Synthesis and cytotoxic activity of novel amidine analogues of bis(2-chloroethyl)amine

Article abstract of DOI:10.1002/ardp.200800231

Novel nitrogen mustard agents 7-12 involving 4-(N,N-bis(2-chloroethyl) aminophenyl)propylamine linked to a 5-(4-N-alkylamidinophenyl)-2-furancarboxylic acid moiety by the formation of an amide bond have been synthesized, characterized, and evaluated for t

Full text of DOI:10.1002/ardp.200800231

484
Arch. Pharm. Chem. Life Sci. 2009, 342, 484–490
Full Paper
Synthesis and Cytotoxic Activity of Novel Amidine Analogues
of Bis(2-chloroethyl)amine
Krzysztof Bielawski, Anna Bielawska, and Bozena Poplawska
Department of Medicinal Chemistry and Drug Technology, Medical University of Bialystok, Bialystok, Poland
Novel nitrogen mustard agents 7–12 involving 4-(N,N-bis(2-chloroethyl)aminophenyl)propyl-
amine linked to a 5-(4-N-alkylamidinophenyl)-2-furancarboxylic acid moiety by the formation of
an amide bond have been synthesized, characterized, and evaluated for their in-vitro cytotoxic
activity against MDA-MB-231 and MCF-7 human breast cancer cells. Evaluation of the cytotoxicity
of 7–12 employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA demon-
strated that these compounds exhibit remarkable cytotoxic effects in comparison with 4-[bis(2-
chloroethyl)amino]benzenebutanoic acid. Compounds 7 and 9, which possess a cationic amidine
and 4,5-dihydro-1H-imidazol function moiety are approximately ten times more potent than 4-
[bis(2-chloroethyl)amino]benzenebutanoic acid. The new compounds were evaluated as DNA top-
oisomerase II inhibitors. The cytotoxicity of the compounds 7–12 correlates with their DNA-
binding affinities and their relative potency as topoisomerase II inhibitors.
Keywords: Alkylating agents / Amidines / Cytotoxicity / DNA binding / Topoisomerase II /
Received: December 22, 2008; accepted: March 24, 2009
DOI 10.1002/ardp.200800231
action of drug and DNA plays a key role in pharmacology
Introduction
and it is of great significance for designing and synthesiz-
ing new drugs targeted to DNA. Their effectiveness
depends on the mode and affinity of their binding.
Recently, there has been increasing interest in the design
and synthesis of DNA-interstrand crosslinking agents as
well as conjugates to enhance the sequence selectivity
and increase selectivity for tumor cells. As DNA minor-
groove binders constitute an important class of deriv-
atives in anticancer therapy, these compounds may be
exploited to understand their potential as carrier com-
pounds of the bis(2-chloroethyl)amine moiety for the
development of anticancer drugs [4–6]. Minor-groove
drugs binding with DNA adopt a characteristic curved
shape isohelical with the target groove and they bind in
the region of rich AT base pairs which is more narrow
than regions rich in GC base pairs [7–9]. Selectively
directing a drug into the vicinity of its intended site of
action has clear therapeutic advantages, including
reduced toxicity, smaller dose levels, and especially
enhanced efficacy. Further, the bifunctional DNA-interac-
tive agents, in which the two functionalities possess two
different mechanisms of interaction with DNA and are
Alkylating drugs such as 4-[bis(2-chloroethyl)amino]ben-
zenebutanoic acid (chlorambucil) are in widespread use
for the treatment of various cancers [1]. Despite its unpar-
alleled clinical success, as evidenced by the high cure
rates of hematological malignancies, chlorambucil-based
chemotherapy suffers from major drawbacks. Tumor
resistance to the drug, probably the most severe obstacle,
is multifactorial in nature and known to be mediated by
reduced cellular uptake of the drug, increased levels of
detoxifying sulfur nucleophiles in the cytoplasm,
enhanced repair or tolerance of drug-DNA adducts, and
lack of functional p53 protein [2, 3]. The development of
thousands of bis(2-chloroethyl)amine derivatives reflects
the common opinion that the formation of bifunctional
covalent adducts on DNA is a pre-requisite for cytotoxic-
ity in nitrogen mustard agents. So, the study on the inter-
Correspondence: Krzysztof Bielawski, Department of Medicinal Chemis-
try and Drug Technology, Medical University of Bialystok, Kilinskiego 1,
15-089 Bialystok, Poland
E-mail: kbiel@umwb.edu.pl
Fax: +48 85 748-5416
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Arch. Pharm. Chem. Life Sci. 2009, 342, 484–490
Amidine Analogues of Bis(2-chloroethyl)amine
485
capable of recognizing heterogeneous DNA sequences –
coupled by a spacer – have also attracted considerable
attention as a new class of antitumor agents. Such target-
ing can also significantly alter the pattern of DNA alkyla-
tion by the nitrogen mustard [4–6]. Chlorambucil and
melphalan preferably alkylate guanine-N7 in the major
groove, whereas the major target of the minor-groove
binders linked mustards is adenine-N3 in the minor
groove.
The present study was designed to extend our recent
findings related to amidine analogues of alkylating
agents [10–13]. In continuation of our efforts in this
direction, we considered it interesting to design and syn-
thesize molecules based on 4-(N,N-bis(2-chloroethyl)ami-
nophenyl)propylamine linked to a 5-(4-N-alkylamidino-
phenyl)-2-furancarboxylic acids 1–6 moiety by the forma-
tion of an amide bond. Several 5-[4-(N-alkylamidino)phe-
nyl]furan compounds are able to bind to double-stranded
DNA, preferentially at AT base pairs along the minor
groove by formation of hydrogen bonds [14–16]. The
introduction of a spacer arm between the carrier and the
drug is a strategy widely used to separate the active moi-
ety from the carrier. Ideally, as a conjugate is meant to
reach the malignant site, the linkage should be suffi-
ciently stable during the circulation in the blood stream
to maintain its chemical integrity until it reaches its tar-
get. However, after uptake into the cells, they should
have an intrinsic activity or release the active compound.
The structure of all the targeted compounds varying by
the nature of their terminal basic side chains is given in
Scheme 1. Our work was especially focused on how these
amidine moieties contributed to DNA binding and the
antitumor activity of the newly synthesized compounds
7–12 in both MDA-MB-231 and MCF-7 breast cancer cells.
The DNA binding efficacy and preferred mode of binding
of a series of amidine analogues of bis(2-chloroethyl)-
amine was investigated by the ethidium displacement
assay using calf thymus DNA, T4 coliphage DNA, poly(dA-
dT)2 and poly(dG-dC)2, and by a topoisomerase I/II inhibi-
tion assay.
Scheme 1. Preparation of target molecules 7–12.
(N,N-bis(2-chloroethyl)aminophenyl)propylamine
was
synthesized by Curtius rearrangement of an azide deriv-
ative of 4-[bis(2-chloroethyl)amino]benzenebutanoic acid
[17]. The amidino acids 1–6 were conjugated with 4-(N,N-
bis(2-chloroethyl)aminophenyl)propylamine in the pres-
ence of N,N9-carbonyldiimidazole (CDI) as condensing
agent in DMF at 08C to give compounds 7–12 in good
yields. Compounds 7–12 were isolated as the hydrochlor-
ide salts by dissolving the free base in acetone and adding
a few drops of hydrochloric acid. These new amidine ana-
logues of bis(2-chloroethyl)amine 7–12 differing by the
nature of their terminal basic side chains were isolated
as the hydrochloride salts. The structures of compounds
7–12 have been proven by 1H-NMR, 13C-NMR, and elemen-
tal analysis.
Pharmacology
The viability of MCF-7 and MDA-MB-231 breast cancer
cells was measured by the method of Carmichael using 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
[18]
Results and discussion
(Fig. 1). In terms of reduction of cell viability, the com-
pounds rank in both MCF-7 and MDA-MB-231 cells in the
order 7 A 9 A 8 A 10 A 11 A 12 A chlorambucil. Although
growth inhibition was concentration-dependent in
either cell line, it was more pronounced at shorter times,
in MCF-7 than MDA-MB-231 (Fig. 1). The values of IC50
were relatively higher for 7 and 9 which possess a cati-
onic 4,5-dihydro-1H-imidazol and amidine function,
respectively. Among these derivatives, compound 12
Chemistry
The new compounds 7–12 were prepared using the
methods outlined in Scheme 1. 5-[4-(N-Alkylamidino)-
phenyl]-2-furancarboxylic acids 1–6, were prepared by
the general method described previously by Bielawski et
al. [16]. The amidino acids 1–6 providing a carboxyl
group are appropriately functionalized to bind to the
alkylating residue by the formation of an amide bond. 4-
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486
K. Bielawski et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 484–490
Mean values l S.D. from three independent experiments done in duplicate are presented.
Figure 1. Viability of MCF-7 (A) and MDA-MB-231 (B) cells treated for 24 h with different concentrations of compounds 7–12 and
chlorambucil.
Mean values l S.D. of 3 independent experiments (n = 4) done in duplicates are presented.
Figure 2. Inhibition of DNA synthesis by compounds 7–12 and chlorambucil in the cultured breast cancer MCF-7 (A) and MDA-MB-
231 (B) cells.
proved to be only slightly more potent than chlorambu- noic acid [10, 11]. The concentrations of 7 and 9 needed
cil in both MDA-MB-231 and MCF-7, with IC50 values of to inhibit [3H]thymidine incorporation into DNA by 50%
66 l 2 and 71 l 2 lM, respectively, compared to 93 l 2 (IC50) in MDA-MB-231 was found to be 5 l 2 lM and
and 97 l 2 lM for chlorambucil. In contrast, compound 7 12 l 2 lM, respectively, suggesting higher cytotoxic
is clearly much more active and showed a high level of potency compared to chlorambucil (IC50 = 49 l 2 lM). The
cytotoxic potency, IC50 7 l 2 and 11 l 2 lM in MCF-7 and concentrations of 7, 9, and chlorambucil needed for a
MDA-MB-231, respectively.
50% reduction in [3H]thymidine incorporation into DNA
We studied the effect of compounds 7–12 and chlor- in breast cancer MCF-7 (IC50) was found to be 8l 2 lM,
ambucil on DNA synthesis in both MDA-MB-231 and MCF- 11 l 2 lM, and 55 l 2 lM, respectively. Among the deriv-
7 breast cancer cells (Fig. 2). All of the tested compounds atives, compound 12 in both MDA-MB-231 and MCF-7
showed concentration-dependent activity, yet with proved to be slightly less potent than chlorambucil, with
different potency. The compounds 7–12 exhibited a IC50 values of 71 l 2 and 75 l 2 lM, respectively.
smooth trend of decreasing cytotoxic potency as the size
The binding affinities of compounds 7–12, netropsin,
of N-terminal amidine group increases. A similar correla- and distamycin to calf thymus DNA, T4 coliphage DNA,
tion was observed in our previous work for other amidine and synthetic polymers poly(dA-dT)2 and poly(dG-dC)2
analogues of 4-[bis(2-chloroethyl)amino]benzenebuta- were compared by using the ethidium displacement
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Arch. Pharm. Chem. Life Sci. 2009, 342, 484–490
Amidine Analogues of Bis(2-chloroethyl)amine
487
Table 1. DNA binding and topoisomerase II inhibitory effect of netropsin, distamycin, and compounds 7–12.
a)
a)
Ligand
Calf thymus DNAa)
Binding
T4 DNAa)
poly(dA-dT)2
Binding
poly(dG-dC)2
Binding
Inhibition of topo II
Activity
(lM)
Binding
(Kapp6105 M– 1
)
(Kapp6105 M– 1
)
(Kapp6105 M– 1
)
(Kapp6105 M– 1
)
Netropsin
Distamycin
7
8
9
10
11
12
8.7
7.5
4.4
2.8
3.8
2.6
0.9
0.7
8.3
6.4
3.8
2.0
3.2
2.2
0.7
0.7
875
340
16.2
12.2
14.8
11.0
2.2
2.5
2.0
1.4
1.2
1.3
1.3
0.6
0.3
5
5
5
20
10
10
60
60
2.0
a)
The error for netropsin, distamycin and compounds 7–12 is l 0.26105 M– 1
.
assay [19, 20]. The concentration of the test compounds for DNA in ethidium bromide displacement assays when
in the study required to quench fluorescence by 50% was compared with compounds 7–10 (Table 1). It is interest-
measured in each case. Using these values, the associa- ing that the cytotoxic activity of 11 and 12 is reduced by
tion constants were determined, and the data are pre- approximately tenfold relative to 9, illustrating the link-
sented in Table 1. The increase in the size of the N-alkyl age between a baseline level of required DNA binding
terminal amidine substituents for the series of 7–12 affinity for biological activity. DNA binding suggests that
decreased the binding affinity, which suggested that the combined effect resulting from alkylation and DNA
steric hindrance might be responsible for a negative role minor-groove binding might be in part responsible for
of the substituents to the DNA-binding ability. The associ- the cytotoxic activity of compounds 7–12. The results
ation constant were relatively higher for 7 and 9, which suggest that crosslink formation is dependent upon the
possess a cationic amidine and 4,5-dihydro-1H-imidazol drug orientation prior to alkylation and the required
function, respectively. The large apparent binding con- deformation of the DNA to permit 1,3 crosslinking can
stants for T4 coliphage DNA for 7–12 gave evidence of largely be achieved in the non-covalent complex.
their minor-groove selectivity, because the major groove
DNA-processing enzymes are important targets for
of T4 coliphage DNA is blocked by a-glycosylation of the anticancer drugs. A large number of agents bind to
5-(hydroxymethyl)cytidine residues [21]. It is known that duplex DNA in a covalent or non-covalent fashion,
compounds that bind solely to the DNA minor groove thereby interfering with its role as a template in replica-
generally show a strong preference (a factor of 10 or tion and transcription. The transcription factors that
greater) for binding to AT-rich sequences relative to GC- have been targeted to date have primarily been DNA
rich sequences due to the occlusion from the GC-rich major groove-binding proteins, but there are also impor-
minor groove by the protruded 2-NH2 group of guanine. tant minor groove-binding transcription factors. The
The preferential binding of these compounds to the AT mammalian topoisomerase II also targets the DNA minor
regions compared to the GC region was substantiated groove and plays critical roles in disease processes. High
experimentally by considering the binding of 7–12 inde- levels of expression of topoisomerases have been
pendently with AT rich and GC rich DNA spectrophoto- observed in a number of tumor cells including breast,
metric titrations (Table 1). These data indicate that com- lung, and pancreatic cancers [23–25]. A number of stud-
pounds 7–12 have more affinity for poly(dA-dT)2 than ies have demonstrated that the overexpression of topoi-
poly(dG-dC)2. The binding constants for 7 were found to somerases associated with transformation and meta-
be 16.26105 M– 1 and 1.46105 M– 1 with AT- and GC-rich static progression of neoplastic cells and the expression
DNA, respectively. The amidines provide H-bond donat- level exhibits a correlation with the degree of malignant
ing -NH groups to interact with H-bond acceptors, N3 of transformation [25]. Many compounds that target the
adenine and O2 of thymidine at the floor of the minor minor groove in AT sequences in DNA are well character-
groove in AT sequences [14, 22]. The flat amidine and 4,5- ized and are promising reagents for use as modulators of
dihydro-1H-imidazole groups of 7 and 9 give these com- topoisomerase–DNA complexes. Such compounds could
pounds the ability to form a number of non-bonding con- compete with topoisomerase II for binding to the same
tacts with the surface of the minor groove, which are AT-rich binding site in the DNA promoter region and,
inherently unavailable to other amidines. Compounds thus, inhibit the topoisomerase II activity as a transcrip-
11–12 were shown to have moderate binding affinities tion factor. The ability of compounds 7–12 to inhibit top-
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488
K. Bielawski et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 484–490
l 0.4% calculated values were obtained. 1H-NMR (200 MHz) and
13C-NMR (150 MHz) spectra were recorded on a Bruker AC 200F
spectrometer (Bruker, Ettlingen, Germany), using TMS as an
internal standard. Chemical shifts are expressed in d value
(ppm). Multiplicity of resonance peaks are indicated as singlet
(s), doublet (d), triplet (t), quartet (q), and multiplet (m). 4-[Bis(2-
chloroethyl)amino]benzenebutanoic acid, dimethylformamide,
triethylamine, N,N'-carbonyldiimidazole, LiOH, and ethylenedi-
amine were purchased from Sigma Chemical Co (St. Louis, MO,
USA).
oisomerase I and II activity was quantified by measuring
the action on supercoiled pBR322 DNA substrate as a
function of increasing concentration of the ligands by
the use of agarose gel electrophoresis. The concentra-
tions of the inhibitor that prevented 50% of the super-
coiled DNA from being converted into relaxed DNA (IC50
values) were determined (Table 1). Chlorambucil as a con-
trol was, as expected, ineffective in this assay. These
results demonstrated that 7–12 have topoisomerase II
inhibitory activity with 50% inhibitory concentrations
(IC50) ranging from 5 to 60 lM. None of the compounds
5–12 inhibited the topoisomerase-I-mediated relaxation
of supercoiled DNA at a concentration of 200 lM (not
shown). Compounds 9 and 7 were the most potent topoi-
somerase II inhibitors, with 50% inhibitory concentra-
tions (IC50) ranging from 5 to 10 lM. Table 1 also shows
that compound 9 is approximately tenfold more active
than 11–12 as a topoisomerase II inhibitor. The fact that
compounds 7–10 are more potent than 11 and 12 indi-
cates that topoisomerase inhibition is selective and sensi-
tive to the nature of the cationic amidine terminal
groups of the compounds. The compounds that were less
effective against topoisomerase II activity were also rela-
tively weaker DNA-binding compounds (Table 1).
We hypothesize on the basis of the present results that
simultaneous DNA binding and inhibition of topoiso-
merase II activity leads to increased cytotoxicity of com-
pounds 7–10 compared to the parent drug, chlorambu-
cil. Detailed biophysical studies of the DNA complexes of
such modified compounds, when correlated to changes
in biological activity, should provide a better understand-
ing of the biological mechanism of these compounds. It
will be important to extend these preliminary findings
and characterize how compounds 7–12 may modulate
chlorambucil mechanisms of cytotoxicity. Development
of this class of agents should be pursued. The further bio-
chemical studies are warranted, such as analysis of enzy-
matic DNA digests and footprinting experiments, to
establish the exact covalent binding sites and sequence
specificity of these compounds. These studies are in prog-
ress.
The compounds 1–6 have been prepared and described previ-
ously [16], and syntheses of all new compounds are given below.
N-(3-(4-Bis(2-chloroethyl)aminophenyl)propyl)-5-(4-
amidinophenyl)-2-furancarboxamide hydrochloride 7
To a solution of 5-(4-amidinophenyl)-2-furancarboxylic acid
hydrochloride 1 (0.96 g, 3 mmol) in 5 mL of anhydrous DMF in
an ice bath was added N,N9-carbonyldiimidazole (0.49 g,
3 mmol). The solution was stirred for 30 min and 4-(N,N-bis(2-
chloroethyl)aminophenyl)propylamine (0.83 g, 3 mmol) was
added. It was stirred at 08C for 2 h, then the temperature was
allowed to rise to the ambient and stirring was continued for
8 h. The volume of the reaction mixture was reduced to 1 mL by
evaporation and the remaining mixture was diluted with ace-
tone. The resulting solid was filtered, washed with acetone, and
dried under vacuum at room temperature. The crude product
was basified with 1 M LiOH to pH above 9. A gummy solid was
filtred, washed with water, and dried under vacuum. The free
base was converted into the salt by taking it up in 3 mL of meth-
anol, treatment with 2 mL of 2 M HCl, and stirring for 30 min.
Acetone was added and the final product was filtered, washed
with acetone, and recrystallized from absolute ethanol to give
1.06 g of 7.
Yield: 65%; m.p.: 236–2388C; 1H-NMR (DMSO-d6) d: 9.38 (br,
2H), 9.06 (br, 2H), 8.02 (d, 2H), 7.92 (d, 2H), 7.39 (d, 1H), 7.37 (d,
1H), 7.05 (d, 2H), 6.69 (d, 2H), 4.59 (m, 4H), 3.69 (m, 4H), 2.72 (m,
2H), 2.50 (m, 2H), 1.81 (t, 2H); 13C-NMR (DMSO-d6) d: 164.8, 158.3,
154.5, 145.3, 133.6, 128.9, 127.5, 124.4, 119.7, 110.5, 144.5,
129.3, 128.0, 112.1, 52.3, 41.2, 38.3, 30.8, 29.0. Anal. calc. for
C25H28N4O2Cl2 N HCl N H2O (541.5): C, 55.40; H, 5.72; N, 10.34.
Found: C, 55.23; H, 5.63; N, 10.28.
N-(3-(4-Bis(2-chloroethyl)aminophenyl)propyl)-5-(4-(N-
isopropylamidino)phenyl)-2-furancarboxamide
hydrochloride 8
The authors have declared no conflict of interest.
This compound was synthesized using a similiar procedure as
for 7.
1
Yield: 70%; m.p.: 224–2268C; H-NMR (DMSO-d6) d: 9.62 (br,
1H), 9.45 (br, 1H), 9.07 (br, 1H), 8.01 (d, 2H), 7.90 (d, 2H), 7.38 (d,
1H), 7.36 (d, 1H), 7.05 (d, 2H), 6.69 (d, 2H), 4.59 (m, 4H), 4.05 (m,
1H), 3.69 (m, 4H), 2.72 (m, 2H), 2.50 (m, 2H), 1.81 (t, 2H), 1.32 (t,
3H), 1.27 (d, 6H); 13C-NMR (DMSO-d6) d: 161.0, 158.0, 154.5, 145.1,
144.5, 133.1, 129.5, 129.2, 128.9, 128.0, 124.2, 119.8, 112.1,
110.3, 52.3, 45.1, 41.2, 38.3, 30.8, 29.0, 21.2. Anal. calc. for
C28H34N4O2Cl2 N HCl N H2O (583.5): C, 57.58; H, 6.34; N, 9.60.
Found: C, 57.39; H, 6.32; N, 9.53.
Experimental
Chemistry
Melting points were determined on Bꢀchi 535 melting-point
apparatus (Bꢀchi, Flawil, Switzerland) and were uncorrected.
Analyses were performed on a Perkin Elmer 2400 analyser (Per-
kin Elmer, Shelton, CT, USA) and satisfactory results within
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Arch. Pharm. Chem. Life Sci. 2009, 342, 484–490
Amidine Analogues of Bis(2-chloroethyl)amine
489
N-(3-(4-Bis(2-chloroethyl)aminophenyl)propyl)-5-[4-(4,5-
dihydro-1H-imidazol-2-yl)phenyl]-2-furancarboxamide
hydrochloride 9
This compound was synthesized using a similar procedure as for
compound 7.
Pharmacology
Ethidium bromide, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenylte-
trazolium bromide (MTT), netropsin, distamycin, calf thymus
DNA, homopolymers poly(dA-dT) N poly(dA-dT), and poly(dG-
dC) N poly(dG-dC) were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Topoisomerase I and II was purchased from
Amersham Pharmacia Biotech (USA). Stock cultures of MDA-MB-
231 and MCF-7 human breast cancer cells were purchased from
the American Type Culture Collection (Rockville, MD, USA). Dul-
becco's minimal essential medium (DMEM) and foetal bovine
serum (FBS) used in cell culture were products of Gibco (Carls-
bad, CA, USA). Glutamine, penicillin and streptomycin were
obtained from Quality Biologicals Inc. (Gaithersburg, MD, USA).
[3H]Thymidine (6.7 Ci/mmol) was the product of NEN (Boston,
MA, USA).
Yield: 74%; m.p.: 217–2198C; 1H-NMR (DMSO-d6) d: 10.66 (br,
1H), 8.07 (dd, 4H), 7.42 (d, 1H), 7.40 (d, 1H), 7.05 (d, 2H), 6.69 (d,
2H), 4.59 (m, 4H), 4.02 (s, 4H), 3.69 (m, 4H), 2.72 (m, 2H), 2.50 (m,
2H), 1.81 (t, 2H); 13C-NMR (DMSO-d6) d: 164.2, 158.0, 154.2, 145.4,
144.5, 134.1, 129.5, 129.0, 128.0, 124.6, 121.7, 119.7, 112.1,
111.0, 52.3, 44.5, 41.2, 38.3, 30.8, 29.0. Anal. calc. for
C27H30N4O2Cl2 N HCl N H2O (567.5): C, 57.09; H, 5.81; N, 9.87.
Found: C, 57.00; H, 5.77; N, 9.80.
N-(3-(4-Bis(2-chloroethyl)aminophenyl)propyl)-5-(4-(N-
cyclopropylamidino)phenyl)-2-furancarboxamide
hydrochloride 10
This compound was synthesized using a similar procedure as for
Cell culture
Human breast cancer MDA-MB-231 and MCF-7 cells maintained
in DMEM supplemented with 10% fetal bovine serum (FBS),
50 U/mL penicillin, 50 lg/mL streptomycin at 378C. Cells were
cultured in Costar flasks and subconfluent cells were detached
with 0.05% trypsin and 0.02% EDTA in calcium-free phosphate
buffered saline, counted in hemocytometers and plated at
56105 cells per well of 6-well plates (Nunc, Denmark) in 2 mL of
growth medium (DMEM without phenol red with 10% CPSR1).
Cells reached about 80% of confluency at day 3 and in most cases
such cells were used for the assays.
compound 7.
1
Yield: 78%; m.p.: 202–2048C; H-NMR (DMSO-d6) d: 9.96 (br,
1H), 9.64 (br, 1H), 9.15 (br, 1H), 8.01 (d, 2H), 7.82 (d, 2H), 7.36 (d,
1H), 7.30 (d, 1H), 7.05 (d, 2H), 6.69 (d, 2H), 4.59 (m, 4H), 3.69 (m,
4H), 2.78-2.50 (m, 5H), 1.81 (t, 2H), 0.94 (m, 2H), 0.83 (m, 2H); 13C-
NMR (DMSO-d6) d: 174.7, 164.0, 158.0, 154.5, 145.1, 144.5, 133.2,
129.3, 128.2, 127.8, 124.2, 119.7, 112.1, 110.4, 52.3, 41.2, 38.3,
30.8, 29.0, 24.7, 6.6. Anal. calc. for C28H31N4O2Cl2 N HCl N H2O
(580.5): C, 57.88; H, 5.86; N, 9.65. Found: C, 57.68; H, 5.69; N, 9.59.
DNA synthesis assay
To examine the effect of the studied compounds on cells' prolif-
eration of MCF-7, cells were seeded in 6-well plates and grown as
described above. Cells in culture were incubated with varying
concentrations of compounds 7–12 or chlorambucil and 0.5 lC
of [3H]thymidine for 24 h at 378C. The cells were then harvested
by trypsinization and washed (with cold phosphate-buffered sal-
ine) with centrifugation for 10 min at 1500 g several times (4–5)
until the dpm in the washes were similar to the reagent control.
Radioactivity was determined by liquid scintillation counting.
[3H]Thymidine uptake was expressed as dpm/cell.
N-(3-(4-Bis(2-chloroethyl)aminophenyl)propyl)-5-(4-(N-
cyclopentylamidino)phenyl)-2-furancarboxamide
hydrochloride 11
This compound was synthesized using a similiar procedure as
for 7.
Yield: 75%; m.p.: 259–2618C; 1H-NMR (DMSO-d6) d: 9.74 (br,
1H), 9.50 (br, 1H), 9.10 (br, 1H), 8.02 (d, 2H), 7.83 (d, 2H), 7.42 (d,
1H), 7.30 (d, 1H), 7.05 (d, 2H), 6.69 (d, 2H), 4.59 (m, 4H), 4.16 (m,
1H), 3.77 (m, 4H), 3.60 (t, 4H), 2.70–2.50 (m, 4H), 2.07 (m, 2H),
1.81 (t, 2H), 1.75 (m, 4H), 1.60 (m, 2H); 13C-NMR (DMSO-d6) d:
162.5, 158.0, 154.5, 144.5, 144.0, 132.8, 129.3, 128.9, 128.0,
124.2, 120.0, 112.1, 110.3, 54.2, 52.3, 41.2, 38.3, 31.2, 30.8, 29.0,
23.6. Anal. calc. for C30H35N4O2Cl2 N HCl N H2O (608.5): C, 59.16; H,
6.24; N, 9.20. Found: C, 59.03; H, 6.19; N, 9.08.
Cell viability assay
The assay was performed according to the method of Carmichael
et al. using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) [18]. Confluent cells, cultured for 48 h with vari-
ous concentrations of studied compounds in 6-well plates were
washed three times with PBS and then incubated for 4 h in 1 mL
of MTT solution (0.5 mg/mL of PBS) at 378C in 5% CO2 in an incu-
bator. The medium was removed and 1 mL of 0.1 mol/L HCl in
absolute isopropanol was added to attached cells. Absorbance of
converted dye in living cells was measured at a wavelength of
570 nm. Cell viability of breast cancer cells cultured in the pres-
ence of ligands was calculated as a per cent of control cells.
N-(3-(4-Bis(2-chloroethyl)aminophenyl)propyl)-5-[4-
(3,4,5,6-tetrahydro-1H-pyrimidine-2-yl)phenyl]-2-
furancarboxamide hydrochloride 12
This compound was synthesized using a similar procedure as for
compound 7.
Yield: 77%; m.p.: 249–2518C; 1H-NMR (DMSO-d6) d: 10.13 (br,
1H), 8.03 (d, 2H), 7.90 (d, 2H), 7.05 (d, 2H), 7.37 (d, 1H), 7.35 (d,
1H), 6.69 (d, 2H), 4.59 (m, 4H), 3.69 (m, 4H), 3.39 (t, 4H), 2.72 (m,
2H), 2.50 (m, 2H), 2.01 (m, 2H), 1.81 (t, 2H); 13C-NMR (DMSO-d6) d:
158.9, 158.3, 154.4, 144.5, 144.1, 132.0, 129.3, 128.4, 128.1,
129.7, 124.3, 112.1, 119.6, 110.2, 52.3, 41.2, 38.6, 38.1, 30.8, 29.0,
17.6. Anal. calc. for C28H32N4O2Cl2 N HCl N H2O (581.5): C, 57.78; H,
6.02; N, 9.63. Found: C, 57.64; H, 5.98; N, 9.70.
Relaxation assay of topoisomerase I and II
PBR322 plasmid DNA (0.083 lg) was incubated with 1 unit of
human topoisomerase
I (reaction buffer: 50 mM Tris-HCl
(pH 7.9), 1mM EDTA, 0.5 M NaCl, 1 mM dithiothreitol) or human
topoisomerase II (reaction buffer: 10 mM Tris-HCl (pH 7.9), 1 mM
ATP, 50 mM KCl, 5 mM MgCl2, 50 mM NaCl, 0.1 mM EDTA, and
15 lg/mL bovine serum albumin) in the presence of varying con-
centrations of the test compound. The mixture was incubated at
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www.archpharm.com

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K. Bielawski et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 484–490
378C for 1 h and the reaction was terminated by addition of 2 lL
of 10% SDS and 2 lL of proteinase K (1 mg/mL). The reaction mix-
ture was subjected to electrophoresis through a 0.8% agarose gel
containing 0.5 mg/mL ethidium bromide in TBE buffer (90 mM
Tris-borate and 2 mM EDTA). The gels were stained with ethi-
dium bromide and photographed under UV light. For the quan-
titative determination of topoisomerase-concentration activity,
photographic negatives were scanned and the area representing
supercoiled DNA, migrating as a single band at the bottom of
the gel was measured using UVI-KS4000i gel documentation and
analysis system (SyngenBiotech, USA). The concentrations of the
inhibitor that prevented 50% of the supercoiled DNA from being
converted into relaxed DNA (IC50 values) were determined by
averaging the data from at least three experiments.
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Ethidium displacement assay
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Fluorescence was measured using a Hitachi spectrophotometer
F-2500 FL (Tokyo, Japan) at room temperature. The DNA-ethi-
dium complex was excited at 546 nm and the fluorescence was
measured at 595 nm. To 2 mL of ethidium bromide (5.0610– 6
M) in 10 mM Tris-HCl (pH 7.4), 75 mM NaCl buffer solution, con-
taining 25 lL of DNA solution (A260 = 2) was added, and the max-
imum fluorescence was measured. Aliquots of a 10 mM stock of
the test compound solution were then added to the DNA-ethi-
dium solution, and the fluorescence was measured after each
addition until a 50% reduction of fluorescence had occurred.
Theoretical curves were fit to the fluorescence intensity data
points with nonlinear least-squares computer routines. The
apparent binding constant was calculated from KEtBr[EtBr] = Kapp
[drug], where [drug] = the concentration of the test compound at
a 50% reduction of fluorescence and KEtBr is known [20]. The com-
pounds 7–12 and their complexes with DNA showed neither
optical absorption nor fluorescence at 595 nm and did not inter-
fere with the fluorescence of an unbound ethidium bromide.
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Statistical analysis
In all experiments, the mean values for three assays l standard
deviations (S.D.) were calculated. The results were submitted to
statistical analysis using the Student's t-test. Differences were
considered significant when p a 0.05. Mean values, the standard
deviations and the number of measurements in the group (n)
are presented in the figures.
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i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com