Triaryl methane derivatives as antiproliferative agents

Article abstract of DOI:10.1016/j.bmcl.2003.11.004

Clotrimazole (CLT) 1, a synthetic anti-fungal imidazole derivative, inhibits tumor cell proliferation and angiogenesis. In the current study, flow cytometric analysis demonstrated that the decrease in tumor cell growth by CLT 1 was associated with inhibition of cell cycle progression at the G ₁-S phase transition, resulting in G₀-G₁ arrest. A series of CLT 1 analogues has been generated in order to develop CLT 1 derivatives that are devoid of the imidazole moiety which is responsible for the hepatoxicity associated with CLT 1 while retaining CLT 1 efficacy. The majority of these analogues demonstrate in vitro antiproliferative activity ranging from submicromolar to micromolar concentrations.

Full text of DOI:10.1016/j.bmcl.2003.11.004

Bioorganic & Medicinal Chemistry Letters 14 (2004) 347–350
Triaryl methane derivatives as antiproliferative agents
Raed A. Al-Qawasmeh,* Yoon Lee, Ming-Yu Cao, Xiaoping Gu, Aikaterini Vassilakos,
Jim A. Wright and Aiping Young
Lorus Therapeutics Inc., 2 Meridian Road, Toronto, ON, Canada M9W 4Z7
Received 18 September 2003; revised 18 September 2003; accepted 4 November 2003
Abstract—Clotrimazole (CLT) 1, a synthetic anti-fungal imidazole derivative, inhibits tumor cell proliferation and angiogenesis. In
the current study, flow cytometric analysis demonstrated that the decrease in tumor cell growth by CLT 1 was associated with
inhibition of cell cycle progression at the G –S phase transition, resulting in G –G arrest. A series of CLT 1 analogues has been
1
0
1
generated in order to develop CLT 1 derivatives that are devoid of the imidazole moiety which is responsible for the hepatoxicity
associated with CLT 1 while retaining CLT 1 efficacy. The majority of these analogues demonstrate in vitro antiproliferative
activity ranging from submicromolar to micromolar concentrations.
#
2003 Elsevier Ltd. All rights reserved.
Cancer remains the second leading cause of death in the
US and as a result there is a pressing need for novel and
effective treatments. One characteristic of cancer cells,
that distinguishes them from most normal cells, is their
high proliferative index. As a result, targeting of pro-
liferative pathways resulting in cell death via apoptosis
or prevention of cell division via cell-cycle arrest, are
considered effective strategies for fighting this disease.
Calcium is one of the most versatile second messengers
involved in cell growth, differentiation and signaling of
zole 4 (Fig. 1), was found to inhibit voltage- and ligand-
+
2
6
stimulated Ca influx in nucleated cells. Recent stud-
ies have demonstrated that CLT 1 has antiproliferative
7
,8
and antiangiogenetic activity.
This has led to the
search for structural analogues of CLT 1 that retain
biological activity after removal of the imidazole moiety
which is responsible for hepatotoxicity that limits clin-
ical application of CLT 1. It has been demonstrated that
the imidazole ring of CLT 1 is not required for
+
+
+
inhibition of human erythrocyte Ca
activated K
1
9
apoptosis. As a result, modulation of calcium signaling
pathways in cells may play an important role in con-
trolling cell growth and directing cell fate. Compounds
channels. Furthermore, other imidazole containing
antimytotic compounds like 2, 3 and 4 have similar
antimycotic activity as CLT 1, but do not possess anti-
2
+
7
designed to block Ca influx have demonstrated inhi-
bition of human prostate cancer cell proliferation in
proliferative activity. Taken together, these observa-
tions lead to the hypothesis that the antiproliferative
activity of CLT 1 results from the non-imidazole part of
its structure, while the imidazole moiety is responsible
for antimycotic activity.
2
vitro and in a mouse model.
Clotrimazole (CLT) 1 (Fig. 1) is a synthetic chlorinated
imidazole derivative first synthesized by BAYER
3
researchers. CLT 1 is a broad-spectrum antimycotic
To test this hypothesis, the imidazole moiety of CLT 1
was removed via refluxing in 1 M HCl for 3 h (Scheme
1) to generate compound 5. Compound 5 was subse-
quently transformed to compound 6 in the presence of
NaI, chlorotrimethylsilane, MeCN, and CH Cl at
agent used to treat pathogenic dermatophytes, yeast
infections, and topical fungal infections caused by sev-
eral species of Candida, Trichophyton, Microsporum,
4
Epidermophyton and Malassezia. The antifungal
activity of CLT 1 is via the inhibition of sterol 14a-
demethylase, a microsomal cytochrome P-450 enzyme.
2
2
room temperature for 48 h. Both compounds 5 and 6
are considered to be the main metabolites of CLT 1 in
5
9
10
humans. A sulforhodamine B (SRB) assay, using
human colon adenocarcinoma cells (HT-29), was per-
formed to compare the antiproliferative activity of CLT
CLT 1, like other antimycotic imidazole containing
compounds, econazole 2, metronidazole 3, and micona-
1
with that of compounds 5 and 6. The IC50 values were
determined to be 5.1, 17.6 and 19.9 mM, respectively. To
corroborate these results, a mitogen-induced cell pro-
liferation assay, using NIH 3T3 cells was performed
*
Corresponding author. Tel.: +1-416-798-1200; fax: +1-416-798-
200; e-mail: ralqawasmeh@lorusthera.com
1
1
2
0
960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.004

348
R. A. Al-Qawasmeh et al. / Bioorg. Med. Chem. Lett. 14 (2004) 347–350
may be the result of a general contribution of electron
density to the molecule and/or from the introduction of
specific functionalities on the aryl groups. These and
other possibilities are currently under investigation. In
addition, further modification and potency screening
may result in a detailed understanding of the underlying
mechanisms by which modification affects potency. The
ultimate goal of these studies is the generation and
characterization of potential therapeutic compounds
that can be advanced to preclinical and clinical
development.
To further investigate the effects of CLT 1 on cell cycle
progression, DNA content analysis of the human non-
small cell lung carcinoma cells (NCI-H460) was deter-
1
3
mined after treatment with CLT 1. Figure 2 shows the
results upon treatment with different concentrations of
CLT 1. When cultures were synchronized by serum-
starvation and subsequently released into complete
medium and treated with vehicle alone (DMSO) for 16
h, cell cycle progression was evident with an increase in
Figure 1. The structures of the antimitotic imidazole derivatives, Clo-
trimazole 1, Econazole 2, Metronidazole 3 and Miconazole 4.
the number of cell progressing into S and G –M phases
2
(A; 54.28 and 13.55%). However, cell cycle progression
was dramatically blocked in G –G when cells were
0
1
treated with CLT 1. In the presence of 10 mM CLT 1,
fewer cells exited out of G –G phase (B; 43.99%) and
0
1
Scheme 1. (a) 1 M HCl, reflux, 3 h; (b) NaI, chlorotrimethylsilane,
MeCN, CH Cl , rt, 48 h.
2 2
consequently fewer cells progressed to S phase (B;
45.99%). Higher concentrations of CLT 1 (25 and 50
mM) resulted in a dose dependent increase in the num-
ber of cells remaining in G –G phase at 68.53% (C)
and 77.78% (D), respectively. There was a concomitant
decrease in the number of cells in S phase. There was no
indication that CLT 1 treatment-induces apoptosis as
and the differences in IC50 values of these compounds
were determined to be comparable to those obtained
from SRB assays at 0.63, 2.1 and 3.9 mM, respectively.
These results demonstrate that removal of the imidazole
moiety does not completely inhibit the antiproliferative
activity of CLT 1, although the 3- to 6-fold increase in
IC50 value illustrates the importance of the imidazole,
either directly or as a result of contributions to the
structural configuration of the molecule. Given the
potential therapeutic benefit from removal of the imi-
dazole moiety, these results warranted a more detailed
investigation of the antiproliferative activity of non-
imidazole structure.
0
1
there was no increase in the sub-G population of cells.
1
Taken together, these results are consistent with pre-
vious findings that CLT 1 inhibits the expression of G1
cyclins, such as cylclin D1 and cyclin E, through deple-
2
+
7
tion of intracellular Ca
stores. This depletion was
shown to result in activation of RNA-dependent protein
kinase (PKR), inducing phosphorylation of eIF2a at
serine 51 and its concomitant inactivation. Inactivation
of eIF2a inhibits formation of the ternary complex
between Met-tRNA, eIF2a, and GTP, the rate-limiting
1
4,15
Subsequently, the non-imidazole portion of CLT 1 was
modified and a series of triaryl methane derivatives
was synthesized using previously described synthetic
step in translation initiation.
In order to assess whether the analogues decrease cell
proliferation via a similar mechanism as CLT 1, the
flow cytometric analysis was repeated with compounds
26 and 27. Table 2 summarizes the results and shows
that both compounds produce a similar pattern of cell
cycle progression as CLT 1. Analysis of additional ana-
logues is ongoing as well as in vitro studies aimed at
understanding the detailed mechanism of action of the
more promising compounds, including analysis of cyclin
expression and induction of apoptosis.
1
2
procedures.
assay was used to evaluate the antiproliferative effect of
A mitogen-induced cell proliferation
1
1
these derivatives in NIH 3T3 cells. Table 1 shown
below summarizes the IC50 values and the percent inhi-
bition at a concentration of 10 mM, for 23 compounds
compared to CLT 1.
The results clearly demonstrate the feasibility of gen-
erating therapeutically useful compounds through
structural modification of the triaryl structure of CLT 1
while completely eliminating the imidazole moiety.
Despite the modifications described in Table 1, all the
compounds are predicted to have a similar conforma-
tion around the central methane atom. Several analo-
gues were shown to possess similar or better
antiproliferative activity than CLT 1 (see compounds 7,
In conclusion, the results of this study, together with
previous findings, demonstrate that the antimycotic
drug CLT 1 is an effective cell cycle inhibitor that spe-
cifically arrests the cell cycle in G –G phase. Several
0
1
analogues of CLT 1, described in the current study,
were also effective in vitro, with growth inhibition in the
submicromolar to micromolar concentration range.
1
1, 13, 15, 23, 24, 25 and 26). The differences in potency

R. A. Al-Qawasmeh et al. / Bioorg. Med. Chem. Lett. 14 (2004) 347–350
349
a
Table 1. Mitogen-induced cell proliferation assay of the triaryl methane derivatives (R
R
1 2
R
3
CR ), CLT 1 analogues
4
Compd
R
1
R
2
R
3
R
4
IC50, mM
% Inhibition at 10 mM
CLT
0.63
2.1
3.9
0.4–2.0
2.2
3.3
1.0
0.8
93.0
31.0
91.8
99.0
99.0
99.0
99.0
99.0
98.0
94.0
98.0
99.0
99.0
93.0
94.0
98.0
96.0
92.0
95.8
99.0
98.0
99.0
98.0
77.0
5
6
7
8
9
2-Cl–Ph
2-Cl–Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
PhPh
Ph
Ph
Ph
Ph
OH
H
OH
OEt
OH
OH
OH
OH
OH
OH
OH
OH
CN
OH
CN
Ph
2-OH–Ph
2-Cl–Ph
Ph
Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
2-OH–Ph
2-OH–Ph
4-CF –Ph
Ph
2-Cl 4-NO
3
3.1
0.3
3.0
2
Ph
Ph
4-Me–Ph
4-Br–Ph
1-Naphthaline
Ph
Cyclohexane
Cyclohexane
Piperidine
Ph
Ph
4-Me–Ph
4-Br–Ph
Ph
4-Me–Ph
Ph
Ph
0.50
1.60
1.70
9.50
0.90
2.20
1.20
2.2
0.2
0.4
0.8
0.4
4-NO
2
–Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CO
2
Et
2-Cl–Ph
2-Cl–Ph
4-NH –Ph
CH
2
2
CO H
Ph
4-NH –Ph
2
CN
OH
OH
OH
OH
2
4-NH –Ph
2
Ph
Ph
Ph
Ph
C
2
Ph
Ph
Ph
4-Cl–Ph
Ph
Ph
C
2
–Ph
Ph
(S)-(+)-5-(Oxymethyl)-2-Pyrrolidinone
3.8
a
The assay has been performed as in ref. 11.
Figure 2. DNA content analysis of NCI-H460 non-small cell lung cancer cells by flow cytometry. NCI-H460 cells were serum-starved for 56 h and
then treated with various concentrations (10–50 mM) of CLT 1 for 16 h. Cells were stained with propidium iodide and analyzed by flow cytometry.
Percentage of cells under different stages of cell cycles (G
0
–G
1
, S or G
2
–M) is indicated below each graph.
Preliminary analysis of two analogues suggests they act
in a similar manner to CLT 1. These results are highly
encouraging and have led to studies aimed at optimiza-
tion of these analogues with a long-term goal of devel-
oping novel anti-cancer therapeutics based on the
isolated triaryl structure of CLT 1.
Table 2. Effects of compounds 26 and 27 on cell cycle progression
Treatmenta
G
S
0
–G
1
2
G –M
DMSO
32.17
46.80
69.91
58.07
80.85
54.28
44.63
25.09
35.29
4.79
13.55
8.57
5.0
6.64
14.36
25 mM 26
50 mM 26
25 mM 27
50 mM 27
a
DNA content analysis of NCI-H460 non-small cell lung cancer cells
by flow cytometry. NCI-H460 cells were serum-starved for 56 h and
then treated with various concentrations (25–50 mM) of CLT 1 ana-
logues for 16 h. Cells were stained with propidium iodide and ana-
lyzed by flow cytometry. Percentage of cells under different stages of
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8
ꢀ
anol at 4 C at least 4 h. The fixed cells were centrifuged at
ꢀ
1500 rpm for 4 min at 4 C, washed twice with cold PBS
containing 2% FBS, and treated with 3 mg/mL ribonu-
9
. Brugnara, C.; Armsby, C. C.; Sakamoto, M.; Rifai, N.;
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iodide (PI) (Sigma Chemical Co.) for 30 min at 37 C.
ꢀ
1
0. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.;
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Flow cytometry analyses were performed on the Becton
Dickinson fluorescence-activated sorter FACScan by
using the Becton Dickinson Cell Quest program. Data
were evaluated using Modfit software (Verity software
House, Topsham, ME, USA).
589.
1
1. The mitogen-induced cell proliferation assays were
performed using NIH 3T3 mouse fibroblast cells (ATCC
#
CRL1658). The assay was performed essentially as
14. Aktas, H.; Fluckiger, R.; Acosta, J. A.; Savage, J. M.;
Palakurthi, S. S.; Halperin, J. A. Proc. Natl. Acad. Sci.
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described in ref 7 and the cited references therein. Briefly,
the cells were exposed to a mitogenic stimulus (such as
3
PDGF or bFGF) for 10 h, and then [ H] thymidine added
ꢀ
15. For the discussion of antiproliferative effect which is
mediated by translation initiation inhibition and other
related references, see: (a) Palakurthi, S. S.; Fluckiger, R.;
Aktas, H.; Changolkar, A. K.; Shahsafaei, A.; Harneit, S.;
Kilic, E.; Halperin, J. A. Cancer Res. 2000, 60, 2919. (b)
Palakurthi, S. S., Aktas, H., Grubissich, L. M., Morten-
sen, R. M., Halperin, J. A. Cancer Res. 2001, 61, 6213,
and references therein.
to the well. The cells were maintained in culture at 37 C
2
and 5% CO for another 21 h, and then washed four
times with DME medium. The acid-precipitable radio-
activity was extracted with cold 10% TCA and after neu-
tralization with 0.3 N NaOH, aliquots were counted in a
Packard Tri-Carb Scintillation counter. For the cell pro-
liferation assay, cells were seeded in culture plates and
allowed to attach to the culture plates for 24 h. Different