Synthesis and biological evaluation of distamycin analogues - New potential anticancer agents

Article abstract of DOI:10.1002/ardp.200800122

Eight of analogues of distamycin, potential minor-groove binders, were synthesized and tested for in-vitro cytotoxicity towards human breast cancer cells MCF-7 and MDA-MB-231. The method of synthesis is simple and convenient. All of the compounds 1 - 8 sh

Full text of DOI:10.1002/ardp.200800122

Arch. Pharm. Chem. Life Sci. 2009, 342, 87–93
D. Drozdowskia et al.
87
Full Paper
Synthesis and Biological Evaluation of Distamycin Analogues
– New Potential Anticancer Agents
Danuta Drozdowska1, Malgorzata Rusak2, Wojciech Miltyk3, and Krystyna Midura-Nowaczek1
1 Department of Organic Chemistry, Medical University, Białystok, Poland
2 Department of Haematological Diagnostics, Medical University, Białystok, Poland
3 Laboratory of Drug Analysis, Medical University, Białystok, Poland
Eight of analogues of distamycin, potential minor-groove binders, were synthesized and tested
for in-vitro cytotoxicity towards human breast cancer cells MCF-7 and MDA-MB-231. The method
of synthesis is simple and convenient. All of the compounds 1–8 showed antiproliferative and
cytotoxic effects against both cell lines in the range 3.47 to 12.53 lM for MDA-MB-231 and 4.35
to 12.66 lM for MCF-7. All compounds demonstrated activity against DNA topoisomerases I and
II at a concentration of 50 lM. The ethidium bromide assay showed that these compounds bind
to plasmid pBR322, yet weaker than distamycin. Further investigations concerning the mecha-
nism of cytotoxicity are now in progress, but the IC50 values suggest that synthetic distamycin
analogues with a free amino group, 3–4 and 7–8, can serve as potential carriers of strong acting
elements, e.g. alkylating groups.
Keywords: Cytotoxic activity / Anticancer activity / Distamycin analogue / DNA binding / DNA topoisomerase /
Received: June 30, 2008; accepted: November 3, 2008
DOI 10.1002/ardp.200800122
pounds with similar interaction with DNA. We searched
a lot of compounds which do not intercalate with DNA,
have a high base-pair specificity, an isohelical shape simi-
lar to the minor groove of B-DNA [3]. The class of synthetic
heteroaromatic oligopeptides, projected after the models
of netropsin and distamycin, received the name lexitrop-
sins [4]. Although a huge progress in designing lexotro-
pins with an extremely selective mode of operation was
made, we did not obtain compounds ready to be applied
in therapy [5].
We focused on the strategy to replace the N-methylpyr-
role rings of distamycin and netropsin with benzene
rings, simultaneously modifying the cationic heads. The
carbocyclic analogues of distamycin with an unsubsti-
tuted N-terminal group NH2 inhibited in-vitro activity of
topoisomerase I and II [6], in the same fashion as the
derivatives of netropsin with an aliphatic linker (four
and six groups of CH2) [7]. Studying the interaction of
compounds with DNA by the ethidium-displacement
assay confirmed that they had a larger specificity for AT
in comparison to GC-rich regions, similarly to netropsin
and distamycin [8, 9]. These carbocyclic analogues of
netropsin and distamycin served as carriers to other
Introduction
The rapidly increasing knowledge in molecular biology
makes it possible to observe that the large family of
sequence-specific ligands non-intercalatively binding
within the minor groove of B-DNA is very important in
antitumour drugs searching [1, 2]. This interaction
results in the occupancy of natural nucleic sequence tar-
geted by enzymes or in DNA distortion nearby these sites.
The cytotoxic effect of these antineoplastic agents and
the inhibition of many cellular processes is determined
mainly by interference with the catalytic activity of
important regulatory proteins, such as topoisomerase I
and II and a number of proteases [1]. Most of the minor-
groove DNA-binding drugs, especially netropsin and dis-
tamycin, exhibit high sequence selectivity.
From the DNA-binding model of netropsin and dista-
mycin came the inspiration to search for new com-
Correspondence: Danuta Drozdowska (Bartulewicz), Department of Or-
ganic Chemistry, Medical University, 15-222 Białystok, Poland.
E-mail: ddrozd@amb.edu.pl
Fax: +48 85 748-5416
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D. Drozdowskia et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 87–93
Figure 1. Structures of distamycin A and furamidine B.
groups. We obtained derivatives with a N-terminal chlor- perature and at atmospheric pressure. Then, a black pre-
ambucyl group which exhibited activity in cultured cipitate of catalyst was filtered off and the resulting solu-
breast cancer MCF-7 [10], and with a 5-[N,N-bis(2-chlore- tion was concentrated under reduced pressure. The
thylo)amine]-2,4-dinitrobenzoyl group – in the face of crude products 3, 4, 7, and 8 were purified by column
hepatoma HEP G2 in hypoxic conditions [11].
chromatography in chloroform with a methanol gra-
This paper is in continuation of rational drug design dient.
program aiming to develop distamycin analogues, poten-
tial minor-groove binders, and inhibitors of topoisomer- Pharmacology
ases. We developed new compounds with skeletons that The described compounds were tested for their in-vitro
combine the structural features of distamycin A and fura- antitumour activity in the human breast cancer cells,
midine B (Fig. 1).
standard MCF-7 and estrogen-independent MDA-MB-231.
The purpose of this work was the synthesis of eight Their cytotoxic activity as percentage of nonviable cells is
new compounds containing benzene connected with het- shown in Tables 1 and 2. All of the tested compounds
eroaromatic rings, in different configurations. Prelimi- showed concentration-dependent activity.
nary evaluation of their biological properties – antiproli-
Fluorescent microscopy assay showed morphological
ferative and cytotoxic activity in MCF-7 and MDA-MB-231 changes of cells to determine the apoptosis process. After
cell lines and the capacity to inhibit human topoisomer- the incubation of MCF-7 and MDA-MB-231 breast cancer
ases I and II in vitro were planned. We also investigated cells with the tested compounds, the cells were dyed.
the binding of compounds 1–8 to plasmid DNA employ- Acridine orange (fluorescent DNA-binding dye) interca-
ing the ethidium-bromide assay [12].
lates into DNA, making it appear green, and binds to
RNA, staining it red-orange. Ethidium bromide is only
taken up by nonviable cells; its fluorescence overpowers
that of the acridine orange, making the chromatin of
necrotic cells appear orange [13].
Results and discussion
Chemistry
Two hundred cells per sample were examined by fluo-
The analogues 1–8 were synthesized by simple acylation rescence microscopy, according to the following criteria:
of the aromatic amines as shown in Scheme 1. The start- viable cells with normal nuclei (fine reticular pattern of
ing materials for synthesis, isophtaloyl chloride B1 and green stain in the nucleus and red-orange granules in the
terephtaloyl chloride B2 were dissolved in methylene cytoplasm); viable cells with apoptotic nuclei (green chro-
chloride and cooled. Aromatic amines A1 –A4, dissolved matin which is highly condensed or fragmented and uni-
in methylene chloride and mixed with TEA (triethyl- formly stained by acridine orange); nonviable cells with a
amine), were added dropwise to the solution of chlorides. normal nuclei (bright orange chromatin with organized
The reaction mixtures were stirred at room temperature structure); and nonviable cells with apoptotic nuclei
for 2 h. The precipitated crude products 1, 2, 5, and 6 (bright orange chromatin which is highly condensed or
were filtered off and washed with chloroform.
fragmented).
The nitro group of compounds A3BA3 and A4BA4 were
In this experiment, we have found that all analyzed
reduced by catalytic hydrogenation (Pd / C) in methanol. compounds induced concentration-dependent apoptosis
The reaction mixture was stirred for 1.5 h at room tem- (Fig. 2). Only compound 2, 3, and 4 at the concentration
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Arch. Pharm. Chem. Life Sci. 2009, 342, 87–93
Analogues of Distamycin – Synthesis / Biological Evaluation
89
Scheme 1. Synthesis of compounds 1–8.
Table 1. Viability of MCF-7 cells treated for 24 h with different concentrations of compounds 1–8.
Concentration
Nonviable cells (% of control l 2)a)
(lM)
1
2
3
4
5
6
7
8
5
36a
56
70
91
100
6.38
23
44
68
100
100
30
56
68
93
100
8.32
24
36
63
100
100
20
52
68
99
100
10.99
44
70
76
100
100
5.83
20
28
84
100
100
12.66
40
52
76
98
100
10
15
30
50
IC50
11.74
11.67
4.35
a)
Mean values l SD from three independent experiments done in duplicate are presented.
Table 2. Viability of MDA-MB-231 cells treated for 24 h with different concentrations of compounds 1–8.
Concentration
Nonviable cells (% of control l 2)a)
(lM)
1
2
3
4
5
6
7
8
5
34a
58
61
83
96
21
60
65
98
100
9.81
49
55
60
67
72
35
47
56
79
98
12.53
49
55
52
69
85
32
46
68
86
100
10.62
44
48
62
87
100
8.07
39
49
52
73
99
12.53
10
15
30
50
IC50
8.79
5.76
3.47
a)
Mean values l SD from three independent experiments done in duplicate are presented.Figure
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D. Drozdowskia et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 87–93
Figure 2. Morphological apoptosis evaluation in the cytotoxic assay on MCF-7 and MDA-MB-231 cells treated for 24 h with 10 lM of
the examined compounds 1–8. White columns represent cells in the apoptotic stage and black columns represent cells in the
necrotic stage. Mean percentages l S.D. from three independent experiments are presented.
Table 3. DNA-binding effect of distamycin and compounds 1–8.
Compound:
EtBr
0
DNA-EtBr DST
100 63.45
1
2
3
4
5
6
7
8
% fluorescence:
92.15
92.54
92.69
91.47
85.67
81.34
92.03
93.14
with ethidium bromide. The lower concentrations did
not show any changes. As can be seen in Fig. 3, the super-
helical plasmid (lane 1) was relaxed by topoisomerases
(lane 3), controls (CT-camtothecin or ET-etoposide,
respectively) inhibited activity of topoisomerases entirely
(lane 2). Figure 3 demonstrates that at a concentration of
50 lM compounds 3 and 8 have a very small effect on the
ability of topo I to transform supercoiled DNA into sev-
eral topoisomer forms of relaxed DNA. At this concentra-
tion, other compounds inhibited the activity on topoiso-
merase I. As we can see, all of the compounds 1–8 showed
inhibitory activity against topoisomerase II.
Figure 3 also shows that the investigated compounds
1–8 are more effective against topoisomerase II. At the
concentration of 50 lM, all of them inhibit topoisomer-
ase I activity only partially.
Under identical conditions, complete inhibition of
DNA cleavage was obtained using 2 lM camptothecin (CT)
and 10 lM etoposide (ET), drugs which are potent inhibi-
tors of topoisomerase Iand topoisomerase II, respectively.
The ethidium bromide assay showed that the investi-
gated compounds can bind to DNA, although relatively
weaker than distamycin (Table 3).
Figure 3. Inhibition of topoisomerases by compounds 1–8.
Native pBR322 plasmid DNA (lane –) was incubated with four
units of topoisomerases I (Topo I) and II (Topo II) in the absence
(lane +) or in the presence of control (line CT–camptothecin or
ET–etoposide) or drug (lanes 1–8). The DNA was analyzed by
1% agarose gel electrophoresis. The gels were stained with ethi-
dium bromide and photographed under UV light.
10 lM caused increased necrotic cell death in MCF-7 cells.
Apoptosis induced by 1 and 5–8 was definitely stronger.
We also observed that the percentage of necrotic cells
was increased in case MDA-MB-231 cells. Only compound
1, 4, and 5 induced apoptotic death of cells.
Purified topoisomerases I and II were incubated with
increased concentrations of compounds 1-8 (5, 10, 15, 30,
and 50 lM) in the presence of supercoiled plasmid DNA.
The products were subjected to electrophoresis to sepa-
rate relaxed and supercoiled circular DNA. Figure 3
shows the results of our electrophoresis analysis of exam-
ined compounds at 50 lM concentration, after staining
Conclusion
We obtained eight new compounds, potential minor
groove binders, and analogues of distamycin. Their syn-
theses were simple, convenient, and allowed to get the
compounds in a good yield. This procedure could be use-
ful to seek other derivatives of distamycin to find the best
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Arch. Pharm. Chem. Life Sci. 2009, 342, 87–93
Analogues of Distamycin – Synthesis / Biological Evaluation
91
prepared on precoated plates (Merck, silica gel 60F-254) and
visualized with UV. 5% NH3aq in methanol was used as a solvent
system. Silica gel 60 (Merck, 230–400 mesh) was used for col-
umn chromatography. Solvents used in the experiments were
dried and distilled. Melting points were determined on Bꢀchi
535 melting-points apparatus (Bꢀchi, Switzerland) and are
configuration of aromatic, benzene, and hetero-aromatic
rings.
The in-vitro experiment findings revealed that all of new
lexitropsins exhibit sufficient tumor cell cytotoxicity
towards the standard cell line of the mammalian tumour
MCF-7 and estrogen-independent MDA-MB-231 breast can-
cer cells. The most interesting compound seems to be 1
1
uncorrected. H-NMR and 13C-NMR spectra were recorded on a
Bruker AC 200F spectrometer (Bruker, 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). The
results of the elemental analyses for C and H were within l 0.4%
of the theoretical values. Syntheses of all new compounds are
given below.
with
a time-dependent reduction in proliferation
observed in both cell lines at concentrations: 6.38 lM for
MCF-7 and 8.79 lM for MDA-MB-231 cells, respectively.
Compound 5 with IC50 10.99 lM for MCF-7 and 3.47 lM for
MDA-MB-231 cells, respectively, is also interesting. All of
the investigated compounds are more potent than chlor-
ambucil whichMCF-7 IC50 averagesof 24.6 lM[6].
N,N9-Bis-thiazol-2-yl-isophthalamide 1
To a cooled solution of 2-aminothiazole (2 g, 19.97 mmol) in
CH2Cl2 (50 mL) was added triethylamine (2.77 mL, 19.97 mmol).
The isophthaloyl chloride (2.03 g, 9.98 mmol) in CH2Cl2 (50 mL)
was then added dropwise during 0.5 h. Then, the reaction mix-
ture was stirred at room temperature over a period of 3 h. The
obtained beige precipitate was filtered off and washed with
CHCl3. After recrystallisation from CHCl3 and drying, we
obtained 2.67 g of compound 1 (80.1%), m.p.: 306–3078C; Rf =
0.72; 1H-NMR (D2O) d [ppm]: 8.31 (s, 4H), 7.31 (d, 2H), 7.58 (d, 2H);
13C-NMR (D2O) d [ppm]: 166.62, 164.52, 137.40, 135.53, 135.52,
129.42, 128.29, 114.00; C14H10N4O2S2: 330.39.
Evaluation of the topoisomerase inhibition provided
additional insight into the structure-activity relationship
associated with these compounds. All of the compounds
inhibited the activity of DNA topoisomerases, but we can
see more activity against topoisomerase II. It would be
interesting to investigate the DNA-binding mode of these
compounds in detail and to determine the topoisomer-
ase IC50, especially for compounds 3 and 8 and to deter-
mine the compounds binding constants for binding
selectivity.
We have shown that compounds 1–8 can bind to DNA
and are potent inhibitors of both topoisomerase I and II.
These compounds inhibit the catalytic activity of the top-
oisomerase at a step prior to the formation of the topo-
DNA complexes. This suggests that DNA binding may be
implicated in the cytotoxicity of the compounds, possi-
bly by inhibiting the interactions between topoisomer-
ases and their DNA targets. Moreover, there might be
other possible targets, such as other enzymes, involved in
DNA metabolism and / or transcription factors because
their activities were inhibited by some minor-groove
binders [15–17]. Further biological studies of the DNA-
binding mode and other biological properties will be
described in due course.
N,N9-Di-pyridin-2-yl-isophthalamide 2
To a cooled solution of 2-aminopyridine (2 g, 21.25 mmol) in
CH2Cl2 (30 mL) was added triethylamine (2.95 mL, 21.15 mmol).
The isophthaloyl chloride (2.16 g, 10.63 mmol) in CH2Cl2 (40 mL)
was then added dropwise during 0.5 h. Then, the reaction mix-
ture was stirred at room temperature over a period of 3 h. The
obtained yellow precipitate was filtered off and washed with
CHCl3. After recrystallisation from CHCl3 and drying, we
obtained 0.31 g of compound 2 (59.14%), m.p.: 296–2978C; Rf =
0.82; 1H-NMR (DMSO-d6) d [ppm]: 12.67 (br, 2H), 8.82 (s, 1H), 8.32
(d, 2H), 7.72 (t, 1H), 7.41 (m, 6H), 7.28 (t, 2H); 13C-NMR (DMSO-d6) d
[ppm]: 164.55, 156.27, 146.32, 144.66, 137.53, 132.12, 131.94,
129.08, 120.86, 114.01; C18H12N4O2: 318.33.
N,N9-Bis-(5-amino-pyridin-2-yl)-isophthalamide 3
To
a
cooled solution of 2-amino-5-nitropyridine (2 g,
14.38 mmol) in CH2Cl2 (60 mL) was added triethylamine
(2.95 mL, 21.15 mmol). The isophthaloyl chloride (1.46 g,
7.19 mmol) in CH2Cl2 (40 mL) was then added dropwise during
0.5 h. Then the reaction mixture was stirred at room tempera-
ture over a period of 20 h. The obtained beige precipitate was fil-
tered off and washed with CHCl3. After recrystallisation from
CHCl3 and drying, we obtained 2.11 g of the nitro-compound
(71.92%). Catalytic hydrogenation of the nitrocompound (0.2 g;
0.57 mmol) was carried out in methanol (20 mL) in the presence
of Pd / C (10%). The aromatic amine was purified using the sol-
vent system CH2Cl2 / MeOH (gradient). After evaporating of frac-
tions with amine, we obtained compound 3 (0.124 g, 0.39 mmol)
(68.42%), m.p.: 319–3208C; Rf = 0.85; 1H-NMR (DMSO-d6) d [ppm]:
10.15 (br, 2H), 8.47 (s, 1H), 8.11 (m, 4H), 7.62 (t, 3H), 6.97 (d; 2H),
5.65 (br, 4H); 13C-NMR (DMSO-d6) d [ppm]: 164.69, 139.61, 139.60,
135.55, 131.92, 131.49, 128.78, 128.56, 109.89, 108.46;
C18H16N6O2: 348.36.
The obtained analogues of distamycin with free amino
groups can also be used as carriers for active groups, e.g.
alkylating fragments. Their therapeutic applications
could be considered after further investigation.
We thank the State Committee for Scientific Research (grant
KBN No. 2P05F 017 27, in 2004-2007) for financial support.
Experimental
Chemistry
Compounds were synthesized with suitable parent substances
(Merck and Aldrich, Germany). Thin-layer chromatograms were
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Arch. Pharm. Chem. Life Sci. 2009, 342, 87–93
N,N9-Bis-(3-amino-phenyl)-isophthalamide 4
Pharmacology
To a cooled solution of 3-nitroaniline (2 g, 14.48 mmol) in
CH2Cl2 (50 mL) was added triethylamine (2.01 mL, 14.48 mmol).
The isophthaloyl chloride (1.47 g, 7.24 mmol) in CH2Cl2 (50 mL)
was then added dropwise during 0.5 h. Then, the reaction mix-
ture was stirred at room temperature over a period of 20 h. The
obtained beige precipitate was filtered off and washed with
CHCl3. After recrystallisation from CHCl3 and drying, we
obtained the nitro-compound (4.31 g, 11.45 mmol) (79.07%). Cat-
alytic hydrogenation of the nitro-compound (0.2 g, 0.53 mmol)
was carried out in methanol (20 mL) in the presence of Pd / C
(10%). The aromatic amine was purified using the solvent system
CH2Cl2 / MeOH (gradient). After evaporating of fractions with
amine, we obtained compound 4 (0.12 g, 0.39 mmol) (70.59%),
m.p. 285–2868C; Rf = 0.92; 1H-NMR (DMSO-d6) d [ppm]: 10.48 (br,
2H), 8.49 (s, 1H), 8.14 (d, 2H), 7.62 (t, 1H), 7.20-6.86 (m, 8H), 6.35
(br, 4H); 13C-NMR (DMSO-d6) d [ppm]: 164.67, 139.61, 139.60,
135.55, 131.92, 131.49, 128.77, 128.57, 109.89, 108.49;
C18H16N6O2: 348.36.
Ethidium bromide was purchased from Carl Roth GmbH, Ger-
many, topoisomerase I and II from Amersham Pharmacia, Bio-
tech (USA). Stock cultures of breast cancer MCF-7 and MDA-MB-
231 were purchased from the American Type Culture Collection,
Rockville, MD, USA. Dulbecco's modified Eagle's medium
(DMEM), fetal bovine serum (FBS), distamycin, streptomycin,
and penicillin were products of Sigma (Germany). Plasmid
pBR322 was purchased from Fermentas Life Science (Germany).
Cell culture
Human breast cancer MDA-MB-231 and MCF-7 cells maintained
in Dulbecco's modified Eagle's medium supplemented with 10%
FBS, 50 lg/mL streptomycin, 100 U/mL penicillin at 378C, atmos-
phere containing 5% CO2. Cells were cultivated in Costar flasks
and subconflluent cells were detached with 0.05% trypsin and
0.02% EDTA in calcium-free phosphate-buffered saline. The
study was carried out using cells from passages 3-7, growing as
monolayer in six-well plates (Nunc) (56105 cells per well) and
pre-incubated 24 h without phenol red.
N,N9-Bis-thiazol-2-yl-terephthalamide 5
Determination of IC50
The procedure was analogous to that for compound 1 with ter-
ephtaloic chloride (2.03 g, 9.98 mmol) in CH2Cl2 (50 mL). Yield:
The compounds were dissolved in DMSO and used at concentra-
tions of 5, 10, and 15 lM. Microscopic observations of cell mono-
layers were performed with a Nikon optiphot microscope.
Wright–Giemsa staining was performed using the Fisher Leuko
Stat Kit (Fisher Scientific). After 24 h of drug treatment, MCF-7
cells were mixed with a dye mixture (10 lM acridine orange and
10 lM ethidium bromide, prepared in phosphate-buffered sal-
ine). At the end of each experimental time point, all of the media
was removed and cells were harvested by incubation with 0.05%
trypsin and 0.02% EDTA for 1 min and washed with the medium.
Then, 250 lL of cell suspension was mixed with 10 lL of the dye
mix and 200 cells per sample were examined by fluorescence
microscopy and we counted the percentage of nonviable (apop-
totic and necrotic) cells. The results were submitted to statistical
analysis using the method of the smallest squares.
1
92.18% (3.04 g, 9.20 mmol), m.p.: 344–3458C; Rf = 0.65; H-NMR
(DMSO-d6) d [ppm]: 10.19 (br, 2H), 8.20 (s, 4H), 7.57 (d, 2H), 7.32 (d,
2H); 13C-NMR (DMSO-d6) d [ppm]: 166.65, 164.56, 137.38, 135.55,
128.32, 114.04; C14H10N4O2S2: 330.39.
N,N9-Di-pyridin-2-yl-terephthalamide 6
The procedure was analogous to that for compound 2 with ter-
ephtaloic chloride (2.16 g, 10.63 mmol) in CH2Cl2 (40 mL). Yield:
61.71% (2.09 g, 6.56 mmol), m.p.: 252–2538C; Rf = 0.74; 1H-NMR
(DMSO-d6) d [ppm]: 10.97 (br, 2H), 8.39 (d, 2H), 8.13 (d, 2H), 7.65 (s,
4H), 7.20 (m, 4H; 13C-NMR (DMSO-d6) d [ppm]: 166.69, 152.01,
147.97, 137.54, 137.34, 128.84, 119.99, 114.79; C18H12N4O2:
318.33.
Relaxation assay of topoisomerase I and II
Native pBR322 plasmid DNA (0.20 lg) was incubated with 4 unit
topoisomerase I (reaction buffer: 50 mM Tris-HCl (pH 7.9), 1 mM
EDTA, 0.5 M NaCl, 1 mM dithiothreitol) or human topoisomer-
ase 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 absence or presence of varying
concentrations of the test compounds (5, 10, 15, 30 or 50 lM), as
well camptothecin (2 lM) or etoposide respectively (10 lM) in a
final volume of 10 lL. The mixture was incubated at 378C for 30
min and the reaction was terminated by addition of 2 lL of 10%
SDS. The reaction mixture was subjected to electrophoresis (3
hour, 90 V) through a 1.0% agarose gel in TBE buffer (90 mM Tris-
borate and 2 mM EDTA). The gels were stained for 30 min with
ethidium bromide solution (0.5 lg/mL). The DNA was visuali-
sated using 312 nm wavelength transilluminator and photo-
graphed under UV light (Canon PowerShot G6, 7.1 mLn mega-
pixels).
N,N9-Bis-(5-amino-pyridin-2-yl)-terephthalamide 7
The procedure was was analogous to that for compound 3 with
terephtaloic chloride (1.46 g, 7.19 mmol) in CH2Cl2 (40 mL).
Obtained nitro-compound: yield: 88.76% (2.61 g, 6.90 mmol).
Hydrogenation of the nitro-compound (0.5 g, 1.32 mmol) gave
compound 7 (0.14 g, 0.40 mmol) (30.30%), m.p. 209–2118C; Rf =
0.90; 1H-NMR (DMSO-d6) d [ppm]: 10.13 (br, 2H), 8.05 (s, 2H), 7.99
(d, 2H), 7.12 (s, 4H), 6.93 (d, 2H), 6.31 (d, 4H); 13C-NMR (DMSO-d6) d
[ppm]: 164.53, 139,53, 137.52, 129,15, 127.57, 109.92;
C18H12N4O2: 318.33.
N,N9-Bis-(3-amino-phenyl)-terephthalamide 8
The procedure was analogous to that for compound 4 with ter-
ephtaloic chloride (1.47 g, 7.24 mmol) in CH2Cl2 (50 mL).
Obtained nitro-compound: yield: 59.39% (1.62 g, 4.30 mmol).
Hydrogenation of the nitro-compound (0.2 g, 0.53 mmol) gave
compound 8 (0.13 g, 0.37 mmol) (69.81%), m.p.: 285–2868C; Rf =
0.92; 1H-NMR (DMSO-d6) d [ppm]: 10.48 (br, 2H), 8.09 (s, 4H), 7.72
(s, 4H), 6.35 (br, 4H); 13C-NMR (DMSO-d6) d [ppm]: 164.22, 141.63,
136.96, 129.14, 127.65, 122.26, 116.02; C18H16N6O2: 348.36.
Ethidium bromide assay
Each well of 96-well plate was loaded with Tris buffer containing
ethidium bromide (0.1 M Tris, 1 M NaCl, pH 8, 0.5 mM EtBr final
concentration, 100 lL). To each well was added 15 lg plasmid
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Arch. Pharm. Chem. Life Sci. 2009, 342, 87–93
Analogues of Distamycin – Synthesis / Biological Evaluation
93
pBR322 as water solution (0.05 lg/lL). Then, to each well was
added distamycin A or compound 1–8 (1 lL of a 1 mM solution
in water, 10 lM final concentration). After incubation at 258C
for 30 min, the fluorescence of each well was read on a Multila-
bel Reader Victor 3V (ex.: 355 nm, em.: 615 nm) in duplicate
experiments with two control wells (no drug = 100% fluores-
cence, no DNA = 0% fluorescence). Fluorescence readings are
reported as% fluorescence relative to the controls.
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Statistical analysis
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deviations (S.D.) were calculated.
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nation in the range 0.9600 a R2 a 1. Mean values, the standard
deviations and the number of measurements in the group are
presented in the figures.
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www.archpharm.com