬁltered. The ﬁltrate was evaporated under reduced pressure, and the

product 16 was isolated using column chromatography (mobile

a Buchi B-545 apparatus (BUCHI Labortechnik AG, Flawil,

̈

1

Switzerland) and are uncorrected. H and ¹³C NMR spectra were

recorded using a Varian Mercury VNMR S500 NMR spectrometer

(Varian, Palo Alto, CA). Chemical shifts were reported as δ values in

parts per million (ppm) and were indirectly referenced to

tetramethylsilane (TMS) via the solvent signal. The elemental

analysis was carried out on an Automatic Microanalyser EA1110CE

(Fisons Instruments S.p.A., Milano, Italy). UHPLC system Acquity

UPLC I-class (Waters, Milford) coupled to a high-resolution mass

spectrometer (HRMS) Synapt G2Si (Waters, Manchester, U.K.)

based on Q-TOF was used for the HRMS spectra measurement.

Chromatography was performed using an Acquity BEH Shield RP18

(2.1 × 100 mm², 1.7 μm) column using gradient elution with 0.1%

formic acid in water (A) and acetonitrile (B) at a ﬂow-rate of 0.3 mL/

min. Electrospray ionization (ESI) was operated in the positive mode.

The ESI spectra were acquired in the range 50−1200 m/z using

leucine−enkephalin as a lock mass reference and sodium formate for

calibration. Tri(tert-butyl) 1,2-diaminoethane-N,N′,N′-triacetate (15)

was prepared as described previously.³⁹ADR-925 was prepared as

described previously.³³

1

phase: hexane/EtOAc, 4:1). Yield: 1.5 g (55%) as a yellowish oil. H

NMR (500 MHz, CDCl₃) δ 5.69 (s, 1H), 3.44 (s, 4H), 3.30 (s, 2H),

3.15 (s, 2H), 2.86−2.60 (m, 6H), 1.45 (s, 27H), 1.43 (s, 9H). ¹³C

NMR (126 MHz, CDCl₃) δ 170.57, 156.21, 80.96, 78.64, 56.10,

55.87, 53.18, 52.32, 51.90, 38.50, 28.44, 28.12.

4-(2-(3-Oxopiperazin-1-yl)ethyl)piperazine-2,6-dione (3). Tri-

(tert-butyl) N-(2-(tert-butoxycarbonylamino)ethyl)-1,2-diamino-

ethane-N,N′,N′-triacetate 16 (1.5 g, 2.75 mmol) and hydrochloric

acid (36%, 4.86 mL, 55 mmol) in H₂O (20 mL) was heated to 90 °C

for 2 h. Then, the volatiles were evaporated under reduced pressure.

The residue was mixed with formamide (5 mL) and heated under

reduced pressure (30 mbar) at 110 °C for 1 h. Then, the reaction

vessel was ﬁlled with argon and the reaction mixture was heated to

150−160 °C for 5 h. Formamide was distilled oﬀ under reduced

Synthesis of 4,4′-(2-Hydroxypropane-1,3-diyl)bis-

(piperazine-2,6-dione) (1). 1,3-Diamino-2-hydroxypropane-

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pressure (30 mbar), and the product 3 was puriﬁed using column

32.11, 32.08. Anal. calcd for C₁₂H₁₈N₄O₄: C, 51.06; H, 6.43; N, 19.85.

Found: C, 50.97; H, 6.2; N, 19.74. HRMS (ESI⁺) calcd for

(C₁₂H₁₈N₄O₄+ H)⁺m/z: 283.14008; found: 283.1414.

chromatography (mobile phase: CHCl₃/MeOH, 20:1). Yield: 0.15 g

1

(24%) as a yellowish solid; mp 175−177 °C. H NMR (500 MHz,

DMSO-d₆) δ 11.07 (s, 1H), 7.70 (s, 1H), 3.36 (s, 4H, overlapped

with water), 3.13−3.10 (m, 2H), 2.93 (s, 2H), 2.59 (t, J = 6.4 Hz,

2H), 2.57−2.52 (m, 2H), 2.51−2.45 (m, 2H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 171.70, 167.89, 57.02, 55.40, 54.12, 52.13, 49.04, 40.49.

Anal. calcd for C₁₀H₁₆N₄O₃: C, 49.99; H, 6.71; N, 23.32. Found: C,

50.24; H, 6.95; N, 23.11. HRMS (ESI⁺) calcd for (C₁₀H₁₆N₄O₃+ H)⁺

m/z: 241.12952; found: 241.1304.

Synthesis of 1,1′-(Propane-1,2-diyl)bis(imidazolidine-2,4-

dione) (4). (Propane-1,2-diyldiimino)diacetonitrile (18). 1,2-Dia-

minopropane 17 (5 g, 0.067 mol) was cooled to 0 °C and

glycolonitrile (55% in H₂O, 12 mL, 0.12 mol) was added dropwise.

The reaction mixture was stirred at rt overnight. The resulting mixture

was extracted with EtOAc (3 × 30 mL). The combined organic

extracts were dried over anhydrous Na₂SO₄and evaporated. The

product 18 was puriﬁed using column chromatography (mobile

phase: CHCl₃/MeOH, 25:1). Yield: 77% as a yellowish oil. ¹H NMR

(500 MHz, CDCl₃) δ 3.71−3.52 (m, 4H), 2.99−2.92 (m, 1H), 2.89

(dd, J = 11.9, 3.5 Hz, 1H), 2.50 (dd, J = 11.9, 8.5 Hz, 1H), 1.09 (d, J

= 6.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 117.93, 117.56, 53.61,

50.77, 37.08, 34.85, 17.61.

Synthesis of 4,4′,4′′-(Propane-1,2,3-triyl)tris(piperazine-

2,6-dione) (7). Propane-1,2,3-triyl Trimethanesulfonate (20).

Methanesulfonyl chloride (43.55 g, 29.5 mL, 0.38 mol) was added

dropwise to a solution of glycerol 19 (10 g, 8 mL, 0.11 mol) and

triethylamine (38.4 g, 52.9 mL, 0.38 mol) in CH₃CN (250 mL) at 3

°C under an argon atmosphere. The reaction mixture was stirred at rt

for 48 h and then ﬁltered. The precipitate on the ﬁlter was washed

with EtOAc (150 mL) and the ﬁltrate was evaporated under reduced

pressure. The crude product was dissolved in EtOAc (200 mL) and

washed with water (2 × 200 mL) and brine (1 × 100 mL). The

organic layer was evaporated to give a crude product 20. Yield: 99% as

a light brown oil. ¹H NMR (500 MHz, DMSO-d₆) δ 5.16 (tt, J = 5.9,

3.4 Hz, 1H), 4.50 (dd, J = 11.6, 3.4 Hz, 2H), 4.43 (dd, J = 11.6, 5.9

Hz, 2H), 3.29 (s, 3H), 3.27 (s, 6H). ¹³C NMR (126 MHz, DMSO-d₆)

δ 76.13, 67.63, 38.27, 37.05.

1,2,3-Triazidopropane (21). The mixture of propane-1,2,3-triyl

trimethanesulfonate (20) (35 g, 0.107 mol) and sodium azide (20.87

g, 0.321 mol) in DMSO (200 mL) was stirred at 85 °C for 5 h under

an argon atmosphere. Upon completion, water (600 mL) was added

and the resulting solution was extracted with CH₂Cl₂(4 × 100 mL).

The combined organic extracts were washed with water (2 × 100 mL)

and brine (1 × 100 mL), dried over anhydrous Na₂SO₄, and

evaporated. The resulting 1,2,3-triazidopropane 21 was used in the

1,1′-(Propane-1,2-diyl)bis(imidazolidine-2,4-dione) (4). (Pro-

pane-1,2-diyldiimino)diacetonitrile (18) (7 g, 0.046 mol) was added

to the solution of NaOH (5.5 g, 0.138 mol) in H₂O (20 mL) and the

resulting mixture was heated to 80 °C for 3 h. Upon cooling, the

reaction mixture was neutralized to pH = 7 using Amberlyst 15,

ﬁltered, and evaporated to dryness under reduced pressure to give 6 g

of the crude 1,2-diaminopropane-N,N′-diacetic acid. The crude 1,2-

diaminopropane-N,N′-diacetic acid (1 g) and KOCN (1.28 g, 15.8

mol) were heated in H₂O (7 mL) to reﬂux for 30 min. The reaction

mixture was acidiﬁed to pH = 3 using aqueous HCl and heated to

reﬂux for another 30 min. Then, the reaction mixture was cooled

down and kept in the refrigerator at 4 °C for 1 week. The resulting

crystalline product was ﬁltered and recrystallized from water. Yield:

1

next step without further puriﬁcation. Yield: 91% as a yellow oil. H

NMR (500 MHz, CDCl₃) δ 3.70−3.64 (m, 1H), 3.53−3.41 (m, 4H).

¹³C NMR (126 MHz, CDCl₃) δ 60.55, 51.94.

Propane-1,2,3-triamine Trihydrochloride (22). A solution of

triazide 21 (16.3 g, 0.0975 mol) in EtOH (300 mL) under an

argon atmosphere was reﬂuxed and hydrazine hydrate (140 mL) and

5% Pd/C (1.5 g) were added in three portions over a period of 1.5 h

at 0.5 h interval. The reaction mixture was heated to reﬂux for another

12 h, and then ﬁltered through a pad of silica gel. The solvent was

evaporated under reduced pressure to give the crude propane-1,2,3-

triamine. The crude propane-1,2,3-triamine was dissolved in ethanol

(150 mL) and 36% hydrochloric acid (20 mL) was added. This

mixture was kept in a refrigerator at 4 °C for 48 h. The resulting

suspension was ﬁltered and propane-1,2,3-triamine trihydrochloride

was dried over P₂O₅. Yield: 70% as a brown crystalline solid. ¹H NMR

(500 MHz, D₂O) δ 3.96 (tt, J = 7.3, 5.2 Hz, 1H), 3.41 (dd, J = 14.4,

5.2 Hz, 2H), 3.35 (dd, J = 14.4, 7.3 Hz, 2H). ¹³C NMR (126 MHz,

D₂O) δ 46.86, 38.69.

Hexa(tert-butyl) 1,2,3-Triaminopropane-N,N,N′,N′,N′′,N′′-hex-

aacetate (23). The solution of propane-1,2,3-triamine trihydro-

chloride (1.5 g, 7.56 mmol), tert-butyl bromoacetate (9.58 g, 7.2 mL,

49.11 mmol), and K₂CO₃(9.9 g, 71.63 mmol) in a mixture of THF

(100 mL) and H₂O (20 mL) was reﬂuxed for 25 h. The majority of

THF was evaporated under reduced pressure and the residue was

extracted with EtOAc (2 × 100 mL). The combined organic extracts

were washed with water (2 × 200 mL), dried over anhydrous Na₂SO₄,

and evaporated under reduced pressure. The product was puriﬁed

using column chromatography (mobile phase: hexane/EtOAc, 15:1−

3:1). Yield: 44% as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 3.55−

3.45 (m, 12H), 3.00−2.93 (m, 3H), 2.77−2.68 (m, 2H), 1.46 (s,

36H), 1.45 (s, 18H). ¹³C NMR (126 MHz, CDCl₃) δ 171.39, 170.91,

80.57, 80.39, 60.37, 56.13, 54.77, 53.55, 28.18, 28.11.

4,4′,4′′-(Propane-1,2,3-triyl)tris(piperazine-2,6-dione) (7). Hexa-

(tert-butyl) 1,2,3-triaminopropane-N,N,N′,N′,N′′,N′′-hexaacetate 23

(6.3 g, 8.1 mmol) and 36% aq. HCl (50 mL) in H₂O (50 mL) was

heated to 90 °C for 4.5 h. Then, all of the volatiles were evaporated

under reduced pressure to give the crude 1,2,3-triaminopropane-

N,N,N′,N′,N′′,N′′-hexaacetic acid trihydrochloride. The crude 1,2,3-

triaminopropane-N,N,N′,N′,N′′,N′′-hexaacetic acid trihydrochloride

was mixed with formamide (24 mL), and the reaction mixture was

heated under reduced pressure (34 mbar) to 115 °C for 1.5 h. Then,

the reaction vessel was ﬁlled with argon and the reaction mixture was

heated for another 5 h at 150−160 °C under an argon atmosphere.

Upon completion, formamide was distilled oﬀ under reduced pressure

1

5% as a white solid; mp 265−267 °C. H NMR (500 MHz, D₂O) δ

4.40−4.33 (m, 1H), 4.28−4.02 (m, 4H), 3.71 (dd, J = 14.9, 10.8 Hz,

1

1H), 3.17 (dd, J = 14.9, 3.7 Hz, 1H), 1.23 (d, J = 7.0 Hz, 3H). H

NMR (500 MHz, DMSO-d₆) δ 10.74 (s, 1H), 10.72 (s, 1H), 4.25−

4.16 (m, 1H), 4.01−3.78 (m, 4H), 3.57 (dd, J = 14.5, 10.8 Hz, 1H),

2.95 (dd, J = 14.5, 3.8 Hz, 1H), 1.10 (d, J = 6.9 Hz, 3H). ¹³C NMR

(126 MHz, D₂O) δ 175.42, 175.16, 159.06, 158.86, 51.97, 47.50,

46.15, 45.26, 14.94. ¹³C NMR (126 MHz, DMSO-d₆) δ 172.09,

171.90, 157.29, 157.13, 50.82, 46.42, 44.27, 44.01, 15.24. Anal. calcd

for C₉H₁₂N₄O₄: C, 45.00; H, 5.04; N, 23.32. Found: C, 44.83; H,

5.18; N, 23.36. HRMS (ESI⁺) calcd for (C₉H₁₂N₄O₄+ H)⁺m/z:

241.09313; found: 241.0937.

3,3′-(Hydrazine-1,2-diyl)bis(pyrrolidine-2,5-dione) (5). The sol-

ution of maleimide (1 g, 10.3 mmol) and hydrazine hydrate (0.257 g,

5.13 mmol) in 96% EtOH (20 mL) was stirred at rt for 72 h. The

precipitated product was ﬁltered and dried over P₂O₅. Yield: 69% as a

white solid; mp 93−95 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 11.06

(s, 2H), 4.65 (s, 0.9H), 4.58 (s, 1.1H), 3.86−3.75 (m, 2H), 2.82−2.67

(m, 2H), 2.61−2.50 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ

179.91, 179.57, 177.82, 177.46, 59.18, 59.00, 34.67, 34.14. Anal. calcd

for C₈H₁₀N₄O₄: C, 42.48; H, 4.46; N, 24.77. Found: C, 42.31; H,

4.29; N, 24.5. HRMS (ESI⁺) calcd for (C₈H₁₀N₄O₄+ H)⁺m/z:

227.07748; found: 227.0783.

N,N′-Bis(2,5-dioxopyrrolidin-3-yl)-N,N′-dimethylethylenedi-

amine (6). N,N′-Dimethylethylenediamine (0.79 g, 0.97 mL, 9 mmol)

was added in 10 portions over a period of 10 h into the stirred

solution of maleimide (2 g, 20 mmol) in THF (5 mL) at rt. The

resulting solution was further stirred for 12 h. THF was evaporated

under reduced pressure and the product was puriﬁed using column

chromatography (mobile phase: acetone). Yield: 38% as a yellowish

1

solid; mp 118−120 °C. H NMR (500 MHz, DMSO-d₆) δ 11.14 (s,

2H), 3.96 (dd, J = 8.9, 5.3 Hz, 2H), 2.68 (dd, J = 18.1, 8.9 Hz, 2H),

2.61−2.51 (m, 6H), 2.20 (d, J = 3.6 Hz, 6H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 178.74, 176.97, 63.22, 63.16, 52.45, 52.36, 37.62, 37.54,

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(34 mbar), the residue was cooled to rt and MeOH (20 mL) was

added. The resulting beige precipitate was ﬁltered, washed with cold

methanol (20 mL), and dried in air. Yield: 40% as a brownish solid;

evaporated under reduced pressure. The product 28 was puriﬁed

using column chromatography (mobile phase: hexane/EtOAc, 20:1

1

→ 5:1). Yield: 29%. H NMR (500 MHz, CDCl₃) δ 3.50−3.38 (m,

1

mp 230−232 °C. H NMR (500 MHz, DMSO-d₆) δ 11.08 (s, 2H),

8H), 2.89 (dd, J = 13.4, 6.0 Hz, 1H), 2.70 (p, J = 6.5 Hz, 1H), 2.54−

2.44 (m, 1H), 1.53−1.37 (m, 38H), 0.95 (t, J = 7.4 Hz, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 171.60, 170.83, 80.71, 80.39, 62.49,

56.39, 56.00, 53.29, 28.14, 28.08, 23.65, 11.43.

10.95 (s, 1H), 3.50 (s, 4H), 3.42−3.25 (m, 8H, overlapped with

water), 2.57−2.50 (m, 3H, overlapped with DMSO), 2.35 (dd, J =

13.2, 5.5 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 172.12, 171.59,

55.29, 55.00, 51.71. Anal. calcd for C₁₅H₂₀N₆O₆: C, 47.37; H, 5.30; N,

22.10. Found: C, 47.41; H, 5.39; N, 22.37. HRMS (ESI⁺) calcd for

(C₁₅H₂₀N₆O₆+ H)⁺m/z: 381.15171; found: 381.1521.

1,2-Diaminobutane-N,N,N′,N′-tetraacetic Acid Dihydrochloride

(29). Ester 28 (12.3 g, 0.0226 mol) and 36% aq. HCl (40 mL, 0.465

mmol) in H₂O (40 mL) was heated to 90 °C for 4 h. All of the

volatiles were evaporated under reduced pressure. The product 29

was dried over P₂O₅. Yield: 97% as a white solid. ¹H NMR (500 MHz,

DMSO-d₆) δ 3.93−3.77 (m, 8H), 3.42−3.32 (m, 1H), 3.24 (dd, J =

14.4, 3.5 Hz, 1H), 3.05 (dd, J = 14.3, 11.9 Hz, 1H), 1.79−1.68 (m,

1H), 1.40−1.25 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 170.76, 170.65, 62.64, 54.60, 53.54, 52.55, 19.61,

11.00.

trans-4,4′-(Cyclohexane-1,2-diyl)bis(piperazine-2,6-dione) (8).

trans-1,2-Cyclohexanediamine-N,N,N′,N′-tetraacetic acid monohy-

drate 24 (10 g, 3.13 mmol) was mixed with formamide (40 mL),

and the reaction mixture was heated under reduced pressure (30

mbar) at 110 °C for 1.5 h. Then, the reaction vessel was ﬁlled with

argon and the reaction mixture was heated for another 5 h at 150−

160 °C under an argon atmosphere. Upon completion, formamide

was distilled oﬀ under reduced pressure (30 mbar), the residue was

cooled to rt and MeOH (20 mL) was added. The resulting suspension

was stirred at rt overnight. The crystalline product was ﬁltered,

washed with another MeOH (25 mL), and dried. Yield: 57% as a

4,4′-(Butane-1,2-diyl)bis(piperazine-2,6-dione) (9). Tetraacetic

acid 29 (8.6 g, 0.022 mol) was mixed with formamide (35 mL),

and the reaction mixture was heated under reduced pressure (30

mbar) at 110 °C for 1.5 h. Then, the reaction vessel was ﬁlled with

argon and the reaction mixture was heated under an argon

atmosphere to 150−160 °C for another 5 h. Upon completion,

formamide was distilled oﬀ under reduced pressure (30 mbar), the

residue was cooled to rt, and MeOH (20 mL) was added. The

resulting suspension was stirred at rt overnight. The crystalline

product was ﬁltered, washed with another MeOH (25 mL), and dried

1

white solid; mp 313−315 °C. H NMR (500 MHz, DMSO-d₆) δ

10.93 (s, 2H), 3.50−3.34 (m, 8H), 2.75−2.65 (m, 2H), 1.74−1.57

(m, 4H), 1.22−1.04 (m, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ

172.29, 60.99, 51.77, 26.52, 24.80. Anal. calcd for C₁₄H₂₀N₄O₄: C,

54.54; H, 6.54; N, 18.17. Found: C, 54.15; H, 6.28; N, 18.09. HRMS

(ESI⁺) calcd for (C₁₄H₂₀N₄O₄+ H)⁺m/z: 309.15573; found:

309.1568.

1

over P₂O₅. Yield: 44% as a white solid; mp 213−215 °C. H NMR

(500 MHz, DMSO-d₆) δ 11.07 (s, 1H), 10.96 (s, 1H), 3.48−3.38 (m,

4H), 3.38−3.26 (m, 4H, overlapped with water), 2.84−2.74 (m, 1H),

2.62 (dd, J = 13.3, 8.5 Hz, 1H), 2.28 (dd, J = 13.3, 4.7 Hz, 1H), 1.36

(dt, J = 14.5, 7.3 Hz, 1H), 1.24 (dt, J = 14.0, 6.9 Hz, 1H), 0.83 (t, J =

7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 172.22, 171.56,

59.34, 55.76, 55.34, 51.72, 21.90, 11.41. Anal. calcd for C₁₂H₁₈N₄O₄:

C, 51.06; H, 6.43; N, 19.85. Found: C, 50.93; H, 6.25; N, 19.74.

HRMS (ESI⁺) calcd for (C₁₂H₁₈N₄O₄+ H)⁺m/z: 283.14008; found:

283.1411.

Synthesis of 4,4′-(Butane-1,2-diyl)bis(piperazine-2,6-dione)

(9). Butane-1,2-diyl Dimethanesulfonate (26). Methanesulfonyl

chloride (55.93 g, 38 mL, 0.488 mol) was added dropwise to a

solution of 1,2-butanediol 25 (20 g, 13.91 mL, 0.222 mol) and

triethylamine (49.3 g, 68 mL, 0.488 mol) in CH₂Cl₂(250 mL) at 5

°C under an argon atmosphere. The reaction mixture was stirred at rt

for 48 h. Upon completion, the reaction mixture was ﬁltered and the

solid on the ﬁlter was washed with another CH₂Cl₂(100 mL). The

organic ﬁltrate was washed with saturated solution of NaHCO₃(1 ×

250 mL) and water (2 × 150 mL), dried over anhydrous Na₂SO₄, and

evaporated under reduced pressure. Yield: 76%. ¹H NMR (500 MHz,

CDCl₃) δ 4.86−4.79 (m, 1H), 4.40 (dd, J = 11.5, 3.0 Hz, 1H), 4.29

(dd, J = 11.5, 6.4 Hz, 1H), 3.09 (s, 3H), 3.08 (s, 3H) 1.84−1.76 (m,

2H), 1.05 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 80.30,

69.20, 38.68, 37.69, 24.37, 9.23.

1,2-Diazidobutane (27). Mixture of butane-1,2-diyl dimethanesul-

fonate 26 (39.4 g, 0.17 mol) and sodium azide (33 g, 0.51 mol) in

DMSO (300 mL) was heated under an argon atmosphere at 85 °C for

5 h. Upon completion, water (600 mL) was added and the resulting

solution was extracted with CH₂Cl₂(4 × 100 mL). The combined

organic extracts were washed with water (2 × 100 mL) and brine (1 ×

300 mL), dried over anhydrous Na₂SO₄, and evaporated. The

resulting 1,2-diazidobutane 27 was used in the next step without

further puriﬁcation. Yield: 60% as a yellowish oil. ¹H NMR (500

MHz, CDCl₃) δ 3.45−3.37 (m, 2H), 3.35−3.29 (m, 1H), 1.69−1.51

(m, 2H), 1.03 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

63.41, 54.42, 24.92, 10.27.

Tetra(tert-butyl) 1,2-Diaminobutane-N,N,N′,N′-tetraacetate

(28). Ten percent of Pd on activated carbon (4.2 g, 3.9 mmol, 5

mol %) was added to the solution of 1,2-diazidobutane 27 (11 g,

0.0785 mol) in MeOH (250 mL) under an argon atmosphere. The

reaction mixture was stirred in a hydrogen atmosphere (1 atm,

balloon) for 14 days (hydrogen was replenished every second day).

Upon completion, the reaction mixture was ﬁltered and the solvent

was evaporated under reduced pressure. The residue was dissolved in

the mixture of THF and H₂O, 5:1 (300 mL) and tert-butyl

bromoacetate (39.6 g, 29.7 mL, 0.203 mol) and potassium carbonate

(29.6 g, 0.214 mol) were added. The reaction mixture was reﬂuxed for

12 h. Upon completion, the reaction mixture was cooled down, the

majority of THF was evaporated and the residue was extracted with

EtOAc (2 × 100 mL). The combined organic extracts were washed

with water (2 × 200 mL), dried over anhydrous Na₂SO₄, and

Synthesis of 4,4′-(2-Methylpropane-1,2-diyl)bis(piperazine-

2,6-dione) (10). 2-Methyl-1,2-diaminopropane-N,N,N′,N′-tetraa-

cetonitrile (31).⁴⁴2-Methyl-1,2-diaminopropane 30 (4.4 g, 0.05 mol)

was cooled in the ice bath and aqueous formaldehyde (37%, 20.3 g,

0.25 mol), 50% aqueous H₂SO₄(35 g, 0.175 mol), and sodium

cyanide (12.25 g, 0.25 mol) in H₂O (22 mL) were subsequently

added dropwise under vigorous stirring. Then, the reaction mixture

was stirred and heated to 35 °C for 40 h. Upon completion, the

reaction mixture was cooled down and kept in the refrigerator

overnight. The resulting crude product was ﬁltered and recrystallized

1

from water. Yield: 19%. H NMR (500 MHz, DMSO-d₆) δ 3.92 (s,

4H), 3.86 (s, 4H), 2.59 (s, 2H), 1.16 (s, 6H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 118.28, 116.48, 59.26, 59.11, 44.13, 37.33, 22.43.

2-Methyl-1,2-diaminopropane-N,N,N′,N′-tetraacetic Acid (32).⁴⁴

Compound 31 (2.33 g, 9.5 mmol) and Ba(OH)₂·8H₂O (7.5 g, 23.8

mmol) were heated in water (30 mL) to reﬂux for 24 h. The reaction

mixture was cooled down, placed in the ice bath, and H₂SO₄(96%,

2.42 g, 23.8 mmol) was added dropwise. A hot mixture with

precipitated BaSO₄was ﬁltered and clear ﬁltrate was evaporated under

reduced pressure to obtain a yellow crystalline product. The ﬁnal

product 32 was recrystallized from water. Yield: 58% as a white solid.

¹H NMR (500 MHz, DMSO-d₆) δ 12.40 (br s, 1H), 3.56 (s, 4H),

3.45 (s, 4H), 2.65 (s, 2H), 0.98 (s, 6H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 175.42, 173.00, 61.91, 58.94, 56.40, 53.16, 23.38.

4,4′-(2-Methylpropane-1,2-diyl)bis(piperazine-2,6-dione) (10).

Compound 32 (1 g, 3.12 mmol) was mixed with formamide (4

mL), and the reaction mixture was heated under reduced pressure (30

mbar) at 110 °C for 1.5 h. Then, the reaction vessel was ﬁlled with

argon and the reaction mixture was heated at 150−160 °C under an

argon atmosphere for another 5 h. Upon completion, formamide was

distilled oﬀ under reduced pressure (30 mbar), the residue was cooled

to rt and MeOH (2 mL) was added. The solvent was evaporated, and

the product 10 was puriﬁed using column chromatography (CHCl₃/

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MeOH, 20:1). The isolated product was washed with MeOH and

dried. Yield: 18% as a white solid; mp 205−207 °C. H NMR (500

The organic layer was dried over anhydrous Na₂SO₄and evaporated

under reduced pressure.

1

MHz, DMSO-d₆) δ 11.09 (s, 1H), 11.08 (s, 1H), 3.41 (s, 4H), 3.34

(s, 4H), 2.44 (s, 2H), 0.99 (s, 6H). ¹³C NMR (126 MHz, DMSO-d₆)

δ 172.19, 171.88, 60.88, 57.35, 57.05, 50.02, 21.51. Anal. calcd for

C₁₂H₁₈N₄O₄: C, 51.06; H, 6.43; N, 19.85. Found: C, 51.17; H, 6.37;

N, 19.62. HRMS (ESI⁺) calcd for (C₁₂H₁₈N₄O₄+ H)⁺m/z:

283.14008; found: 283.1407.

The product 37a was puriﬁed using column chromatography

(mobile phase: hexane/EtOAc, 20:1 → 1:1). Yield: 76% (21.1 g) as a

1

white solid; R_f0.75 (hexane/EtOAc, 4:1); mp 79-80 °C. H NMR

(500 MHz, CDCl₃) δ 3.34 (s, 8H), 2.65−2.58 (m, 2H), 1.45 (s,

36H), 1.14 (d, J = 6.0 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ

171.56, 80.49, 61.92, 53.87, 28.11, 12.69.

Synthesis of meso-4,4′-(Butane-2,3-diyl)bis(piperazine-2,6-

dione) (11) and rac-4,4′-(Butane-2,3-diyl)bis(piperazine-2,6-

dione) (12). Butane-2,3-diyl Dimethanesulfonate (34). To a

solution of 2,3-butanediol 33 (mixture of isomers, 20 g, 0.22 mol)

and triethylamine (56 g, 77 mL, 0.55 mol) in CH₂Cl₂(600 mL),

methanesulfonyl chloride (63.55 g, 43 mL, 0.55 mol) was added

dropwise under an argon atmosphere. The reaction mixture was

stirred for 2 h. The reaction mixture was washed with water (2 × 400

mL), saturated NaHCO₃(1 × 400 mL), and again with water (1 ×

400 mL). The organic layer was dried over anhydrous sodium sulfate

and the solvent was evaporated under reduced pressure. Yield: 99% as

a yellowish oil. The ratio of meso-butane-2,3-diyl dimethanesulfonate

and rac-butane-2,3-diyl dimethanesulfonate was 2.5:1 according to ¹H

The product 37b was obtained as the by-product. Yield: 4% as a

white solid; R_f0.1 (hexane/EtOAc, 4:1); mp 45−47 °C. H NMR

1

(500 MHz, CDCl₃) δ 3.52 (d, J = 17.4 Hz, 4H), 3.42 (d, J = 17.4 Hz,

4H), 2.96−2.89 (m, 2H), 1.45 (s, 36H), 1.04 (d, J = 6.7 Hz, 6H). ¹³C

NMR (126 MHz, CDCl₃) δ 171.82, 80.47, 59.93, 54.27, 28.12, 12.99.

meso-2,3-Diaminobutane-N,N,N′,N′-tetraacetic Acid Dihydro-

chloride (38a). The solution of compound 37a (4.58 g, 8.41

mmol) and 36% aq. HCl (14.7 mL, 0.166 mol) in H₂O (25 mL) was

heated to 85 °C for 3 h. The solvent was evaporated and the resulting

product 38 was dried over P₂O₅. Yield: 99% as a white solid. ¹H NMR

(500 MHz, D₂O) δ 3.95 (d, J = 17.7 Hz, 4H), 3.72 (d, J = 17.7 Hz,

4H), 3.46−3.38 (m, 2H), 1.14 (d, J = 6.5 Hz, 6H). ¹³C NMR (126

MHz, D₂O) δ 172.86, 62.95, 54.22, 9.77.

1

NMR. Meso form; H NMR (500 MHz, CDCl₃) δ 4.94−4.86 (m,

rac-2,3-Diaminobutane-N,N,N′,N′-tetraacetic Acid Dihydro-

chloride (38b). The solution of compound 37b (0.85 g, 1.56

mmol) and 36% aq. HCl (2.76 mL, 31.2 mmol) in H₂O (10 mL) was

heated to 85 °C for 3 h. The solvent was evaporated and the resulting

2H), 3.09 (s, 6H), 1.44 (d, J = 6.6 Hz, 6H). ¹³C NMR (126 MHz,

CDCl₃) δ 78.93, 38.61, 15.88. Rac form; ¹H NMR (500 MHz,

CDCl₃) δ 4.81−4.74 (m, 0.78H), 3.08 (s, 2.32H), 1.47 (d, J = 6.2 Hz,

2.15H). ¹³C NMR (126 MHz, CDCl₃) δ 78.66, 38.75, 17.19.

1

product 38b was dried over P₂O₅. Yield: 99% as a white solid. H

2,3-Diazidobutane (35). The mixture of butane-2,3-diyl dimetha-

nesulfonate (mixture of isomers, 54 g, 0.22 mol) and sodium azide

(61 g, 0.94 mol) in DMF (550 mL) was heated to 85 °C for 6 h. The

reaction mixture was cooled down and diluted with water (1000 mL).

The mixture was extracted with Et₂O (5 × 200 mL). The combined

organic extracts were additionally washed with water (2 × 100 mL).

Most of the solvent was evaporated on a rotary evaporator at 40 °C

(water bath) and 300 mbar. As 2,3-diazidobutane 35 is volatile and

potentially explosive, it was not completely dried from the rest of the

solvents and was used without further puriﬁcation in the next step.

Yield: 32.7 g of a crude product as a yellowish oil. The ratio of meso-

2,3-diazidobutane and rac-2,3-diazidobutane was 2.8:1 according to

¹H NMR. Meso form; ¹H NMR (500 MHz, CDCl₃) δ 3.52−3.46 (m,

NMR (500 MHz, DMSO) δ 3.81 (d, J = 17.5 Hz, 4H), 3.67 (d, J =

17.6 Hz, 4H), 3.36−3.29 (m, 2H), 1.08 (d, J = 5.4 Hz, 6H). ¹³C

NMR (126 MHz, DMSO) δ 170.18, 60.16, 10.12.

meso-Tetramethyl 2,3-diaminobutane-N,N,N′,N′-tetraacetate

(39). Thionyl chloride (10 g, 6 mL, 0.084 mol) was added dropwise

to a suspension of meso-2,3-diaminobutane-N,N,N′,N′-tetraacetic acid

dihydrochloride 38a (3.28 g, 0.0083 mol) in CH₃OH (100 mL) at 5

°C. The reaction mixture was stirred at rt for 24 h. The volatiles were

evaporated under reduced pressure. The residue was partitioned

between EtOAc (100 mL) and a saturated solution of NaHCO₃(100

mL). The organic layer was separated, washed with water (2 × 70

mL), dried over anhydrous Na₂SO₄, and evaporated under reduced

1

pressure. Yield: 92% as a white solid; mp 82−84 °C. H NMR (500

2H), 1.29 (d, J = 6.5 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 60.91,

15.01. Rac form; ¹H NMR (500 MHz, CDCl₃) δ 3.45−3.40 (m,

0.7H), 1.31 (d, J = 6.5 Hz, 2.15H). ¹³C NMR (126 MHz, CDCl₃) δ

61.07, 15.90.

MHz, CDCl₃) δ 3.68 (s, 12H), 3.49 (s, 8H), 2.72−2.64 (m, 2H), 1.13

(d, J = 6.4 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 172.53, 61.96,

52.60, 51.48, 12.46.

meso-4,4′-(Butane-2,3-diyl)bis(piperazine-2,6-dione) (11). The

solution of compound 39 (2.3 g, 6.11 mmol) and formamide (2.75

g, 2.44 mL, 61.06 mmol) in dioxane (25 mL) was added to the

suspension of NaH (60% in mineral oil, 2. g, 72.5 mmol) in dioxane

(25 mL) at 10 °C under an argon atmosphere. The reaction mixture

was stirred at rt for 24 h. Then, the reaction mixture was mixed with

hexane (90 mL), stirred for 20 min, and ﬁltered. The precipitate was

carefully dissolved in water with crushed ice (70 mL) and the

resulting solution was treated with 36% aq. HCl to pH 4−5. The

formed precipitate was ﬁltered and dried to give 0.64 g of the product

11. The ﬁltrate was partially evaporated under reduced pressure and

the formed precipitate was ﬁltered to give an additional 0.27 g of

product 11. Yield: 53% (0.91 g) as a white solid; 55% (8.25 g) when

stared from 20.06 g of meso-tetramethyl 2,3-diaminobutane-

2,3-Butanediamine Dihydrochloride (36). 10% Pd on activated

carbon (11.7 g, 0.011 mmol, 5 mol %) was added to a 1000 mL ﬂask

and the ﬂask was ﬁlled with Ar. Then, the solution of the crude 2,3-

diazidobutane 35 (32.7 g) in MeOH (500 mL) was added. The

reaction mixture was stirred in a hydrogen atmosphere (hydrogen was

replenished once a week) for 1 month. The reaction was ﬁltered and

the ﬁltrate was saturated with HCl (gas). The reaction mixture was

kept in the refrigerator at 4 °C for 2 days. The resulting crystalline

product was ﬁltered and washed with Et₂O to obtain 13.25 g of meso-

2,3-butanediamine dihydrochloride as a yellow solid. Yield: 38% over

two steps from compound 34. The ratio of meso-2,3-butanediamine

and rac-2,3-butanediamine dihydrochlorides was 12:1 according to ¹H

NMR. Meso-2,3-butanediamine dihydrochloride; ¹H NMR (500

MHz, D₂O) δ 3.62−3.55 (m, 2H), 1.35 (d, J = 6.8 Hz, 6H). ¹³C

NMR (126 MHz, D₂O) δ 49.62, 14.36. rac-2,3-Butanediamine

dihydrochloride; ¹H NMR (500 MHz, D₂O) δ 3.72−3.65 (m,

0.18H), 1.30 (d, J = 6.7 Hz, 0.5H). ¹³C NMR (126 MHz, D₂O) δ

48.50, 12.17.

meso-Tetra(tert-butyl) 2,3-Diaminobutane-N,N,N′,N′-tetraace-

tate (37a) and rac-Tetra(tert-butyl) 2,3-Diaminobutane-

N,N,N′,N′-tetraacetate (37b). The solution of meso-2,3-butanedi-

amine dihydrochloride 36 (8.2 g, 0.051 mol), tert-butyl bromoacetate

(44.68 g, 33.6 mL, 0.23 mol) and K₂CO₃(42 g, 0.3 mol) in the

mixture of THF (250 mL) and H₂O (50 mL) was reﬂuxed for 24 h.

The majority of THF was evaporated under reduced pressure. The

residue was extracted with EtOAc (3 × 100 mL). The combined

organic extracts were additionally washed with water (2 × 100 mL).

1

N,N,N′,N′-tetraacetate; mp 315−317 °C (with decomp.). H NMR

(500 MHz, DMSO-d₆) δ 11.01 (s, 2H), 3.32 (dd, J = 16.4, 1.7 Hz,

4H), 3.22 (dd, J = 16.4, 1.6 Hz, 4H), 2.78−2.72 (m, 2H), 0.89 (d, J =

5.9 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ 172.27, 58.86, 51.87,

9.44. Anal. calcd for C₁₂H₁₈N₄O₄: C, 51.06; H, 6.43; N, 19.85. Found:

C, 50.84; H, 6.12; N, 19.64. HRMS (ESI⁺) calcd for (C₁₂H₁₈N₄O₄+

H)⁺m/z: 283.14008; found: 283.1407.

rac-4,4′-(Butane-2,3-diyl)bis(piperazine-2,6-dione) (12). Com-

pound 38b (1.3 g, 3.3 mmol) was mixed with formamide (5 mL),

and the reaction mixture was heated under reduced pressure (30

mbar) at 110 °C for 1.5 h. Then, the reaction vessel was ﬁlled with

argon and the reaction mixture was heated for another 5 h at 150−

160 °C under an argon atmosphere. Upon completion, formamide

was distilled oﬀ under reduced pressure (30 mbar), the residue was

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cooled to rt, and MeOH (5 mL) was added. The resulting suspension

was stirred at rt overnight. The crystalline product was ﬁltered,

washed with another MeOH (5 mL), and dried. Yield: 20% as a

brownish solid; mp 270−272 °C with decomposition. ¹H NMR (500

MHz, DMSO-d₆) δ 10.93 (s, 2H), 3.40 (d, J = 16.6 Hz, 4H), 3.32 (d,

J = 16.6 Hz, 4H, overlapped with H₂O), 2.85−2.80 (m, 2H), 0.86 (d,

J = 6.0 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ 172.25, 57.86,

51.58, 12.36. HRMS (ESI⁺) calcd for (C₁₂H₁₈N₄O₄+ H)⁺m/z:

283.14008; found: 283.1405.

Lonza, Belgium) in 75 cm²tissue culture ﬂasks (TPP, Switzerland) at

37 °C in a humidiﬁed atmosphere of 5% CO₂. For the cytotoxicity

assays, the cells were plated on 96-well plates (TPP, Switzerland) in a

density of 10 000 cells per well (100 000 cells/mL). The cells were

incubated with examined agents or their combinations for 72 h. The

proliferation was determined using the XTT assay. Brieﬂy, 25 μL of

XTT/phenazine methosulfate (PMS) solution in phosphate-buﬀered

saline (PBS) (1 mg/mL 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-

2H-tetrazolium-5-carboxanilide (XTT; Serva, Germany), 50 μM

phenazine methosulfate (PMS); ﬁltered through 0.22 μm ﬁlter) was

added to each well, and after 4 h of incubation at 37 °C (5% CO₂) the

absorbance was measured at 450 nm.

General Method for the Synthesis of Hydrolysis Products 1-

hp, 2-hp, 11-hp, and 12-hp. The mixtures of compound 1, 2, 11,

or 12 (0.7 mmol) and 1 M NaOH (1.75 mL, 2.5 equiv) in H₂O (4

mL) were stirred at rt for 24 h. The reaction mixtures were acidiﬁed

with Amberlyst 15 (hydrogen form) to pH 4. Amberlyst 15 was

ﬁltered oﬀ and the clear aqueous ﬁltrate was evaporated to dryness

under reduced pressure. The crude products 1-hp, 2-hp, 11-hp, or

12-hp were further dried under reduced pressure over P₂O₅and used

without further puriﬁcation.

Inhibition of Human Recombinant Topoisomerase II Iso-

forms. The TOP2 activity assay was performed using recombinant

human TOP2A or TOP2B (Inspiralis, U.K.) incubated with kDNA

isolated in house as described previously⁶⁶in a reaction buﬀer

containing 55 mM Tris−HCl (pH 7.5), 135 mM NaCl, 10 mM

MgCl₂, 5 mM DTT, 0.1 mM EDTA, 3 mM ATP, 100 μg/mL bovine

serum albumin, and 1/10 volume of each compound diluted in 10%

DMSO (ﬁnal DMSO concentration 1%) for 30 min at 37 °C. The

reaction was then stopped by the addition of gel loading buﬀer (equal

volume; 40% (w/v) sucrose, 10 mM EDTA, 0.5 mg/mL

bromophenol blue, 100 mM Tris−HCl (pH 8)), and the samples

were analyzed on a 1% agarose gel in TAE buﬀer (40 mM Tris base,

20 mM acetic acid, 1 mM EDTA, pH 8.3) at 3 V/cm for

approximately 2 h. Gels were stained with SYBR Safe (Thermo

Fisher Scientiﬁc) for 15 min and visualized using a Gel Doc EZ with

Image Lab software (Bio-Rad, Hercules, CA). The signal of the

treated samples was normalized to the value of the control (untreated

sample; 100%) present on the same gel. The normalized signal of

three independent measurements was then expressed as mean SD.

Western Blot Analysis of Topoisomerase II in Cardiomyo-

cytes. The NVCMs plated on 24-well plates were incubated with

DEX or its analogues for 24 h, washed with PBS, and lysed in 75 μL of

lysis buﬀer (2% sodium dodecyl sulfate (SDS) in 50 mM Tris−HCl

(pH 6.8), 1 mM EDTA). Total protein in the lysate was assessed by

the bicinchoninic acid (BCA) protein assay (Cyanagen, Italy), and 10

μg of protein in sample buﬀer (0.05% bromophenol blue, 0.1 M DTT,

10% glycerol, 50 mM Tris−HCl (pH 6.8)) was loaded into each lane

of 1 mm/15 well 7.5% TGX Mini-PROTEAN Stain-Free gel (Bio-

Rad). After the separation (100 V constant, approx. 90 min), the

proteins were electrically transferred using Trans-Blot Turbo (Bio-

Rad; 10 min, 2.5 A const, approx. 15−20 V) onto a nitrocellulose

membrane (Bio-Rad). Rabbit monoclonal anti-TOP2A/B (EPR

3577; Abcam, U.K.; dilution 1:2000) and horseradish peroxidase-

conjugated goat anti-rabbit IgG (Goat F(ab′)2 Anti-Rabbit IgG

F(ab′)2 (horseradish peroxidase (HRP)) preadsorbed (ab6112),

Abcam, U.K.; dilution 1:5000) were used for immunostaining with

enhanced chemiluminescence detection (Clarity, Bio-Rad). Densito-

metric quantiﬁcation was performed using Image Lab software (Bio-

Rad) with normalization to total protein detected using stain-free

technology (Bio-Rad). The integrated signal of the treated samples

was further normalized to the respective control (untreated sample;

100%) present on the same membrane. The normalized signal of four

1-hp. MS (ESI⁺): m/z (%) = 323.07 (100) [M + H]⁺; 345.00 (50)

[M + Na]⁺; MS (ESI⁻): m/z (%) = 321.2 (45) [M − H]⁻; 343.20

(100) [M − 2H + Na]⁻.

2-hp. MS (ESI⁺): m/z (%) = 278.07 (100) [M + H]⁺; 300.09 (25)

[M + Na]⁺.

11-hp. MS (ESI⁺): m/z (%) = 318.93 (100) [M + H]⁺; 341.07

(15) [M + Na]⁺.

12-hp. MS (ESI⁺): m/z (%) = 318.92 (100) [M + H]⁺; 341.08

(17) [M + Na]⁺.

Toxicities of Studied Compounds on the Neonatal Rat

Cardiomyocytes and Their Ability to Prevent DAU-Induced

Cardiotoxicity. The NVCMs were isolated from 1- to 3-day-old

Wistar rats. Brieﬂy, the hearts were minced in a buﬀer (1.2 mM

MgSO₄·7H₂O; 116 mM NaCl; 5.3 mM KCl; 1.13 mM NaH₂PO₄·

H₂O; 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

(HEPES)) on ice, then serially digested (20 min, six repetitions) at 37

°C with collagenase type II (60 U/mL, Gibco). After 2 h preplating

on 150 mm Petri dish per approx. 20 hearts to minimize nonmyocyte

contamination, the cells were plated on 24-well gelatin-coated plates

in a density of 400 000 cells per well. NVCMs were cultured at 37 °C

and 5% CO₂in the Dulbecco’s modiﬁed Eagle’s medium (DMEM)/

F12 culture medium supplemented with 10% horse serum, 5% fetal

bovine serum (FBS), 20 mM sodium pyruvate, 50 U/mL penicillin,

and 50 μg/mL streptomycin. Freshly isolated NVCMs were left for 40

h to attach and form a culture of spontaneously beating

cardiomyocytes, then the medium was changed to DMEM/F12

medium supplemented with 5% fetal bovine serum, 20 mM sodium

pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin. For the

experiments, the medium was changed to serum- and pyruvate-free

DMEM/F12 with 50 U/mL penicillin and 50 μg/mL streptomycin.

In this medium, the cells were maintained to the end of the

experiment. The myocytes were pretreated with DEX or the novel

analogues for 3 h, and then coincubated with DAU for 3 h or

incubated without DAU (own toxicity of compounds). After the DAU

treatment period, the culture medium was changed for the drug-free

DMEM/F12 with 50 U/mL penicillin and 50 μg/mL streptomycin

for the 48 h follow-up. Subsequently, the sample of the culture

medium was taken from each well for the assessment of the lactate

dehydrogenase (LDH) activity; the control wells were treated with

lysis buﬀer (0.1 M potassium phosphate, 1% Triton X-100, 1 mM

dithiothreitol (DTT), 2 mM EDTA, pH 7.8, 15 min in rt) for total

LDH. All of the samples were analyzed immediately in Tris−HCl

buﬀer (pH 8.9) containing 35 mM lactic acid and 5 mM NAD⁺. The

rate of NAD⁺reduction was monitored spectrophotometrically at 340

nm for 2 min. The slope of the linear region was calculated, and the

data were expressed as the percent of total LDH.

independent measurements was then expressed as mean

SD.

Precision Plus Protein Standards Kaleidoscope (Bio-Rad) was used as

a molecular weight marker.

Molecular Modeling and Dynamic Simulation Studies.

Molecular docking was used for binding poses calculations. The

three-dimensional (3D) structure ligands were built using Open

Babel, v.2.3.2,⁶⁷and optimized using Avogadro, v.1.2.0 using the

general AMBER force ﬁeld (GAFF).⁶⁸They were converted into

pdbqt-format using Open Babel, v. 2.3.2. The yeast Top2B structure

was gained from the RCSB database (PDB ID: 1QZR, the crystal

structure of the ATPase region of Saccharomyces cerevisiae topoisomer-

ase II bound to ICRF-187 (dexrazoxane), resolution 1.90 Å) and

prepared for docking by the function DockPrep of the software

Chimera, v.1.14,⁶⁹and by MGLTools, v.1.5.4.⁷⁰The docking

calculation was performed using Vina, v.1.1.2,⁷¹as semiﬂexible with

a ﬂexible ligand and a rigid receptor.

Assessments of the Antiproliferative Activity. The HL-60 cell

line, derived from a single patient with acute promyelocytic

leukemia,⁶⁵was purchased from the American Type Culture

Collection. The cells were cultured in Roswell Park Memorial

Institute (RPMI)-1640 supplemented with 10% fetal bovine serum

(FBS), 50 U/mL penicillin and 50 μg/mL streptomycin (all from

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J. Med. Chem. 2021, 64, 3997−4019

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

The docking poses of DEX, meso-11, (R,R)-12, and (S,S)-12 were

improved by the MD simulation. The receptor structure was prepared

by the software Chimera. The best-scored docking pose was taken as

the initial state for MD. The force-ﬁeld parameters for ligands were

assessed using Antechamber,⁷²v.20.0 using GAFF.⁷³MD simulations

were carried out using Gromacs, v. 2018.1.⁷⁴The complex receptor−

ligand was solvated in the periodic water box using the TIP3P

model.⁷⁵The system was neutralized by adding Na⁺and Cl⁻ions to a

concentration of 10 nM. The system energy was minimalized and

equilibrated in a 100 ps isothermal-isochoric NVT and then a 100 ps

isothermal-isobaric NPT phase. Then, a 10 ns MD simulation was run

at a temperature of 300 K. The molecular docking and MD results

were 3D visualized using The PyMOL Molecular Graphics System,

Iuliia Melnikova − Faculty of Pharmacy in Hradec Králové,

Charles University, 50005 Hradec Králové, Czech Republic

̌

́

Jan Korábecny − Biomedical Research Center, University

Hospital Hradec Kralove, 50005 Hradec Králové, Czech

Republic; Faculty of Military Health Sciences, University of

Defence, 50005 Hradec Králové, Czech Republic

̌

Tomás Kucera − Faculty of Military Health Sciences,

University of Defence, 50005 Hradec Králové, Czech

Republic; orcid.org/0000-0001-6650-1738

́

Eduard Jirkovsky − Faculty of Pharmacy in Hradec Králové,

Charles University, 50005 Hradec Králové, Czech Republic

Lucie Nováková − Faculty of Pharmacy in Hradec Králové,

Charles University, 50005 Hradec Králové, Czech Republic

̈

version 2.0.6, Schrodinger, LLC.

Displacement of Iron Ions from the Complex with DAU. The

rates of Fe³⁺displacement from the DAU-Fe³⁺complex by DEX and

its selected analogues 1, 2, and 11 and their degradation products

were measured using a modiﬁed spectrophotometric assay described

previously.⁷⁶A complex of DAU and Fe³⁺(3:1 in 15 mM HCl) was

added to the reaction buﬀer (50 mM Tris/150 mM KCl, pH = 7.4, rt)

in one well of a 96-well microplate to yield a ﬁnal concentration of 45

μM DAU and 15 μM Fe³⁺. The absorbance was measured at λ = 600

nm using a Tecan Inﬁnite 200 plate reader. After 3 min of

equilibration, the studied substances (or the reference chelators

EDTA and SIH, all 100 μM) have been added and the absorbance

was measured for further 10 min.

̌

Hana Bavlovic Piskácková − Faculty of Pharmacy in Hradec

Králové, Charles University, 50005 Hradec Králové, Czech

Republic

̌

Josef Skoda − Faculty of Pharmacy in Hradec Králové,

Charles University, 50005 Hradec Králové, Czech Republic

Martin Sterba − Department of Pharmacology, Faculty of

Medicine in Hradec Králové, Charles University, 50003

Hradec Králové, Czech Republic

Caroline A. Austin − Biosciences Institute, Faculty of Medical

Sciences, Newcastle University, Newcastle upon Tyne NE2

4HH, United Kingdom

Tomás Simunek − Faculty of Pharmacy in Hradec Králové,

̊

Charles University, 50005 Hradec Králové, Czech Republic

̌

ASSOCIATED CONTENT

■

sı

* Supporting Information

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02157

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02157.

Determination of ICRF-193 (11) in racemate 12, copies

of NMR and HRMS spectra of all ﬁnal compounds

(Figures S1−S6) (PDF)

Notes

The authors declare no competing ﬁnancial interest.

PDB ﬁle for DEX docking (PDB)

ACKNOWLEDGMENTS

■

PDB ﬁle for compound 10 docking (PDB)

PDB ﬁle for compound 11 docking (PDB)

PDB ﬁle for compound (R,R)-12 docking (PDB)

PDB ﬁle for compound (S,S)-12 docking (PDB)

Molecular formula strings and associated biological data

(CSV) (CSV)

PDB ﬁle for DEX MD simulation (PDB)

PDB ﬁle for compound meso-11 MD simulation (PDB)

PDB ﬁle for compound (S,S)-12 MD simulation (PDB)

PDB ﬁle for compound (R,R)-12 MD simulation (PDB)

This study was supported by the Czech Science Foundation

̌

(GACR) 18-08169S and 21-16195S and by the project

INOMED (no. CZ.02.1.01/0.0/0.0/18_069/0010046) co-

funded by the ERDF. J.K., V.S., and J.S. thank Charles

University (projects GAUK 246219, SVV 260 549, and SVV

260 550). J.K. and T.K. acknowledge support by MH CZ-DRO

(University Hospital Hradec Kralove, Nr. 00179906) and by

long-term development plan (Faculty of Military Health

Sciences). The authors would like to thank Prof. Julius

̌

Lukes and Eva Kriegová for the culture of Crithidia fasciculata.

ABBREVIATIONS

■

AUTHOR INFORMATION

Corresponding Authors

■

ANT, anthracycline; DAU, daunorubicin; DEX, dexrazoxane;

DMSO, dimethyl sulfoxide; DMEM, Dulbecco’s modiﬁed

Eagle’s medium; DTT, dithiothreitol; EtOAc, ethyl acetate;

HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid;

LDH, lactate dehydrogenase; NVCMs, neonatal rat ventricular

cardiomyocytes; PMS, phenazine methosulfate; ROS, reactive

oxygen species; SAR, structure−activity relationships; SIH,

salicylaldehyde isonicotinoyl hydrazone; THF, tetrahydrofur-

an; TOP2A, topoisomerase IIα; TOP2B, topoisomerase IIβ;

XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-

lium-5-carboxanilide

Anna Jirkovská − Faculty of Pharmacy in Hradec Králové,

Charles University, 50005 Hradec Králové, Czech Republic;

Email: jirkovan@faf.cuni.cz

Jaroslav Roh − Faculty of Pharmacy in Hradec Králové,

Charles University, 50005 Hradec Králové, Czech Republic;

orcid.org/0000-0003-4698-8379; Email: jaroslav.roh@

faf.cuni.cz

Authors

Galina Karabanovich − Faculty of Pharmacy in Hradec

Králové, Charles University, 50005 Hradec Králové, Czech

Republic

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Structure-Activity Relationship Study of Dexrazoxane Analogues Reveals ICRF-193 as the Most Potent Bisdioxopiperazine against Anthracycline Toxicity to Cardiomyocytes Due to Its Strong Topoisomerase IIβ Interactions

DOI: 10.1021/acs.jmedchem.0c02157

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