Synthesis and biological evaluation of nimesulide based new class of triazole derivatives as potential PDE4B inhibitors against cancer cells

Article abstract of DOI:10.1016/j.bmcl.2013.10.035

A new class of 1,2,3-triazol derivatives derived from nimesulide was designed as potential inhibitors of PDE4B. Synthesis of these compounds was carried out via a multi-step sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step in aqueous media. The required azide was prepared via the reaction of aryl amine (obtained from nimesulide) with α-chloroacetyl chloride followed by displacing the α-chloro group by an azide. Some of the synthesized compounds showed encouraging PDE4B inhibitory properties in vitro that is >50% inhibition at 30 μM that were supported by the docking studies of these compounds at the active site of PDE4B enzyme (dock scores ～ -28.6 for a representative compound). Two of these PDE4 inhibitors showed promising cytotoxic properties against HCT-15 human colon cancer cells in vitro with IC₅₀ ～ 21-22 μg/mL.

Full text of DOI:10.1016/j.bmcl.2013.10.035

Accepted Manuscript
Synthesis and biological evaluation of nimesulide based new class of triazole
derivatives as potential PDE 4B inhibitors against cancer cells
Jyoti Mareddy, N. Suresh, A. Jayasree, Yumnam Priyadarshini Devi, Lakshmi
Narasu Mangamoori, Ravikumar Kapavarapu, Sarbani Pal
PII:
S0960-894X(13)01241-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.10.035
BMCL 20989
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
14 July 2013
31 August 2013
18 October 2013
Please cite this article as: Mareddy, J., Suresh, N., Jayasree, A., Devi, Y.P., Mangamoori, L.N., Kapavarapu, R.,
Pal, S., Synthesis and biological evaluation of nimesulide based new class of triazole derivatives as potential PDE
4
B inhibitors against cancer cells, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/
j.bmcl.2013.10.035
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Synthesis and biological evaluation of nimesulide based new class of triazole derivatives as
potential PDE 4B inhibitors against cancer cells
a,b
b
b
c
Jyoti Mareddy , N. Suresh, A. Jayasree , Yumnam Priyadarshini Devi, Lakshmi Narasu
c
d
a,
Mangamoori, Ravikumar Kapavarapu, Sarbani Pal *
a
Department of Chemistry, MNR Degree & PG College, Kukatpally, Hyderabad-500085, India.
b
Centre for Chemical Sciences and Technology, Institute of Science & Technology, Jawaharlal
Nehru Technological University Hyderabad, Kukatpally, Hyderabad-500085, India.
c
Department of Biotechnology, Jawaharlal Nehru Technological University Hyderabad,
Kukatpally, Hyderabad-500085, India.
Doctoral Programme in Experimental Biology and Biomedicine, Center for Neuroscience and
d
Cell Biology, Institute for Interdisciplinary Research, University of Coimbra (IIIUC), 3004-517
Coimbra, Portugal.
---------------------------------------------------------------------------------------------------------------------
Abstract: A new class of 1,2,3-triazol derivatives derived from nimesulide was designed as
potential inhibitors of PDE4B. Synthesis of these compounds was carried out via a multi-step
sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step in
aqueous media. The required azide was prepared via the reaction of aryl amine (obtained from
nimesulide) with -chloroacetyl chloride followed by displacing the -chloro group by an azide.
Some of the synthesized compounds showed encouraging PDE4B inhibitory properties in vitro
i.e. > 50% inhibition at 30 µM that were supported by the docking studies of these compounds at
the active site of PDE4B enzyme (dock scores ~ -28.6 for a representative compound). Two of
these PDE4 inhibitors showed promising cytotoxic properties against HCT-15 human colon
cancer cells in vitro with IC ~ 21-22 µg/mL.
5
0
Keywords: nimesulide, 1,2,3-triazole, cycloaddition, PDE4B, cytotoxic activities
---------------------------------------------------------------------------------------------------------------------
-
*
----------------------------
Corresponding author: Tel.: +91 40 23041488; fax: +91 040 23067380
E-mail: sarbani277@yahoo.com
1

The design and construction of compound library based on novel “drug like” molecules via
structural modifications of a known drug by taking advantages of modern synthetic
methodologies is the central focus of current medicinal chemistry and new drug discovery. Due
1
to its simplicity the use of “click chemistry” has attracted particular attention for this purpose.
2
Cancer being the second leading cause of death worldwide after cardiovascular diseases requires
urgent attention and thus discovery and development of suitable agents possessing novel
mechanism of action is highly desirable to treat various types of cancer. Recently, studies have
3
indicated that phosphodiesterases 4 (PDE4, a member of super family of enzymes e.g. PDEs)
that degrade cyclic adenosine monophosphate (cAMP) specifically is widely expressed in tumor
cells. Thus, inhibition of PDE4 in cancerous cells can be a new therapeutic target for cancer.
This is exemplified by the reports on ability of PDE4 inhibitors to (i) inhibit brain tumor cell
4
,5
6
growth, (ii) reduce proliferation and angiogenesis of lung cancer cell lines and (iii) cause
7
selective apoptosis of malignant cells without affecting the normal cells.
Nimesulide (Fig. 1), a well known non-steroidal anti-inflammatory drug (NSAID) is presently
8
available in several countries for the treatment of acute pain, the symptomatic treatment of
osteoarthritis and primary dysmenorrhoea in adolescents and adults above 12 years old. It is a
cycloxygenase-2 (COX-2) inhibitor possessing analgesic and antipyretic properties and shows a
fast onset of action due to its multi-factorial mode of action. However, nimesulide has been
withdrawn from the market in many countries due to concerns about the risk of its
9
hepatotoxicity. Studies have indicated that the observed hepatotoxicity of nimesulide can be
linked to its uncoupling effects on mitochondria via a protonopheretic mechanism and oxidation
1
0
of mitochondrial NADH and NADPH. Since the chemical reduction of nitro group of
nimesulide to amine completely suppressed the above mitochondrial responses hence it is
reasonable to use the reduced form i.e. the corresponding amine derived from nimesulide for the
1
1
design of new chemical entities. In view of reported anticancer properties of nimesulide and
1
2
anti-inflammatory activities 1,2,3-triazoles we anticipated that combining the structural features
of nimesulide and 1,2,3-triazole in a single molecule (Fig. 1) may afford novel template A for the
design and synthesis of new anticancer agents that may inhibit PDE4. This was supported by the
docking studies of a representative molecule e.g. 2-(4-((4-chlorophenoxy)methyl)-1H-1,2,3-
triazol-1-yl)-N-(4-(methylsulfonamido)-3-phenoxyphenyl)acetamide (B) into PDE4B protein in
silico which showed strong interactions (Glide score -26.18) involving –OCH - and -NHSO Me
2
2
groups of B with the Gln443 and Asn283 residues of PDE4B respectively (Fig. 2). Due to our
1
3-18
continuing interest in the structural modifications of nimesulide,
we now report the synthesis,
1
9
in vitro PDE4 inhibition and cytotoxic effects of a series of novel 1,2,3-triazoles A derived
from nimesulide. To the best of our knowledge synthesis and pharmacological evaluation of
1
,2,3-triazoles derived from nimesulide is not known in the literature. Additionally, evaluation of
novel PDE4 inhibitors for the potential treatment of cancer is not common in the literature.
2

NHSO Me
2
NHSO Me
2
O
PhO
N
N
N
HN
R
N
N
NO2
N
O
1
,2,3-triazole
Nimesulide
A
R
Fig. 1. Design of novel and potential anticancer agents based on nimesulide and 1,2,3-triazole
3

Fig. 2. Binding of designed molecule B in the PDE4B pocket
The synthesis of our target compounds 3 (or A) was carried out by adopting a multi-step
sequence as outlined in Scheme 1. The 1,2,3-triazoles are generally synthesized by using click
chemistry approach that involves copper (I)-catalyzed 1,3-cycloaddition reaction between a
1
terminal alkyne and an azide. Thus, the copper-catalyzed azide-alkyne cycloaddition (CuAAC)
in aqueous media was used as a key step in our synthesis. A mixture of CuSO and sodium
4
ascorbate was used as a precatalyst system which generated the Cu(I) species in situ. The
2
0
required azide was prepared from nimesulide that was reduced to amine (1) which on reaction
with 2-chloroacetyl chloride afforded the corresponding chloro derivative. Treating this chloro
compound with KI followed by sodium azide (1) provided the corresponding azide derivative
(2). The azide (2) was then coupled with a variety of terminal alkynes (2a-i) in the presence of
CuSO and sodium ascorbate smoothly under mild conditions in water to afford the desired
4
1
,2,3-triazoles (3). All these compounds were well characterized by spectral (NMR, IR and MS)
1
and analytical data. For example the appearance of two singlets near δ 5.2 & 5.1 ppm in the H
NMR spectrum of 3b (see ESI) indicated the presence of two methylene groups. The methyl
protons of NHSO Me group appeared as a singlet at δ 3.0 ppm. The NH hydrogens appeared as
2
two singlets at δ 10.5 and 9.3 ppm that disappeared during D O exchange experiment. As
2
expected from its molecular structure, a total of 20 signals appeared for twenty chemically
1
3
nonequivalent carbon atoms when broad band decoupled CNMR spectrum of 3b (Fig. 3) was
recorded. The peak at δ 164.2 ppm confirmed amide C=O group. Additionally, 16 out of 20
signals were assigned for different aromatic carbons which include both quaternary (C) and
methine (CH) carbon atom. The methyl carbon of -NHSO Me appeared at δ 29.3 ppm. In order
2
to assign the chemical shift values correctly DEPT spectrum was also recorded (Fig. 3). This
showed signals at (i) δ 60.6 and 51.8 ppm (inverted peaks) due to the presence of two CH (ii) δ
2
2
9.3 ppm due to a methyl and (iii) δ 129.8, 129.2, 127.6, 125.9, 123.8, 120.5, 119.2, 114.3, 113.7
and 108.4 ppm because of ten CH groups. Additionally, the signals for six aromatic quaternary
4

carbons and one C=O group appeared in the broad band decoupled spectrum disappeared
completely in the DEPT spectrum.
NHSO CH3
2
NHSO CH3
NHSO CH
NHSO CH
3
2
2
3
2
OPh
OPh
OPh
OPh
(
i)
(ii), (iii), (iv)
(v)
N
NH
N
NH
N
R
NO2
nimesulide
NH2
N3
O
2
O
3
1
R (time; % yield)
3
3
3
3
3
3
3
3
3
a: Ph (50 min; 86)
b: PhOCH (1h; 83)
2
c: C H OCH (2h; 97)
1
0
7
2
d: p-CH C H (2h; 83)
3
6
4
e; p-CH C H OCH (10 min; 71)
3
6
4
2
f; p-ClC H OCH (10 min; 83)
6
4
2
g; p-Cl(m-CH ) C H OCH (10 min; 85)
3
6
3
2
h; o-ClC H OCH (10 min; 95)
6
4
2
i; p-NO C H OCH (30 min; 85)
2
6
4
2
Scheme 1. Reagents and conditions: (i) Sn/HCl (ii) ClCH COCl, Et N, CHCl , room temp, 1.5 h,
2
3
3
8
5%; (iii) KI, acetone, room temp, 3h, 91%; (iv) NaN , acetone, room temp, 6h, 90%; (v) R-
3
C≡CH, CuSO , Na-ascorbate, H O, room temp, 10 min-2h.
4
2
5

1
13
Fig. 3. H and C NMR spectra of compound 3b in DMSO-d .
6
6

All the 1,2,3-triazoles synthesized were assessed for their PDE4B inhibitory potential using an
2
1
enzyme based assay. Rolipram was used as a reference compound in this assay. A number of
compounds e.g. 3a-c, 3e and 3f showed significant inhibition (> 50%) of PDE4 when tested at 30
µM (Table 1) whereas nimesulide did not show any inhibition at the same concentration. To
understand the nature of interactions of this class of heterocycles with the PDE4B protein
docking studies were performed using selected compounds e.g. 3a, 3c, 3e, 3f and 3i that showed
~
50% inhibition of PDE4B. The dock scores and molecular interactions of compound 3 after
docking into the PDE4B protein are presented in Table 2. It is evident that the compound 3a
showed highest dock score which is in agreement with its in vitro data generated using the
enzyme assay. Additionally, all these compounds showed similar docking score which are also in
agreement with their PDE4B inhibition data (Table 1). While all these 1,2,3-triazoles interacted
with the same and /or similar amino acid residues at the active site of the PDE4B protein their
orientation of interactions were largely found to be somewhat dissimilar depending on the
substituents present on the triazole ring (Fig. 2, 4 and 5, see also Fig. S-1 and S-2 in the ESI). All
these molecules showed common interactions with Gln443 of PDE4B protein similar to
Rolipram (see Fig S-3 in the ESI). In general, the moieties or substitutents that participated
simultaneously or individually in the interactions include the triazole ring, central benzene ring,
the -NHSO - group, -CH O- moiety etc. In a dose response study the compound 3a showed dose
2
2
dependent inhibition of PDE4B with IC value of 4.92 ± 0.53 µM.
5
0
Table 1. Inhibition of PDE4B by compound 3 at 30 µM.
Entry
Compound 3
Average % Inhibition
SD
1
2
3
4
5
6
7
8
9
1
.
3a
64.09
51.17
59.69
36.01
56.67
51.32
32.91
42.53
47.11
94.12
5.20
1.31
5.61
2.32
1.94
2.01
0.88
1.90
2.43
1.01
.
3b
.
3c
.
3d
.
3e
.
3f
3g
.
.
3h
.
3i
0.
Rolipram
SD = standard deviation
7

Table 2. Dock scores and summary of molecular interactions of compound 3 after docking into
PDE4B
Summary of interactions
Compound 3
Dock scores
3
3
3
3
3
a
c
e
f
i
-28.62
-26.62
-24.92
-26.18
-25.16
Gln443, His234,His278
Gln443, Asn283
Gln443
Gln443, Asn283
Gln443
8

Fig. 4. Docking of 3a at the active site of PDE4B.
9

Fig. 5. Docking of 3c at the active site of PDE4B.
1
0

Having identified a number of 1,2,3-triazoles (3) as novel inhibitors of PDE4B we performed in
vitro cytotoxic evaluation of some of these compounds using human colon cancer cell line HCT-
2
2
1
5. Colon (or colorectal) cancer is widespread in the Western world and is the focus of our
current research. Moreover, recent study suggested that PDE4B is upregulated by oncogenic
KRAS, and inhibition of PDE4 catalytic activity by rolipram can induce both epithelial cell
2
3
polarity and luminal apoptosis in human colorectal cancer (CRC) HCT116 cells. The test
compounds were examined for their ability to inhibit the cancerous cells (HCT-15) based on an
MTT assay. Doxorubicin, a known anthracycline antibiotic was used as a reference compound in
our assay. All these compounds were tested at five different concentrations e.g. 5, 10, 15, 20 and
2
5µg/mL and the percentage of cell death measured for each compound along with IC values
50
of active compounds are summarized in Table 3. It is evident that 3a and 3d showed good
activities especially at higher dose. Both these compounds showed IC values in the range 21-22
5
0
µg/mL (Table 3) compared to doxorubicin’s IC value of 50 µg/mL (0.09 µM). Thus the present
5
0
1
,2,3-triazole based PDE4 inhibitors have potential for the development of new anticancer agents
especially for colon cancer.
Table 3. The in vitro anticancer properties of some of the 1,2,3-triazoles (3) synthesized.
Entry Compounds
% of cell death at various concentrations
IC50
(µg/mL)
5
µg /mL 10 µg /mL 15 µg /mL 20 µg /mL 25 µg /mL
1
2
3
3a
3c
3d
10.8
10.6
37.0
15.2
14.3
40.2
31.0
21.1
45.6
42.7
25.3
49.6
60.1
33.6
53.0
22.4
n.d.
21.0
All the values are the average of the experiments done in triplicates. The cell line used is HCT-
5 human colon cancer cell line. Doxorubicin [IC50 = 50 µg /mL (0.09 µM)] was used as a
1
reference compound. n.d. = not done.
In conclusion, novel 1,2,3-triazole derivatives derived from nimesulide were designed as
potential inhibitors of PDE4. Synthesis of these compounds was carried out via a multi-step
sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step in
aqueous media. The required azide was prepared via the reaction of aryl amine (obtained from
nimesulide) with -chloroacetyl chloride followed by displacing the -chloro group by an azide.
All the 1,2,3-triazole derivatives were prepared in 71-97% yield. Some of the synthesized
compounds showed encouraging PDE4B inhibitory properties in vitro i.e. > 50% inhibition at 30
µM that were supported by the docking studies of these compounds at the active site of PDE4B
enzyme. All these compounds showed dock scores in the range of -28.6 to -24.9. Two of these
PDE4 inhibitors i.e. 3a and 3d showed promising cytotoxic properties against HCT-15 human
colon cancer cells in vitro with IC50 ~ 21-22 µg/mL. Thus, the present 1,2,3-triazole class of
compounds represent new templates for the identification and development of novel PDE4
inhibitors / potential anti-cancer agents.
Acknowledgements. The authors (J. M. and S. P.) thank Mr. M. N. Raju, the chairman of
M.N.R. Educational Trust for his constant encouragement.
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2

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Synthesis and biological evaluation of nimesulide based new Leave this area blank for abstract info.
class of triazole derivatives as potential PDE 4B inhibitors
against cancer cells
Jyoti Mareddy, N. Suresh, A. Jayasree, Yumnam Priyadarshini Devi, Lakshmi Narasu Mangamoori, Ravikumar
Kapavarapu, Sarbani Pal*
2
NHSO CH3
2
NHSO CH3
NHSO CH
2
3
OPh
OPh
OPh
N
NH
N
NH
N
R
NO2
nimesulide
N3
O
O
Binding mode with PDE4B (R = Ph)