Novel dual cyclooxygenase and lipoxygenase inhibitors targeting hyaluronan-CD44v6 pathway and inducing cytotoxicity in colon cancer cells

Article abstract of DOI:10.1016/j.bmc.2013.02.033

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzyme have been found to play a role in promoting growth in colon cancer cell lines. The di-tert-butyl phenol class of compounds has been found to inhibit both COX-2 and 5-LOX enzymes with proven effectiveness in arresting tumor growth. In the present study, the structural analogs of 2,6 di-tert-butyl-p-benzoquinone (BQ) appended with hydrazide side chain were found to inhibit COX-2 and 5-LOX enzymes at micromolar concentrations. Molecular docking of the compounds into COX-2 and 5-LOX protein cavities indicated strong binding interactions supporting the observed cytototoxicities. The signaling interaction between endogenous hyaluronan and CD44 has been shown to regulate COX-2 activities through ErbB2 receptor tyrosine kinase (RTK) activation. In the present studies it has been observed for the first time, that three of our COX/5-LOX dual inhibitors inhibit proliferation upon hydrazide substitution and prevent the activity of pro-angiogenic factors in HCA-7, HT-29, Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressed in colon cancer cells, through inhibition of the hyaluronan/CD44v6 cell survival pathway. Since there is a substantial enhancement in the antiproliferative activities of these compounds upon hydrazide substitution, the present work opens up new opportunities for evolving novel active compounds of BQ series for inhibiting colon cancer.

Full text of DOI:10.1016/j.bmc.2013.02.033

Bioorganic & Medicinal Chemistry 21 (2013) 2551–2559
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel dual cyclooxygenase and lipoxygenase inhibitors targeting
hyaluronan–CD44v6 pathway and inducing cytotoxicity in colon
cancer cells
Suniti Misra a, , Shibnath Ghatak a, , Neha Patil b, Prasad Dandawate b, Vinita Ambike c,
Shreelekha Adsule c, Deepak Unni d, K. Venkateswara Swamy d, Subhash Padhye b,c,
⇑
a Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA
b ISTRA, Department of Chemistry, Abeda Inamdar Senior College, University of Pune, Pune 411001, India
c Department of Chemistry, University of Pune, Pune 411007, India
d Department of Bioinformatics and Computer Science, Dr. D.Y. Patil Biotechnology and Bioinformatics Institute, Dr. D.Y. Patil Vidyapeeth, Pune 411044, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzyme have been found to play a role in pro-
moting growth in colon cancer cell lines. The di-tert-butyl phenol class of compounds has been found
to inhibit both COX-2 and 5-LOX enzymes with proven effectiveness in arresting tumor growth. In the
present study, the structural analogs of 2,6 di-tert-butyl-p-benzoquinone (BQ) appended with hydrazide
side chain were found to inhibit COX-2 and 5-LOX enzymes at micromolar concentrations. Molecular
docking of the compounds into COX-2 and 5-LOX protein cavities indicated strong binding interactions
supporting the observed cytototoxicities. The signaling interaction between endogenous hyaluronan
and CD44 has been shown to regulate COX-2 activities through ErbB2 receptor tyrosine kinase (RTK) acti-
vation. In the present studies it has been observed for the first time, that three of our COX/5-LOX dual
inhibitors inhibit proliferation upon hydrazide substitution and prevent the activity of pro-angiogenic
factors in HCA-7, HT-29, Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-
expressed in colon cancer cells, through inhibition of the hyaluronan/CD44v6 cell survival pathway. Since
there is a substantial enhancement in the antiproliferative activities of these compounds upon hydrazide
substitution, the present work opens up new opportunities for evolving novel active compounds of BQ
series for inhibiting colon cancer.
Received 8 November 2012
Revised 12 February 2013
Accepted 21 February 2013
Available online 27 February 2013
Keywords:
Cyclooxygenase
Lipoxygenase
Colon cancer
Dual COX–LOX inhibitor
Hyaluronan
CD44v6
Cell proliferation and drug resistance
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
metastatic CRC.2 Adjuvant chemotherapy based on 5-fluorouracil
(5-FU) combined with radiotherapy after the radical resection of
Colorectal cancer (CRC) is the third most common cause of can-
cer related deaths in the United States with an estimated 103,107
new cases and 51,690 number of deaths in 2012.1 Surgery remains
the only treatment with curative potential for both primary and
primary CRC has been found to prolong the survival of patients.3
In cases of metastases improvements have been observed by com-
bining 5-FU with irinotecan and oxaliplatin. Attempts in designing
novel drugs have led to discovery of potential chemopreventive
agents that are effective at the pre-clinical and clinical levels.4,5
The cyclooxygenase enzyme exists in two different isoforms,
viz. COX-1, and COX-2, respectively which uses arachidonic acid
(AA) as a substrate to produce prostaglandins (PGs). AA is also a
substrate for another enzyme, viz. 5-lipoxygenase (5-LOX), which
generates leukotrienes (LTs).6 The roles of both downstream prod-
ucts of COX and 5-LOX, viz. PGE2 and LTB4, in the inflammatory
process have been extensively explored in the past.6 It has been
demonstrated that the metabolism of AA, by either the COX or
LOX pathway, generates a host of pro-inflammatory metabolites
called eicosanoids including PGs, thromboxanes and leukotrienes
(LTs), respectively.6 Consequently increased levels of COX and
Abbreviations: COX-2, cyclooxygenase; CD44v6, variant exon 6 of CD44; CD44s,
CD44 standard form; HA, hyaluronan; Has2, hyaluronan synthase-2; RTK, receptor
tyrosine kinase; PGE2, prostaglandin E2; LOX, lipoxygenase; AA, arachidonic acid;
Darbufelone, 5-((Z)-3,5-di-tert-butyl-4-hydroxybenzylidene)-2-imino-4-thiazolidi-
none; Licofelone, [6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-
pyrrolizin-5-yl]acetic acid; CRC, colon rectal cancer; 5-FU, 5-fluorouracil; LTB4,
leukotriene B4; 5-HETE, 5-hydroxyeicosatetranoic acid; NSAID, Non-Steroidal Anti-
Inflammatory Drugs.
⇑
Corresponding author. Tel.: +918390025533.
E-mail addresses: misra@musc.edu (S. Misra), ghatak@musc.edu (S. Ghatak),
sbpadhye@hotmail.com, bhash46@hotmail.com (S. Padhye).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.033

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S. Misra et al. / Bioorg. Med. Chem. 21 (2013) 2551–2559
LOX enzymes have been implicated in adenomatous polyposis7,8
and in experimentally induced colon tumors in rodents.9 The can-
cer growth-promoting effects of downstream products of 5-LOX
pathway such as 5-hydroxyeicosatetranoic acid (5-HETE) and leu-
kotriene B4 (LTB4) have been reported10 which forms the basis of
the use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), differ-
ent COX inhibitors and 5-LOX inhibitors such as REV5901, Zileuton,
and Minocyclines.11–13 Since these two arachidonic acid-metabo-
lizing enzymes are so closely related in their substrates and mech-
anisms of action, it can be envisaged that blocking one enzymatic
pathway may activate the other.14 This phenomenon might ex-
plain, at least in part, the observed limited efficacy of COX-2 inhib-
itors as anticancer agents.15,16 At higher concentrations, the anti-
carcinogenic effects of Celecoxib have been explained by both
COX-2-dependent and -independent mechanisms.16,17 These in-
ter-relationship of their biological functions suggest that blocking
both COX-2 and 5-LOX pathways using designed synthetic mole-
cules may provide a promising integrated approach for prevention
and therapy of colon cancer.
A number of studies have aimed at identifying molecules that
are specifically expressed by epithelial tumor cells which correlate
with tumor growth and drug resistance. Among such molecules,
hyaluronan (HA) is a major component in the extra-cellular matrix
of most mammalian tissues and animal models,18,19 while CD44 is
a trans-membrane glycoprotein that communicates with the envi-
ronment by binding to its major ligand HA20 and thereby regulat-
ing cell survival and motility.21–23 In the process of growth and
spread of tumors, CD44/HA signaling employs at least three differ-
ent functions of CD44.24 Firstly, CD44 needs to stimulate tumor cell
proliferation, motility, and/or invasiveness through recruitment
and enabling the activity of membrane-associated matrix metallo-
proteinases25,26 or receptor tyrosine kinases.27,28 Secondly, the
variants of CD44 may function as co-receptors for the activation
of growth-promoting tumor receptor tyrosine kinases.29 Finally,
the variant isoform CD44v6 is over-expressed in colon tumors30–
39 and the endogenous binding of HA to CD44v6 is more avid than
to CD44.18,40,41 We have shown that CD44v6/HA interaction
regulates COX-2-induced prostaglandins E2 (PGE2) which in turn
controls HA synthesis and hence the HA/CD44v6 signaling.42–46
We and other groups have shown that both HA and CD44 are in-
volved in chemotherapeutic drug resistance in many cancer
types,47–49 and the reversal of HA/CD44v6 signaling has been
shown to modulate the cancer phenotype and adenoma growth
in Apc Min/+ mice suggesting potential of HA/CD44v6 as targets
for anti-cancer/chemoprevention drugs.18,50,51 Conceptually,
agents that target COX/LOX or CD44v6 are noteworthy because
of their clinical efficacy in the prevention of polyp formation.18,52
Surprisingly, only few dual COX–LOX inhibitors have been stud-
ied clinically for their efficacy as the anti-inflammatory agents.
Amongst them Licofelone is in phase III of clinical trials, while
Tenidap developed by Pfizer has been withdrawn because of its
high hepatotoxicity.53 The di-tert-butyl phenols represent a potent
class of dual COX/5-LOX inhibitors54–56 which is represented by
Darbufelone (1), CI987 (2), S2474 (3) and KME4 (4) (Table 1). Dar-
bufelone was recently found to inhibit growth of non-small cell
lung cancer cell lines, inducing cell cycle arrest at G0/G1 phase
and apoptosis by activation of caspase-3 and caspase-8, respec-
tively.57 However, these compounds or their pro-drugs di-tert-bu-
tyl benzoquinones, have remained inadequately explored for the
inhibition of colon cancer in spite of their low ulcerogenicity and
potent COX–LOX inhibitory activity.58 Hence, in the present study,
we have synthesized and structurally characterized three di-tert-
butyl benzoquinone COX/LOX inhibitors appended with hydrazinic
side chain, viz. BQBH, BQNH and BQIH as shown in Scheme 1. The
compounds were docked into COX-2 and 5-LOX protein cavities
and evaluated for their cyclooxygenase-1, -2 (COX-1, -2) and 5-
lipoxygenase (5-LOX) enzyme inhibitory activities followed by
evaluation of their cytotoxic effects against colon cancer cell line.
Further, we also examined whether the COX/5-LOX pathway and
the CD44v6/HA interactions co-operate in promoting proliferative
stimulus in human colon cancer cells HT29, HCA7 and pre-neoplas-
tic Apc10.1 cells isolated from Apc Min/+ mice.59 It was found that
these compounds were able to inhibit cell proliferation dramati-
cally and differentially by increasing apoptosis in HT29 and HCA7
Table 1
Examples of the dual COX–LOX inhibitors
O
O
O
O
O
N
S
N
O
N
S
S
HO
HO
HO
HO
O
N
OH
S2474 (3)
Darbufelone (1)
KME-4 (4)
CI-987 (2)
O
N
N
O
N
O
O
O
OH
Cl
NH2
Licofelone (5)
ER-34122 (6)
Chebulagic acid

S. Misra et al. / Bioorg. Med. Chem. 21 (2013) 2551–2559
2553
ligands signals in the region of 7.097–7.856 ppm were due to aro-
matic protons confirming the formation of expected Schiff bases.
The sharp singlet in the region of 12.027–12.374 ppm was ascribed
to the imine (–NH) proton. Two signals appearing as singlets due to
tert-butyl groups in the region of 1.271–1.281 and 1.320–
1.334 ppm suggest that hydrazide-substitution induced asymme-
try in the hydrazonate ligands.70 The 13C NMR showed two prom-
inent signals at 187.16 and 189.03 ppm ascribed to two quinone
carbonyls in the parent BQ compound. In the hydrazonate ligands
one of these carbonyl signals (189.03 ppm) disappeared with
emergence of a new signal in the region 151.79–152.09 ppm corre-
sponding to the imino carbon. The carbons of the tert-butyl
groups71,72 in BQ with signals at 26.36 and 35.55 ppm undergo
downfield shift to 29.59–29.89 and 35.15–36.41 ppm, respectively
upon Schiff base formation, while their aromatic carbon atoms can
be observed in the region of 119.47–152.86 ppm, respectively.
O
O
NH2
R
N
H
N
O
N
H
R
ethanol, 60-650C,
conc HCl
O
O
BQ
R=
N
N
BQBH
BQNH
BQIH
Scheme 1. Synthesis of 2,6-di-tert-butyl-benzoquinone-4-hydrazone ligands.
2.2. Molecular docking study
cells and hyaluronan synthase-2 over-expressed Apc10.1 cells.59 It
was also observed that CD44v6shRNA sensitizes these cells to che-
motherapeutic drugs like Celecoxib and Licofelone as well as pres-
ent COX–LOX dual inhibitor, viz. BQNH. Finally, it is reasonable to
speculate that COX-2/PGE2 may regulate CD44v6, and HA synthe-
sis since HA/CD44v6 signaling regulates COX-2/PGE2 signaling42,43
and COX-2 and 5-LOX have been closely related in mechanisms of
action and their substrates.60,61 Our results suggest that COX/5-
LOX dual inhibitors with hydrazinic side chain represent a potent
class of chemopreventive agents for colon cancer.
In order to identify the possible binding sites of the hydrazonate
ligands, the compounds were docked into the pre-defined binding
site of COX-2 and 5-LOX proteins. All presently synthesized com-
pounds as well as standard dual COX–LOX inhibitor Darbufelone
compound, exhibited extensive hydrogen bonding interactions in
the COX-2 cavity. The binding energies calculated for the new ana-
logs were in the range of ꢀ6.8 to ꢀ7.2 Kcal/mol compared to Dar-
bufelone’s binding energy of ꢀ6.8 Kcal/mol (Table 2 and Fig. 1:
Panel 1). The best binding energy was exhibited by BQNH ligand.
Darbufelone exhibited three hydrogen bonding interactions with
residues VAL228 (3.4 Å), ASN537 (2.1 Å) and GLY533 (2.0 Å),
respectively, while the most potent compound BQNH showed
interactions with ASN87 (3.3 Å) and HIS90 (2.0 Å), respectively.
The Van der Waals interactions between nitrogen of the pyridine
ring and oxygen of the nicotylhydrazide moiety were also found
to be involved in the stabilization of ligand–enzyme complexes.
In 2011 Gilbert et al.64 have reported on the crystal structure of
human 5-LOX. The amino acid residues like HIS367, LEU368,
HIS372, LEU373, ILE406, LEU414, HIS550, ASN554, LEU607 and
ILE673 were identified in the active site of 5-LOX protein.73 When
docked into the protein cavity of 5-LOX, BQNH exhibited best bind-
ing energy of ꢀ8.4 Kcal/mol followed by compound BQIH, Darbufe-
lone and BQBH with binding energies of ꢀ8.2, ꢀ7.3 and ꢀ7.0 Kcal/
mol, respectively indicating their tight binding in the protein cav-
ity. Darbufelone was found to interact with ASP285 (2.5 Å),
GLU287 (2.6 Å), ILE283 (3.3 Å), LEU244 (3.5 Å), respectively, while
compound BQNH interacted with GLN549 (2.9 Å) and ARG370
(2.2 Å), suggesting that these interactions are involved in stabiliz-
ing these compounds in the protein cavity (Table 2 and Fig. 1: Pa-
nel 2). Our docking study confirmed that the newly synthesized
2. Results and discussion
2.1. Chemistry
The compounds BQBH, BQNH and BQIH were shades of yellow-
orange. The IR spectrum of BQ exhibited a sharp intense band at
1654 cmꢀ1 due to the quinone carbonyl group flanked by the butyl
groups and shoulder absorption at 1599 cmꢀ1 due to other free car-
bonyl, respectively.68 Upon Schiff base formation the free carbonyl
stretch disappeared accompanied by the appearance of the C@N
stretch in the region 1633–1680 cmꢀ1 which overlaps with the
remaining quinone carbonyl and the hydrazinic carbonyl fre-
quency resulting in broadening of the band. The hydrazinic N–N
stretch occurred in the range 1252–1258 cmꢀ1, while the aromatic
C@C stretches can be observed in the region 1521–1546 cmꢀ169
The electronic spectra of the ligands in DMSO showed intra-ligand
transitions in the range 26,300–27,700 cmꢀ1, respectively.
The 1H NMR spectra of the parent BQ compound showed sharp
singlets at 1.286 and 6.512 ppm ascribed to di-tert-butyl protons
and protons of the quinone moiety.68 In case of the hydrazonate
Table 2
Docking results and consensus scores of 2,6 di-tert-butyl benzoquinone-4-hydrazones in COX-2 and 5-LOX protein cavity
Molecule
COX-2
5-LOX
logP
B.E. (Kcal/mol)
H bond
H bonding residues
Distance (Å)
B.E. (Kcal/mol)
H bond
1
H bonding residues
ASP170
Distance (Å)
2.3
BQBH
5.47
ꢀ6.8
3
2
1
3
HIS351,
GLN192
ASN87,
HIS90
3.3
2.2 and 3.4
3.3, 2.0
ꢀ7.0
BQNH
3.34
3.28
4.98
ꢀ7.2
ꢀ7.0
ꢀ6.7
ꢀ8.4
ꢀ8.2
ꢀ7.3
2
2
4
GLN549,
ARG370
ARG47
ARG401
ASP285,
GLU287,
ILE283,
LEU244
2.9
2.2
2.3,
2.6
2.5,
2.6,
3.3,
3.5
BQIH
GLN192
2.2
Darbufelone
VAL228,
ASN537,
GLY533
3.4, 2.1, 2.0
COX-2 = cyclo-oxygenase-2, 5-LOX = 5-lipoxygenase, B.E. = binding energy, H bond = hydrogen bond, H bonding residues = hydrogen bonding residues.

2554
S. Misra et al. / Bioorg. Med. Chem. 21 (2013) 2551–2559
Panel 1: BQBH
BQNH
BQI
H
Darbufelone
Panel 2: BQBH
BQNH
BQIH
Darbufelone
Figure 1. Docking features of 2,6 di-tert-butyl benzoquinone-4-hydrazones in COX-2 (Panel 1) and 5-LOX (Panel 2).
compounds can interact with COX-2 and 5-LOX protein cavities
wherein the heterocyclic hydrazide moiety containing nitrogen
contributes towards enhancing interaction with amino acid
residues. BQNH showed best binding energy in both COX-2 and
5-LOX protein cavities which correlate well with its superior
antiproliferative activity in vitro against the colon cancer cell lines
employed in the present study.
established that constitutive hyaluronan–CD44 interaction stimu-
lates a signaling pathway involving ErbB2, phosphoinositide-3-ki-
nase/AKT, b-catenin, and cyclooxygenase-2/prostaglandin E(2) in
HCA7 colon carcinoma cells.43 Consequently in the present study,
we sought to determine whether hyaluronan also regulates 5-
LOX expression employing two human CRC HT29, HCA-7 cells,
and Has2 over-expressing Apc10.1 cells with different basal COX-
2 protein expression (Fig. 2A). In our studies 5-LOX expression
was detected in all three cell lines (Fig. 2A), while Has2 over-
expression in Apc 10.1-Has2 cells exhibited high levels of COX-2
compared to moderate levels expressed by HT29 and HCA-7 cells
(Fig. 2A, lane 2 vs lane 1). In agreement with our previous study,
Apc10.1 cells expressed low levels of COX-2 and 5-LOX protein,
suggesting that COX-2 as well as 5-LOX is both regulated by hyalu-
ronan. In further studies we used HT29 cells wherein our results
indicated (Fig. 2B) that very low levels of COX-2 and 5-LOX are
present in the CD44v6shRNA transfected HT29 cells with a sub-
stantial inhibition by BQBH, BQNH and BQIH as well as Licofelone
(standard COX/LOX inhibitor) and Celecoxib (COX-2 inhibitor).
Subsequently, we determined that HA over-expression induces
the recruitment of a significant amount of COX-2 and 5-LOX into
the HA signaling cascade. This led us to conclude that just as HA/
CD44-linked COX-2 activation is correlated with tumorigenic
behavior, the 5-LOX expression is also regulated by HA and
CD44v6 (Fig. 2B, lane 8 compared to lane 2) and present COX–
LOX dual inhibitors are able to target the surface adhesion mole-
cules like CD44v6, (Fig. 2B, lanes 3, 4, 5 and 7 vs lane 2).
2.3. Biological activity
2.3.1. In vitro COX–LOX assay
The potential of all synthesized compounds to inhibit COX-1,
COX-2 isoforms and 5-LOX was determined on pure enzymes.
Amongst the new compounds BQBH and BQIH exhibited most po-
tent 5-LOX and COX-2 inhibitory activities (Table 3). These results
indicated that the hydrazonate pharmacophore in the side chain
plays an important role in deciding the preferences for these en-
zyme sites. The present work thus confirms that 2,6 di-tert-bu-
tyl-p-benzoquinone hydrazone ligands are effective COX/LOX
dual inhibitors which can serve as promising therapeutic agents
against colon cancers.
2.3.2. COX-2 and 5-LOX expression in colon tumor cells and
Has2-overexpressing Apc10.1 cells
COX-2 and 5-LOX have been shown to over-express during the
process of colonic adenoma formation and have been implicated as
promoter for tumor development.61 In our earlier studies, we have
2.3.3. Inhibition of COX-2, and COX-2/5-LOX attenuate HA-
stimulated cell survival in colorectal cells
Table 3
COX–LOX inhibitory potential of 2,6-di-tert-butyl-benzoquinone-4-hydrazone
Although the role of the COX pathway in colonic cancer devel-
opment has been widely studied, investigation of the role of the
LOX pathway in colon cancer has been limited. We have previously
shown that COX-2 induced PGE2 regulates HA production, and
hence HA/CD44 induced signaling. The question is whether COX/
LOX pathways and HA/CD44 pathways are co-regulated or act
independently in the tumor specific behavior. Experiments in Fig-
ure 2 show that there exists a relationship between HA/CD44v6
compounds
Compound
IC50 COX-2
M)
IC50 COX-1
M)
IC50 COX-2/IC50
COX-1
IC50 5-LOX
M)
(
l
(l
(l
BQBH
BQNH
BQIH
9.5
9.3
7.6
14.0
48.6
55.7
35.00
0.68
0.19
0.14
0.014
5.1
7.4
20
Darbufelone 0.48
0.77

S. Misra et al. / Bioorg. Med. Chem. 21 (2013) 2551–2559
2555
Figure 2. Analyses of HA-induced CD44V6 associated COX-2 and 5-LOX expression in colorectal carcinoma (CRC) cell lines.
and COX-2 and 5-LOX. Therefore, we investigated the anti-carcin-
ogenic effect of dual inhibition of COX-2 and 5-LOX in the process
of cell proliferation. The functional effect of 5-LOX and COX-2 on
CRC cell proliferation and survival was analyzed by exposing cells
to either colon cancer chemotherapeutic drug Celecoxib (inhibits
COX-2), or Licofelone (inhibits 5-LOX), or the three COX-2/5-LOX
inhibitors along with control shRNA or CD44v6shRNA transfection
at various doses. Results in Figure 3 indicate that these antagonist
drugs dose-dependently inhibited colon cancer cell proliferation.
They also indicate that 5-LOX and COX-2 in addition to HA/
CD44v6 could be involved in the mechanisms of CRC cell carcino-
genesis because our new COX–LOX dual inhibitors (Scheme 1) de-
crease CRC proliferation similar to CD44v6shRNA, with the effect of
the COX-2 inhibitor Celecoxib showing somewhat stronger inhibi-
tory effect compared with the 5-LOX inhibitor Licofelone.
sence of Has2 over expression (Table 4). In the absence of Has2,
HT29, HCA-7 and Apc10.1 cells treated with BQBH or BQIH dis-
played a low level of tumor cell survival, with IC50 values of 12–
20
BQNH exhibited a relatively low level of tumor cell survival, with
IC50 values between 6.5 and 11 M (Table 4). The cytotoxic activity
lM (Table 4). HT29, HCA-7 and Apc10.1 cells treated with
l
of parent building blocks of the new inhibitors was also tested
against HT29 cells which revealed higher IC50 values in the range
26.5 3.3 to 175.0 28.3 lM, respectively. This suggests that the
cytotoxic activity was due to intact compounds inside the cell.
Celecoxib-treated cells exhibited a very low level of IC50 values be-
tween 2.0 and 4.2
role in cell survival. Licofelone-treated cells showed higher IC50
values between 45 and 75 M indicating that 5-LOX may have a
lM, indicating that COX-2 may have a greater
l
greater role in apoptotic resistance. CD44v6shRNA-transfected
cells exhibited the highest inhibition of cell survival (Table 4), indi-
cating that CD44v6 is necessary for cell survival. However, the
over-expression of Has2 enhances cell survival in untreated con-
trols (i.e., without chemotherapeutic drugs) and decreases the abil-
ity of BQBH, or BQNH, or BQIH, or the standard inhibitors to induce
To further confirm whether the chemotherapeutic drug re-
sponses of Apc10.1, HT29, and HCA7 cells might be regulated by
the HA/CD44v6 interaction, cell growth assays were done using co-
lon cancer chemotherapeutic drugs Celecoxib and Licofelone along
with BQBH, BQNH, BQIH, and CD44v6shRNA in the presence or ab-
Figure 3. Effect of COX/5-LOX, and CD44v6 inhibition of proliferation on colorectal carcinoma (CRC) cell lines.

2556
S. Misra et al. / Bioorg. Med. Chem. 21 (2013) 2551–2559
Table 4
IC50 analyses of drugs (COX-2/5-LOX inhibitor BQBH, BQNH, BQIH, Licofelone, COX-2 inhibitor Celecoxib and HA/CD44v6 antagonist CD44v6shRNA) in cell proliferation of HT29,
HCA7, and Has2 over-expressed Apc10.1 cells
Cell lines
BQBH
BQNH
BQIH
Celecoxib
Licofelone
IC50 M)
CD44v6shRNA
IC50 (pmole)
IC50
(l
M)
IC50
(l
M)
IC50
(
l
M)
IC50
(
lM)
(l
Has2
HT29
HCA-7
Apc10.1
ꢀ
+
ꢀ
+
ꢀ
+
ꢀ
+
+
ꢀ
+
15 1.5
12.5 0.9
20 3.7
38.5 3.6
26.4 1.9
31 4.2
11 1.3
6.5 1.2
7.5 2.2
28.6 3.2
34.5 4.3
12.2 1.8
15.2 1.9
12 1.5
8.9 1.2
5.4 5.7
38.6 3.9
18.3 2.0
3.0 0.8
2.0 0.9
4.2 1.2
18.3 2.3
17.5 2.5
6.0 1.5
75 8.3
48 5.2
45 7.2
158 9.3
186 11.9
86 7.2
80.0
7
240 18
280 19
100 9.5
110.0 11
180 17.2
IC50 values are presented as the means S.D (n = three replicates of 4 independent analyses).
IC50 is designated as the concentration (lM) of these drugs that causes 50% inhibition of colon tumor cell proliferations.
Figure 4. The inhibition curves of BQNH at different test concentrations using MTT assay.
tumor cell death (Table 4). These observations strongly suggest
that HA causes an increase in tumor cell survival, leading to
enhancement of chemo-resistance (i.e., resistance to cell death)
to all the drug/inhibitors treatments. Furthermore, pre-treatment
of these CRC cells with CD44v6shRNA followed by Has2 over-
expression significantly reduces the HA-mediated resistance to cell
death (Table 4). Thus, inhibition of HA/CD44v6 interaction, or inhi-
bition of COX-2/5-Lox appears to be functionally linked to anti-
apoptotic effects in CRC cells. These results indicate that HA/
CD44v6 interaction promotes resistance to apoptosis (anti-apopto-
sis) in the presence of novel dual COX–LOX inhibitors (BQBH,
BQNH and BQIH) and chemotherapeutic drugs, Celecoxib and
Licofelone.
CD44v6. The probable mechanism of action involves targeting
COX-2/5-LOX proteins and abrogating their interaction with
CD44v6 receptors thereby interfering with HA/CD44v6 signaling
through autocrine/paracrine mechanism which offers a novel alter-
native therapeutic strategy for rational design of anticancer agents
for colon cancer.
4. Material and methods
All reagents used for synthesis were of analytical grade and
were used without further purification. Solvents employed were
purified by standard procedures prior to use. The reaction progress
was monitored through thin-layer chromatography (TLC) on pre-
coated silica gel on aluminum plates (Merck), while the reaction
products were separated by column chromatography on silica gel
60 (Merck 60–120 mesh).
2.3.4. The growth inhibitory effects of BQNH at different test
concentrations by MTT assay
Inordertofurtherconfirmthe cytotoxicactivityof themostpotent
compound BQNH, we have carried out another set of growth inhibi-
tion measurement using MTT assay (Fig. 4). Since the tumor cells,
such as HT29, HCA7 and Apc-Has2 express COX-2, 5-LOX, and
CD44v619 (and Fig. 2), the compounds active in this assay are indi-
cated to be selective toward the tumor cells, and their cell growth-
inhibiting activity isattributedtoCD44v6–COX–LOX axis. In addition,
the proliferation assay results of Figure 3 go parallel with MTT assay
for the HT29, HCA7 and Apc-Has2 tumor cells. Importantly, this assay
also showed that the new inhibitors have no effect on normal intesti-
nal epithelial cells IEC6, which do not exhibit COX-2,61a LOX and
CD44v6 expression. Since pre-neoplastic Apc10.1 cells59 express both
COX and LOX, the compound BQNH has reasonable cytotoxic activity.
These results further confirm the potential of CD44v6–COX–LOX as
targets in colon cancer therapy and prevention using our newly syn-
thesized COX–LOX dual inhibitor compounds.
4.1. General procedure for preparation of compounds (2–4)
The compounds were synthesized as shown in Scheme 1 by
condensation of equimolar amount of 2,6-di-tert-1,4-benzoqui-
none (BQ) and respective isonicotyl (BQIH), nicotyl (BQNH) and
benzoyl (BQBH) hydrazide in ethanol in presence of catalytic
amount of concd HCl with continuous stirring at temperature of
60–65 °C. The reaction mixture was poured on crushed ice and
then purified by silica gel column chromatography using chloro-
form/methanol as solvent system.
4.1.1. BQ: 2,6-di-tert-butylcyclohexa-2,5-diene-1,4-dione
1H NMR (400 MHz, DMSO-d6): 1.286 (s, 18H), 6.512 (s, 2H). 13
C
NMR (100 MHz, DMSO-d6): 26.36, 35.55, 130.13, 157.84, 187.16,
and 189.03. IR (KBr, cmꢀ1): 1654 (
–C@O, adjacent to di-tert-butyl
groups), 1654 ( –C@O, free)
m
m
3. Conclusions
4.1.2. BQBH: N0-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-
dienylidene) benzohydrazide
Thus, our present studies indicate that dual COX–LOX inhibitors
are potent inhibitors of colon cancer growth and their consequent
effect on drug resistance and CRC growth are regulated by HA, and
Yield: 73%; mp: 160 °C; 1H NMR (400 MHz, DMSO-d6): 1.281–
1.329 (s, 18H), 7.108 (s, 1H), 7.505 (t, 2H, J = 7.64 Hz), 7.584 (t,

S. Misra et al. / Bioorg. Med. Chem. 21 (2013) 2551–2559
2557
1H, J = 7.36 Hz), 7.66 (d, 1H, J = 11.3 Hz), 7.856 (d, 2H, J = 7.6 Hz),
12.027 (s, 1H, –NH), 13C NMR (125.75 MHz, DMSO-d6): 29.614,
29.892, 35.151, 36.369, 119.523, 128.818, 129.273, 132.513,
133.849, 134.101, 149.196, 151.407, 159.958, 187.048. IR (KBr,
resulting docking conformations were clustered into families of
similar conformation, with root mean square deviation (RMSD)
clustering tolerance as 1.0 Å. The lowest docking energy conforma-
tions were included as a rule in the largest cluster. Flexible torsion
in the ligands was assigned with Autotors, an auxiliary module for
AutoDockTools. Each ligand was docked individually with 5-LOX to
obtain the best binding conformation.
cmꢀ1): 1651(
m–C@O), 1535 (mꢀC@N); UV–Vis: kmax(nm, DMSO):
369; Anal. Calcd for C21H26N2O2: C, 74.55; H, 7.69; N, 8.28. Found:
C, 73.87; H, 7.09; N, 7.73. ESIMS (m/z): Calcd 338.44, found 339
(M++H) in accordance with MF C21H26N2O2.
4.3. In vitro cyclooxygenase (COX) inhibition assay
4.1.3. BQNH: N0-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-
dienylidene) nicotinohydrazide
The ability of the synthesized compounds to inhibit the conver-
sion of arachidonic acid (AA) to prostaglandin H2 by ram seminal
vesicle COX-1 and sheep placental COX-2 was determined using
a COX-2/COX-2 inhibitor screening assay kit (No. 560101; Cayman
Chemical, Ann Arbor, MI). Cyclooxygenase catalyzes the first step
Yield: 65%; mp: 197 °C; 1H NMR (400 MHz, DMSO-d6): 1.278–
1.334 (s, 18H), 7.141 (s, 1H), 7.489 (t, 1H, J = 6.8 Hz), 7.618 (d,
1H, J = 15.84 Hz), 8.24 (s, 1H), 8.7709 (s, 1H), 9.107 (s, 1H),
12.178 (s, 1H, –NH). 13C NMR (125.75 MHz, DMSO-d6): 29.592,
29.890, 35.160, 36.416, 119.478, 123.904, 129.802, 133.906,
137.251, 149.452, 149.977, 151.728, 152.865, 187.024. IR (KBr,
in the biosynthesis of AA to PGH2. PGF2 produced from PGH2 by
a
reduction with stannous chloride was measured by enzyme immu-
noassay. This assay is based on the competition between PGs and a
PG-acetyl cholinesterase conjugate (PG tracer) for limited amount
of PG antiserum. The amount of PG tracer that is able to bind to the
PG antiserum is inversely proportional to the concentration of PGs
in the wells since the concentration of PG tracer is held constant
while that of PGs varies. This antibody–PG complex binds to a
mouse anti-rabbit monoclonal antibody that has been previously
attached to the well. The plate is washed to remove any unbound
reagents and the Ellman’s Reagent is added to the well, which con-
tains the substrate to acetyl cholinesterase the product of this
enzymatic reaction produces a distinct yellow color that absorbs
at 405 nm. The intensity of this color is determined spectrophoto-
metrically and is proportional to the amount of the well that in
turn is inversely proportional to the amount of PGs present in
the well during the incubation. Percent inhibition was calculated
by comparison of compound treated to various control incubations.
The concentration of the test compound causing 50% inhibition
cmꢀ1): 1666(
m C@O), 1539 (mꢀC@N); UV–Vis: kmax(nm, DMSO):
366; Anal. Calcd for C20H25N3O2: C, 70.79; H, 7.37; N, 12.38. Found:
C, 70.07; H, 7.37; N, 12.09. ESIMS (m/z): Calcd 339.43, found 340
(M++H) in accordance with MF C20H25N3O2.
4.1.4. BQIH: N0-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-
dienylidene) isonicotinohydrazide
Yield: 77%; mp: 170 °C; 1H NMR (400 MHz, DMSO-d6): 1.271–
1.320 (s, 18H), 7.097 (d, 1H, J = 13 Hz), 7.658–7.943 (m, 3H),
8.777 (s, 2H), 12.374 (s, 1H, –NH). 13C NMR (125.75 MHz, DMSO-
d6): 29.587, 29.879, 35.178, 36.419, 119.486, 122.968, 133.887,
149.612, 150.519, 151.820, 187.032. IR (KBr, cmꢀ1): 1666(
mC@O),
1541 (mꢀC@N); UV–Vis: kmax(nm, DMSO): 363; Anal. Calcd for
C
20H25N3O2: C, 70.79; H, 7.37; N, 12.38. Found: C, 68.87; H, 7.09;
N, 11.973. ESIMS (m/z): Calcd 339.43, found 339 (M+) in accor-
dance with MF C20H25N3O2.
4.2. Molecular docking study
(IC50, lM) was calculated from the concentration–inhibition re-
sponse curve.65,66
All calculations were performed using AutoDock-Vina soft-
ware.62 The crystal structure of COX-2 protein was obtained from
PDB ID (6COX).63 The active site of the enzyme was defined to
include residues of the COX-2 within the grid size of
50 Å ꢁ 50 Å ꢁ 50 Å to any of the inhibitory atoms. The AutoDock-
Vina program which is an automated docking program was used
to dock all three 2,6 di-tert-butyl-p-benzoquinone hydrazone ana-
logs as well as Darbufelone molecule in the active site of COX-2 en-
zyme. For each compound the most stable docking model was
selected based upon conformation of best scored predicted by Auto
Dock scoring function. The compounds were energy minimized
with MMFF94 force field. From the histogram relevant parameters
such as binding energy, total number of hydrogen bond formed
and hydrogen bonding pattern were determined.
4.4. In vitro lipoxygenase (5-LOX) inhibition assay
The ability of the test compounds to inhibit potato 5-LOX (cat-
alog number 60401, Cayman Chemical, Ann Arbor, MI) was deter-
mined using an enzyme immunoassay (EIA) kit (catalog number
760700, Cayman Chemical, Ann Arbor, MI) according to the manu-
facturer’s instructions. The Cayman Chemical Lipoxygenase inhib-
itor screening assay detects and measures the hydroperoxides
produced in the lip oxygenation reaction using a purified lipoxy-
genases. Stock solutions of test compounds were dissolved in a
minimum volume of DMSO and were diluted using the supplied
buffer solution. The lipoxygenases reaction was initiated by the
addition of 10
lL arachidonic acid. After maintaining the 96-well
The 3D crystal structure of stable 5-lipoxygenase was obtained
from Protein Data Bank (PDB ID: 3O8Y).64 The ligands were energy
minimized and partial charges were added using PRODRG algo-
rithms. Each ligand was then docked individually into the active
site of 5-LOX using AutoDock-Vina. The AutoDockTools graphical
user interface was used to add polar hydrogens and partial charges,
using Kollman United charges, to 5-LOX. Atomic solvation param-
eters and fragment volumes were assigned using the ADDSOL sub-
routine. The grid map was calculated using the auxiliary program
Autogrid3. Grid maps of 50 Å ꢁ 50 Å ꢁ 50 Å points centered on
the active site of the ligand were calculated for each atom types
found on the adducts. Lamarckian Genetic Algorithm (LGA) was se-
lected for ligand conformational search. The Genetic Algorithm
(GA) population size was set to 150, the maximum number of GA
energy evaluations as 2,500,000, GA mutation rate as 0.02, GA
crossover rate as 0.8 and GA docking runs was set as 100. The
plate on a shaker for 5 min, 100
l
L of chromogen was added and
the plate was retained on a shaker for 5 min. The lipoxygenases
activity was determined after measuring absorbance at a wave-
length of 500 nm. Percent inhibition was calculated by the compar-
ison of compound-treated to the control incubations. The
concentration of the test compound causing 50% inhibition (IC50
,
l
M) was calculated from the concentration–inhibition response
curve (duplicate determinations).67
4.5. Cell proliferation assay
Cell proliferation was measured by CellTiter 96Ò AQueous Assay
(Promega). The reagent consists of solutions of a novel tetrazolium
compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) and
an electron coupling reagent (phenazine methosulfate; PMS).

2558
S. Misra et al. / Bioorg. Med. Chem. 21 (2013) 2551–2559
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Conflicts of interest
37. Misra, S.; Hascall, V.; Karamanos, N.; Markwald, R.; Ghatak, S. De Gruyter,
2012.
38. Misra, S.; Hascall, V. C.; Karamanos, N. K.; Markwald, R. R.; Ghatak, S.
DeGruyter: Berlin, Germany, 2012.
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Author declare no conflicts of interest
Acknowledgments
We thank Dr. Vincent C. Hascall (Cleveland Clinic, Cleveland,
Ohio), and Dr. Roger R. Markwald (Medical University of South Car-
olina, Charleston, SC) for their support and academic discussions in
this work. We also thank Dr. Carla De Giovanni (University of Bolo-
gna, Bologna, Italy) for Apc 10.1 cells. This work was supported, in
whole from the grant ‘‘1R03CA167722-01A1’’ (to S.M. and S.G.).
This work was supported, in whole or in part, by National Institutes
of Health Grants P20RR021949 (to S.G.), P20RR016434 (to S.M.,
S.G., and R.R.M.), P20RR16461-05 (to S.G., and R.R.M), HL033756-
24 and 1 P30AR050953 (to V.C.H.). This work was also supported
by Mitral-07 CVD 04 (to R.R.M.), Medical University of South Caro-
lina University Research Project 2204000-24330 (to S.M.) and
2204000-24329 (to S.G.). P.D. acknowledges CSIR, New Delhi, India
for providing Senior Research Fellowship. S.P. acknowledges Mr. P.
A. Inamdar and Dr. E. M. Khan for lab facilities for this project.
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