A novel series of potent cytotoxic agents targeting G2/M phase of the cell cycle and demonstrating cell killing by apoptosis in human breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2007.06.033

Breast cancer, a leading cause of mortality in women, warrants the development and biological evaluation of new anticancer agents. A novel series of thiopyridine triazine derivatives was synthesized and investigated in the human breast cancer cell line, MDA-MB-468. SM40, the most potent derivative, induced a G2/M arrest and apoptosis with a possible involvement of p53. The cytotoxicity of SM40 was also examined against the NCI 60 cell line panel and its potency was rationalized using molecular modeling. Results suggest that SM40 is a promising cytotoxic agent.

Full text of DOI:10.1016/j.bmcl.2007.06.033

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4955–4960
A novel series of potent cytotoxic agents targeting G2/M phase
of the cell cycle and demonstrating cell killing by apoptosis
in human breast cancer cells
a
b
b
d
´
Soma Mandal, Gervais B e´ rub e´ , Eric Asselin, Iqbal Mohammad,
a
b
c
c
Vernon J. Richardson, Atul Gupta, Saroj K. Pramanik, Arthur L. Williams
a,d,
*
and Sanat K. Mandal
a
Faculty of Medicine, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3V6
D e´ partement de Chimie-Biologie, GRBCM, Universit e´ du Qu e´ bec a` Trois-Rivi e` res, C.P. 500, Trois-Rivi e` res, Que., Canada G9A 5H7
b
c
Department of Biology Morgan State University, Baltimore, MD 21251, USA
Division of Science & Technology, College of the North Atlantic, Clarenville Campus, Clarenville, NL, Canada A5A 1V9
d
Received 16 April 2007; revised 7 June 2007; accepted 7 June 2007
Available online 12 June 2007
Abstract—Breast cancer, a leading cause of mortality in women, warrants the development and biological evaluation of new anti-
cancer agents. A novel series of thiopyridine triazine derivatives was synthesized and investigated in the human breast cancer cell
line, MDA-MB-468. SM40, the most potent derivative, induced a G2/M arrest and apoptosis with a possible involvement of
p53. The cytotoxicity of SM40 was also examined against the NCI 60 cell line panel and its potency was rationalized using molecular
modeling. Results suggest that SM40 is a promising cytotoxic agent.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Breast cancer carries a high mortality rate, globally,
regardless of therapeutic advances. Among the treat-
ment choices available, chemotherapy is one of them de-
spite its many disadvantages including toxic side effects
and the development of resistance. Ideal drugs would
be small molecules, soluble and relatively stable in aque-
ous media, more potent, less toxic, tissue specific and
have increased bioavailability. Thus successful treat-
ment of cancer demands the synthesis and characteriza-
tion of new drugs with novel mechanism of action.
cancer cell lines (NCI). The NCI has selected compound
C (Chart 1), SM40, for further studies.
Compounds were synthesized in a straightforward one
or two-step high yielding procedure as outlined in
Scheme 1. All compounds were characterized by
NMR, mass, and elemental analysis, respectively.
SM40 (compound C, Chart 1), 2,4,6-tris(pyridin-2-
ylthio)-1,3,5-triazine, proved to be the most potent in
this group of compounds (Chart 1).
We have proposed our rationale in designing simple
–4
1
molecules to improve specificity and potency.
Here,
we report the synthesis of new thiopyridine derivatives
of 1,3,5-triazine (Scheme 1) and their biological response
in an estrogen receptor a (ERa) negative human breast
cancer cell line, MDA-MB-468. We have also examined
the cytotoxic potency of these derivatives in 60 human
Keywords: Cytotoxic agent; MDA-MB-468; Cell cycle; TUNEL assay;
Electron micrographs; Apoptosis; Immunohistochemistry; Subcellular
localization; p53273.His; Molecular modeling; Triazine; Thiopyridine
triazine.
*
Corresponding author. E-mail: sanat.mandal@cna.nl.ca
Chart 1.
0
960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.033

4956
S. Mandal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4955–4960
Additionally, as a selective inhibitor, 1,3,5-triazine
based molecules have been found to act in many differ-
9
ent targets which include: HIV-1 reverse transcriptase;
1
0
11
estrogen receptor beta; glutathione S-transferase;
1
2
M. tuberculosis dihydrofolate reductase; photosyn-
14
thetic reaction center, and urate oxidase.
1
3
As exemplified in the above targets, proper modification
of 1,3,5-triazine ring system could lead to the develop-
ment of novel target-specific drugs. Careful evaluation
using molecular modeling and docking tools revealed that
none of these targets are suitable for SM40. With the
exception of the 1,3,5-triazine ring, nothing is structurally
common to any of the other inhibitors that are bound to
the above targets and SM40 is no exception. Therefore,
the target for SM40 is unknown and remains to be identi-
fied. To the best of our knowledge, triazine derivatives are
numerous but thiopyridine derivatives of triazine possess-
Scheme 1. Synthetic strategy for compound 2 (SM40) and other
derivatives. Reagents and conditions: For 2, (a) 1 in dry acetone
(
25 ꢁC) and 2-thiopyridine [9 equivalents (equiv) in acetone], stirred for
2
–3 h, 70%. For 3, (a) 1 and excess 2-aminopyridine in methanol,
stirred for 6–7 h, 50%. For 4, (a) 1 and excess benzenethiol in refluxing
benzene (5–6 h), 60%. For 5, (b) 1 in dry acetone, cooled to À15 ꢁC and
2-thiopyridine in dry acetone added drop-wise, separated as solid
1
,2
ing novel biological properties are rare.
product, hydrochloride salt. For 6, (c) 5 and dimethylamine (6 equiv)
in methanol/water, stirred 7–8 h at 25 ꢁC, 70%. For 8, (c) 1 in dry
acetone, cooled to À15 ꢁC and dimethylamine (1 equiv) in dry acetone
added dropwise; For 9, (c) 8 (1 equiv) and excess 2-thiopyridine in
The preparation of some of the derivatives has been
1
described elsewhere. S-2-pyridyl, NH-2-pyridyl, and
1
acetone stirred for 2–3 h at 25 ꢁC, 60%. For SM40: H NMR
S-phenyl derivatives (2, 3, and 4; Scheme 1) were pre-
pared to establish the requirement of nitrogen atom in
second position of the thioheterocyclic ring. In a rela-
tively small number of derivatives, it has been observed
that both the nitrogen atom in a specific position of the
heterocyclic ring (second position with respect to sulfur
atom) and the sulfur atoms are essential for activity. For
this series of compounds, the concentration required for
50% growth inhibition (GI ) against 60 cell lines ranged
(
400 MHz, CDCl3) d 8.50 (m, 3H, 3· 6-pyridyl), 7.50 (m, 6H, 3·
,4-pyridyl), 7.19 (m, 3H, 3 · 5-pyridyl); m/z 408; Anal. Calcd for
: C 52.92%, H 2.96%, N 20.57%; found C 52.69%, H 3.00%
3
18 12 6 3
C H N S
and N 20.49%.
It is well documented that compounds containing the
1,3,5-triazine moiety have many interesting properties
and this moiety has been used to develop molecules in
5
0
1
,2,5–14
a number of research fields
1
including cancer drug
5
from 0.146 to 100 lM as evaluated by the NCI. Preli-
minary results of cytotoxic potency show an interesting
structure–activity relationship in these derivatives given
by the mean GI50 of 60 human cancer cell lines (NCI,
USA); A (GI50 = 96.99 lM, B (GI50 = 99.19 lM), C
,2,5,7–10
design;
(
for example, hexamethylmelamine 10
Scheme 1) is a potent cytotoxic agent against a number
of cancers including breast cancer but its use is limited
due to its undesirable side effects such as vomiting and
nausea after administration.
(GI50 = 3.17 lM),
D
(GI50 > 100 lM), and
E
Figure 1. Conformational analysis for C and D; I is the lowest energy (À107.65 kcal/mol) structure for C that has similar conformation, with respect
to heterocyclic ring nitrogen atoms (as shown using arrows), with D shown on the right, IV, but IV (À126.94 kcal/mol) is not the lowest energy
structure for D; III is the lowest energy (À130.89 kcal/mol) structure for D that has similar conformation with C as shown on the left, II. Conversion
of III to IV requires 3.95 kcal/mol of energy.

S. Mandal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4955–4960
4957
(
GI50 = 49.77 lM) (Chart 1). Due to the interesting bio-
nitrogen atoms in the pyridine ring number three (3)
are oriented oppositely to each other in I and III, but
also the position of the other nitrogen atoms in the pyr-
idine rings number one (1) and ring number two (2) are
significantly different as well. Therefore, the availability
of the pyridine ring nitrogen atoms in the two structures
will be different to make contact with the active site of
their target.
logical profile in 60 human cancer cell lines, compound
C has been selected by the NCI for extensive biological
evaluation.
To understand the observed differences in the potency of
C, D, and E, we performed conformational analysis
(
(
1
5
using molecular mechanics, CONFLEX with MM3)
Fig. 1, I, II for C, III and IV for D). It is interesting
to note that the lowest energy structure (Fig. 1, I) of C
is significantly different, with respect to the heterocyclic
ring nitrogen atoms (shown by arrows) from the lowest
energy structure (Fig. 1, III) of D. In I, the lowest energy
structure of C, one of the ring nitrogen atoms is pointing
outside (Fig. 1, bottom, on the left side, red arrow)
whereas in III, the lowest energy structure of D, ring
nitrogen atom is inside (Fig. 1, top, on the right side,
black arrow).
None of the triazine based ligands in the above-men-
tioned targets utilize the triazine ring nitrogen atoms
to form hydrogen bond(s) with their appropriate targets.
As the target of SM40 is unknown, at present, it is un-
clear about the involvement of the triazine ring atoms.
To show the possible differences (availability of the
nitrogen atoms to make possible contact to their targets)
in these compounds, we used the space filling model
(Fig. 3) of the lowest energy structures (in vacuum) of
C, D, and E.
The cytotoxic potency of D is significantly lower than
that of C. According to conformational analysis of C
and D, their lowest energy structures are different. To
visualize the structural differences and to understand
their impact on potency, we superimposed the lowest en-
ergy structures of C (Fig. 1, conformer I) and D (Fig. 1,
conformer III). In conformer I, one of the nitrogen
atoms of the pyridine ring is directed in opposite direc-
tion with respect to the conformer III. The ring nitrogen
atoms are encapsulated in the conformer III. Therefore,
these nitrogen atoms may not be available for bonding
with its target. Figure 2 shows the superimposition of
I and III. From Figure 2, it is clear that not only the
Among the different types of drug-receptor interactions,
certainly, C can form hydrogen bonds with its target be-
cause of the availability of the heterocyclic ring nitrogen
atoms and possibly they are located in appropriate dis-
tance from the hydrogen atom of the target. In C, het-
erocyclic ring nitrogen atoms could make contact with
binding sites on the target and this could be one of the
possible reasons for C to be most potent. As shown in
D, the pyridine ring nitrogen atoms are encapsulated
by the ring (ring number 2 and 3) atoms and thus these
nitrogen atoms may be unavailable for bonding with its
Figure 3. Space filling model; showing the lowest energy structures for
compounds C to E (Chart 1) and possible contacts (shown using
arrows) of C with its target.
Figure 2. Superimposition of the lowest energy structures; C (I, sky
blue) and D (III, pink); Pyridine rings are numbered (1–3), nitrogen
and sulfur atoms are colored as blue and yellow, respectively.
Figure 4. Examination of Shape of the chemical samples using
DGauss/DFT; Electron density isosurface for the lowest energy
structures of C (I) and D (III).

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S. Mandal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4955–4960
Figure 5. Effect of SM40 on cell cycle distribution and induction of apoptosis. (a) Representative DNA histograms of MDA-MB-468 cells exposed to
SM40 for 24 h. At higher concentrations, the sub-G1 peak (A ) was observed. (b) TUNEL assay of SM40 treated cells (24 h) showed that condensed and
fragmented nuclei incorporated TACS-blue labeled dNTP, indicating DNA strand breaks, in high (8.0 lM) as well as in low concentrations (0.5 lM)
scale bar–10 lm). Fragmentation increasedin a concentration-consistent manner. (c) Representative electron micrographs of control and 8.0 lM SM40
treated cells (24 h). Drug treated cells exhibit blebbing (b), apoptotic bodies (ab), vacuolization (v), and nuclear condensation (nc) (scale bar–3 lm).
0
(
target. This could be one of the possible reasons for the
D to be less potent. Also, aqueous solubility of D is poor
as compared to other derivatives. Perhaps the solvation
energy of D is greater than that of other derivatives.

S. Mandal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4955–4960
4959
Also, D can form intermolecular hydrogen bonding due
to the presence of the NH group. Figure 3 shows the
availability of pyridine ring nitrogen atoms in all deriv-
atives. In C, pyridine ring nitrogen atoms are freely
available and perhaps these nitrogen atoms are at an
appropriate distance to form hydrogen bonds with its
target. Possible contacts of C with its target are shown
by arrows (Fig. 3).
To understand other factors that may influence the po-
tency of C compared to D, we examined the electron
density isosurface which gives a more accurate represen-
tation of the true shape of chemical sample (Fig. 4). The
electron density isosurface is tabulated from a DGauss/
DFT wavefunction for lowest structures of C and D, at
a geometry determined by performing a geometry opti-
mization calculation in DGauss using the B88-LYP
GGA functional with the DZVP basis sets. Proper shape
of a drug molecule is critically important for binding
selectively with its target. Figure 4 shows that the shapes
of C and D are significantly different. The surface area of
Figure 6. Effect of SM40 on the protein expression and subcellular
localization of p53. (a) SM40 treated cells (8 h) showing total p53
protein expression (FL-393 antibody reacts with both mutant and
wild-type p53). (b) Representative images of SM40 treated cells
showing alterations in the subcellular localization of p53. Arrows
indicate the presence of nuclear p53 (mutant) in control and 0.5 lM
treated cells, but total absence in 8.0 lM treated cells. Arrows in the
lower panel show absence of nuclear p53 (wild-type) in control and
2
2
˚
˚
D is smaller (367.03 A ) than that of C (380.42 A ).
Therefore, D may not fit properly in the active site of
the target and may not form a stable drug-target com-
2
˚
plex. The surface area of E is much larger (398.99 A )
0
.5 lM treated cells and presence of the protein in 8.0 lM treated cells
than that of either C or D. In addition to the availability
of ring nitrogen atoms that can form hydrogen bonds,
the shape and size of C may be suitable for binding to
its target. Therefore, Figures 1–4 clearly explain the
greater potency of SM40 over other derivatives.
(
scale bar—10 lm).
firmed that SM40 induced apoptosis in MDA-MB-468
cells.
There are many widely used inhibitors for G1, S, and M
phases used for cell synchronization, but no effective G2
To explain how SM40 induced growth arrest and cell
death in MDA-MB-468 cells, we studied the possible
involvement of the phosphoprotein p53, a major player
of these cellular events. Western blotting analysis of pro-
tein level and subcellular localization by indirect immu-
nofluorescence were done in SM40 treated cells for 8 h,
prior to the onset of apoptosis.
1
6
phase inhibitors that could be used for cell cycle re-
search. Here, we report the synthesis of a series of novel
class of compounds of which SM40 (C, Chart 1) was
found to be most the potent due to its structural shape,
size, availability of ring nitrogen atoms, and their posi-
tions in appropriate locations among other properties.
To this end, we performed biological characterization
of SM40 using the highly aggressive ERa negative hu-
man breast cancer cell line, MDA-MB-468. In agree-
ment with the structural attributes, SM40 was the
most potent among the other derivatives as shown by
the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltet-
razolium bromide] assay (IC50 = 1.6 lM) in these cells.
The biological efficacy of SM40 was further evaluated
by its ability to induce growth arrest and cell death in
MDA-MB-468 cells. Since the IC50 value of SM40 was
This protein primes cells to die of apoptosis on exposure
1
7
to cytotoxic drugs and mediates its tumor suppressor
effects by the processes of growth arrest and apoptosis
1
8
to prevent the propagation of damaged DNA.
1
regulator of both the G1 and G2 checkpoints,
A
the
9,20
nuclear localization of p53 is essential for its growth-
suppressive (wild-type p53) and oncogenic (mutant
2
1
p53) actions.
p53
MDA-MB-468 cells possess the
mutant, which becomes growth suppressive
similar to its wild-type counterpart under certain condi-
2
73.His
1
.6 lM, we used a concentration range of 0.5–8.0 lM
for all experiments. Flow cytometry showed that at
4 h, SM40 induced a strong G2/M arrest at lower con-
2
2–25
tions.
There was no change in the total p53 protein
2
6
2
level even at 8.0 lM SM40 (Fig. 6a). Interestingly
though, when drug treated cells were stained with mu-
tant (PAb 240) and wild-type specific p53 (PAb 1620)
antibodies, we observed alterations in the subcellular
p53 localization in a concentration-dependent manner
(Fig. 6b). Representative images of SM40 treated cells
show that the PAb 1620 reactive p53 species was local-
ized to the nucleus, which was concomitant to the ab-
sence of nuclear localization of the PAb 240 reactive
p53 species; in contrast, control cells had a nuclear local-
ization of the PAb 240 reactive p53 species (Fig. 6b).
Thus, SM40 induced the nuclear localization of
centrations (1.0–2.0 lM, not shown); at higher concen-
trations, DNA histogram showed the presence of a
sub-G1 peak (A ) indicative of apoptosis induction by
0
SM40 (Fig. 5a). Using the TUNEL assay, we observed
a concentration dependent increase in the number of
cells with fragmented DNA (TUNEL positive)
(
Fig. 5b). Electron microscopy showed morphological
changes such as blebbing; nuclear condensation and
vacuolization of cells exposed to higher SM40 concen-
trations (Fig. 5c). Taken together, the presence of the
sub-G1 peak, morphology, and TUNEL assay con-

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S. Mandal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4955–4960
wild-type p53, independent of changes in the total pro-
tein level. We conclude that SM40 induced growth arrest
and apoptosis with a possible involvement of p53.
7. Coley, H. M.; Brooks, N.; Phillips, D. H.; Hewer, A.;
Jenkins, T. C.; Jarman, M.; Judson, I. R. Biochem.
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as a promising cytotoxic agent.
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Acknowledgments
We thank the Basic Medical Sciences, Memorial Univer-
sity, and College of the North Atlantic for their support.
We sincerely appreciate the free gift of all antibodies and
TUNEL assay kit from Santa Cruz Biotechnology, Inc.
1
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