Identification of an anti-inflammatory derivative with anti-cancer potential: The impact of each of its structural components on inflammatory responses in macrophages and bladder cancer cells

Article abstract of DOI:10.1016/j.ejmech.2015.04.026

Inflammation plays a crucial role in many types of cancer and is known to be involved in their initiation and promotion. As such, it is presently recognized as an important risk factor for several types of cancers such as bladder, prostate and breast cancers. The discovery of novel anti-inflammatory compounds can have a huge implication not only for the treatment of cancer but also as preventive and protective treatment modalities. We have recently identified a new compound (1) that presents interesting anti-inflammatory activity. In order to better understand its biological action, we have divided the molecule in its basic components and verified their respective contribution towards the anti-inflammatory response of the whole molecule. We have discovered that only the combination of the maleimide function together with the tert-butyloxycarbonylhydrazinamide function lead to important anti-inflammatory properties. The main derivative 1 can decrease the activating effects of INFγ or IL6 on human (hMωs) macrophages by 38% or by 64% at a concentration of 10 μM as indicated by a decrease of STAT1 or STAT3 activation. The expression of pro-inflammatory markers CD40 and MHCII in INFγ stimulated hMωs were reduced by 87% and 49%, respectively with a 3 h pretreatment of 1 at 10 μM. The cell motility assay revealed that 1 at 10 μM can reduce relative cell motility induced by IL6 by 92% in comparison with the untreated control hMω monolayers. Compound 1 reduced by 91% the inflammatory response induced by the cytokines (INFγ + TNFα) in the macrophage-like J774A.1 cells at a concentration of 25 μM, as measured by the detection of NO production with the Griess reagent. Furthermore, upon removal of the tert-butyloxycarbonyl protective group the unprotected derivative as a hydrochloride salt (1A) retains interesting anti-inflammatory activity and was found to be less toxic than the parent compound (1).

Full text of DOI:10.1016/j.ejmech.2015.04.026

European Journal of Medicinal Chemistry 96 (2015) 259e268
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of an anti-inflammatory derivative with anti-cancer
potential: The impact of each of its structural components on
inflammatory responses in macrophages and bladder cancer cells
,
,
Jovane Hamelin-Morrissette a 1, Suzie Cloutier b 1, Julie Girouard a, Denise Belgorosky c,
c
d
a,
*
, *
ꢀ b
ꢀ
ꢀ
Ana María Eijan , Jean Legault , Carlos Reyes-Moreno , Gervais Berube
a
ꢀ
ꢀ
Departement de Biologie Medicale et, Canada
b
c
ꢀ
ꢀ
ꢀ
ꢁ
ꢁ
ꢁ
ꢀ
Departement de Chimie, Biochimie et Physique, Universite du Quebec a Trois-Rivieres, C.P. 500, Trois-Rivieres, Quebec G9A 5H7, Canada
Instituto de Oncologia “Angel H. Roffo” Area Investigacion, Ciudad de Buenos Aires, C1417DTB, Argentina
Departement des Sciences Fondamentales, Universite du Quebec a Chicoutimi, 555 boulevard de l'Universite, Chicoutimi, Quebec G7H 2B1, Canada
ꢀ
ꢀ
ꢀ
d
ꢀ
ꢀ
ꢀ
ꢁ
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Inflammation plays a crucial role in many types of cancer and is known to be involved in their initiation
and promotion. As such, it is presently recognized as an important risk factor for several types of cancers
such as bladder, prostate and breast cancers. The discovery of novel anti-inflammatory compounds can
have a huge implication not only for the treatment of cancer but also as preventive and protective
treatment modalities. We have recently identified a new compound (1) that presents interesting anti-
inflammatory activity. In order to better understand its biological action, we have divided the mole-
cule in its basic components and verified their respective contribution towards the anti-inflammatory
response of the whole molecule. We have discovered that only the combination of the maleimide
function together with the tert-butyloxycarbonylhydrazinamide function lead to important anti-
Received 29 October 2014
Received in revised form
9 April 2015
Accepted 10 April 2015
Available online 13 April 2015
Keywords:
Bladder cancer
Inflammation
Interferon gamma
Interleukine 6
Nitric oxide
inflammatory properties. The main derivative 1 can decrease the activating effects of INF
human (hM s) macrophages by 38% or by 64% at a concentration of 10 M as indicated by a decrease of
STAT1 or STAT3 activation. The expression of pro-inflammatory markers CD40 and MHCII in INF
stimulated hM s were reduced by 87% and 49%, respectively with a 3 h pretreatment of 1 at 10 M. The
cell motility assay revealed that 1 at 10 M can reduce relative cell motility induced by IL6 by 92% in
comparison with the untreated control hM monolayers. Compound 1 reduced by 91% the inflammatory
response induced by the cytokines (INF ) in the macrophage-like J774A.1 cells at a concentration
þ TNF
of 25 M, as measured by the detection of NO production with the Griess reagent. Furthermore, upon
g or IL6 on
f
m
g
STAT pathway
f
m
m
f
g
a
m
removal of the tert-butyloxycarbonyl protective group the unprotected derivative as a hydrochloride salt
(1A) retains interesting anti-inflammatory activity and was found to be less toxic than the parent
compound (1).
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
urogenital tract. In fact, it is the seventh most frequent cancer in the
world and the fourth in developed countries [1]. In Canada, BCa
Bladder cancer (BCa) is the second most common tumor of
ranked fifth among all cancer cases diagnosed in the Canadian
population with about 8000 new cases diagnosed during 2014 [2].
Most of detectable BCa tumors are initially non-muscle-invasive at
diagnosis and are generally curable by means of chirurgical resec-
tion. For BCa patients with non-muscle-invasive tumors, intra-
vesical immunotherapy with the immunomodulator Bacillus
Calmette-Guerin (BCG) or chemotherapeutic agents is recom-
mended for reducing the risk of tumor recurrence [3]. However, the
current treatments are still far from satisfactory due to low
Abbreviations list: BCa, bladder cancer; macrophage, M
* Corresponding authors.
f; NO, nitric oxide.
E-mail addresses: jovane.hamelin@outlook.com (J. Hamelin-Morrissette), Suzie.
Cloutier@uqtr.ca (S. Cloutier), Julie.Girouard@uqtr.ca (J. Girouard), d_belgo@
ꢀ
hotmail.com (D. Belgorosky), anamariaeijan@fibertel.com.ar (A.M. Eijan), jean.
legault@uqac.ca (J. Legault), Carlos.Reyes-Moreno@uqtr.ca (C. Reyes-Moreno),
ꢀ
ꢀ
Gervais.Berube@uqtr.ca (G. Berube).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.04.026
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

260
J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
response rate and/or severe side effects [4]. In fact, it is estimated
that about 27e30% of new cases diagnosed will die from that dis-
ease due to recurrent tumors exhibiting a lethal phenotype char-
acterized by high histological grade and muscle invasion [1,2].
At present, as the mechanisms of acquisition of highly malignant
phenotype of BCa tumors are not yet well understood, very few
therapeutic alternatives have been proposed and tested in the
clinic. Nevertheless, compelling data actually suggest that chronic
inflammation acts as an important mediator of human carcino-
genesis and cancer disease progression [5e10]. In the case of BCa,
evolution into different stages of cancer development is strongly
associated with episodes of chronic inflammation caused by to-
bacco smoke, urinary tract infections or chronic cystitis [11e13]. For
instance, higher levels of pro-inflammatory cytokines and tumor-
promising approach to decrease cancer development [6,21,23,27,28].
Recently, it was reported that veratric acid, a natural benzoic
acid derivative, possesses anti-oxidant, anti-inflammation and
blood pressure-lowering effects [29]. Based on the latter study, it
was decided to screen a series of benzoic acid analogs made in our
laboratory for their potential anti-inflammatory activity. It was
discovered that a maleimide-hydrazinecarboxylic acid tert-butyl
ester derivative possesses good anti-inflammatory properties. This
compound was an important intermediate for the construct of
heterobifunctional cross-linking reagents allowing the synthesis of
doxorubicin-monoclonal antibody immunoconjugates [30,31].
Hence, in order to better understand its unsuspected biological
anti-inflammatory action, the molecule was divided into its various
components that were individually tested on NO production in
bladder cancer cells (MB49-I) along with the product of interest.
This study aims at establishing the possible repurposing to this
molecule as an anti-inflammatory. This communication reports the
synthesis and biological properties of this new anti-inflammatory
compound (1), its unprotected analog (1A) and that of its various
structural components (see Fig. 1).
infiltrating macrophages (Mfs) were found to be associated with
more aggressive tumors, low responsiveness to intravesical BCG
therapy, and poor prognosis [5e7,14e18]. In our recent studies,
in vitro models of functional interactions between human cancer
cells and two different subtypes of human M
-1 and anti-inflammatory M -2, were developed in an attempt
to investigate the role of polarized M s in human cancer cell
behavior changes [19,20]. Our findings that both types of M s can
fs, pro-inflammatory
Mf
f
f
f
2. Results and discussion
regulate tumor cell survival and invasion further support the idea
that, via alterations in the expression of inflammatory mediators
and inflammatory cell function, the inflammatory microenviron-
ment may contribute to the development of a highly malignant
phenotype of tumors in vivo [19,20].
2.1. Chemistry
Using para-amino benzoic acid (2) as the starting material de-
rivative 1 was made in a three-step reaction sequence (Scheme 1)
[30]. Para-amino benzoic acid (2 or PABA) was first reacted with
maleic anhydride (MA) in dry acetone to give the diacid (3) with 90%
yield. Cyclisation to form the maleimide was accomplished with
acetic anhydride and sodium acetate to give compound 4 with 89%
yield after hydrolysis of the mixed anhydride intermediate with
water. Finally, activation of acid 4 with iso-butyl chloroformate in
the presence of pyridine was done followed by treatment with tert-
butyl carbazate gave the desired anti-inflammatory derivative 1
with 54% yield. Deprotection of 1 with hydrochloric acid in ether
gave the hydrochloride salt 1A with 46% yield after recrystallization.
The left and right hands of derivative 1 were also synthesized in
order to assess the respective contribution of the remaining func-
tional group towards the observed anti-inflammatory activity. The
left hand of 1 was prepared in a two-step reaction sequence from
aniline (5). The reaction of aniline (5) with MA followed by cycli-
sation of the amido-acid intermediate gave the desired maleimide
7 in about 38% overall yield. The right hand of 1 was also prepared
in a single step form benzoic acid upon activation with iso-butyl
chloroformate followed by treatment with tert-butyl carbazate to
yield compound 9 in 48% yield. Of note, these reactions were not
optimized as derivatives 7 and 9 were only needed in small
quantities to assess their anti-inflammatory properties in compar-
ison to 1 and 1A. The compounds were characterized by their
respective infrared (IR), nuclear magnetic resonance spectroscopy
(proton and carbon NMR) and by high resolution mass analysis.
Among the major regulator of systemic and local inflammation,
the cytokines tumor necrosis factor alpha (TNF
(IL6) have been shown to promote BCa development [5]. In theory,
TNF and IL6 exert pro-tumoral effects on tumor cells through
activation of the transcription factors nuclear factor kappa B (NF B)
and signal transducer and activator of transcription 3 (STAT3),
respectively [21]. The signaling pathways TNF /NF B and IL6/STAT3
a) and interleukin 6
a
k
a
k
were identified as the main factors that transform the intra-
tumoral leukocytes into pro-tumor cells [5]. Such functional
phenotype control is critical to prevent tumor immune surveillance
and instead, to turn away the responses of adaptive and innate
immune cells toward the promotion of highly invasive, lethal forms
of BCa.
Nitric oxide (NO), another important component of the in-
flammatory response, is often deregulated and associated with
numerous types of malignancies, including BCa [22e26]. It is
known that NO can have dichotomous effects on tumor growth,
depending on its concentration, time of exposure and tumor
microenvironment. These dual effects of NO on cancer arise from its
ability to regulate different events such as induction of DNA dam-
age, suppression of DNA repair enzymes, posttranslational modi-
fications of proteins, enhancement of cell proliferation, inhibition of
apoptosis and antitumor immunity [23e25]. It is known that one of
the isoforms of the enzyme that produces NO (inducible nitric
oxide synthase (iNOS)) is not expressed in normal bladder, but is
detected in approximately 50% of bladder tumors, being a poor
prognosis factor associated with tumor invasion in patients with
BCa [26]. Treatment with an inhibitor of NO production in a murine
model, inhibited properties in vitro and in vivo, related to tumor
growth, progression, and viability [22]. Among anticancer strate-
gies, the modulation of NO production for therapeutic benefit has
gained interest over the past decade [23,27].
2.2. Evaluation of anti-inflammatory properties in activated
macrophages
This set of experiments were planned to investigate whether the
main two derivatives 1 and 1A have the ability to modulate the
activating effects of potent pro-inflammatory cytokines, namely
Considering the critical functions of inflammatory mediators in
BCa growth, dissemination and resistance to cell death, they repre-
sent potential drug targets to improve the efficacy of immuno-
therapy and chemotherapy agents. The use of chemopreventive
agents against inflammatory mediators or key transcription factors
involved in the inflammatory proteins expression might thus be a
INF
(mM
g
, TNF
a
, and IL6, on the behavior of human (hM
fs) and murine
f
s) macrophages. IFN
g, through the activation of the tran-
scription factor signal transducer and activator of transcription 1
(STAT1), is known to induce the expression of several pro-
inflammatory genes in M
fs, such as the cytokines TNFa and IL6,
and the cell surface receptors cluster of differentiation 40 (CD40)

J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
261
O
O
O
O
N
N
O
O
NHNH2 * HCl
NHNH
O
O
1
1A
Fig. 1. Molecular structure of N'-[4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-benzoyl]-hydrazinecarboxylic acid tert-butyl ester (1) and of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic
acid hydrazide hydrochloride (or 4-maleimidbenzoic acid hydrazide hydrochloride) (1A).
and the major histocompatibility complex II (MHC-II) [32]. The
cytokine IL6 is a key regulator of systemic and local inflammation
[33,34]. Functionally, IL6 mediates its effects through the binding of
IL6 receptor subunits IL6R and gp130 which leads to the activation
of STAT3 [35]. Activation of the IL6/STAT3 signaling pathway is
critical to M migration and differentiation [36]. On the other hand,
TNF and IFN are considered as the most important cytokines
f
a
g
involved in NO production by mouse Mfs [37].
O
O
H2N
H2N
OH
OH
8
2 (PABA)
5
a
a
O
O
O
HN
HN
CO2H
CO2H
OH
6 (86%)
3 (90%)
b
b
O
O
N
OH
O
4 (89%)
c
c
O
O
O
O
O
O
N
O
O
N
NHNH
NHNH
O
O
9 (48%)
7 (44%)
1 (54%)
Right hand of 1
Left hand of 1
d
O
O
N
NHNH2 * HCl
O
1A (46%)
Scheme 1. Reagents and conditions: a) Maleic anhydride (MA), dry acetone, methanol, 22 ꢀC, 1 h; b) 1) Ac2O, AcONa, 50 ꢀC, 2 h; 2) H2O, 70 ꢀC, 2 h; c) 1) iso-butyl chloroformate,
Et3N, CH2Cl2, 0 ꢀC, 1 h and 22 ꢀC, 1 h; 2) tert-butyl carbazate, CH2Cl2, 22 ꢀC, 12 h; d) HCl, dioxane, 22 ꢀC, 5 h.

262
J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
Fig. 2a shows representative activation status of STAT1 by IFN
g
However, the stimulatory effect of IL6 on cell motility was signifi-
cantly blocked when hM s were pretreated with derivatives 1A
and STAT3 by IL6 in hM
f
s pretreated for 30 min with derivatives 1A
f
and 1. hM
f
s express low basal levels of pSTAT1 and pSTAT3
and 1; the relative number of motile cells being decreased to 53.7%
of control with 1A, and to 13.5% of control with 1 (Fig. 4b).
Finally, anti-inflammatory activity was assessed by measuring
(t ¼ 0 min) when pretreated with derivatives 1A or 1 (Fig. 2a). Very
little or no variation in total STAT1 and STAT3 expression was
observed, irrespective of the treatments. As expected, IFN
vates STAT1, the levels of pSTAT1 being increased with IFN
t ¼ 30 min (Fig. 2b). These increased effects in pSTAT1 were effi-
ciently reduced when hM s were pretreated with 1A (from 14.8
folds to 12.4 folds at 10 M, and 5.1 folds at 50 M) and even more
with 1 (from 9.9 folds to 6.1 folds at 10 M, and 2.8 folds at 50 M).
Similar to pSTAT1 by IFN , the induction of pSTAT3 by IL6 was
significantly reduced when hM s were pretreated with 1A (from
23.1 folds to 19.7 folds at 10 M, and 14.2 folds at 50 M) and even
more with 1 (from 32.9 folds to 11.9 folds at 10 M, and 4.9 folds at
50 M).
To further investigate the influence of derivatives 1 and 1A on
the biological responses induced by inflammatory mediators in
hM s, two set of experiments were designed to evaluate the
expression level of the pro-inflammatory markers CD40 and MHC-
II in IFN -stimulated hM s, by flow cytometry, and cell motility in
IL6-stimulated hM s, by the scratch wound healing assay. Consis-
tently with the pro-inflammatory role of IFN , M CD40 and MHC-
II expressions were enhanced by 16.7 folds and 15.3 folds, respec-
tively (Fig. 3). However, the rise in CD40 expression in IFN -acti-
vated M s was blocked when they were pretreated with 1A (9.5
g
acti-
NO production in mM
f
s
stimulated by two potent pro-
g
at
inflammatory signals, INF
g
and TNF , and using the Griess reac-
a
tion method according to the published procedure [22]. In brief, the
NO produced by the cells is converted into nitrite ion (NOꢁ2 ). The
latter is reacted with the Griess reagent to form an azo dye that can
be spectrophotometrically quantitated based on its absorbance at
f
m
m
m
m
g
548 nm. As shown in Fig. 5, in response to combined INF
activation, the production of NO were increased by 8.7 folds (from
M in unstimulated cells up to 61 M in stimulated cells).
However, when mM s were activated after pretreatment with
derivatives 1A and 1 both at a concentration of 10 M, NO pro-
duction was only induced by 6.6 folds (45.9 M) and 4.7 folds
(32.8 M), respectively. Also, in the presence of derivatives 1A and
1 at a concentration of 50 M, the induction of NO production was
respectively reduced to 2.7 folds (18.6 M) and 0.8 folds (5.5 M).
g
þ TNF
a
f
m
m
7
3
m
9 m
m
f
m
m
m
m
f
m
m
m
g
f
These results represent a reduction of about 69% and 91%, respec-
tively of the inflammatory response induced by the cytokines
(Fig. 5).
Taken together, these results clearly demonstrated that the main
derivatives 1 and 1A presents significant anti-inflammatory activ-
ities, and that the anti-inflammatory properties of 1 are obviously
greater than that of 1A.
f
g
f
g
f
folds) and even more with 1 (2.2 folds). For MHC-II, the expression
inductions were reduced to 11.8 folds with 1A and 7.8 folds with 1.
Scratch assays were performed to further demonstrate the deacti-
2.3. Evaluation of anti-proliferative activities in tumor cells
vating effects of derivatives 1 and 1A on IL6-activated hM
(Fig. 4a). We found that stimulation with exogenous IL6 for 48 h
significantly enhanced hM motility, the ratio of motile cells being
increased up to 177% compared with control cells (Fig. 4b).
fs
In this set of experiments, the anti-proliferative activity of the
compounds 1 and 1A in the murine BCa cell line MB49-I was
evaluated in vitro using the MTT cell viability/proliferation assay as
f
Fig. 2. Representative images (a) and graphical analysis (b) showing the immunodetection of phosphorylated STAT1 and STAT3 in hM
fs pretreated for 30 min with vehicle (DMSO)
or compounds 1 and 1A, and then washed and recovered immediately (t ¼ 0) or after 30 min of activation with either 50 U/mL IFN
g
or 25 ng/mL IL6. The ratio of phosphorylated/no
phosphorylated proteins was calculated from densitometric analysis of each sample to evaluate the relative activation of pSTAT1 or pSTAT3. *p < 0.05 and **p < 0.01 denote
significant differences between treatments.

J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
263
Fig. 3. Representative images (a) and graphical analysis (b) showing flow cytometry analysis to determine the expression level of MHC-II and CD40 surface antigens in resting and
IFNg
-activated hM
fs untreated and pretreated with compounds 1 (10
m
M) and 1A (25
m
M). *p < 0.05 and **p < 0.01 denote significant differences between treatments.
previously described [19,20,38,39]. The MTT assay was performed
in cells stimulated with INF and TNF , over an incubation period of
0.55 AU and 0.35 AU, respectively in the absence and in the pres-
g
a
ence of INF
cells, derivative 1 is not toxic at a concentration of 10
induces a significant reduction in cell proliferation of about 19% at
25 M, 45% at 37.5 M and 50% at 50 M (Fig. 3A). In contrast to 1,
derivative 1A remains not toxic up to a concentration of 37.5
with INF stimulation (Fig. 6).
þ TNF
g
þ TNF
a
stimulation. In INF
g
- and TNF
a
-stimulated
24 h, after pretreatment with vehicle (DMSO) and the compounds 1
and 1A. Relative cell viability data are presented as values of
absorbance units (AU) measured at 580 nm. As shown in Fig. 6,
derivative 1 was significantly more toxic than its analog 1A. Indeed,
the results demonstrate that pro-inflammatory stimulation inhibits
MB49-I cell proliferation, the value of absorbance at t ¼ 24 h being
mM or less but
m
m
m
mM
g
a
2.4. Evaluation of anti-inflammatory properties of the structural
components of compound 1
The next objective of this study was to evaluate individually the
anti-inflammatory properties of the various structural components
(MA, 2, 3, 4, 6, 7 and 9, see Scheme 1) of compound 1. In this set
experiments, the various derivatives were tested for their ability to
modulate NO production in BCa cells (MB49-I) stimulated by INF
and TNF relative to control cells (with cytokines INF and TNF
alone). The results found in Fig. 7 show that combined INF
þ TNF
induces an 11-fold increase in the production of NO (from 6
in unstimulated tumor cells up to 65 M in stimulated tumor
g
a
a
a
g
g
2 mM
7
m
Fig. 4. Representative images (a) and graphical analysis (b) showing scratch wound
healing assays performed in hM
f
monolayers cultured for 3 h with vehicle (DMSO) or
M), and then activated for 48 h with vehicle (PBS)
compounds 1 (10 M) and 1A (25
m
m
or 25 ng/mL IL6. The images of the scratch were acquired at t ¼ 0 h and t ¼ 48 h by
fluorescence microscopy. Five fields were taken randomly for each different treatment.
All observations were performed at 5 ꢂ magnification. Cell motility was expressed as
percent (%) of control of motile cells at t ¼ 48 h relative to motile cells at t ¼ 0 h
*p < 0.05 and **p < 0.01 denote significant differences between treatments.
Fig. 5. Graphical representation of NO production in the macrophage-like J774A.1 cells
following a pro-inflammatory stimulation by IFN and TNF after pretreatment with
vehicle (DMSO) and the derivatives 1 and 1A. *p < 0.05 and **p < 0.01 denote sig-
nificant differences between treatments.
g
a

264
J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
Fig. 6. Graphical representation of relative cell viability on the murine BCa cell line MB49-I following a pro-inflammatory stimulation by IFNg and TNFa after pretreatment with
vehicle (DMSO) and anti-inflammatory derivatives 1 and 1A at different concentrations. *p < 0.05 and **p < 0.01 denote significant differences between treatments.
cells). Of note, at a concentration of 50
production was respectively reduced to 2-folds (12
presence of 1, and 4-folds (23 M) in the presence of 1A. These
results represent a reduction of about 81% and 72%, respectively of
the inflammatory response induced by the cytokines. However, it is
worth mentioning that the observed reduction of 81% for derivative
1 might be produced by a combination of both cytotoxic and anti-
inflammatory activity (see Fig. 6). Also, in the presence of de-
m
M, the induction of NO
in order to better understand the anti-inflammatory activity of
derivatives 1 and 1A. It is now apparent that the anti-inflammatory
activity requires the presence of the maleimide function together
with a hydrazine amide function for optimal activity. It is also
proven that the various precursor intermediates (3, 4, 6), reagents
(2, PABA)) and maleic anhydride (MA) as well as the left hand (7)
and right hand (9) of the main anti-inflammatory compound 1
were inactive. Significant reductions of STAT1 or STAT3 activation
by several pro-inflammatory cytokines were observed with com-
pounds 1 and 1A. Also, significant decreased of CD40 and MHC-II
expressions were observed using 1 and 1A. Cell motility assays
also revealed that 1 and 1A can induce a drastic reduction of
mM) in the
m
rivatives 1 and 1A at a concentration of 10
m
M, NO production in-
M),
duction was reduced to 7-folds (39 M) and 9-folds (51
m
m
respectively. Interestingly, the reagents MA and 2 (PABA) and the
synthetic intermediates 3, 4 and 6 do not present any anti-
inflammatory properties (Fig. 7). Furthermore, derivative
7
motility as illustrated by a 92% reduction in cell motility at 10 mM
bearing only the maleimide function (left hand of 1) and derivative
9 bearing only the tert-butyloxycarbonyl function (right hand of 1)
did not show any anti-inflammatory activity (Fig. 7). Hence, the
combination of the maleimide function together with the hydra-
zine amide (either BOC protected (1) or as a hydrochloride salt (1A))
are necessary for anti-inflammatory activity.
for derivative 1. Relative cell viability revealed that derivative 1A
was less toxic than derivative 1. Taken together, these results sug-
gest that the development of such type of anti-inflammatory de-
rivative must include the basic carbon skeleton of 1A as it was
proven less toxic that its precursor derivative 1. Hence, this simple
compound might be of interest as a non-toxic anti-inflammatory
derivative to investigate the role of inflammation in BCa and other
cancers in the future. The results confirm the anti-inflammatory
properties of 1, its analog 1A and provide an alternative use for 1
that was initially prepared as a heterobifunctional cross-linking
3. Conclusion
In this study, we prepared and characterized several compounds
Fig. 7. Graphical representation of NO production by the murine BCa cell line MB49-I following a pro-inflammatory stimulation by IFNg and TNFa after pretreatment with vehicle
(DMSO) and derivatives 1 and 1A and various precursor intermediates (3, 4, 6), reagents (2, PABA) and maleic anhydride (MA) as well as the left hand (7) and right hand (9) of the
main anti-inflammatory compound 1. *p < 0.05 and **p < 0.01 denote significant differences between treatments.

J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
265
reagent. Further studies using an in vivo mouse model for BCa in-
4.1.4. Motility assays
The in vitro scratch wound healing assay was performed to study
the effects of compounds 1 (10 M) and 1A (25 M) in IL6-induced
hM cell migration, as described [46]. Briefly, THP1-derived hM
vasion and metastasis are currently underway to demonstrate the
anticancer effects of these new anti-inflammatory molecules. Once
completed, the results from these studies will be reported
elsewhere.
m
m
f
fs
(750 ꢂ 103 cells/mL) were seeded into 24-well tissue culture plate
to reach ~70e80% confluence as a monolayer. The cell monolayers
were scraped in a straight line in one direction to create a “scratch”
with a p200 pipet tip. To obtain the same field during the image
acquisition, another straight line was scratched perpendicular to
the first would line to create a cross in each well. Cell debris were
removed and the edges of the scratch were smoothed by washing
the cells once with 1 mL of Hank's buffer. Cell monolayers were
4. Experimental protocols
4.1. Biological evaluation
4.1.1. Cell culture
Biological assays were performed using the human monocytic
cell line THP1, the murine macrophage-like cell line J774A.1, and the
murine BCa cell line MB49-I. The cells were maintained in RPMI
medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and containing 1 mM sodium pyruvate, 10 mM 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and
pretreated for 3 h with vehicle (DMSO) or compounds 1 (10
mM)
and 1A (25 M), and then left untreated (control) or treated for 48 h
m
with 25 ng/mL IL6. Using the cross as reference points the plate was
placed under an inverted fluorescence microscope, and the images
of the scratch were acquired at t ¼ 0 h and t ¼ 48 h. The number of
motile cells was determined using Java-based image processing
program ImageJ (National Institutes of Health) and relative cell
motility was expressed as percent (%) of control of motile cells at
t ¼ 48 h relative to motile cells within the initial wound (at t ¼ 0 h).
50 mg/mL gentamycin (referred as 10% FBS RPMI-1640). The cells
were maintained at 37 ꢀC in a moisture-saturated atmosphere
containing 5% CO2.
THP1 cells and J774A.1 cells are the most widely used cell lines
to investigate the function and differentiation of monocytes and
4.1.5. Evaluation of NO production by the Griess reagent method
The MB49-I cells and the J774A.1 cells (25 ꢂ 103 cells/well) were
grown and pretreated, as indicated, with various anti-inflammatory
derivatives, precursors and mono-functional derivatives for a
period of 3 h. Afterwards the cells were washed twice with 10% FBS
RPMI-1640 and then activated to produce NO for a period of 24 h
M
f
s in response to various inflammatory mediators [40,41]. Un-
differentiated THP1 cells resemble primary monocytes/M s iso-
lated from healthy donors or donors with inflammatory diseases,
such as diabetes mellitus and atherosclerosis [42]. After treatment
with phorbol esters, THP1 cells differentiate into M
which mimic native monocyte-derived M s in several respects
[43]. As we previously described, green fluorescent protein-
f
f-like cells
f
with cytokines INFg and TNFa. NO production was measured using
the Griess reagent method as previously described [22]. This
method involves the detection of nitrite ions (NO-2) formed by the
spontaneous oxidation of NO under physiological conditions. Ac-
cording the manufacture procedure (Life Technologies; # G-7921),
equal volumes of sulfanilic acid and N-(1-naphthyl)ethylenedi-
amine are mixed together to form the Griess reagent. In the pres-
ence of NOꢁ2 , sulfanilic acid is converted to a diazonium salt, which
in turn is coupled to N-(1-naphthyl)ethylenediamine to produce a
pink coloration that is measured with a spectrophotometer (Biotek,
synergy HT) at 548 nm.
expressing (GFP)-THP1 cells were cultured for 18 h in 50 nM
phorbol 12-myristate 13-acetate to induce monocyte-to-M
f dif-
ferentiation [19,20,39]. The J774A.1 cell line was kindly provided by
ꢀ
ꢀ
ꢁ
ꢀ
Dr Tatiana Scorza (Universite du Quebec a Montreal, Canada). This
cell line is a macrophage-like cell model which produce large
amount of NO in response to IFNg, TNFa, bacterial infection and
bacterial products, such as lipopolysaccharide (LPS) [40]. The cell
line MB49-I is a highly invasive and tumorigenic BCa cell model
that was developed by successive in vivo passages of MB49 primary
tumors [44].
4.1.6. Evaluation of cell proliferation by the MTT assay
4.1.2. Cell signaling studies
To evaluate the anti-proliferative activity, cell viability/prolifer-
ation MTT assays were performed as previously described
[19,20,38,39,45]. Briefly, MB49-I cells (5 ꢂ 103 cells/well) were
THP1-derived hM
f
s (750 ꢂ 103 cells/mL) were pretreated for
30 min with vehicle (DMSO) or compounds 1 and 1A (both at 10 mM
and 50
mM), and then washed and recovered immediately
plated in 96-well plates in 100 mL 10% FBS RPMI-1640 and cultured
(t ¼ 0 min) or after 30 min of activation with 50 U/mL IFN
g
and
for 24 h at 37 ꢀC and 5% CO2. Cells were pretreated for a period of 3 h
25 ng/mL IL6. Cell lysates were prepared and analyzed by immu-
noblotting as described [19,20,39,45]. Briefly, protein samples were
resolved by SDS-PAGE under reducing conditions and transferred
onto a PVDF membrane. Blots were first probed with rabbit poly-
clonal antibodies against phospho-STAT1 (pSTAT1) and pSTAT3
(both at 1:2000) overnight at 4 ꢀC. Blots were then incubated with
HRP-conjugated goat anti-rabbit IgG Ab (1:3000) for 1 h at room
temperature. The same blots were stripped and then probed with
anti-STAT1 and anti-STAT3 Abs (both at 1:1000). In both cases,
probed molecules were visualized using an enhancement chem-
iluminescence detection kit (Thermo Fisher Scientific).
with vehicle (DMSO) or derivatives 1 and 1A at 0, 10, 25, 37.5, and
50
mM, and then incubated for 24 h in the absence or the presence
of INF
g
and TNF . At the end of the culture period, 10 L of 5 mg/mL
a
m
methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution was
added to each well. After a 3-h incubation period with MTT reagent,
100 mL of MTT solubilization buffer (10% SDS in 10 mM HCl) was
added and plates were placed overnight in the cell incubator before
absorbance measure. The optical density was read at 580 nm using
the Microplate Reader Manager (from Bio-Rad Laboratories).
4.2. Chemistry
4.1.3. Surface antigen expression analysis
Anhydrous reactions were performed under an inert atmo-
sphere of nitrogen. The starting material, reactant and solvents
were obtained commercially and were used as such or purified and
dried by standard means [47]. Organic solutions were dried over
magnesium sulfate (MgSO4), filtered and evaporated on a rotary
evaporator under reduced pressure. All reactions were monitored
by UV fluorescence. Commercial TLC plates were Sigma T 6145
To study membrane receptor expression, THP1-derived hMfs
(750 ꢂ 104 cells/mL) were pretreated for 3 h with DMSO or com-
pounds 1 (10 mM) and 1A (25 mM), and then left untreated (control)
or treated for 48 h with 50 U/mL IFNg. The expression level of MHC-
II and CD40 was evaluated by flow cytometry as described
[19,20,39].

266
J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
(polyester silica gel 60 Å, 0.25 mm). Flash column chromatography
was performed according to the method of Still et al. on Merck
grade 60 silica gel, 230e400 mesh [48]. All solvents used in chro-
matography were distilled.
125.8 (2); ESI þ HRMS: (M þ H)þ calculated for C11H8NO4
218.0448; found ¼ 218.0447.
¼
Step 3: Synthesis of N'-[4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-
benzoyl]-hydrazinecarboxylic acid tert-butyl ester (1)
The infrared spectra were taken on a Nicolet Impact 420 FT-IR
spectrophotometer. Mass spectral assays were obtained using a
MS model 6210, Agilent technology instrument. The high resolu-
tion mass spectra (HRMS) were obtained by TOF (time of flight)
using ESI (electrospray ionization) using the positive mode (ESIþ)
Derivative 1 was synthesized using a modified procedure re-
ported by Willner et al. [51] as it is also described by Lau et al. [31].
A cooled suspension (0 ꢀC) of molecule 4 (211 mg, 0.97 mmol) in
methylene chloride (4.5 mL) was treated with triethylamine
ꢀ
ꢀ
ꢁ
ꢀ
(Universite du Quebec a Montreal). Nuclear magnetic resonance
(NMR) spectra were recorded on a Varian 200 MHz NMR apparatus.
Samples were dissolved in deuterated acetone (acetone-d6) or
deuterated dimethylsulfoxide (DMSO-d6) for data acquisition using
(190 mL,1.36 mmol) and isobutyl chloroformate (175 mL,1.34 mmol).
The mixture was stirred for 1 h at 0 ꢀC and at room temperature
(22 ꢀC) for about 1 h. Afterwards, tert-butyl carbazate (128 mg,
0.97 mmol) dissolved in methylene chloride (0.8 mL) was added
dropwise to the mixture and stirred for an additional 12 h at 22 ꢀC.
The reaction mixture was diluted with ethyl acetate (55 mL) and
methylene chloride (20 mL) and washed twice with saturated
NaHCO3 (2 ꢂ 50 mL), twice with 0.1 N HCI (2 ꢂ 50 mL), twice with
saturated NaCl (2 ꢂ 50 mL), and finally with H2O (50 mL). The
organic phase was dried (MgSO4) and evaporated to give crude
derivative 1. The product was purified by flash chromatography,
using a mixture of hexanes/acetone (3/2), to yield 173 mg (54%) of 1.
The spectral data of this derivative correspond to those reported in
the residual solvent signal as internal standard (acetone,
for 1H NMR and 29.84 ppm for 13C NMR; dimethylsulfoxide,
2.50 ppm for 1H NMR and 39.52 ppm for 13C NMR). Chemical
) are expressed in parts per million (ppm), the coupling
d 2.05 ppm
d
d
d
shifts (
d
constants (J) are expressed in hertz (Hz). Multiplicities are
described by the following abbreviations: s for singlet, d for
doublet, t for triplet, m for multiplet, #m for several multiplets and,
br s for broad singlet.
4.2.1. Synthesis of anti-inflammatory 1 and 1A
4.2.1.1. Synthesis of derivatives 3, 4 and 1. The synthesis of de-
rivatives 3, 4 and 1 were performed as described earlier for the
same or similar types of compounds [30,31,49,50]. Only, derivatives
4 and 1 were fully characterized earlier [30,31]. So, the spectral data
of intermediate 3 and derivative 1A are given herein. Note: The
complete spectral data of 4 and 1 are also provided in order to
facilitate the comparision between the various molecules described
in this manuscript.
the literature [31]. IR (
2988 (CH, aliphatic),1733 (C]O),1706 (C]O). 1H NMR (acetone-d6,
ppm): 9.05 (s, 1H, NH), 8.02 and 7.53 (2 ꢂ d, J ¼ 8.6 Hz, 4H, aro-
matic), 7.07 (s, 2H, maleimide), 2.84 (br s, 1H, NH), 1.45 (s, 9H, 3 ꢂ
CH3); 13C RMN (acetone-d6,
ppm): 169.3 (2), 166.0, 155.7, 135.1,
n
, cmꢁ1): 3360e3240 (NH), 3087 (C]C),
d
d
134.6 (2), 131.6, 127.9 (2), 125.9 (2), 79.6, 27.5 (3); ESI þ HRMS:
(M þ Na)þ calculated for C16H17N3NaO5 ¼ 354.1060; found ¼
354.1072; (M -2-methylpropene þ H)þ calculated for C12H11N3O5 ¼
276.0620; found ¼ 276.0627.
Step 1: Synthesis of 4-(3-carboxy-acryloylamino)-benzoic acid
(3)
4.2.1.2. Synthesis of derivative 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-
yl)-benzoic acid hydrazide hydrochloride (1A). The hydrolysis of 1
was performed using a similar procedure reported by Heindel et al.
for the cleavage of maleimidoacetic acid (tert-butyloxycarbonyl)
hydrazide with hydrochloric acid to form maleimidoacetic acid
hydrazide hydrochloride [52]. To a solution of 1 (2.41 g, 7.27 mmol)
dissolved in dry dioxane (30 mL) was added a solution of hydro-
chloric acid (60 mL, 1.0 M in diethyl ether, 60 mmol). The mixture
was stirred at room temperature for a period of 5 h. Afterwards,
150 mL of hexanes were added to complete the precipitation of the
hydrochloride salt 1A. The crude precipitated was filtered, washed
with hexanes and recrystallized twice with a mixture of methanol/
isopropyl alcohol/hexanes (8/3/10) to yield 1.7 g (46%) of the
Briefly, para-aminobenzoic acid (2, 5.34 g, 38.93 mmol) was
dissolved in dry acetone (6 mL) to which was added methanol
(1 mL). Maleic anhydride (1.05 eq.) dissolved in dry acetone was
added to the first solution. The reaction mixture was stirred for a
period of 2 h allowing sufficient time for the complete precipitation
of the diacid 3. The precipitate was filtered and washed twice with
acetone (2 ꢂ 2 mL) and dried in a desiccator overnight. The crude
diacid 3 (9.16 g, 90%) was sufficiently pure to be use without further
purification at the next step. IR (
n
, cmꢁ1): 3500e2500 (CO2H),
ppm): 12.79 (br s, 2H,
1686 cmꢁ1 (C]O); 1H NMR (DMSO-d6,
d
2 ꢂ CO2H), 10.58 (s, 1H, NH), 7.89 and 7.71 (2 ꢂ d, J ¼ 8.6 Hz, 4H,
aromatic), 6.48 and 6.30 (2 ꢂ d, J ¼ 12.2 Hz, 2H, maleimide); 13C
desired material. IR (n
, cmꢁ1): 3200e2500 (CO2H), 3269 (NH), 1702
NMR (DMSO-d6,
d
ppm): 167.4, 167.3, 164.1, 143.2, 132.1, 130.9 (2),
(C]O), 1693 (C]O); 1H NMR (DMSO-d6,
d
ppm): 8.06 and 7.52 (2 ꢂ
130.6, 126.0, 119.2 (2); ESI þ HRMS: (M þ H)þ calculated for
d, J ¼ 8.8 Hz, 4H, aromatic), 7.21 (s, 2H, maleimide); 13C MNR
C
11H10NO5 ¼ 236.0553; found ¼ 236.0558.
(DMSO-d6,
d ppm) 170.0 (2), 165.6, 135.9, 135.4 (2), 129.6, 129.0 (2),
126.8 (2); ESI
þ
HRMS: (M
þ
H)þ calculated for
Step 2: Synthesis of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-
benzoic acid (4)
C
11H10N3O3 ¼ 232.0717; found ¼ 232.0717 and ESI þ HRMS:
(M þ H)þ calculated for C11H11ClN3O3 ¼ 268.0483; found ¼
268.0483.
The diacid 3 (2.01 g, 8.54 mmol) was treated with acetic anhy-
dride (4.0 mL, 36.28 mmol) and anhydrous sodium acetate (350 mg,
4.27 mmol) and the mixture heated at 50 ꢀC for 2 h. Afterwards, the
solution was evaporated to dryness and stirred with water at 70 ꢀC
for a period of 2 h. The resulting precipitate was filtered and dried
in a desiccator overnight to yield 1.65 g (89%) of maleimide 4. The
spectral data of this derivative correspond to those reported in the
4.2.2. Synthesis of derivatives 6.7 and 9
4.2.2.1. Synthesis of derivative 7
Step 1: Synthesis of 3-phenylcarbamoyl-acrylic acid (6)
Following the procedure described earlier for the preparation of
derivative 3 using aniline (3.0 g, 2.96 mL, 32.4 mmol), MA (3.33 g,
34.0 mmol) and dry acetone (21 mL). The precipitated was filtered
and washed with dry acetone (2 ꢂ 3 mL) and dried in a desiccator
overnight. The reaction yielded acid 6 (5.36 g, 86%) that was used as
literature [30]. IR (n
, cmꢁ1): 3475e2600 (CO2H), 1715 (C]O), 1704
(C]O); 1H NMR (acetone-d6,
d
ppm): 8.14 and 7.57 (2 ꢂ d,
J ¼ 8.6 Hz, 4H, aromatic), 7.08 (s, 2H, maleimide); 13C MNR
(acetone-d6, d ppm) 169.3 (2),166.2,136.2,134.7 (2),130.1 (2),129.3,

J. Hamelin-Morrissette et al. / European Journal of Medicinal Chemistry 96 (2015) 259e268
267
Cancer Statistics 2014, Canadian Cancer Society, Toronto, ON, 2014.
such in the next step. IR (
n
, cmꢁ1): 3273 and 3208 (NeH),
ppm):
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logical basis in the pathogenesis and treatment of bladder cancer, Expert Rev.
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3230e2900 (CO2H), 1694 cmꢁ1 (C]O); 1H NMR (DMSO-d6,
d
12.87 (br s, 1H, CO2H), 10.37 (s, 1H, NH), 7.60, 7.31 and 7.07 (3 ꢂ m,
2H, 2H and 1H, aromatic), 6.46 and 6.28 (2 ꢂ d, J ¼ 12.1 Hz, 2H,
maleimide); 13C NMR (DMSO-d6,
d ppm): 167.3, 163.7, 139.0, 132.1,
130.9, 129.3 (2), 124.3, 120.0 (2); ESI þ HRMS: (M þ H)þ calculated
for C10H10NO3 ¼ 192.0655; found ¼ 192.0650.
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Step 2: Synthesis of 1-phenyl-pyrrole-2,5-dione (7)
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Epidemiol. 119 (1984) 510e515.
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Following the procedure described above for the preparation of
derivative 4 using acid 6 (823 mg, 4.28 mmol), acetic anhydride
(2.10 mL, 22.2 mmol), sodium acetate (175 mg, 2.13 mmol), 50 ꢀC for
2 h, then H2O (80 mL), 70 ꢀC for 2 h. The final product was filtered,
dried overnight in a desiccator to give 325 mg (44%) of final
product. This compound is also commercially available from Sig-
maeAldrich Inc. IR (
ppm): 7.75, 7.34 and 7.13 (3 ꢂ m, 2H, 2H and 1H, aromatic), 6.43 (s,
2H, maleimide); 13C MNR (acetone-d6,
ppm) 163.1 (2), 138.7, 133.4
(2), 128.8 (2), 124.2, 119.8 (2); ESI þ HRMS: (M þ H)þ calculated for
10H8NO2 ¼ 174.0550; found ¼ 174.0554.
n
, cmꢁ1): 1703 (C]O); 1H NMR (acetone-d6,
d
d
C
4.2.2.2. Synthesis of derivative N0-benzoyl-hydrazinecarboxylic acid
tert-butyl ester (9). Following the procedure described earlier for
the preparation of derivative 1 using benzoic acid (321 mg,
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2.62 mmol), triethylamine (513
mL, 3.68 mmol), isobutyl chlor-
oformate (480 L, 3.68 mmol), tert-butyl carbazate (347 mg,
m
2.62 mmol) and methylene chloride (8 mL in total). The crude
material was purified by flash chromatography using a mixture of
hexanes/ethyl acetate (9/1 and 7/3) to yield the desired material 9
(300 mg, 48%). IR (
n
, cmꢁ1): 3475e3150 (NH), 3070 (C]C), 2979
(CH, aliphatic), 1713 (C]O), 1659 (C]O). 1H NMR (acetone-d6,
d
ppm): 9.43 (s, 1H, NH), 7.92 and 7.50 (2 ꢂ m, 5H, aromatic), 2.84
(br s, 1H, NH), 1.44 (s, 9H, 3 ꢂ CH3); 13C RMN (acetone-d6,
d ppm):
166.5, 155.7, 133.1, 131.7, 128.4 (2), 127.3 (2), 79.5, 27.5 (3);
ESI þ HRMS: (M þ Na)þ calculated for C12H16N2NaO3 ¼ 259.1053;
found ¼ 259.1058; (M -2-methylpropene þ H)þ calculated for
C8H9N2O3 ¼ 181.0613; found ¼ 181.0612.
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4.3. Statistical analyses
For all biological assays, data were presented as mean SD from
three independent experiments. Data were analyzed by one-way
ANOVA followed by Bonferonni post-test using Prism software,
version 3.03 (GraphPad, San Diego, CA). p values of ꢃ0.05 were
considered to indicate statistical significance.
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Acknowledgments
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This work was supported by grants from the Natural Sciences
and Engineering Research Council of Canada (NSERC #2014-06516),
ꢀ ꢀ
the Fonds Quebecois de la Recherche sur la Nature et les Technol-
ꢀ
ꢀ ꢀ
ogies (FQRNT), and the Reseau Quebecois en Reproduction (RQR) to
C. Reyes-Moreno. S. Cloutier is a recipient of a NSERC undergrad-
uate summer scholarship award. J. Hamelin-Morrissette is sup-
ported by the RQR-CREATE scholarships program. J. Girouard holds
ꢀ
a postdoctoral fellowship from the Fonds de la Recherche en Sante
ꢀ
du Quebec (FRSQ). D. Belgorosky holds a doctoral scholarship from
the Emerging Leaders in the Americas Program from Canada.
[29] W.-S. Choi, P.-Y. Shin, J.-H. Lee, G.-D. Kim, The regulatory effect of veratric acid
on NO production in LPS-stimulated RAW264.7 macrophage cells, Cellular
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