3
18
S. Ghatak et al. / Bioorg. Med. Chem. Lett. 24 (2014) 317–324
experimental rodent model.6 The cancer growth-promoting
effects of downstream products of 5-LOX pathway such as
the potential of HA/CD44v6 as target for anticancer/chemopreven-
3
0,33,34
tion therapy.
The di-tert-butyl phenols represent a potent class of antioxi-
5
-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene E4
7
35,36
(
LTB4) have also been recorded. It thus appears logical that inhi-
dants and dual COX/LOX inhibitors
which are represented by
bition of both COX and LOX pathways may lead to more effective
control of colon cancer growth and associated signal transduction
processes. Use of non-steroidal anti-inflammatory agents (NSA-
IDs) as chemopreventive strategy for colorectal cancers has
generated great deal of interest as they have been shown to offer
protective effects during different stages of colorectal tumorigen-
esis including apoptosis, angiogenesis and cell-cycle progression.
Most of these effects have been attributed to the inhibition of
COX enzymes responsible for prostaglandin biosynthesis as these
have been found to be elevated in majority of colorectal cancers.
However, safety considerations restrict their use for cancer pre-
vention. Similarly, although selective COX-2 inhibitors exhibit
good benefits in controlling various types of cancers including
those of colon, the compounds also show increased risk of cardio-
vascular events thereby limiting their use in the clinics. Under
such circumstances inhibition of lipoxygenase pathway (LOX)
involving leukotrienes, might be useful for targeting the CRC.
More recently, involvement of the 5-LOX enzyme and its prod-
ucts have been implicated in development and progression of co-
experimental drugs like Darbufelone, Licofelone and KME4, respec-
tively. Darbufelone has recently been found to inhibit growth of
non-small cell lung cancer cell lines, inducing cell cycle arrest at
G0/G1 phase and apoptosis by activating caspase-3 and caspase-
3
7
8, respectively. However, this class of potent COX–LOX dual
inhibitors has remained inadequately explored for inhibition of
colon cancer. In the present work, we describe synthesis, charac-
terization of four 3,5-di-tert-butyl phenols appended with hydrazi-
nic chain, viz. DTPBHZ, DTPSAL, DTPNHZ and DTPINH as shown in
Scheme 1 where the side chain contributes towards enhanced lipo-
philicity and metabolic stability. The compounds were evaluated
for their antiproliferative potential against COX-positive human
3
0
colon cancer HT-29, HCA7 cells,
as well as COX-negative
SW480 human colon cancer cells and Has2 over-expressed murine
3
0
intestinal epithelial Apc10.1 Has2 cells. An in vitro assay was also
performed to elucidate their potential against inhibition of COX-1,
COX-2 and 5-LOX enzymes. The compounds were docked into
COX-2 and 5-LOX protein cavities in order to estimate their bind-
ing energies in these protein cavities which revealed that the syn-
thesized compounds had higher binding energies than the
standard drugs like Celecoxib and Licofelone, respectively. It was
also observed that CD44v6shRNA sensitizes these cells to DTPSAL
as well as to the chemotherapeutic drugs like Celecoxib and Licofe-
lone. Our results suggest that the di-tert-butyl moiety with hydra-
zinic side chain represents an active class of COX/5-LOX dual
inhibitors with chemo-preventive potential against colon cancer.
The di-tert-butyl-phenylhydrazone ligands DTPBHZ, DTPSAL,
DTPNHZ and DTPINH (Scheme 1) were prepared by mixing equi-
molar amounts of 3,5-di-tert-butyl-2-hydroxy benzaldehyde
(DPS) with respective hydrazides in methanolic solvent and
re-crystallizing the resulting compounds using methanol–water
mixtures. The details of spectroscopic assignments are given in
Supplementary data. The mass spectral (MS) data for the ligand
8
,9
lon cancer.
Both COX-2 as well as 5-LOX are up-regulated
1
0
during colon carcinogenesis and are related to tumor size,
depth and vessel invasion. Since both these enzymes use arachi-
8
donic acid as the substrate for eicosanoids synthesis, controlling
the amount of free arachidonic acid as well as the activity of
the metabolizing enzymes can potentially alter the production
of prostaglandin and leukotriene.11 It may also perhaps explain
why the anti-inflammatory and chemopreventive properties of
Celecoxib may be compromised in cells and tissues in which ara-
chidonic acid is shunted into the 5-LOX pathway contributing to
1
2
its cardiovascular complications. Such cardiovascular risks can
be minimized by employing dual COX/5-LOX inhibitors rather
than COX-2 inhibitors alone.
Several clinically effective NSAIDs have been structurally mod-
ified to yield potent dual COX–LOX inhibitors. Knaus and co-work-
ers have reported novel Aspirin analogs recently for their in vitro
as well as in vivo anti-inflammatory efficacy.13 This group has also
studied several analogs of other known NSAIDs including
ꢀ
DTPBHZ showed molecular ion peak at 352 corresponding to M1
peak. Similarly the MS data for other ligands DTPSAL, DTPNHZ
and DTPINH revealed peaks at 368, 353 and 353, respectively, con-
firming formation of the respective Schiff bases. The IR spectrum of
1
4
15
16,17
Indomethacin, Rofecoxib and Celecoxib
which have exhib-
the parent DPS compound exhibited a sharp intense band at
À1
ited higher efficacy as COX–LOX dual inhibitors. Among the dual
COX/LOX inhibitors studied so far, Licofelone has advanced into
1663 cm
due to aldehyde carbonyl group which disappeared
upon Schiff base formation replaced by the appearance of the
1
8
À1
clinical trial. Its safety and efficacy, in comparison with clinically
used NSAIDs like Naproxen and Rofecoxib have recently been
C@N imines stretching vibration in the region 1560–1552 cm
3
8
confirming successful reaction. All ligands showed a band due
to hydroxyl stretching vibration of the aldehyde in the region
3348–3230 cm . The hydrazinic NAH stretches were located in
1
9,20
reviewed.
Rao and co-workers have shown that Licofelone is
À1
able to inhibit both small intestinal and colon tumorigenesis in
Min/+
21
À1
À1
Apc
mice, where it is reported to trigger apoptosis in a dose-
the region 3002–3196 cm , while the hydrazinic carbonyl was
2
2
and time-dependent manner in human colon cancer cell HCA-7.
observed at 1641–1652 cm , respectively.
1
Subsequently in an attempt to identify molecules that are specifi-
cally expressed by epithelial tumor cells which correlate with
The H NMR signal at d 9.19–11.87 ppm was attributed to the
hydroxyl group on the di-tert-butyl phenyl ring. The sharp signal
of the aldehyde proton appearing at d 10.00–10.50 ppm was found
to be absent upon hydrazone formation. The signal for the protons
of one of the tert-butyl groups was observed as a singlet with
downfield shift between 1.41 and 1.46 ppm due to presence of
adjacent hydroxyl group while signal for the other tert-butyl group
tumor growth and drug resistance it was found that a major
extra-cellular component (ECM) hyaluronan (HA)2
3,24
and its
interaction with its receptor variant isoform CD44v62
3,25–27
play
crucial role in regulating COX-2/PGE2 mediated cell survival,
motility, and drug resistance.2
6,28–32
We and others have demon-
strated that the reversal of HA/CD44v6 signaling modulates the
appears as a singlet at 1.27–1.34 ppm. This suggests that hydrazide
Min/+
39
cancer phenotype and adenoma growth in Apc
mice by inhib-
substitution induced asymmetry in the hydrazonate ligands.
A
iting CD44v6/ErbB2/COX-2-PGE2 pathway. Additionally, our study
indicates that CD44v6/HA interaction has a regulatory effect on
COX-2-induced prostaglandins E2 (PGE2) which in turn controls
HA synthesis and hence the HA/CD44v6 signaling. Further it was
also observed that, elevated HA production resulted in the increase
of COX-2 expression (3 to 4-fold) as evident by RT-PCR, suggesting
new signal appearing at d 7.14–8.24 ppm confirmed the Schiff base
formation. All ligands exhibit a broad signal at d 8.49–10.66 ppm
due to presence of the hydrazinic–NH proton. The aromatic
protons appeared in the range of d 6.0–8.2 ppm for all compounds.
The C NMR spectra for the present ligands showed a singlet in the
range d 136.72–154.53 ppm ascribed to the imines carbon (–C@N)
1
3