Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards with potential enzymes inhibitors based on melamine framework

Article abstract of DOI:10.3109/14756366.2011.604482

The new class of hybrid anticancer drugs were obtained by selective functionalization of the triazine scaffold. These were prepared by rearrangement of mono-, bis- and/or tris-(1,3,5-triazin-2-yl)-1,4-diazabicyclo[2.2.2]octanium chlorides leading to forma

Full text of DOI:10.3109/14756366.2011.604482

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(5): 619–627
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.604482
ReseaRch aRtIcle
Synthesis and cytotoxicity studies of bifunctional
hybrids of nitrogen mustards with potential enzymes
inhibitors based on melamine framework
Beata Kolesinska1, Konrad Barszcz1, Zbigniew J. Kaminski1, Danuta Drozdowska2,
Joanna Wietrzyk3, and Marta Switalska3
1Institute of Organic Chemistry, Technical University of Lodz, Poland, 2Department of Organic Chemistry,
Medical University, Bialystok, Poland, and 3Ludwik Hirszfeld Institute of Immunology and Experimental erapy,
Polish Academy of Science, Wroclaw, Poland
abstract
The new class of hybrid anticancer drugs were obtained by selective functionalization of the triazine scaffold. These
were prepared by rearrangement of mono-, bis- and/or tris-(1,3,5-triazin-2-yl)-1,4-diazabicyclo[2.2.2]octanium
chlorides leading to formation of 2-chloroethylamino fragments attached to 1,3,5-triazine via one, two or three
piperazine rings respectively. Their inhibitory effect was found strongly dependent on the structure of substituents in
triazine ring.The anti-proliferative activity of the hybrids evaluated in vitro by using mammalian tumour cells estimated
as IC was in the range 0.62–139,78 µM. Both cytotoxicity and alkylating activity depended on the substituents of
triazi5n0e ring, however, also the mono-functional analogues of nitrogen mustards, which are unable to form liaisons
between two DNA strands, induced apoptosis and necrosis in the tested cells.
Keywords: Proliferation inhibitors, apoptosis, hybrid anticancer drugs, nitrogen mustard, triazine scaffold,
alkylating agent
Introduction
resistance less likely2. Since cancer is a genetic disease
with several mutations altering the replication system of
the cell, the strategy based on the construction of hybrid
anticancer drugs possessing different toxicity profiles
seems to be very promising.
In the last decade, several new highly potent cytostatic
agents, derived from 1,3,5-triazine, have been presented
that are active as inhibitors of enzymes involved in the
cell proliferation cycle (see Figure 1).
A melamine derivative, ZSTK474 [2-(2-difluorom-
ethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-
triazine] (1) has been found, which inhibits the growth
of tumour cells with its molecular target identified
as phosphatidylinositol 3-kinases (PI3K) with strong
antitumour activity against human cancer xenografts
without toxic effects on critical organs3. For melamine
2 it has been suggested that its antimetastatic and
e recent therapeutic approach in which drug candi-
dates are designed to possess diverse pharmacological
properties and act on multiple targets has stimulated
development of the hybrid drugs. Such dual action drugs
with two dissimilar drug molecules combined together
by direct linking of the two molecular entities are already
known and found to be useful tools in the therapy of
complex diseases such as cardiovascular and inflamma-
tory diseases and might be used as antibacterial agents
useful in the treatment of drug resistant pathogens1.
In several cases the collected data provide a conclu-
sive molecular mechanism for the independent modes
of action of both drug-like fragments, amplification of
effects of fragments or even the extraordinary biological
effects not attributed to any of the individual partner of
the hybrid construct that make the emergence of drug
Address for Correspondence: Prof Zbigniew J. Kaminski, Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116,
90–924 Lodz, Poland. Tel: 48426313151; Fax: 48426365530. E-mail: zbigniew.kaminski@p.lodz.pl
(Received 27 May 2011; revised 04 July 2011; accepted 05 July 2011)
619

620 B. Kolesinska et al.
Figure 1. Structures of triazine based antitumour agents and enzyme inhibitors.
antitumour activity is due in part to inhibition of angio-
genesis, rather than direct anti-proliferative action on
tumour cells4.
A series of thiopyridine 5 and aminopyridine
6 substituted triazine derivatives has been found
inducing G2/M arrest and apoptosis with a possible
involvement of p537. Triazine 7 has been found to be
efficient topoisomeraze inhibitors8 and terpene sub-
stituted triazine 8 is active as a cytostatic, but with an
unknown molecular target9. Additionally, as a selective
inhibitor, 1,3,5-triazine based molecules have been
found to act on many different targets which include:
HIV-1 reverse transcriptase10, estrogen receptor beta11,
glutathione S-transferase12, M. tuberculosis dihydro-
folate reductase13, photosynthetic reaction centre14,
guanosine-5´-triphosphate binding site15, ATP com-
petitive inhibitor of mammalian target of rapamycin16
and urate oxidase17. Considering enzyme inhibition
as an intrinsic property of the complete melamine
structure, still remains vast potential of decoration of
triazine scaffold by introducing “address” fragment
increasing selectivity, components facilitating trans-
port through the cell membranes, and last but not least,
components of classic anticancer drugs with well-doc-
umented therapeutic competence. erefore recent
approaches involve development of hybrid cancer
drugs18 or application of 1,3,5-triazine as inductor of
new specific molecular targets19. In these studies it has
been attempted to introduce into 1,3,5-triazine scaffold
Melamine derivatives 3 were also found to be some of
the best histone deacetylase inhibitors with anti-prolif-
erative activities with IC50 values below the micromolar
range. Some of these compounds can also significantly
reduce tumour growth in human tumour xenograft mod-
els in mice. In general, derivatives of the triazine series
show better profiles than their pyridine, pyrimidine, and
purine analogues and moreover, these compounds are
selective to the cancer cell line tested (HCT116) with a
toxicity index that in some cases is over 100-times higher
than that of a normal cell line (HMEC5). Melamine deriv-
ative 4 were also found to be the potent G-quadruplex
ligand, which is shown to induce both telomere shorten-
ing and apoptosis in the human A549 cell line as a func-
tion of its concentration and time exposure. Several other
melamine analogues were also considered as potential
antitumour agents that block telomere replication stabi-
lizing the telomeric G-rich single stranded DNA overhang
into G-quadruplex and consistently inducing telomere
erosion, which results in senescence after prolonged
contact. ey are also able to induce apoptotic or non-
apoptotic cell death, alteration of cell cycle progression,
and depression of telomerase activity6.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards 621
one, two or three fragments bearing 2-chloroethylam-
2-[4-(2-Chloroethyl)-piperazin-1-yl]-4,6-bis-(2,2,2-
trifluoroethoxy)-[1,3,5]triazine (12c)
ine moiety characteristic for nitrogen mustards20,21 and
to confirm anti-proliferative activity of the obtained
hybrids.
Starting materials: 2-chloro-4,6-bis-(2,2,2-trifluoroethoxy)-
[1,3,5]triazine (9c) (1.56 g, 5 mmol), DABCO (1.12 g,
5 mmol). Product: 2-[4-(2-chloroethyl)-piperazin-1-yl]-4,6-
bis-(2,2,2-trifluoroethoxy)-[1,3,5]triazine (12c) (2.03 g,
96%), oil.
Materials and methods
General Information
1H-NMR (CDCl3): 2.67 (t, 4H, J = 5.5 Hz); 2.88 (t, 2H,
J = 8.0 Hz); 3.66 (t, 2H, J = 8.0 Hz); 3.94 (t, 4H, J = 5.5 Hz);
4.75 (qw, 4H, J = 7.5 Hz). 13C-NMR (CDCl3): 36.31; 40.08;
43.15; 52.26; 59.13; 63.30; 165.95; 170.67. IR (film/NaCl):
2980; 2940; 2870; 2820; 2250; 1720; 1670; 1590; 1525; 1420;
1375; 1300; 1270; 1160; 1130; 1070; 990; 950. Anal. Calcd
for C13H16ClF6N5O2: C, 36.85; H, 3.81; N, 16.53. Found: C,
36.71; H, 3.75; N, 16.42.
in layer chromatographies (TLC) were carried out on
SiO2 (Merck; 60 Å F254) and spots located with: UV light
(254 and 366 nm) and 1% ethanolic 4-(4´-nitrobenzyl)-
pyridine (NBP). Melting points were determined on a
Büchi apparatus, model 510. IR spectra were recorded
as KBr pellets or film on a Infracord 137 E spectrometer.
1H-NMR, 13C-NMR, spectra were recorded on a Bruker
Avance DPX 250 (250 MHz) spectrometer. Chemical
shifts (ppm) are relative to TMS used as an internal
standard. e multiplicity were marked as s = singlet,
d = dublet, t = triplet, q = quartet, qu = quintet, m = multip-
let. Triazines 9a-d were obtained from cyanuric chloride
according to standard procedure described22.
2-[4-(2-Chloroethyl)-piperazin-1-yl]-4,6-diphenoxy-[1,3,5]
triazine (12d)
Starting materials: 2-chloro-4,6-diphenoxy-1,3,5-triazine
(9d) (1.50 g, 5 mmol), DABCO (1.12 g, 5 mmol). Product:
2-[4-(2-chloroethyl)-piperazin-1-yl]-4,6-diphenoxy-
[1,3,5]triazine (12d) (1.73 g, 84%), m.p. = 138–140˚C.
1H-NMR (CDCl3): 2.48 (t, 4H, J= 7.5 Hz); 2.73 (t, 2H,
J = 5.0 Hz); 3.57 (t, 2H, J = 5.0 Hz); 3.72 (t, 4H, J = 7.5 Hz);
7.12–7.37 (m, 10H). 13C-NMR (CDCl3): 40.53; 43.20; 52.48;
59.39; 121.58; 125.90; 129.04; 151.92; 166.42; 172.13. IR
(film/NaCl): 2970; 2920; 2805; 1740; 1670; 1595; 1575;
1530; 1490; 1445; 1390; 1375; 1310; 1280; 1260; 1210;
1160; 1125; 1070; 1020; 995. Anal. Calcd for C21H22ClN5O2:
C, 61.24; H, 5.38; N, 17.00. Found: C, 61.04; H, 5.41; N,
16.95.
2,4-Bis-methoxy-6-[4-(2-chloroethyl)-piperazin-1-yl]-[1,3,5]
triazine (12a).
General procedure: 1,4-Diazabicyclo[2.2.2]octane (10)
(DABCO) (1.12 g, 10 mmol) was added to a vigorously
stirred solution of 2-chloro-4,6-dimethoxy-1,3,5-triazine
(9a) (1.76 g, 10 mmol) in dichloromethane (20 mL),
cooled to 5°C. e mixture was stirred at 5°C for 0.5h and
then under reflux condition for 1 h. Progress of reaction
was monitored by TLC (Rf = 0 for salt 11a, Rf = 0.15 for 12a,
DCM, 1% solution of (NBP) for visualization of spots).
e organic layer was concentrated under evaporated
reduced pressure. 2,4-Bis-methoxy-6-[4-(2-chloroethyl)-
piperazin-1-yl]-[1,3,5]triazine (12a) was obtained (2.81 g,
yield 98%), m.p. = 88–90˚C.
2,4-Bis-[4-(2-chloroethyl)-piperazin-1-yl]-6-methoxy-[1,3,5]
triazine (12e)
Starting materials: 2,4-dichloro-6-methoxy-1,3,5-tri-
azine (9e, DCMT) (0.90 g, 5 mmol), DABCO (2.24 g, 10
mmol). Product: 2,4-bis-[4-(2-chloroethyl)-piperazin-
1-yl]-6-methoxy-[1,3,5]triazine (12e) (2.02 g, 80%),
m.p. = 278–281˚C.
1H-NMR (CDCl3): 2.55 (t, 4H, J = 5.2 Hz); 2.77 (t, 2H,
J = 7.5 Hz); 3.61 (t, 2H, J = 7.5 Hz); 3.87 (t, 4H, J = 5.2 Hz);
3.95 (s, 6H). 13C-NMR (CDCl3): 40.58; 43.13; 52.50; 54.20;
55.60; 59.41; 166.23; 172.06. IR (film/NaCl): 2952; 2840;
2808; 1584; 1536; 1472; 1368; 1308; 1280; 1255; 1190; 1135;
1040; 990. Anal. Calcd for C11H18ClN5O2: C, 45.92; H, 6.31;
N, 24.34. Found: C, 45.81; H, 6.33; N, 24.39.
1H-NMR (CDCl3): 2.53 (t, 8H, J = 5.1 Hz); 2.76 (t, 4H,
J = 8.0 Hz); 3.63 (t, 4H, J = 8.0 Hz); 3.84 (t, 8H, J = 5.1 Hz);
3.87 (s, 3H). 13C-NMR (CDCl3): 38.99; 41.23; 51.10; 53.01;
57.97; 160.68; 164.01; 169.66. IR (film/NaCl): 3000; 2950;
2860; 2810; 2770; 2230; 1675; 1590; 1580; 1525; 1490; 1380;
1350; 1300; 1245; 1190; 1150; 1120; 1090; 1045; 995. Anal.
Calcd for C16H27Cl2N7O: C, 48.81; H, 6.99; N, 23.44. Found:
C, 48.54; H, 6.75; N, 23.40.
2,4-Bis-benzyloxy-6-[4-(2-chloroethyl)-piperazin-1-yl]-[1,3,5]
triazine (12b)
Starting materials: 9b (1.64 g, 5 mmol), DABCO (1.12 g, 5
mmol). Product: 2,4-bis-benzyloxy-6-[4-(2-chloroethyl)-
piperazin-1-yl]-[1,3,5]triazine (12b) (2.09 g, 95%), oil.
1H-NMR (CDCl3): 2.54 (t, 4H, J = 5.0 Hz); 2.76 (t, 2H,
J = 7.5 Hz); 3.61 (t, 2H, J = 7.5 Hz); 3.88 (t, 4H, J = 5.0 Hz);
5.38 (s, 4H); 7.26–7.45 (m, 10H). 13C-NMR (CDCl3): 40.60;
43.23; 52.53; 59.44; 68.80; 127.94; 128.06; 128.25; 135.97;
166.35; 171.61. IR (film/NaCl): 3050; 2960; 2815; 2255;
1830; 1695; 1580; 1525; 1495; 1445; 1420; 1345; 1305;
1270; 1155; 1105; 1040; 995. Anal. Calcd for C23H26ClN5O2:
C, 62.79; H, 5.96; N, 15.92. Found: C, 62.29; H, 5.71;
N, 15.98.
2,4,6-Tris-[4-(2-chloroethyl)-piperazin-1-yl]-[1,3,5]triazine
(12f)
Starting materials: cyanuric chloride (0.92 g, 5 mmol),
DABCO (3.36 g, 15 mmol). Product: 2,4,6-Tris-[4-(2-
chloroethyl)-piperazin-1-yl]-[1,3,5]triazine (12f) (2.11 g,
81%), oil.
1H-NMR (CDCl3): 2.35–2.95 (m, 22H); 3.59-3.77
(m, 14H). 13C-NMR (CDCl3): 40.47; 42.69; 52.57; 59.26;
164.69. IR (film/NaCl): 3020; 2930; 2820; 2350; 1725;
1670; 1575; 1540; 1525; 1475; 1440; 1375; 1310; 1260;
© 2012 Informa UK, Ltd.

622 B. Kolesinska et al.
1220; 1180; 1140; 1090; 1010; 950. Anal. Calcd for
C21H36Cl3N9: C, 49.39; H, 7.16; N, 23.56. Found: C, 49.31;
H, 7.11; N, 23.49.
MCF-7 cultures
Stock cultures of breast MCF-7 cancer cells (purchased
from the American Type Culture Collection, Rockville,
MD) 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 atmosphere containing 5% CO2. Cells were cul-
tivated in Costar flasks and subconfluent detached with
0.05% trypsin and 0.02% EDTA in a calcium-free phos-
phate buffered saline. e study was carried out using
cells from passages 3 to 7, growing as monolayer in 6-well
plates (Nunc) (5 × 105 cells per well and preincubated 24
hours without phenol red.
Cytotoxicity determined by SRB method
e following established in vitro human cancer cell
lines were applied: SW707 (colorectal adenocarcinoma),
Jurkat (leukemia), A549 (lung cancer), LNCaP (pros-
tate cancer) and T47D (breast cancer). All lines were
obtained from the American Type Culture Collection
(Rockville, Maryland, USA) and maintained at the Cell
Culture Collection of the Institute of Immunology and
Experimental erapy, Wroclaw, Poland.
Twenty-four hours before addition of the tested agents,
the cells were plated in 96-well plates (Sarstedt, USA) at a
density of 104 cells per well in 100 μL of culture medium.
e cells were cultured in the opti-MEM medium supple-
mented with 2 mM glutamine (Gibco, Warsaw, Poland),
streptomycin (50 μg/mL), penicillin (50 U/mL) (both
antibiotics from Polfa, Tarchomin, Poland) and 5% fetal
calf serum (Gibco, Grand Island, USA). e cell cultures
were maintained at 37°C in humid atmosphere saturated
with 5% CO2.
Solutions of compounds were prepared by dilu-
tion of 1 mg of 12a,b,f in DMSO (100 μL) and diluted
subsequently to 1000 μL with culture media or 12c,e
in DMSO (1000 mL). Compound 12f was sonicated for
1 minute to enhance dilution. Compounds were tested
in final concentration of: 100, 10, 1 and 01 μg/mL for
12a,c,e or 10, 1, 0.1 and 0.01 μg/mL for 12b,d,f in cul-
ture media.
Determination of apoptotic index and cell vialibility
e compounds were dissolved in sterile water and used
atconcentrationsof1,10,50,100and150μM.Microscopic
observations of cell monolayers were performed with
a Nikon optiphot microscope. Wright-Giemsa staining
was performed using the Fisher Leuko Stat Kit. Adherent
MCF-7 cells grown in 6-well plates were stained after
induction of apoptosis with a dye mixture (10 μM acridine
orange and 10 μM ethidium bromide, prepared in phos-
phate 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. en, 250
μL of cell suspension was mixed with 10 μL of the dye mix
and 200 cells per sample were examined by fluorescence
microscopy, according to the following criteria:
•
viable cells with normal nuclei (a fine reticular pat-
tern stained green in the nucleus and red-orange
granules in the cytoplasm);
SRB assay
e details of this technique were described by Skehan23.
e cytotoxicity assay was performed after 72-hour
exposure of the cultured cells to varying concentrations
(0.1–100 μg/mL) of the tested agents. e cells attached
to the plastic were fixed by gently layering cold 50% TCA
(trichloroacetic acid, Aldrich-Chemie, Germany) on the
top of the culture medium in each well. e plates were
incubated at 4°C for 1 h and then washed five times with
tap water. e background optical density was measured
in the wells filled with culture medium, without the cells.
e cellular material fixed with TCA was stained with
0.4% sulforhodamine B (SRB, Sigma, Germany) dissolved
in 1% acetic acid (POCh, Gliwice, Poland) for 30 minutes.
Unbound dye was removed by rinsing (4×) with 1% acetic
acid. e protein-bound dye was extracted with 10mM
unbuffered Tris base (POCh, Gliwice, Poland) for deter-
mination of optical density (at 540 nm) in a computer-
interfaced, 96-well microtiter plate reader Multiskan RC
photometer (Labsystems, Helsinki, Finland). Each com-
pound in given concentration was tested in triplicates in
each experiment, which was repeated 3–5 times.
•
viable cells with apoptotic nuclei (green chromatin
which is highly condensed or fragmented and uni-
formly stained by the acridine orange);
non-viable cells with normal nuclei (bright orange
chromatin with organised structure);
non-viable cells with apoptotic nuclei (bright
orange chromatin witch is highly condensed or
fragmented).
•
•
Antitumour activity investigated compounds expressed
as percentage of non-viable MCF-7 (summarized both
apoptotic and necrotic) mammal tumour cells was shown
in Table 1.
Determination of alkylating properties (Preussmann test)
e tested compounds (0.01 mmol) were dissolved in
2-metoxyethylether (200 μL) and solution of NBP in
2-metoxyethylether(5%,200μL)wereadded.esamples
were heated at 100 0.5°C for 1 h and then quickly cooled
to 20°C. 2-Metoxyethylether (500 μL) and piperidine (100
μL) were added to the samples to give a total volume of
1 mL. e final concentration of the tested compounds
was 10 μM. After 90 s, the absorbance was measured at
λ= 560 nm in a quartz cell (1 cm). 2-Metoxyethylether
was used as a reference solvent.
e results of cytotoxic activity in vitro were expressed
as an IC50 (μg/mL), i.e. the concentration of compound,
which inhibits the proliferation of 50% of tumour cells as
compared to the control untreated cells.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards 623
one, two, or three chloroethylamine groups to surround
Ethidium bromide (EtBr) assay
triazine substituted respectively with one (Scheme 1),
two or three piperazine rings (Scheme 2).
Each well of 96-well plate was loaded with Tris buffer
containing ethidium bromide (0.1M Tris, 1M NaCl, pH 8,
0.5mM EtBr final concentration, 100 μL). To each well was
added 15 μg plasmid. pBR322 as water solution (0.05 μg/
μL). en, to each well was added chlorambucil (CHL) or
compound 12a–f (1 μL of a 1mM solution in water, 10 μM
final concentrations). After incubation at 25°C for 30min,
the fluorescence of each well was read on a Multilabel
Reader Victor 3V (ex.: 355nm, em.: 615nm) in duplicate
experiments with two control wells (no drug=100% fluo-
rescence, no DNA=0% fluorescence). Fluorescence read-
ings are reported as % fluorescence relative to the controls.
In order to prepare such constructs we made use of
the observation26,27 that some N-triazinylammonium
chlorides 11a-f easy accessible from a broad range of tri-
azines 9a–d and 1,4-diazabicyclo[2.2.2]octane (DABCO)
(10) rearranged with a formation of 12a–f bearing 2-chlo-
roethylamino fragment. Most N-triazinylammonium
salts 11 were obtained in almost quantitative yield.
e rearrangement of 11a–f to 12a–f proceeds at room
temperature relatively slowly and is accelerated in non-
aqueous media and at elevated temperatures.
e anti-proliferative activity of the hybrids 12a–f has
been evaluated in vitro by using six tumour cell lines.
e colorimetric tests were performed in 96-well culture
plate and the activity of the cytotoxic drugs presented as
IC50 values were determined after 72 h of drug exposures
(Table 1).
Statistical analysis
In all experiments, the mean values for three indepen-
dent assays standard deviations (S.D) were calculated.
e results were submitted to statistical analysis using the
Student’s test. Differences were considered significant
when p< 0.05. Mean values, the standard deviations and
the number of measurements in the group are presented
in the figures.
Inhibitory effects of 12a–f was found strongly depen-
dent on the structure of substituents in triazine ring.
Unexpectedly, for five tumour cell lines the crosslinking
ability, which obligatory required the presence of two or
tri chloroethylamino group were found not crucial for
activity. Inhibitory effects of 12b,d, bearing only single
chloroethylamino group, expressed as IC50 in the range
0.62–3.45 μg/mL on the growth of human tumour cell lines
has been found much stronger than bi- and trifunctional
analogues 12e,f (IC50 in the range 4.50–6.22 μg/mL or
totally negative). is suggests that cell proliferation may
be inhibited by mono-alkylation involving chloroethylam-
ino functionality or by the other mechanisms involving,
unknown as yet, inhibitory effect of triazine structure.
In the further studies inhibition of proliferation, apop-
totic index and cell vialibility was determined in vitro
Results and discussion
As exemplified above, the appropriate modification of
1,3,5-triazine ring system could lead to numerous novel
and diverse target-specific inhibiors of enzymes. us, the
diversity of action mode of triazine based inhibitors and
presence of three reactive centres which could be used
for the selective functionalization of the triazine scaffold
have paved the way to a new class of hybrid anticancer
drugs with a lowered toxicity and broader antitumour
spectrum24. erefore, while designing the structure of
new hybrid anticancer drugs25 we attempted to attach
Scheme 1. Formation of triazines 12a-d bearing one 2-chloroethylamine fragment.
Table 1. Inhibitory effects of 12a–f on the growth of human tumour cell lines expressed as IC50 in μg/mL.
cell line/IC50 [μg/mL] mean SD
12
breast cancer T47D
27.86 0.03
prostate cancer LNCaP
15.78 6.68
colorectal cancer SW707
32.38 0.89
lung cancer A549
34.66 1.55
lymphoblastic leukemia Jurkat
31.29 0.84
a
b
c
1.40 0.33
0.99 0.52
3.45 0.28
2.06 0.66
0.62 0.15
96.84 4.47
18.27 14.32
1.47 0.95
96.84 5.47
61.04 20.31
2.67 1.33
25.60 2.43
d
e
2.60 0.99
2.93 0.81
2.93 0.36
6.22 3.34
4.50 1.98
36.22 15.85%*
negative**
25.35 10.82%*
negative**
31.58 10.43%*
negative**
f
9.01 0.86
negative**
*inhibition of cell proliferation in vitro expressed as percent of proliferation inhibition at concentration of 12e 10 μg/mL.
**no inhibition to the concentration of 12e 10 μg/mL.
© 2012 Informa UK, Ltd.

624 B. Kolesinska et al.
Scheme 2. Synthesis 1,3,5-triazine derivatives bearing two 2-chloroethylamino residues 12e and three 2-chloroethylamino residues 12f.
on breast MCF-7 cancer cells. e obtained results were
compared with the anti-proliferative effect of chloram-
bucil and summarized in Table 2.
Table 2. Viability of MCF-7 cells treated for 24h with different
concentrations of chlorambucil (CHL) and compounds 12a–f.
Non-viable cells (% of control 2)
Conc.
All the compounds 12a–f showed cytotoxic and anti-
proliferative effect. e concentration which inhibits 50%
of colony formation was in the range 18.70–139.78 µM.
It is worthy to notice that in the case MCF-7 cancer the
activity increased with the amount of 2-chloroethylamino
fragments. e analogue 12f with IC50 = 18.70 µM has
three such fragments and is the most active against the
investigated cells. It is more active than chlorambucil
with IC50 = 29.14 µM, the therapeutics with alkylating
properties belonging to the first class of cytostatics used
for cancer therapy28.
Our other aim was to determine the in vitro alkylat-
ing activity of the novel mustards 12a–f. For this purpose
an in vitro Preussmann test29 was used. All of the tested
compounds demonstrate their alkylating activity toward
NBP molecule. Alkylating activity results are presented
in Table 3.
Only compound 12c has a low alkylating activity (+)
and is less active than chlorambucil, the other ones are
more active and can be included in the group of high (++)
alkylating activity.
One can see a correlation between the alkylating
activity of the tested compounds and their cytotoxicity
on the breast cancer MCF-7 cell line. Both cytotoxic-
ity and alkylating activity increase with the amount of
2-chloroethylamino fragments.
[μM]
0
CHL
1a
12a
3
12b
2
12c
3
12d
4
12e
2
12f
2
1
11
6
9
8
23
46
57
63
74
25
31
52
69
72
23
52
62
65
73
10
50
100
150
IC50
31
20
24
42
48
24
39
51
61
27
46
52
75
48
96
100
29.14 139.78 92.02 67.47 32.13 43.95 18.70
aMean values SD from three independent experiments done in
duplicate are presented.
Table 3. Alkylating activity of compounds 12a–f on comparison
to chlorambucil; NBP test results.
Molar extinction
coefficient (ε)
Absorbance
Alkylation
activityb
Compound
(A)a 560 nm
12a
103.6
119.5
88.3
0.1036
0.1195
0.0883
0.1193
0.1302
0.1488
0.0927
++
++
+
12b
12c
12d
119.3
130.2
148.9
92.7
++
++
++
+
12e
12f
Chlorambucil
aAverage data from three determinations.
bAccording to Preussmann: (−) A < 0.05, (+) A=0.05–0.1, (++)
A=0.1-0.5, (+++) A > 0.5.
showed that all of new compounds induced concentra-
tion-dependent apoptosis of MCF-7 cells. e percent-
age of apoptotic and necrotic MCF-7 cells after treatment
with 50 μM solutions of CHL and compounds 12a–f is
shown in Figure 2.
e analyzed triazines 12a–f, analogously to standard
chlorambucil (CHL), inhibited tumour cell growth by
exerting direct anti-proliferative affects with the cytotoxic
(apoptosis/necrosis) consequences. e investigations
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards 625
Figure 2. Percentage of apoptotic and necrotic MCF-7 cells after treatment with 50 μM solutions of chlorambucil (CHL) and compounds
12a–12f. 100%={apoptotic(%) + necrotic(%) + viable cells}.
Figure 3. e relationship of apoptotic cells amount from increasing concentration of chlorambucil (CHL) and compounds 12a–f.
Apoptosis is the main way of the cell death at this con-
centration for all 12a–f.
to DNA although relatively weaker than chlorambucil
(Table 4).
Interestingly, the apoptotic cell death was mainly
observed for every of them in the range of concentration
from 1 to 100 µM, as we can see in Figure 3. ese results
also show that necrosis is predominant cell death for all
of compounds at concentration 150 µM. e ethidinium
displacement assay showed that triazines 12a–e can bind
conclusions
New dual action drugs combining together by direct link-
ing melamine based potential inhibitors of enzymes with
mustard alkylating groups were obtained. e method
© 2012 Informa UK, Ltd.

626 B. Kolesinska et al.
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96.74
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triazines 12b,d substituted with single chloroethylamino
fragment only. ese exclude formation of bifunctional
lesions and cross-link of nucleobases within the DNA
duplex, but could be attributed to the additional inhibi-
tory activity of triazine scaffold.
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cell line shown that in this case bi- trifunctional analogues
12e,f of nitrogen mustards, which are capable to form
liaisons between two DNA strands, induced apoptosis
and necrosis in the tested cells more effectively than
mono-functional 12a–d. e data from ethidium dis-
placement assay suggests that DNA binding may be
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Declaration of interest
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