Novel ametantrone-amsacrine related hybrids as topoisomerase IIβ poisons and cytotoxic agents

Article abstract of DOI:10.1002/ardp.201400111

The precise definition of the structural requirements for effective topoisomerase II poisoning by drug molecules is still an elusive issue. In the attempt to better define a pharmacophoric pattern, we prepared several conjugates combining the chemical features of two well-known topoisomerase II poisons, amsacrine and ametantrone. Indeed, an appropriate fusion geometry, which entails the anthracenedione moiety of ametantrone appropriately connected to the methanesulfonamidoaniline side chain of amsacrine, elicits DNA-intercalating properties, the capacity to inhibit the human topoisomerase IIβ isoform, and cytotoxic activity resembling that of the parent compounds. In addition, the properties of the lateral groups linked to the anthracenedione group play an important role in modulating DNA binding and cell cytotoxicity. Among the compounds tested, 10, 11, and 19 appear to be promising for further development. The structural requirements for cleavable complex stabilization by topoisomerase II poisons are still poorly understood. To better define a pharmacophoric pattern, several conjugates were prepared combining the chemical features of two well-known topoisomerase II poisons, amsacrine and ametantrone. Neither the planar chromophore nor the side chains alone are able to direct the strength of the interaction between the small molecule and the macromolecular complex.

Full text of DOI:10.1002/ardp.201400111

7
28
Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
Full Paper
Novel Ametantrone–Amsacrine Related Hybrids as
Topoisomerase IIb Poisons and Cytotoxic Agents
1
2
1
1
1
Giuseppe Zagotto , Alessandra Gianoncelli , Claudia Sissi , Cristina Marzano , Valentina Gandin ,
1
3
1
1
Riccardo Pasquale , Giovanni Capranico , Giovanni Ribaudo , and Manlio Palumbo
1
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
Chemistry for Technologies Laboratory, University of Brescia, Brescia, Italy
Department of Biochemistry G. Moruzzi, University of Bologna, Bologna, Italy
2
3
The precise definition of the structural requirements for effective topoisomerase II poisoning by drug
molecules is still an elusive issue. In the attempt to better define a pharmacophoric pattern, we
prepared several conjugates combining the chemical features of two well-known topoisomerase II
poisons, amsacrine and ametantrone. Indeed, an appropriate fusion geometry, which entails the
anthracenedione moiety of ametantrone appropriately connected to the methanesulfonamidoaniline
side chain of amsacrine, elicits DNA-intercalating properties, the capacity to inhibit the human
topoisomerase IIb isoform, and cytotoxic activity resembling that of the parent compounds. In
addition, the properties of the lateral groups linked to the anthracenedione group play an important
role in modulating DNA binding and cell cytotoxicity. Among the compounds tested, 10, 11, and 19
appear to be promising for further development.
Keywords: Ametantrone / Amsacrine / DNA / Intercalation / Topoisomerase II
Received: March 19, 2014; Revised: May 7, 2014; Accepted: May 28, 2014
DOI 10.1002/ardp.201400111
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
cleaving a phosphodiester bond by a transesterification
reaction in which an enzymatic tyrosine plays an essential
role; then, they force the passage of another duplex through
the break and, as a final step, rejoin the breaks by the
reverse transesterification reaction. The precise mechanism of
the reaction steps is still largely unknown as regards the
stereochemistry [4, 5] and, mainly, the time course [6]. Several
drugs, among which anthracenediones and acridines
deserve special mention, are reported to interfere with the
covalent DNA–enzyme intermediate, called the “cleavage
complex” [7–9], stabilising it and triggering apoptotic cell
death [10–12]. On the other hand, an extensive and irrefutable
description of the drug–DNA interaction motif has not
been provided yet, and molecular modeling strategies are
still widely used to investigate a realistic model [12b]. In
addition to this, despite the presence of several drugs reported
to be effective against the complex, the quest for new
candidates is still open to provide a consistent aid both in the
antibacterial and in the anticancer field [12c]. To support this
research, structural informations concerning the ligand and,
The concept of the pharmacophore as “the ensemble of steric
and electronic features necessary to ensure the optimal
supramolecular interactions with a specific biological target
and to trigger (or block) its biological response” [1, 2] urges the
medicinal chemist to identify the essential features that favor
selective drug-receptor recognition. In this connection, the
case of the enzyme topoisomerase II [3] represents an
interesting example where a “transient” pharmacophoric
receptor is generated at the interface between a nucleic acid
and a protein, when the latter is covalently linked to the
former during the catalytic cycle. DNA topoisomerases
regulate DNA supercoiling in all living organisms. Type II
enzymes introduce double-strand breaks in a DNA duplex
Correspondence: Dr. Giuseppe Zagotto, Department of Pharmaceutical
and Pharmacological Sciences, University of Padova, Via Marzolo, 5,
3
5131 Padova, Italy.
E-mail: giuseppe.zagotto@unipd.it
Fax: þ39 049 827 5366
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
Ametantrone–Amsacrine Related Hybrids
729
in particular, the pharmacophore are of a primary relevance.
Given the importance of understanding the molecular
mechanism(s) by which topoisomerase II poisons produce
anticancer effects [13], we considered two currently used
drugs, ametantrone and amsacrine. Ametantrone interacts
with the double helix of the deoxyribonucleic acid likely by
positioning the two side chains in the DNA major groove and
the anthraquinone chromophore intercalated into the double
helix with its long axis almost perpendicular to the long axis
of base pairs [14–19]. For amsacrine, evidence shows that the
characteristic of amsacrine, was linked in position 6. Then,
assuming that the anthracene-9,10-dione major axis is
perpendicular to the bases major axis as it is in mitoxantrone
and anthracycline with DNA [23, 24], the new compounds
should interact with DNA positioning the twin chains
within the major groove and the 4-methanesulfonamidoani-
line substituent in the minor groove, thus reinforcing
complex formation and, possibly, exhibiting effective poison-
ing of topoisomerase II; while if, as stated by Wu [22a], the
anthracene-9,10-dione major axis essentially parallels the
bases major axis we should observe a decrease in activity in
respect to the one of the parent compounds [25]. If so the
interactions of side chains with the polynucleotide and
the enzyme, and not the stacking of the anthraquinone with
the bases, give the major stabilizing contribution to the
interaction.
4-methanesulfonamidoaniline chain lies in the minor groove
tilted approximately 70° with respect to the base pair plane
and the acridine is intercalated with its long axis parallel to
the long axis of base pairs with the heterocyclic nitrogen in
the center of the major groove [20, 21]. Despite these
observations, eukaryotic topoisomerase II-DNA–drug struc-
tures are still being investigated and some models were
recently described [22a]. In this paper, while the position of Results
amsacrine relative to the nucleic acid confirms previous data,
mitoxantrone and ametantrone show the anthraquinone
parallel to the long axis of base pairs in contrast with previous
reports [22b]. To better understand the relevance of the side
chains relative to the one of the chromophore in the
interaction of the drug with the polynucleotide we decided
to design hybrid compounds derived from the combination of
side chains from ametantrone with amsacrine moieties or
their analogs maintaining the anthraquinone scaffold. This
project can be summarized as in Fig. 1.
Chemistry
Synthesis
The chemistry was quite straightforward: the starting
synthon, 1,4-dihydroxy-6-carboxyanthracene-9,10-dione, was
synthesized from trimellitic anhydride and hydroquinone by
a known Friedel Crafts followed by a mild air oxidation [26] as
shown in Scheme 1.
Subsequently, the two phenolic groups were protected, the
carboxy group activated as chloride and 4-methanesulfon-
amidoaniline or 2-methoxy-4-methanesulfonamidoaniline
added to obtain the desired amide. After deprotecting the
phenolic groups we obtained the intermediate 4 or 5,
respectively.
Two identical side chains were attached, as in ametantrone,
in positions 1 and 4 of the anthracene-9,10-dione planar
chromophore while the 4-methanesulfonamidoaniline sub-
0
stituent (with and without a methoxy group in position 2 ),
This was reduced to the dihydroderivative, then reacted
with the proper primary amine, and finally mildly oxidized to
give the compounds 6–19. The side chains were selected
according to literature-derived criteria [23–25] and keeping
the ametantrone side chain (compounds 14–15) and its
oxygen homologous (compounds 12–13) since the basic
nitrogen forms an important electrostatic bond between
its protonated form and the phosphate anion in the DNA
backbone (Scheme 2).
The purity of all final compounds was determined by HPLC
as reported in the Supplementary Material. The NMR spectra
to check for particular constraints in the side chains positions
were also run; an example is reported in the Supplementary
Material.
Cytotoxic activity
Ametantrone–amsacrine hybrids were tested for their cyto-
toxic activity toward four human tumor cell lines including
examples of ovarian (2008) and breast (MCF-7) cancer,
melanoma (A375), and promyelocytic leukemia (HL-60).
Figure 1. Conjugation of ametantrone and amsacrine structural
features to give an hybrid derivative.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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G. Zagotto et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
Scheme 1. Synthesis of compounds 1–5. Reagents and conditions: (a) AlCl
c) SOCl , DMF, reflux, 3 h; (d ) 4-methanesulfonamidoaniline, triethylamine, CH
amidoaniline, triethylamine, CH Cl , reflux, 1.5 h; (e) K CO 5% solution in water, 80°C, 2 h.
3
, NaCl, 150°C, 7 h; (b) acetic anhydride, H
2 4
SO , reflux, 3 h;
(
2
1
2
Cl , reflux, 1.5 h; (d ) 2-methoxy-4-methanesulfon-
2
2
2
2
2
3
Cytotoxicity was evaluated by means of MTT test after 72 h of
treatment with increasing concentrations of the tested
compounds. For comparison purposes, cytotoxicities of
ametantrone and amsacrine were evaluated under the same
experimental conditions. IC50 values, calculated from dose-
survival curves, are reported in Table 1.
Among all newly synthesized ametantrone–amsacrine
hybrids, derivatives 9, 12, 13, 17, and 18 proved to be
completely ineffective against all cancer cell lines whereas
compounds 7 and 8 showed a negligible in vitro antiprolifer-
ative activity. Compounds 6, 14, 15, and 16 as well as the
intermediates 4 and 5 showed a detectable cytotoxic potency,
with IC50 values more than 2 orders of magnitude higher than
those of ametantrone and roughly 20-fold higher than those
recorded with amsacrine. Conversely, compounds 10, 11, and
19, all three bearing tertiary non-cyclic amine side chain,
despite retaining a lower antiproliferative activity than
ametantrone, showed a cytotoxicity response similar to that
of amsacrine with IC50 values in the low micromolar range.
Cytotoxic activity of the most promising derivatives 10, 11,
and 19 was also assessed against two additional human
cancer cell lines, LoVo and LoVo MDR, the latter possessing a
MultiDrug Resistant (MDR) phenotype. It is well known that
acquired MDR, whereby cells become refractory to multiple
drugs, poses a most important challenge to the success of
anticancer chemotherapy. The resistance of LoVo MDR cells to
doxorubicin, a drug belonging to the MDR spectrum, is
associated with an overexpression of the multi-specific drug
transporters, such as the 170 kDa P-glycoprotein (P-gp) [26].
Scheme 2. Synthesis of compounds 6–19. Reagents and con-
ditions: (a) Na , N , H O, 70–80°C; (b) NH R, N , H O, 100°C,
6 h; (c) O , H O, 25°C, 6 h.
2
S
2
O
2 4
2
2
2
2
2
1
2
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Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
Ametantrone–Amsacrine Related Hybrids
731
Table 1. Cytotoxicity of the new derivatives on cancer cell lines.
IC50 [mM] ꢀ SD
extensive drug aggregation processes prevented a precise
quantification of the binding affinity. Qualitatively, com-
pound 10 appeared to exhibit the most effective binding to the
nucleic acid and no precipitation was observed in the range of
measured concentrations, so it was considered to study its
interaction with DNA. In addition, the same compound was
able to intercalate into DNA at micromolar concentration as
shown by the unwinding assays performed in the presence of
the enzyme topoisomerase I. In fact, the effects of intercalation
antagonize enzyme-induced relaxation of the supercoiled
plasmid. An example is reported in Fig. 2. Indeed, compound
10 shows interference with the relaxation process at concen-
trations between 1 and 2 mM, whereas compound 14 along with
the other congeners (not shown) is not effective up to 20 mM.
Compounds
2008
A375
MCF-7
HL60
4
5
6
7
8
9
54.23 ꢀ 2.12
>100
64.34 ꢀ 2.23 81.32 ꢀ 2.12
47.54 ꢀ 1.23 54.56 ꢀ 2.11 47.45 ꢀ 2.23 67.54 ꢀ 3.11
66.43 ꢀ 2.21
>100
57.98 ꢀ 3.32 69.65 ꢀ 2.54
79.43 ꢀ 1.23 84.43 ꢀ 1.98 96.52 ꢀ 3.43 89.22 ꢀ 3.54
73.71 ꢀ 2.11
>100
>100
>100.
85.43 ꢀ 3.33
>100
>100
>100
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
6.33 ꢀ 2.53 5.28 ꢀ 1.33 6.71 ꢀ 2.02 4.34 ꢀ 0.99
2.56 ꢀ 1.85 2.01 ꢀ 1.12 3.33 ꢀ 1.75 1.06 ꢀ 0.76
>100
>100
>100
>100
>100
>100
>100
>100
>100
63.33 ꢀ 3.25
55.43 ꢀ 2.12 71.14 ꢀ 1.86
42.22 ꢀ 2.87 51.21 ꢀ 3.64 61.31 ꢀ 2.93 63.01 ꢀ 2.23
59.11 ꢀ 3.60
>100
>100
>100
69.68 ꢀ 2.46 72.32 ꢀ 2.11
>100
>100
>100
>100
>100
Topoisomerase II poisoning
>100
Topoisomerase II poisoning tests showed that only derivative 10
was a good inhibitor of the topoisomerase II activity, producing
100% inhibition at a concentration below 5 mM (same as
amsacrine), the others not being effective up to 25 mM.
Compound 10 was further investigated for activity against
the human enzyme isoforms a and b. An in vitro DNA cleavage
assay was performed and the fraction of linearized plasmid
DNA was quantified in the presence of different drug
concentrations.
We compared the test compound to the known topoisom-
erase poison VM26 as a positive control and to derivative 4,
lacking the ametantrone side chains, as a negative control.
The latter compound had no effect on topoisomerase IIb
(Fig. 3A, lanes 3–5). On the opposite, derivative 10 has a clear
effect producing 30–40% of the linearized form of plasmid
DNA. Moreover, 10 appears to be remarkably more potent
than VM26 since 1 mM of the former produces an effect similar
to 50 mM of the latter (compare lane 2 to lane 6 in Fig. 3A).
Much lower DNA cleavage was detected when using the
topoisomerase IIa isoform (not shown). Hence, compound 10
shows selectivity for the b isofom of the enzyme.
9.31 ꢀ 2.11 5.25 ꢀ 2.53 7.35 ꢀ 2.63 6.17 ꢀ 2.43
Ametantrone 0.03 ꢀ 0.01 0.99 ꢀ 0.12 0.51 ꢀ 0.23 0.36 ꢀ 0.09
Amsacrine
4.22 ꢀ 0.76 2.32 ꢀ 0.69 4.53 ꢀ 0.94 1.15 ꢀ 0.64
IC50 values were calculated by four parameter logistic model (p < 0.05).
4
Cells (3–8 ꢁ 10 /mL) were treated for 72 h with increasing
concentrations of the tested compounds. SD, standard deviation.
Cytotoxicity was assessed after a 48-h exposure by the MTT
test. Cross-resistance profiles were evaluated by means of the
resistance factor (RF), which is defined as the ratio between
IC50 values calculated for resistant cells and those arising
from chemosensitive ones (Table 2).
Derivatives 10, 11, and 19 showed a similar pattern of
response across the parental (LoVo cells) and the resistant
(
LoVo MDR) subline and allowed the calculation of RF values
about 25 times lower than that obtained with doxorubicin,
suggesting that these new derivatives may not be deemed as
potential MDR substrates.
DNA binding mode
Spectroscopic data indicated that the tested derivatives
efficiently interact with calf thymus DNA. Unfortunately,
Table 2. MDR cross-resistance profiles.
LoVo IC50
[mM] ꢀ SD
LoVo MDR IC50
Compounds
[mM] ꢀ SD
RF
1
1
1
0
1
9
4.34 ꢀ 1.12
3.42 ꢀ 1.01
5.23 ꢀ 1.44
0.74 ꢀ 1.23
1.48 ꢀ 0.33
5.32 ꢀ 1.15
4.43 ꢀ 1.65
5.32 ꢀ 2.36
5.77 ꢀ 2.65
43.46 ꢀ 3.32
1.2
1.3
1.0
7.8
29.4
Ametantrone
Doxorubicin
Figure 2. Topoisomerase I relaxation reactions of supercoiled
pBR322 performed in the presence of increasing concentrations of
compounds 10 and 14 (lane 1, 1.25 mM; lane 2, 2.5 mM; lane 3,
5 mM; lane 4, 10 mM; and lane 5, 20 mM). S, supercoiled pBR 322;
C, relaxed pBR 322 obtained incubating the plasmid DNA with the
enzyme in the absence of drug.
IC50 values were calculated by four parameter logistic model (p <
4
0
.05). Cells (5 ꢁ 10 /mL) were treated for 48 h with increasing
concentrations of tested compounds. Cytotoxicity was assessed by
MTT test. SD, standard deviation.
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G. Zagotto et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
and mechanism of action [27–31] and so contribute to a better
definition of the yet ill-defined pharmacophore of topoisom-
erase II poisons and possibly identify novel useful leads. Two
intercalating drugs, ametantrone and amsacrine, were
chosen since the details of their molecular mechanism of
action are still only partially understood, and their modes of
DNA binding foresee a different intercalation geometry and
location of the side-chain groups [17–19, 31], hence generat-
ing different contact points at the nucleic acid–enzyme
interface. The hybrid derivatives maintained the anthraqui-
none ring system, showing side chains of the amsacrine
and ametantrone type bound at positions 2, 5, and 8 of the
aromatic intercalating chromophore.
Preliminary modeling studies using a simplified pharma-
cophore consensus strategy [32, 33] suggest that the hybrids
would fit within the cleavable complex. Moreover, the
amide bond connecting the 4-methanesulfonamidoaniline
substituent to the anthraquinone appears to represent a
reasonable linker. The hybrid compounds were assembled
by a convergent synthesis: the anthraquinone chromophore
and the 4-methanesulfonamidoaniline were prepared first
and then joined; the 1 and 4 side chains were introduced
into the anthraquinone nucleus using a known reaction
strategy.
Figure 3. DNA cleavage results. (A) Lanes are: 1, top IIb; 2, top
IIb þ 50 mM VM26; 3, top IIb þ 1 mM 4; 4, top IIb þ 10 mM 4; 5, top
IIb þ 100 mM 4; 6, top IIb þ 1 mM 10; 7, top IIb þ 10 mM 10; 8, top
IIb þ 100 mM 10; 9, supercoiled pBR322 DNA; 10, HindIII-linearized
pBR322 DNA. (B) DNA cleavage levels as determined by analyses
of gels such as the one shown in panel (A).
Effective poisoning was also observed with compounds 11
and 19, which did not cause substantial DNA precipitation in
the presence of the enzymes.
To assess the contribution of the planar part of the molecule
with respect to the side chains the novel hybrids were tested
for their DNA-binding efficiency, topoisomerase II inhibition,
and cell cytotoxicity against a panel of cancer cell lines. The
results showed that a precise combination of the side chain
substituents can give effective compounds despite their lower
cytotoxicity compared to the parent compounds ametantrone
and amsacrine as shown in Table 1. Indeed, derivatives 10, 11,
and 19 were able to poison topoisomerase II and kill cancer
cells. In fact, a clear-cut connection exists between DNA
binding, topoisomerase II poisoning, and antiproliferative
activity, which suggests that the new drugs are characterized
by the same mechanism of action as the parent drugs. If these
findings confirm that the hybrid strategy can be successful to
obtain active congeners, the overall outcome shown by the
new compounds points to the requirement of strict spatial
relationships to elicit a biological response. SAR analysis
shows that a protonable group must be present at positions 1
Next, we determined the ability of the new drugs to inhibit
the catalytic activity of both human topoisomerase II isoforms
by a relaxation assay (Fig. 4).
In this assay, similar catalytic amounts of the two human
isozymes were used, corresponding to about fivefold less
proteins than in the DNA cleavage assay. Again, 10 was
substantially more active than 4 on topoisomerase IIb.
Moreover, the latter isoform was more sensitive to 10 than
the topoisomerase IIa form. Altogether, the biochemical tests
show that 10 is an effective poison of human topoisomerase II
with selective recognition of the b form.
Discussion
A set of new molecules was designed, synthesized, and tested
starting from the idea of rationally combining the features of
two chemical structures sharing common molecular target(s)
Figure 4. Inhibition of DNA topoisomerase II catalytic activity. Lanes are: 1, top IIa; 2, top IIa þ 50 mM VM26; 3, top IIa þ 1 mM 4; 4, top
IIa þ 5 mM 4; 5, top IIa þ 10 mM 4; 6, top IIa þ 1 mM 10; 7, top IIa þ 5 mM 10; 8, top IIa þ 10 mM 10; 9, top IIb; 10, top IIb þ 50 mM VM26; 11, top
IIb þ 1 mM 4; 12, top IIb þ 5 mM 4; 13, top IIb þ 10 mM 4; 14, top IIb þ 1 mM 10; 15, top IIb þ 5 mM 10; 16, top IIb þ 10 mM 10; 17, supercoiled
pBR322 DNA; 18, HindIII-linearized pBR322 DNA.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
Ametantrone–Amsacrine Related Hybrids
733
and 4 located at a distance of three methylene groups (in
compounds 10 and 11) or two methylene groups (in
compound 19) from the nitrogen linked to the planar ring
system. Interestingly, the presence of the ametantrone side
chain hydroxyethylamino ethyl groups (compounds 14 and
disposable cartridges made of silica gel as stationary phase and
bench solvents as mobile phase.
1
13
1
H and C{ H} NMR spectra were recorded on a Bruker Avance
III 400 MHz and a Bruker AMX 300 MHz spectrometers. All spectra
were recorded at room temperature, and the solvent for a
particular spectrum is given in parentheses. Chemical shifts are
reported in ppm and are relative to TMS internally referenced to
the residual solvent peak. The multiplicity of signals is reported as
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad
1
5) does not produce better results. Addition of a second
protonable nitrogen in the side chains (compound 16) is also
detrimental. The cytotoxicity data show a close matching
between compounds 10, 11, and 19 and amsacrine. This is in
line with the suggestion that the side-chain contacts between
drug and enzyme (the planar portion stacks on the DNA) play
an important role in modulating drug potency and implies
that the moiety located in the minor groove represents a key
determinant. Biochemical studies show that compounds 10,
(br), or a combination of any of these.
High-resolution mass spectra were recorded on a ESI-TOF
Mariner from Perseptive Biosystem (Stratford, Texas, USA), using
electrospray (ES) ionization.
The degree of purity of the compounds synthesized throughout
this investigation was assayed by HPLC, using a Varian Pro-Star
system equipped with a Biorad 1706 UV–VIS detector and an
Agilent C-18 column (5 mm, 4.6 mm ꢁ 150 mm). Water (A) and
acetonitrile (B) were used as mobile phase with an overall flow
rate of 1 mL/min and starting (90% A–10% B), then 15 min (10% A–
1
1, and 19 specifically inhibit and poison the b isoform of
human topoisomerase II, but behave as poor inhibitors of the
a isoform of the enzyme. This is in line with the isolation of
amsacrine-resistant mutants with modified b isoforms [34]
and further supports the role played by the amsacrine side
chain even when connected to a different planar system. In
addition, the presence of the b isoform in non-proliferating
cells suggests that drug preferentially aimed at this isoenzyme
might be active in slow-growing tumors, generally poorly
responsive to chemotherapy [35]. Finally, it is worth recalling
the lack of cross-resistance (Table 2) with anthracycline
resistant cells, which confirms the prevalence of the
amsacrine related portion of the hybrids in recognizing
the topoisomerase II-DNA cleavable complex and, as a
consequence, in affecting cytotoxic response. The biochemical
and biological data collected show that neither the planar
chromophore nor the side chains alone are able to direct the
strength of the interaction between the small molecule and
the macromolecular complex. The above data on active
derivatives prove the concept that building new interfacial
inhibitors based on the combination of fragments from
active molecules is viable, but in this case the concept of
pharmacophore must be extended beyond the one of a
decorated nucleus.
9
0% B), then 20 min (10% A–90% B), then 21 min (90% A–10% B),
finally 25 min (90% A–10% B).
5
,8-Dihydroxy-9,10-dioxo-9,10-dihydro-anthracene-2-
0
carboxylic acid(4 -methanesulfonylamino-phenyl)-amide
(
4)
A
suspension of 5,8-diacetoxy-9,10-dioxo-9,10-dihydro-anthra-
cene-2-carboxylic acid(4-methanesulfonylamino-phenyl)-amide
4.4 g, 8.1 mmol) in 80 mL of 5% aqueous potassium carbonate
(
was heated at 80°C for 2 h. The reaction mixture was cooled and
made acidic with hydrochloric acid, then extracted with ethyl
acetate. The combined organic phases were dried over Na SO
2 4
and the solvent was evaporated to afford compound (4) as red
solid (3.4 g, yield 92%).
1
3 2
H NMR [300 MHz, (CD ) SO] d 10.74 (s, 1H), 9.63 (s 1H), 8.82
(
(
(
d, J ¼ 1.7 Hz, 1H), 8.48 (dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, 1H), 8.41
d, J ¼ 8.2 Hz, 1H), 7.78 (d, J ¼ 8.9 Hz, 2H), 7.49 (s, 2H), 7.24
13
d, J ¼ 8.9 Hz, 2H), 2.98 (s, 3H); C NMR [75 MHz, (CD
3 2
) SO] d 182.2,
181.2, 164.8, 154.6, 154.5, 137.6, 137.1, 133.8, 133.4, 127.6, 126.5,
125.9, 123.5, 122.6, 122.4, 120.7, 120.4, 117.2, 116.5, 41.5; HMRS
calcd. for C22
m.p. >350°C.
þ
H
17
N
2
O
7
S [MþH] , 453.0605; found, 453.0632;
5
,8-Dihydroxy-9,10-dioxo-9,10-dihydro-anthracene-2-
0
0
carboxylic acid(2 -methoxy-4 -methanesulfonylamino-
phenyl)-amide (5)
Experimental
A
suspension of 5,8-diacetoxy-9,10-dioxo-9,10-dihydro-anthra-
cene-2-carboxylic acid(2-methoxy-4-methanesulfonylamino-phen-
yl)-amide (5.1 g, 9 mmol) in 80 mL of 5% aqueous potassium
carbonate was heated at 80°C for 2 h. The reaction mixture was
cooled and made acidic with hydrochloric acid, then extracted
with ethyl acetate. The combined organic phases were dried over
Chemistry
Commercially available chemicals were purchased from Aldrich,
and used as received, unless stated. All air-sensitive manipu-
lations were conducted according to Schlenk line techniques,
using dry nitrogen and glassware. If required, solvents were dried
prior to use. For work-up and chromatographic purification,
commercial grade solvents were used; chromatographic separa-
tions were carried out using silica gel 60 (230–400 mesh, Grace
Davisil). In addition, semi-preparative and preparative purifica-
tion of the derivatives was carried out on Isolera One, an
automated flash chromatography system provided by Biotage
Na
2 4
SO and the solvent was evaporated to afford compound 5 as
1
red solid (4.22 g, yield 97%). H NMR [300 MHz, (CD ) SO] d 10.05
3 2
(s, 1H), 9.97 (s, 1H), 8.77 (d, J ¼ 1.7 Hz, 1H), 8.37 (dd, J ¼ 8.2 Hz,
J ¼ 1.7 Hz, 1H), 8.27 (d, J ¼ 8.2 Hz, 1H), 7.56 (d, J ¼ 8.6 Hz, 1H), 7.30
(s, 2H), 6.93 (d, J ¼ 2.3 Hz, 1H), 6.82 (dd, J ¼ 8.6 Hz, J ¼ 2.3 Hz, 1H),
13
3 2
3.80 (s, 3H), 2.99 (s, 3H); C NMR [75 MHz, (CD ) SO] d 182.2, 182.0,
164.8, 154.6, 154.5, 153.6, 137.5, 137.0, 134.4, 133.8, 130.8, 130.1,
127.5, 123.6, 123.5, 123.4, 120.7, 120.6, 114.2, 108.8, 99.7, 55.9,
(Upsala, Sweden); the chromatography was carried out using
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

7
34
G. Zagotto et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
þ
4
1.5; HMRS calcd. for C23
H
19
N
2
O
8
S [MþH] , 483.4745; found,
5,8-Bis-(2-acetylamino-ethylamino)-9,10-dioxo-9,10-
dihydro-anthracene-2-carboxylic acid(2 -methoxy-4 -
4
83.4723; m.p. 316–318°C.
0
0
methanesulfonylamino-phenyl)-amide (9)
1
Yield: 300 mg (44%). H NMR [300 MHz, (CD
3
)
2
SO] d 11.07 (t,
General procedure for the synthesis of derivatives 6–19
J ¼ 5.3 Hz, 2H), 11.04 (s, 1H), 9.89 (s, 1H), 8.87 (d, J ¼ 1.3 Hz, 1H),
from 4 or 5
8
.35 (dd, J ¼ 8.2 Hz, J ¼ 1.3 Hz, 1H), 8.28 (d, J ¼ 8.2 Hz, 1H), 7.82 (d,
To a suspension of 4 or 5 (0.5 g, 1.1 mmol) in 50 mL water and
J ¼ 8.4 Hz, 1H), 7.66 (s, 2H), 6.96 (d, J ¼ 2.3 Hz, 1H), 6.85 (dd,
J ¼ 8.4 Hz, J ¼ 2.3 Hz, 1H), 4.09–4.04 (m, 2H), 3.82 (s, 3H), 3.65 (t,
potassium carbonate (0.5%) in N
.7 mmol) was added. The reaction mixture was heated at 100°C
and Na (0.5 g, 2.9 mmol) and the proper primary amine were
2 2 2 4
atmosphere, Na S O (1.0 g,
5
13
J ¼ 5.3 Hz, 4H), 3.56–3.51 (m, 4H), 2.98 (s, 3H), 2.29 (s, 6H); C NMR
2 2 4
S O
3 2
[75 MHz, (CD ) SO] d 182.0, 181.9, 162.5, 155.2, 151.9, 138.9, 138.6,
added (11 mmol). The reaction mixture was then refluxed
overnight. Then the mixture was cooled at room temperature
and air was introduced for 6 h. Water was added until complete
precipitation of the product and the solid was filtered out
and dried. The crude product was then purified by column
chromatography.
1
1
4
33.2, 133.1, 132.6, 132.1, 131.2, 130.9, 130.8, 129.4, 126.8, 126.4,
25.6, 125.0, 123.1, 118.6, 115.5, 110.2, 62.6, 61.2, 56.0, 44.6, 44.0,
þ
1.1, 40.6, 40.2; HMRS calcd. for C31
H
35
N
6
O
8
S [MþH] , 651.7586;
found, 651.7545; m.p. 229–230°C.
5
,8-Bis-(3-dimethylamino-propylamino)-9,10-dioxo-9,10-
0
dihydro-anthracene-2-carboxylic acid(4 -
methanesulfonylamino-phenyl)-amide (10)
Yield: 300 mg (44%). H NMR [300 MHz, (CD ) SO] d 11.09 (t,
5
,8-Bis-(2-hydroxy-ethylamino)-9,10-dioxo-9,10-dihydro-
0
1
anthracene-2-carboxylic acid(4 -methanesulfonylamino-
3
2
phenyl)-amide (6) 1
J ¼ 5.5 Hz, 2H), 10.64 (s, 1H), 9.64 (s, 1H), 8.79 (d, J ¼ 1.7 Hz, 1H),
.38 (d, J ¼ 8.3 Hz, 1H), 8.28 (dd, J ¼ 8.3 Hz, J ¼ 1.7 Hz, 1H), 7.68 (d,
J ¼ 8.9 Hz, 2H), 7.57 (s, 2H), 7.22 (d, J ¼ 8.9 Hz, 2H), 3.53–3.48 (m,
H), 2.97 (s, 3H), 2.39 (t, J ¼ 6.3 Hz, 4H), 2.20 (s, 12H), 1.81–1.73 (m,
8
3 2
Yield: 300 mg (50%). H NMR [300 MHz, (CD ) SO] d 11.15–11.10
(
m, 2H), 10.64 (s, 1H), 9.86 (s, 1H) 8.83 (d, J ¼ 1.7 Hz, 1H), 8.37 (d,
J ¼ 8.2 Hz, 1H), 8.28 (dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, 1H), 7.78 (d, J ¼ 8.9,
H), 7.56 (s, 2H), 7.22 (d, J ¼ 8.9 Hz, 2H), 5.00 (br s, 2H), 3.69
t, J ¼ 5.3 Hz, 4H), 3.56 (dt, J ¼ 5.5 Hz, J ¼ 5.3 Hz, 4H), 2.97 (s, 3H);
4
13
2
4H); C NMR [75 MHz, (CD
3 2
) SO] d 180.2, 180.1, 164.8, 147.4,
(
147.0, 138.6, 137.0, 135.4, 134.9, 134.7, 131.7, 131.6, 126.9, 125.7,
1
3
1
22.5, 121.8, 121.3, 108.7, 108.5, 56.8, 56.4, 45.7, 45.6, 45.5, 45.4,
3 2
C NMR [75 MHz, (CD ) SO] d 182.1, 180.8, 162.3, 139.8, 139.2,
þ
1
1
32.5, 132.0, 131.9, 131.2, 130.0, 129.6, 127.9, 126.3, 125.9, 125.2,
22.3, 117.8, 116.0, 110.0, 61.7, 61.0, 45.2, 45.1, 41.3; HMRS calcd.
45.3, 27.5, 27.4; HMRS calcd. for C32
found, 621.8188; m.p. 250–252°C.
H
41
N
6
O
5
S [MþH] , 621.8163;
þ
for C26
H
25
N
4
O
7
S [MþH] , 540.1521; found, 540.1429; m.p. 246–
2
49°C.
5
,8-Bis-(3-dimethylamino-propylamino)-9,10-dioxo-9,10-
0
0
dihydro-anthracene-2-carboxylic acid(2 -methoxy-4 -
5
,8-Bis-(2-hydroxy-ethylamino)-9,10-dioxo-9,10-dihydro-
0
0
methanesulfonylamino-phenyl)-amide (11)
anthracene-2-carboxylic acid(2 -methoxy-4 -
methanesulfonylamino-phenyl)-amide (7)
Yield: 400 mg (63%). H NMR [300 MHz, (CD
1
Yield: 350 mg (48%). H NMR [300 MHz, (CD
3
)
2
SO] d 11.05
1
(t, J ¼ 5.3 Hz, 2H), 9.88 (s, 1H), 9.70 (s, 1H), 8.81 (d, J ¼ 1.3 Hz,
3
)
2
SO] d 11.13 (t,
1
H), 8.37 (d, J ¼ 8.2 Hz, 1H), 8.28 (dd, J ¼ 8.2 Hz, J ¼ 1.3 Hz, 1H), 7.63
J ¼ 5.5 Hz, 2H), 10.44 (s, 1H), 9.87 (s, 1H), 8.80 (d, J ¼ 1.8 Hz, 1H),
(
(
3
d, J ¼ 8.4 Hz, 1H), 7.58 (s, 2H), 6.96 (d, J ¼ 2.3 Hz, 1H), 6.85
dd, J ¼ 8.4 Hz, J ¼ 2.3 Hz, 1H), 3.82 (s, 3H), 3.53–3.44 (m, 4H),
8
.35 (d, J ¼ 8.1 Hz, 1H), 8.27 (dd, J ¼ 8.1 Hz, J ¼ 1.8 Hz, 1H), 7.76 (d,
J ¼ 8.7 Hz, 1H), 7.54 (s, 2H), 6.96 (d, J ¼ 2.2 Hz, 1H), 6.83 (dd,
J ¼ 8.7 Hz, J ¼ 2.2 Hz, 1H), 5.01 (br s, 2H), 3.82 (s, 3H), 3.72 (t,
.04 (s, 3H), 2.36 (t, J ¼ 5.1 Hz, 4H), 2.18 (s, 12H), 1.86–1.81 (m, 4H);
13
13
3 2
C NMR [75 MHz, (CD ) SO] d 182.2, 182.1, 164.8, 153.6,
J ¼ 5.2 Hz, 4H), 3.56 (dt, J ¼ 5.5 Hz, J ¼ 5.2 Hz, 4H), 3.02 (s, 3H);
C
1
1
5
C
2
38.2, 138.0, 137.6, 137.0, 134.4, 133.8, 130.8, 130.1, 127.6,
23.4, 117.7, 117.6, 114.2, 110.8, 110.7, 108.8, 99.7, 56.5, 56.3,
NMR [75 MHz, (CD
3
)
2
SO] d 181.2, 181.1, 162.2, 139.6, 139.0, 132.3,
1
1
32.0, 131.6, 131.2, 131.0, 130.5, 130.2, 129.4, 127.8, 126.3, 125.5,
25.1, 122.4, 117.9, 116.0, 110.1, 61.1, 61.0, 55.4, 45.2, 45.0, 41.0;
5.9, 45.9, 45.8, 42.8, 42.7, 41.5, 26.6, 26.5; HMRS calcd. for
þ
þ
33
H
19°C.
43
N
6
O
6
S [MþH] , 651.8463; found, 651.8452; m.p. 218–
HMRS calcd. for
C
27
H
29
N
4
O
8
S
[MþH] , 570.3548; found,
5
70.3429; m.p. >350°C.
5
,8-Bis-(2-acetylamino-ethylamino)-9,10-dioxo-9,10-
5,8-Bis-[2-(2-hydroxy-ethoxy)-ethylamino]-9,10-dioxo-
0
0
dihydro-anthracene-2-carboxylic acid(4 -
methanesulfonylamino-phenyl)-amide (8)
Yield: 300 mg (44%). H NMR [300 MHz, (CD
9,10-dihydro-anthracene-2-carboxylic acid(4 -
methanesulfonylamino-phenyl)-amide (12)
1
1
3
)
2
SO] d 10.97 (t,
Yield: 350 mg (50%). H NMR [300 MHz, (CD
3 2
) SO] d 11.09 (t,
J ¼ 5.5 Hz, 2H), 10.67 (s, 1H), 9.78 (s, 1H), 8.80 (d, J ¼ 1.7 Hz, 1H),
.37 (d, J ¼ 8.3 Hz, 1H), 8.29 (dd, J ¼ 8.3 Hz, J ¼ 1.7 Hz, 1H), 7.79 (d,
J ¼ 8.9 Hz, 2H), 7.62 (s, 2H), 7.23 (d, J ¼ 8.9 Hz, 2H), 3.58 (t,
J ¼ 5.5 Hz, 2H), 10.85 (s, 1H), 9.89 (s, 1H), 8.83 (d, J ¼ 1.6 Hz, 1H),
8
8
.38 (d, J ¼ 8.2 Hz, 1H), 8.29 (dd, J ¼ 8.2 Hz, J ¼ 1.6 Hz, 1H), 7.78 (d,
J ¼ 8.9 Hz, 2H), 7.58 (s, 2H), 7.24 (d, J ¼ 8.9 Hz, 2H), 4.65 (m, 2H),
1
3
J ¼ 5.5 Hz, 4H), 3.44–3.39 (m, 4H), 2.97 (s, 3H), 1.83 (s, 6H); C NMR
75 MHz, (CD SO] d 181.9, 181.1 162.6, 155.3, 152.9, 139.8, 139.0,
32.8, 132.7, 131.2, 131.0, 130.6, 129.4, 127.8, 126.5, 125.8, 125.1,
3.72 (t, J ¼ 4.9 Hz, 4H), 3.66 (t, J ¼ 4.9 Hz, 4H), 3.54–3.45 (m, 4H),
13
[
3 2
)
2
3 2
.97 (s, 3H); C NMR [75 MHz, (CD ) SO] d 182.2, 182.1, 164.8,
1
1
147.3, 138.2, 134.5, 133.4, 132.9, 128.2, 126.6, 122.9, 122.4, 122.1,
22.1, 117.4, 116.5, 110.5, 62.7, 61.0, 44.9, 45.0, 41.2, 40.9, 40.2;
121.9, 121.0, 120.5, 108.7, 108.4, 72.4, 72.3, 69.7, 69.4, 61.5, 61.4,
þ
þ
HMRS calcd. for
6
C
30
H
33
N
6
O
7
S
[MþH] , 621.2126; found,
4
3.1, 43.0, 42.5; HMRS calcd. for C30
H
35
N
4
O
9
S [MþH] , 627.1974;
21.2106; m.p. 248–250°C.
found, 627.2019; m.p. 261–263°C.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
Ametantrone–Amsacrine Related Hybrids
735
5
9
,8-Bis-[2-(2-hydroxy-ethoxy)-ethylamino]-9,10-dioxo-
,10-dihydro-anthracene-2-carboxylic acid(2 -methoxy-4 -
5,8-Bis-[N-amino-propyl-morpholinyl]-9,10-dioxo-9,10-
dihydro-anthracene-2-carboxylic acid(4 -
0
0
0
methanesulfonylamino-phenyl)-amide (13)
methanesulfonylamino-phenyl)-amide (17)
1
1
Yield: 290 mg (40%). H NMR [300 MHz, (CD
3
)
2
SO] d 11.07
Yield: 400 mg (51%). H NMR [300 MHz, (CD
3
)
2
SO] d 11.05 (t,
(
1
(
(
3
1
1
7
t, J ¼ 5.3 Hz, 2H), 11.04 (s, 1H), 10.64 (s, 1H), 8.79 (d, J ¼ 1.3 Hz,
J ¼ 6.5 Hz, 1H), 10.62 (s, 1H), 9.98 (s, 1H), 8.85 (d, J ¼ 1.7 Hz, 1H),
8.37 (d, J ¼ 8.2 Hz, 1H), 8.28 (dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, 1H), 7.80 (d,
J ¼ 8.9, 2H), 7.58 (s, 2H), 7.22 (d, J ¼ 8.9 Hz, 2H), 3.60–3.56 (m, 8H),
H), 8.35 (dd, J ¼ 8.2 Hz, J ¼ 1.3 Hz, 1H), 8.28 (d, J ¼ 8.2 Hz, 1H), 7.62
d, J ¼ 8.4 Hz, 1H), 7.65 (s, 2H), 6.96 (d, J ¼ 2.3 Hz, 1H), 6.85
dd, J ¼ 8.4 Hz, J ¼ 2.3 Hz, 1H), 3.82 (s, 3H), 3.68–3.61 (m, 8H), 3.54–
3.55 (d, J ¼ 5.5 Hz, 4H), 2.97 (s, 3H), 2.39–2.34 (m, 8H), 1.83 (t,
1
3
13
.48 (m, 8H), 3.03 (s, 3H); C NMR [75 MHz, (CD
3
)
2
SO] d 182.2,
J ¼ 6.5 Hz, 4H), 1.66–1.58 (m, 4H); C NMR [75 MHz, (CD
3 2
) SO] d
82.0, 164.9, 153.6, 138.2, 138.1, 137.6, 137.1, 134.4, 133.8, 130.8,
30.1, 127.6, 123.4, 117.7, 117.6, 114.2, 110.7, 110.6, 108.8, 99.7,
182.2, 182.1, 164.9, 138.2, 138.0, 137.6, 137.0, 133.8, 130.7, 130.2,
127.6, 125.9, 122.4, 117.7, 117.6, 116.3, 116.1, 110.7, 110.6, 66.8,
2.4, 72.3, 70.6, 70.5, 61.5, 61.4, 55.9, 44.2, 44.0, 41.5; HMRS calcd.
66.7, 53.7, 53.5, 51.9, 51.8, 42.8, 42.7, 41.5, 27.2, 27.1: HMRS calcd.
þ
þ
for C31
2
H
37
N
4
O
10S [MþH] , 625.1974; found, 625.2019; m.p. 220–
for C36
240°C.
H
45
N
6
O
7
S [MþH] , 705.1569; found, 705.1547; m.p. 238–
21°C.
5
9
,8-Bis-[2-(2-hydroxy-ethylamino)-ethylamino]-9,10-dioxo-
,10-dihydro-anthracene-2-carboxylic acid(4 -
5,8-Bis-[N-amino-propyl-morpholinyl]-9,10-dioxo-9,10-
dihydro-anthracene-2-carboxylic acid(2 -methoxy-4 -
0
0
0
methanesulfonylamino-phenyl)-amide (14)
methanesulfonylamino-phenyl)-amide (18)
1
1
Yield: 300 mg (43%). H NMR [300 MHz, (CD
3
)
2
SO] d 11.60
Yield: 400 mg (49%). H NMR [300 MHz, (CD
3
)
2
SO] d 11.05 (t,
(
1
7
(
(
1
1
6
C
2
t, J ¼ 5.5 Hz, 2H), 10.59 (s, 1H), 10.21 (s, 1H), 8.81 (d, J ¼ 1.7 Hz,
J ¼ 6.5 Hz, 1H), 10.79 (s, 1H), 9.89 (s, 1H), 8.81 (d, J ¼ 1.7 Hz, 1H),
8.37 (d, J ¼ 8.2 Hz, 1H), 8.26 (dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, 1H), 7.80 (d,
J ¼ 8.9 Hz, 1H), 7.58 (s, 2H), 7.22 (dd, J ¼ 8.9 Hz, J ¼ 2.3 Hz, 1H), 6.98
(d, J ¼ 2.3 Hz, 1H), 3.65–3.61 (m, 8H), 3.55–3.49 (m, 4H), 3.40 (s, 3H),
2.97 (s, 3H), 2.37–2.32 (m, 8H), 1.81–1.76 (m, 4H), 1.63–1.56 (m,
4H); C NMR [75 MHz, (CD ) SO] d 182.2, 182.1, 164.9, 138.2,
3 2
138.0, 137.4, 137.1, 134.4, 133.8, 130.9, 130.1, 127.6, 123.4, 117.7,
117.5, 114.2, 110.7, 110.6, 108.8, 99.7, 66.8, 66.7, 55.9, 53.8 53.7,
H), 8.35 (d, J ¼ 8.2 Hz, 1H), 8.22 (dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, 1H),
.79 (d, J ¼ 8.9 Hz, 2H), 7.57 (s, 2H), 7.24 (d, J ¼ 8.9 Hz, 2H), 4.95
t, J ¼ 4.9 Hz, 2H), 3.65–3.56 (m, 8H), 3.48–3.41 (m, 4H), 2.97
13
s, 3H), 2.87–2.81 (m, 4H); C NMR [75 MHz, (CD
3
)
2
SO] d 182.2,
13
82.1, 164.8, 138.2, 137.6, 133.8, 133.4, 130.8, 130.2, 127.6,
25.9, 122.4, 122.3, 117.7, 117.5, 116.5, 116.4, 110.8, 110.7, 61.6,
1.5, 52.1, 52.0, 49.5, 49.4, 49.1, 49.0, 41.5; HMRS calcd. for
þ
30
H
20°C.
37
N
6
O
7
S [MþH] , 625.2439; found, 625.2453; m.p. 219–
53.6, 51.9, 51.8, 42.8, 42.7, 41.5, 27.2, 27.1; HMRS calcd. for
þ
C
37
H
47
N
6
O
8
S [MþH] , 735.4124; found, 735.4128; m.p. 219–
220°C.
5
9
,8-Bis-[2-(2-hydroxy-ethylamino)-ethylamino]-9,10-dioxo-
,10-dihydro-anthracene-2-carboxylic acid(2 -methoxy-4 -
5
9
,8-Bis-[2-N,N-dimethylamino-ethylamino]-9,10-dioxo-
,10-dihydro-anthracene-2-carboxylic acid(4 -
0
0
0
methanesulfonylamino-phenyl)-amide (15)
Yield: 340 mg (50%). H NMR [300 MHz, (CD
methanesulfonylamino-phenyl)-amide (19)
1
3
)
2
SO] d 11.72 (t,
1
Yield: 400 mg (61%). H NMR [300 MHz, (CD
J ¼ 5.2 Hz, 1H), 10.64 (s, 1H), 8.98 (s, 1H), 8.65 (d, J ¼ 1.7 Hz, 1H),
.38 (d, J ¼ 8.2 Hz, 1H), 8.26 (dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, 1H), 7.80 (d,
J ¼ 8.9, 2H), 7.54 (s, 2H), 7.21 (d, J ¼ 8.9 Hz, 2H), 2.97 (s, 3H), 2.39–
3 2
) SO] d 11.12 (t,
J ¼ 5.4 Hz, 1H), 11.54 (s, 1H), 10.89 (s, 1H), 8.28 (d, J ¼ 1.3 Hz, 1H),
8
.15 (dd, J ¼ 8.2 Hz, J ¼ 1.3 Hz, 1H), 7.68 (d, J ¼ 8.2 Hz, 1H), 7,22 (d,
8
J ¼ 8.4 Hz, 1H), 7.05 (s, 2H), 6.55 (d, J ¼ 2.1 Hz, 1H), 6.42 (dd,
J ¼ 8.4 Hz, J ¼ 2.1 Hz, 1H), 3.82 (s, 3H), 3.65–3.58 (m, 8H), 3.01 (s,
13
1
3
2.34 (m, 8H), 1.83 (s, 12H); C NMR [75 MHz, (CD
3
)
2
SO] d 182.2,
3
H), 2.97–2.92 (m, 4H), 2.01–1.96 (m, 4H); C NMR [75 MHz,
1
1
4
82.1, 164.7, 138.3, 138.2, 137.5, 137.0, 133.4, 130.8, 127.6, 125.9,
22.5, 122.4, 117.7, 117.5, 116.6, 116.4, 110.7, 110.5, 58.1, 58.0,
6.7, 45.7, 45.6, 45.5, 41.5; HMRS calcd. for C30
3 2
(CD ) SO] d 182.2, 182.1, 164.9, 153.6, 138.2, 138.0, 137.5, 137.1,
1
1
4
6
34.4, 133.8, 130.7, 130.1, 127.6, 123.4, 117.7, 117.6, 114.2, 110.9,
10.7, 108.8, 99.7, 61.6, 61.5, 55.9, 52.1, 52.0, 50.2, 49.3, 49.1, 49.0,
1.4; HMRS calcd. for C31
55.8345; m.p. 235–238°C.
þ
H
37
N
6
O
5
S [MþH] ,
þ
593.7853; found, 593.7547; m.p 243–245°C.
H
39
N
6
O
8
S [MþH] , 655.8499; found,
Experiments with human cells
Chemicals
5
,8-Bis-[2-piperazin-1-yl-ethylamino]-9,10-dioxo-9,10-
Compounds 3–19 obtained were dissolved in dimethyl sulfoxide
just before the experiments; calculated amounts of drug solution
were added to the growth medium containing cells to a final
solvent concentration of 0.5%, which had no discernible effect on
cell killing. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide), amsacrine, and ametantrone were obtained from
Sigma Chemical Co. (St. Louis, USA).
0
dihydro-anthracene-2-carboxylic acid(4 -
methanesulfonylamino-phenyl)-amide (16)
Yield: 300 mg (40%). H NMR [300 MHz, (CD
1
3
)
2
SO] d 11.09 (t,
J ¼ 6.4 Hz, 1H), 10.62 (s, 1H), 8.81 (d, J ¼ 1.7 Hz, 1H), 8.37 (d,
J ¼ 8.2 Hz, 1H), 8.26 (dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, 1H), 7.79 (d,
J ¼ 8.9 Hz, 2H), 7.58 (s, 2H), 7.22 (d, J ¼ 8.9 Hz, 2H), 2.97 (s, 3H),
13
2
.89–2.84 (m, 8H), 2.68–2.63 (m, 4H), 1.85–1.82 (m, 12H); C NMR
[
1
1
3 2
75 MHz, (CD ) SO] d 182.3, 174.5, 165.1, 146.1, 137.5, 137.0, 136.1,
33.4, 127.2, 126.2, 126.4, 123.4, 122.2, 121.8, 121.5, 121.4, 121.2,
Cell cultures
Human breast (MCF-7) carcinoma along with melanoma (A375)
and promyelocytic leukemia (HL-60) cell lines were obtained from
ATCC (Rockville, MD). Human ovarian cancer cell line (2008) was
kindly provided by Prof. G. Marverti (Department of Biomedical
08.4, 108.0, 56.9, 53.8, 53.2, 53.1, 46.7, 46.3, 46.0, 45.8, 45.7;
þ
HMRS calcd. for
6
C
34
H
41
N
8
O
5
S
[MþH] , 675.4124; found,
75.4128; m.p. 247–249°C.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

7
36
G. Zagotto et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 728–737
Science of Modena University, Italy). LoVo human colon-
carcinoma cell line and its derivative multidrug-resistant sub-
line (LoVo MDR) were kindly provided by Prof. F. Majone
adding 7 mM EDTA (3 mL) containing 0.77% SDS. Bromophenol
blue (2 mL) containing 15% of glycerol was added to the samples,
which were then analyzed by agarose gel (0.7%) electrophoresis
containing TAE (40 mM Tris-sodium acetate, pH 8.2; 1 mM EDTA)
for 90 min; the gel was stained for 1 h in aqueous ethidium
bromide (0.5 mg/mL). The bands were detected by exposure to an
UV transilluminator TM36 (UVP, Inc., San Gabriel, CA, USA) and
recorded with an Uvitec GAS9200 camera equipped with UVIdoc
software.
(Department of Biology of Padova University, Italy).
Cell lines were maintained in the logarithmic phase at
3
7°C in a 5% carbon dioxide atmosphere using the following
culture media containing 10% fetal calf serum (Euroclone,
Milan, Italy), antibiotics (50 units/mL penicillin and 50 mg/mL
streptomycin), and 2 mM L-glutamine: (i) RPMI-1640 medium
(
(
Euroclone) for HL60, 2008, and MCF-7 cells; (ii) DMEM
Dulbecco’s modified Eagle’s) medium (Euroclone) for A375
DNA cleavage assays
cells; (iii) F-12 Ham’s (Sigma Chemical Co) for LoVo and LoVo
MDR cells (LoVo MDR culture medium also contained 0.1 mg/mL
doxorubicin).
DNA cleavage reactions were performed with recombinant
human topoisomerase II isoforms, purified as described [25].
pBR322 plasmid (230 ng) was incubated with the same enzyme
amounts in the presence of different drug concentrations,
1 mM ATP, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM MgCl2,
0.01 mg/mL BSA, 10 mM DTT, and 150 mM KCl. The reactions were
incubated for 30 min at 37°C, and then stopped with 0.1% SDS
and 0.5 mg/mL proteinase K (Sigma) for 30 min at 53°C. DNA
samples were then run in 1% agarose gels, and stained with
ethidium bromide. DNA bands were quantified with ImageQuant
5.2 software (Amersham) with a local average background
correction.
Cytotoxicity assay
The growth inhibitory effect toward tumor cell lines was
evaluated by means of MTT (tetrazolium salt reduction) assay [23].
3
Briefly, between 3 and 8 ꢁ 10 cells, dependent upon the growth
characteristics of the cell line, were seeded in 96-well microplates
in growth medium (100 mL). After 24 h, cells were treated for 48 or
7
2 h with the appropriate concentrations of the compound. Each
well was added with 10 mL of a 5 mg/mL MTT (3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide) saline solution, and
after additional 5 h of incubation, 100 mL of a sodium dodecyl-
sulfate (SDS) solution in HCl 0.01 M was added. After overnight
incubation, the absorbance of each well was detected by a
microplate reader (Biorad 680, Milan, Italy) at 570 nm. Mean
absorbance for each drug dose was expressed as a percentage of
the control untreated well absorbance and plotted versus drug
concentration. IC50 values represent the drug concentrations that
reduced the mean absorbance at 570 nm to 50% of those in the
untreated control wells.
This work was in part supported by grants from the Ministero
dell’Università e della Ricerca (PRIN program), Rome, Italy and from
the University of Padova. Special thanks to Prof. Stefano Moro for his
invaluable help.
The authors have declared no conflict of interest.
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