Synthesis and cytotoxic effect of carbocyclic potential minor groove binders

Article abstract of DOI:10.1016/j.farmac.2003.11.005

New carbocyclic potential minor groove binders were synthesised, using 3-nitrobenzoyl chloride and aliphatic α,ω-diamines with three, four and five methylene fragments. The half structures, compounds IV-VI can be compared to bis-amidines, compounds X-XII

Full text of DOI:10.1016/j.farmac.2003.11.005

IL FARMACO 59 (2004) 211–214
www.elsevier.com/locate/farmac
Synthesis and cytotoxic effect of carbocyclic potential
minor groove binders
Danuta Bartulewicz a,*, Tomasz Anchim b, Milena DTbrowska c, Krystyna Midura-Nowaczek a
a Department of Organic Chemistry, Medical University of Białystok, Mickiewicza 2a, Białystok 15222, Poland
b Department of Gynaecological Endocrinology, Medical University of Białystok, Mickiewicza 2a, Białystok 15222, Poland
c Department of Haematological Diagnostics, Medical University of Białystok, Mickiewicza 2a, Białystok 15222, Poland
Received 26 February 2003; accepted 8 November 2003
Abstract
New carbocyclic potential minor groove binders were synthesised, using 3-nitrobenzoyl chloride and aliphatic a,x-diamines with three,
four and five methylene fragments. The half structures, compounds IV–VI can be compared to bis-amidines, compounds X–XII to
bis-netropsin. All of the compounds were investigated antiproliferative and cytotoxic effects in the standard cell line of mammalian tumour
MCF-7.
© 2003 Elsevier SAS. All rights reserved.
Keywords: Minor groove binders; Bis-netropsin; Carbocyclic analogues of netropsin
1. Introduction
lines of mammalian tumour MCF-7 [5]. Data from the
ethidium displacement assay showed that these compounds
were able to bind in the minor groove-binding mode in AT
sequences of DNA [6]. They can use as carriers of alkylating
elements—carbocyclic analogues with chlorambucil moiety
were synthesised and investigated [7]. Comparison values of
Kapp tri- and dibenzene analogues suggested that the opti-
mum number of benzene units for binding to the DNA could
be two. In addition, in order to rationalise the experimental
findings, computer molecular modelling studies were per-
formed with an appropriate B-DNA on the basis of molecular
mechanics and molecular dynamics calculations.
Cell-permeable small molecules with the ability to target
predetermined DNA sequences would be valuable tools in
molecular biology and potentially in human medicine. A
number of natural and synthetic compounds are known to
bind to DNA double helix in a non-intercalative manner [1].
This is possible because most DNA-binding molecules pos-
sesses cationic functional groups, complementary in size to
one of the grooves, have an aromatic ring system, or a
combination factors of these [1]. Netropsin, bis-netropsin or
bis-amidines (e.g. pentamidine), DNA minor groove binders,
could provide a significant improvement in cancer manage-
ment increasing gene specificity. There are known a lot of
synthetic compounds that binds specifically to many differ-
ent DNA sequences—they all have the name lexitropsins [2].
Due to the high selectivity of DNA interaction, lots of lex-
itropsin were used in the recent past as a DNA sequence-
selective vector of alkylating functions [3,4].
As a part of ongoing rational drug design programme
aiming at development of carbocyclic minor groove binders,
six novel compounds IV–V and X–XII were synthesised and
evaluated (Scheme 1).
2. Experimental
Studies on benzene-containing and C-terminus-modified
analogues of netropsin and distamycin, afforded compounds
with antiproliferative and cytotoxic effects in standard cell
2.1. Chemistry
Compounds IV–VI and X–XII were prepared according
to the general procedure described in this paper (Scheme 1).
Yields and physico-chemical properties of synthesised com-
pounds are reported in Table 1.
* Corresponding author.
E-mail address: dbartule@amb.edu.pl (D. Bartulewicz).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.11.005

212
D. Bartulewicz et al. / IL FARMACO 59 (2004) 211–214
) NHO
O2N
CONH(CH
C
NO2
n
2
O2N
COCl
i
+
H2N(CH2)nNH2
n = 3,4,5
I , n = 3
II, n = 4
III, n = 5
i i
H
2N
CONH(CH ) NHOC
2 n
NH2
IV , n = 3
V, n = 4
VI, n = 5
iii
VII, n = 3
VIII, n = 4
IX, n = 5
iv
H2N
COHN
CONH(CH ) NHOC
2 n
NHOC
NH
2
X, n = 3
XI, n = 4
XII, n = 5
Scheme 1. Synthesis of carbocyclic analogues of minor groove binders. Reagents and conditions: (i) CH2Cl2, 2 h, room temperature; (ii) H2/Pd, MeOH, 1.5 h,
room temperature; (iii) 3-nitrobenzoyl chloride, Py/CH2Cl2, DMAP, 5 h, room temperature; (iv) H2/Pd, MeOH, 2.5 h, room temperature.
The structures of synthesised compounds were confirmed
filtered off and crystallised from acetone to give pure com-
pound III with good yield (85.02%).
1
by analyses of their H NMR and 13C NMR spectra. The
spectra were recorded on a Bruker AC 200F spectrometer,
using TMS as an internal standard. Chemical shifts are ex-
pressed in d value (ppm). The results of elemental analyses
for C and H were within 0.4% of the theoretical values.
Melting points were determined on Buchi 535 melting points
apparatus and were uncorrected.
The reaction progress was controlled on thin-layer chro-
matography (TLC) plates and spraying dimethylaminoben-
zaldehyde (DMAB) solution. TLC was performed on Silica
Gel 60 F254 (Merck) and visualised with UV. The identifica-
tion of aromatic primary amine was confirmed with solution
containing 1 g DMAB, 10 ml 95% EtOH, 30 ml concentrated
hydrochloric acid (HCl) and 180 ml n-butanol; 5% NH3aq in
methanol was used as a solvent to TLC.
The nitro groups were reduced by catalytic hydrogenation
of compound III (0.85 mmol), which was dissolved in
methanol. The reaction mixture was stirred in room tempera-
ture and at atmospheric pressure for 1.5 h. Then a black
precipitate of catalyst was filtered off, and HCL (10 ml in
100 ml of MeOH) was added to filtrate until the pH reached
5. The resulting solution was concentrated under reduced
pressure. The crude product was purified by column chroma-
tography in chloroform with a MeOH gradient. The concen-
tration of fractions containing product of reduction yielded
compound VI (80.60%).
To solution of diamine VI (0.68 mmol) in pyridine with
DMAP was added dropwise 3-nitrobenzoyl chloride
(1.50 mmol) dissolved in methylene chloride. The mixture
was stirred at room temperature for 5 h and gave nitro
compounds IX. Recrystallisation of this product gave pure
solid of IX (yield 56%). Catalytic reduction of the nitro
groups to the amine XII preceded satisfactorily under the
reaction conditions described earlier (yield 73.87%).
Solvents used in the experiments were dried and distilled.
Silica gel 60 (Merck, 230–400 mesh) was used for column
chromatography.
2.1.1. General procedure for preparation of compound XII
The starting materials for synthesis, 3-nitrobenzoyl chlo-
ride (2.2 mmol), was dissolved in methylene chloride and
added dropwise to the solution 1,5-diamine (1 mmol) in dry
pyridine with 4-dimethylamino-pyridine (DMAP). The reac-
tion mixture was stirred in room temperature for 2 h. After
finishing the reaction, the precipitated crude product was
2.2. Pharmacology
2.2.1. MCF-7 cells
Stock cultures of breast MCF-7 cancer cells (purchased
from the American Type Culture Collection, Rockville, MD)

D. Bartulewicz et al. / IL FARMACO 59 (2004) 211–214
213
Table 1
Physico-chemical properties of compounds (in the form of dihydrochlorides)
Compound
Formula
(molecular
weight)
Yield
(%)
Melting
point (°C)
Rf
1H NMR, d (ppm) (d6-DMSO)
13C NMR, d (ppm) (d6-DMSO)
IV
C
17H20N4O2
78.70
82.84
80.60
84–85
0.44
1.88 (m, 2H, C–CH2–C); 3.37 (m, 2 × 25.01 (C–CH2–C); 36.01 (2C,
2H, CONHCH2); 4.58 (bs, 2 × 2H, CONHCH2); 121.96 (C2); 125.69 (C6);
NH2); 7.72–8.68 (2 × 4H, Ar-H); 9.10 130.04 (C4); 133.60 (C5); 135.88 (C1);
(t, 2 × 1H, CONH) 147.74 (C3); 163.97 (CONH)
1.54 (m, 2H, C–CH2–C); 3.22 (m, 2 × 26.79 (C–CH2–C); 38.91 (2C,
2H, CONHCH2); 5.47 (bs, 2 × 2H, CONHCH2); 113.41 (C2); 115.00 (C6);
NH2); 6.71–7.10 (2 × 4H, Ar-H); 8.29 116.83 (C4); 128.62 (C5); 135.71 (C1);
(t, 2 × 1H, CONH) 147.49 (C3); 166.84 (CONH)
(312.37)
V
C
18H22N4O2
163–164
246–247
0.43
0.34
(326.40)
VI
C
19H24N4O2
1.40 (m, 2H, C–CH2–C); 1.54 (m, 2 × 23.99 (C–CH2–C); 28.81 (2C–CH2–C);
2H, C–CH2–C); 3.15 (s, 2 × 2H, NH2); 39.22 (2C, CONHCH2); 119.61 (C2);
(340.43)
3.26 (m, 2 × 2H, CONHCH2);
7.20–8.35 (2 × 4H, Ar-H); 8.59 (t, 2 ×
1H, CONH)
122.66 (C6); 122.92 (C4); 129.35 (C5);
136.03 (C1); 137.21 (C3); 165.60
(CONH)
X
C
31H30N6O4
72.12
69.59
73.87
155–156
195–196
209–211
0.27
0.25
0.19
1.80 (m, 2H, C–CH2–C); 3.36 (m, 2 × 29.32 (C–CH2–C); 39.74 (2C,
2H, CONHCH2); 5.36 (bs, 2 × 2H, CONHCH2); 116.73 (C2); 119.70 (C6);
NH2); 6.75–8.23 (2 × 8H, Ar-H); 8.52 119.80 (C4); 122.96 (C4′); 128.87 (C5′);
(550.62)
(t, 2 × 1H, CONH); 10.23 (s, 2 × 1H,
CONH)
129.16 (C5); 130.25 (C3′); 131.44 (C1);
139.47 (C1′); 147.71 (C6′); 148.86 (C3);
166.47 (CONH); 167.90 (CONH)
XI
XII
C
32H32N6O4
1.61 (m, 2 × 2H, C–CH2–C); 3.28 (m, 26.71 (C–CH2–C); 38.98 (2C,
2 × 2H, CONHCH2); 6.03 (bs, 2 × 2H, CONHCH2); 113.78 (C2); 115.68 (C6);
NH2); 6.85–8.50 (2 × 8H, Ar-H); 8.93 117.62 (C4) 119.71 (C4′); 121.88 (C5′);
(562.63)
(t, 2 × 1H, CONH); 10.23 (s, 2 × 1H,
CONH)
122.78 (C5); 128.81 (C3′); 135.35 (C1);
135.65 (C1′); 139.32 (C6′); 147.48 (C3);
166.21 (CONH); 167.69 (CONH)
C
33H32N6O4
1.38 (m, 2H, C–CH2–C); 1.55 (m, 2 × 23.99 (C–CH2–C); 28.86 (2C–CH2–C);
(576.66)
2H, C–CH2–C); 3.28 (m, 2 × 2H,
CONHCH2); 5.51 (s, 2 × 2H, NH2);
6.76–7.92 (2 × 4H, Ar-H); 8.22 (t, 2 ×
39.34 (2C, CONHCH2); 113.20 (C2);
114.98 (C6); 117.09 (C4); 119.72 (C4′);
121.91 (C5′); 122.81 (C5); 128.34 (C3′);
1H, CONH); 10.24 (s, 2 × 1H, CONH) 135.39 (C1); 135.61 (C1′); 139.35 (C6′);
148.52 (C3); 166.24 (CONH); 167.82
(CONH)
were maintained in continuously exponential growth by
weekly passage in Dulbecco’s modified Eagle’s medium
(Sigma) supplemented with 10% FBS (Sigma), 50 µg/ml
streptomycin, 100 U/ml penicillin at 37 °C in a humid atmo-
sphere containing 5% CO2. Cells were cultivated in Costar
flasks and subconfluent detached with 0.05% trypsin and
0.02% EDTA in a calcium-free phosphate-buffered saline.
The study was carried out using cells from passages 3–7,
growing as monolayer in six-well plates (Nunc) (5 × 105 cells
per well and preincubated 24 h without phenol red.
of cell suspension was mixed with 10 µl of the dye mix and
200 cells per sample were examined by fluorescence micros-
copy, according to the following criteria:
• viable cells with normal nuclei (a fine reticular pattern
stained green in the nucleus and red-orange granules in
the cytoplasm);
• non-viable cells with normal nuclei (bright orange chro-
matin with organised structure).
Antitumour activity investigated compounds expressed as
percentage of non-viable MCF-7 mammal tumour cells was
shown in Table 2.
2.2.2. Determination of apoptotic index and cell viability
The compounds were dissolved in sterile water and used
at concentrations of 25, 50, 100 and 150 µM.
2.2.3. Statistical analysis
The results were submitted to statistical analysis using the
method of the smallest squares, accepting coefficient of de-
termination in the range 0.9800 <R2 < 1. The IC50 data are
presented in Table 2.
Microscopic observations of cell monolayers were per-
formed with a Nikon optiphot microscope. Wright–Giemsa
staining was performed using the Fisher Leuko Stat Kit.
Adherent MCF-7 cells grown in six-well plates were stained
after induction of apoptosis with a dye mixture (10 µM
acridine orange and 10 µM ethidium bromide, prepared in
phosphate-buffered saline). 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 µl
3. Results and discussion
Described compounds were tested for their cytotoxic and
antiproliferative activity in the standard cell line of mamma-
lian tumour MCF-7. The compound concentration that inhib-

214
D. Bartulewicz et al. / IL FARMACO 59 (2004) 211–214
Table 2
Antitumour activity of tested compounds
Compound
Concentration (µM)
IC50 (µM)
Statistical data (model y =ax + b)
25
50
100
150
a
b
R2
IV
V
VI
X
XI
XII
3%a
5%
5%
3%
5%
7%
5%
20%
20%
25%
20%
15%
20%
35%
30%
30%
30%
20%
30%
209.80
250.00
235.89
238.64
406.62
258.30
0.2664
0.2000
0.2102
0.2224
0.1153
0.1878
–5.8983
0.0000
0.4237
–3.0678
0.7565
1.4915
0.9815
1.0000
0.9581
0.9953
0.9796
0.9950
10%
10%
8%
10%
10%
a Percent of non-viable MCF-7 mammalian tumour cells.
its 50% of colony formation is in the range 209.80–
406.62 µM.
References
The binding of all compounds, as well analogues of bis-
amidines IV–VI, as products IX–XII, is weaker than that of
DAPI (IC50 = 176 µM) and Hoechst 33258 (IC50 = 55 µM)
[8], likewise carbocyclic mono-lexitropsin (24.43 µM) [5].
As can be seen analogues X–XII produced related reduction
in cell viability in breast cancer MCF-7 cells to compounds
IV–VI. This suggests that only one part of molecules of
analogues of bis-netropsin X–XII binds to minor groove, but
second one stays behind minor groove. Conformation of
introduced linkers is such inflexible that bis(two-benzene)
compounds X–XII do not have shapes that fits to minor
groove. It can be explained also by the fact that the com-
pounds are weakly basic anilines, at variance with strong
basic netropsin and pentamidine.
The IC50 values for synthetic analogues of minor groove
binders, IV–VI, suggest that these binders can serve as po-
tential carriers of other strong acting elements, e.g. alkylating
groups. Free amino group of these compounds can be con-
nected with such elements. Compounds X–XII do not give a
lot of hope to be useful.
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