Nicotinic acid conjugates of nonsteroidal anti-inflammatory drugs (NSAID's) and their anti-inflammatory properties

Article abstract of DOI:10.1016/j.ejps.2013.02.007

A series of nicotinic acid conjugates with non-steroidal anti-inflammatory drugs (NSAID's) have been effectively synthesized using TBTU in high yield and purity. All the synthesized conjugates were evaluated for their in vitro anti-inflammatory activity.

Full text of DOI:10.1016/j.ejps.2013.02.007

European Journal of Pharmaceutical Sciences 49 (2013) 227–232
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Nicotinic acid conjugates of nonsteroidal anti-inflammatory drugs (NSAID’s)
and their anti-inflammatory properties
b
a
Machhindra Gund a, Fazlur-Rahman Nawaz Khan a, , Amit Khanna , Varadhan Krishnakumar
⇑
a Organic and Medicinal Chemistry Research Laboratory, Organic Chemistry Division, School of Advanced Sciences, VIT-University, Vellore 632 014, Tamil Nadu, India
b School of Biosciences and Technology, VIT-University, Vellore 632 014, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 March 2012
Received in revised form 25 January 2013
Accepted 15 February 2013
Available online 7 March 2013
A series of nicotinic acid conjugates with non-steroidal anti-inflammatory drugs (NSAID’s) have been
effectively synthesized using TBTU in high yield and purity. All the synthesized conjugates were evalu-
ated for their in vitro anti-inflammatory activity.
Ó 2013 Elsevier B.V. All rights reserved.
Keywords:
Nicotinic acid conjugates
Coupling
Anti-inflammatory
1. Introduction
We are making mutual prodrugs of NSAID’s with Nicotinic acid
and principle of mutual prodrug is combining two therapeutic
Variety of inflammatory diseases including rheumatoid arthri-
tis, ankylosing spondylitis, osteoarthritis of large joints, etc. are
treated by use of the nonsteroidal antiinflammatory drugs NSAID’s
including diclofenac (Skoutakis et al., 1998; Moser et al., 1990;
Sallmann, 1986); antirheumatic drugs (Lee et al., 1976; Mason
et al., 1967; Bandarage et al., 2000); aspirins’ antiplatelet proper-
ties (Cena et al., 2003; Zadrazil, 2006), nonsteroidal anti-inflamma-
tory drugs gastropathy (Laine, 1996), ibuprofen glucopyranoside
conjugates (Zhao et al., 2006; Shanbhag et al., 1992) indomethacin,
farsenil (Kumakura et al., 1990), prodrugs of flufenamic acid and
diclofenac (Ribeiro et al., 2007) derivatives of flurbiprofen (Halen
et al., 2006; Kakuta et al. (2008)). NSAID’s act by inhibiting cyclo-
oxygenase derived prostaglandin synthesis and through local ac-
tion exerted by direct contact of the drugs with gastric mucosa
due to the acidic nature. However upper GI irritation, ulceration,
dyspepsia, bleeding, gastrotoxicity and in some cases death are
the major side effects observed in patients undergoing a long-term
NSAID’s treatment (Albert, 1958). A major drawback with use of
NSAID’s is development of gastric ulcers during treatment. Nico-
tinic acid is used for conjugation with NSAID’s because it has been
known in literature that nicotinic acid helps to reduce gastric irri-
tation associated with use of anti-inflammatory agents. Nicotinic
acid is a vitamin constituent and usually prescribed along with
the NSAID’s for pain, inflammation and arthritis treatment.
agents in a single molecule using a linker. Linker has to cleave in
the biological system under biological pH to release both the
agents. We have used the simplest linker i.e. 1,3-propane diol. As
both partners of mutual prodrugs i.e. NSAID’s and Nicotinic acid
contain carboxylic acid functional groups in the molecule, the sim-
plest linkage we can make is ester linkage. As there are enough
concentration of esterases present in body, they act on the ester
linkage and cleave the components at a time in effective concentra-
tion. Also as we are connecting both components in a single mole-
cule they traverse through all the biological membranes together
and will be probably targeted to the actual site of action in effective
concentration. Thus our principle of mutual prodrug worked as
there is no reduction in biological activity. Also, we can expect
these ester containing conjugates to release the drugs over a longer
period and in a sustained manner, and thus, reducing the doses and
the intervals of the medication. To overcome these side effects
associated with such drugs, converting carboxylic acid containing
drugs into their prodrugs containing ester linkages is the most suc-
cessful approach (Bundgaard, 1985, 1986, 1989, 1991; Manon and
Sharma, 2009; Gund et al., 2011).
Such derivatives are expected to release active parent drug
upon administration either chemically or enzymatically which in
turn exert the desired biological action (Bundgaard, 1985; Bundg-
aard, 1986). Several literature reports containing ester containing
prodrugs of NSAID’s having improved bioavailability, anti-inflam-
matory activity and reduced gastric toxicity are well documented
(Bundgaard, 1991; Bundgaard, 1989). Diclofenac–antioxidant con-
jugates devoid of ulcerogenic side effects are known in literature
⇑
Corresponding author. Tel.: +91 94442 34609; fax: +91 416 2243092.
E-mail address: nawaz_f@yahoo.co.in (Fazlur-Rahman Nawaz Khan).
0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2013.02.007

228
M. Gund et al. / European Journal of Pharmaceutical Sciences 49 (2013) 227–232
(Manon and Sharma, 2009). In continuation of our research inter-
est on C-C, C-O and C-N bond formation (Nawaz Khan et al.,
2002, Khan et al., 2010; Hathwar, et al., 2007a, 2007b; Tajudeen,
2007; Maiyalagan, 2009; Prabakaran, 2010). The present work
was initiated with the aim to develop prodrugs of indomethacin
(Gund et al., 2011). Thus with the promising results in hands we
have prepared a series of novel ester containing prodrugs (3a–f)
of other NSAID’s as depicted in the scheme below (Scheme 1) in
good to excellent yields. Condensation of nicotinic acid with the
appropriate hydroxyl derivative of NSAIDs in the presence of TBTU
(O-(Benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoro-
borate) reagent gave the desired nicotinic acid conjugates which
were evaluated for anti-inflammatory activity. A nicotinic acid is
mainly play a role to reduce the gastric irritation associated with
use of NSAID’s (Nylander, 2011). Further investigation on the ac-
tual quantitative reduction in gastric ulcers associated with use
of NSAID’s is underway and will be communicated soon. The struc-
tures of all prodrugs were established by 1H NMR, 13C NMR and
mass spectral techniques with HPLC purities more than 95%. All
the synthesized conjugates were stable at room temperature and
up to 40 °C and at different pH.
supplemented with 10% FBS. Prior to cell-contact experiments,
cells were cultured for 24 h in the presence of 10 ng/mL of PMA
to enhance their response to activation stimuli.
2.3. Cell based assay for THP-1 cytokine release assay
The human monocytic cell line THP-1 (American Type Culture
Collection, Manassas, Va.) was maintained in RPMI supplemented
with 2 mM L-glutamine, 100 mg of penicillin per mL, 100 mg of
streptomycin per mL with 25 mM HEPES and 10% fetal bovine ser-
um. Induction of cell differentiation was obtained with 100 nM
PMA for 24 h. After incubation, non-adherent cells were removed
by aspiration and the adherent cells were washed with RPMI three
times. For cell stimulation, the cells were further incubated with or
without LPS for 24 h, in fresh complete medium with 10% fetal bo-
vine serum. After cell plating, the test compounds or vehicle (0.5%
DMSO) were added to each well and the plate was incubated for
30 min at 37 °C. Finally, 20
l
L (10
lg/mL) per well of LPS was
added for a final concentration of 1
lg/mL.
2.3.1. General synthesis procedure
Synthesis of 2a: To a stirred solution of naproxen, 1a (1.0 g,
4.34 mmol) in DMF (8 mL) at 0 °C under nitrogen was added Cs2-
CO3 (1.41 g, 4.34 mmol) and stirred for 20 min. A solution of 3-bro-
mo-1-propanol (0.38 mL, 4.34 mmol) in DMF (2 mL) was added to
above mixture at 0 °C. Reaction mixture was stirred at room tem-
perature for ꢀ10 h, poured in ice water (70 mL) and extracted with
ethylacetate (2 ꢁ 40 mL). Ethyl acetate layer was washed with
satu. NaHCO3 (40 mL), brine (40 mL), dried over Na2SO4 and con-
centrated. Crude compound was purified by silica gel column chro-
matography using 30% EtOAc/Pet.ether to afford the hydroxy
derivative, 2a as a colorless oil. (1.04 g, 84%, Rf = 0.50 in 40%
2. Materials and methods
2.1. Materials
Penicillin, streptomycin, Roswell Park Memorial Institute med-
ium1640 (RPMI-1640), fetal bovine serum (FBS), phorbol myristate
acetate (PMA), lipopolysaccharide (LPS), Ficoll Hypaque-1077 and
phosphate buffered saline (PBS) were from Sigma. Duosets for
TNF-a, IL-6 enzyme-linked immunosorbent assays (ELISAs) were
from BD, Biosciences. CCK-8 reagent was from DoJindo and di-
EtOAc/Pet.ether) 1H NMR (300 MHz, CDCl3):
d 1.60 (d, 3H,
methyl sulfoxide (DMSO) is obtained from Sigma Aldrich, USA.
J = 7.2 Hz), 1.77–1.85 (m, 2H), 3.55 (t, 2H, J = 6.0 Hz), 3.88 (q, 1H,
J = 7.2 Hz), 3.93 (s, 3H), 4.25 (t, 2H, J = 5.8 Hz), 7.13–7.18 (m, 2H),
7.40, 7.42 (dd, 1H, J = 1.5 Hz), 7.71 (t, 3H, J = 8.7 Hz). 13C NMR
2.2. Cell lines
The human monocytic cell line THP-1 (American Type Culture
Collection, Manassas, VA.) was maintained in RPMI-1640
(75 MHz, CDCl3): d 17.95, 31.16, 45.02, 54.84, 58.63, 61.28,
105.13, 118.58, 125.46, 125.61, 126.73, 128.78, 133.23, 135.07,
N
a
O
OH
Cl
NSAID
=
NSAID COOH
1
b
NSAID
O
O
2
O
3
O
O
NSAID
COOH
Cl
O
OH
OH
O
NH
CH3
N
O
O
O
Cl
Diclofenac
O
S
OH
Naproxen
H3C
O
Indomethacin
OAc
CH3
H3C
OH
OH
O
OH
O
O
Aspirin
F
Sulindac
Ibuprofen
Where: a)Cs2CO3, 3-bromo 1-propanol, DMF; b) Nicotinic acid, TBTU, Diisopropylamine, THF.
Scheme 1. Synthesis of Nicotinic acid-NSAID’s conjugates. Where (a) Cs2CO3, 3-bromo 1-propanol, DMF; and (b) nicotinic acid, TBTU, diisopropylamine, TMF.

M. Gund et al. / European Journal of Pharmaceutical Sciences 49 (2013) 227–232
229
151.19, 174.68. MS m/z: 289 [M + H]+, 311 [M + Na]+. HRMS ESI m/
z: [M + H]+ calculated for C17H21O4: 289.1434, found: 289.1446.
Synthesis of 3a: To a stirred solution of nicotinic acid (1.1 eq)
and NSAID-alcohol 2a (1.0 eq) in acetonitrile was added TBTU
(1.2 eq) followed by diisopropyl ethylamine (1.3 eq) at room tem-
perature under nitrogen atmosphere. Reaction mixture was then
stirred vigorously for 30–120 min. The reaction completion was
monitored by TLC analysis, the reaction mixture was poured in
ice cold water and extracted with ethyl acetate. Ethyl acetate layer
was successively with saturated NaHCO3 solution and brine. Or-
ganic layer was dried over Na2SO4 and concentrated. Crude com-
pound was purified by silica gel column chromatography using
50% EtOAc/Pet.ether to afford the compound 5a as a colorless oil.
(0.60 g, 89%, Rf = 0.39 in 50% EtOAc/Pet.ether) 1H NMR (300 MHz,
CDCl3): d 1.59 (d, 3H, J = 6.9 Hz), 2.03–2.12 (m, 2H), 3.87 (q, 1H,
J = 7.2 Hz), 3.92 (s, 3H), 4.26 (t, 2H, J = 6.3 Hz), 4.31–4.36 (m, 2H),
7.13–7.16 (m, 2H), 7.35–7.42 (m, 2H), 7.69 (t, 3H, J = 6.6 Hz),
8.22, 8.24 (dt, 1H, J = 1.9 Hz), 8.77, 8.79 (dd, 1H, J = 1.8, 1.5 Hz),
9.19 (d, 1H, J = 1.5 Hz). 13C NMR (75 MHz, CDCl3): d 18.44, 27.90,
31.66, 45.49, 55.30, 59.05, 61.76, 105.59, 119.05, 123.30, 125.93,
126.10, 127.20, 129.26, 133.70, 135.57, 137.02, 150.82, 153.42,
157.66, 175.12. MS m/z: 394.2 [M + H]+, 416.2 [M + Na]+. HRMS
ESI m/z: [M + H]+ Calculated for C23H24NO5: 394.1649, found:
394.1682.
2d: Yellow solid, yield: 63%, MP: 177–179 °C, Rf = 0.42 in 10%
ACN/CH2Cl2. 1H NMR (300 MHz, CDCl3): d 1.76 (t, 1H, J = 5.5 Hz),
1.84–1.92 (m, 2H), 2.23 (s, 3H), 2.82 (s, 3H), 3.59 (s, 2H), 3.67 (1,
2H, J = 5.7 Hz), 4.29 (t, 2H, J = 6.1 Hz), 6.58 (t, 1H, J = 8.7 Hz), 6.88,
6.91 (dd, 1H, J = 2.4 Hz), 7.14–7.19 (m, 2H), 7.67–7.75 (q, 4H,
J = 8.4 Hz). 13C NMR (75 MHz, CDCl3): d 10.52, 31.66, 31.78,
43.85, 59.08, 62.18, 105.88, 106.20, 110.65, 110.95, 123.61,
123.73, 123.82, 128.28, 129.48, 129.51, 130.25, 131.71, 131.74,
170.56. MS m/z: 415.2 [M + H]+, 437.1 [M + Na]+. HRMS ESI m/z:
[M + H]+ Calculated for C23H24FO4S: 415.1374, found: 415.1364.
3d: Yellow solid, yield: 74%, MP: >200 °C, Rf = 0.47 in 10% ACN/
CH2Cl2. 1H NMR (300 MHz, CDCl3): d 2.10–2.19 (m, 2H), 2.22 (s,
3H), 2.83 (s, 3H), 3.59 (s, 2H), 4.30 (t, 2H, J = 6.3 Hz), 4.41 (t, 2H,
J = 6.3 Hz), 6.56 (t, 1H, J = 9.0 Hz), 6.88, 6.91 (dd, 1H, J = 2.4,
2.1 Hz), 7.13–7.17 (m, 2H), 7.38–7.43 (m, 1H), 7.67–7.75 (q, 4H,
J = 8.3 Hz), 8.27, 8.30 (dt, 1H, J = 1.95 Hz), 8.78, 8.80 (dd, 1H,
J = 1.5, 1.8 Hz), 9.21 (d, 1H, J = 1.5 Hz). 13C NMR (75 MHz, CDCl3):
d 10.53, 27.92, 31.73, 43.89, 61.51, 61.74, 105.86, 106.18, 110.66,
110.96, 123.32, 123.62, 123.81, 125.90, 128.34, 129.34, 130.25,
131.59, 137.02, 138.24, 139.62, 141.57, 145.50, 150.86, 153.51,
170.10. MS m/z: 520.2 [M + H]+.
2e: Colorless oil, yield: 83%, Rf = 0.48 in 40% EtOAc/Pet. ether. 1H
NMR (300 MHz, CDCl3): d 0.91 (d, 6H, J = 6.6 Hz), 1.51 (d, 3H,
J = 7.2 Hz), 1.63 (bs, 1H), 1.76–1.90 (m, 3H), 2.46 (d, 2H,
J = 7.2 Hz), 3.55 (t, 2H, J = 5.4 Hz), 3.71 (q, 1H, J = 7.2 Hz), 4.22–
4.27 (m, 2H), 7.11 (d, 2H, J = 8.1 Hz), 7.21 (d, 2H, J = 7.8 Hz). 13C
NMR (75 MHz, CDCl3): d 18.34, 22.36, 30.18, 31.65, 45.01, 45.17,
59.11, 61.63, 127.09, 129.37, 137.63, 140.66, 175.24. MS m/z:
265.1 [M + H]+, 287.2 [M + Na]+. HRMS ESI m/z: [M + Na]+ Calcu-
lated for C16H24O3: 287.1618, found: 287.1643.
The spectral analysis data of compounds 2b–2f and 3b–3f are
given below along with spectral traces as
document.
a Supplementary
2b: Colorless oil, yield: 69%, Rf = 0.50 in 50% EtOAc/Pet.ether. 1H
NMR (300 MHz, CDCl3): d 1.87–1.95 (m, 2H), 3.68 (t, 2H, J = 6.0 Hz),
3.48 (s, 2H), 4.34 (t, 2H, J = 6.1 Hz), 6.57 (d, 1H, J = 8.1 Hz), 6.89 (bs,
1H), 6.93–7.03 (m, 2H), 7.15 (dt, 1H, J = 7.6 Hz), 7.24 (d, 1H,
J = 6.9 Hz), 7.36 (d, 1H, J = 8.1 Hz). 13C NMR (75 MHz, CDCl3): d
30.42, 31.65, 35.04, 36.54, 38.60, 58.96, 60.33, 62.30, 118.23,
122.03, 124.09, 128.04, 128.88, 129.50, 130.82, 137.73, 142.66.
MS m/z: 354.1 [M + H]+, 376.0 [M + Na]+. HRMS ESI m/z:
[M + Na]+ Calculated for C17H17ClNO3: 376.0478, found: 376.0459.
3b: Colorless oil, yield: 80%, Rf = 0.39 in 50% EtOAc/Pet.ether. 1H
NMR (300 MHz, CDCl3): d 2.14–2.23 (m, 2H), 3.84 (s, 2H), 4.32–
4.37 (m, 2H), 4.45 (t, 2H, J = 6.1 Hz), 6.56 (d, 1H, J = 8.1 Hz), 6.90
(bs, 1H), 6.94–7.03 (m, 2H), 7.11–7.17 (m, 1H), 7.22–7.25 (m,
1H), 7.34–7.42 (m, 3H), 8.28, 8.30 (dt, 1H, J = 1.9 Hz), 8.79, 8.80
(dd, 1H, J = 1.8, 1.5 Hz), 9.24 (d, 1H, J = 1.5 Hz). 13C NMR (75 MHz,
CDCl3): d 27.99, 31.69, 38.53, 58.99, 61.90, 62.31, 118.30, 122.07,
123.33, 124.08, 125.94, 128.07, 128.88, 129.47, 130.83, 137.05,
137.74, 142.68, 150.89, 153.52, 165.11, 172.34. MS m/z: 459.1
[M + H]+, 481.1 [M + Na]+. HRMS ESI m/z: [M + H]+ Calculated for
3e: Colorless oil, yield: 66%, Rf = 0.47 in 50% EtOAc/Pet.ether. 1H
NMR (300 MHz, CDCl3): d 0.89 (d, 6H, J = 6.6 Hz), 1.50 (d, 3H,
J = 7.2 Hz), 2.04–2.12 (m, 2H), 2.44 (d, 2H, J = 7.2 Hz), 3.71 (q, 1H,
J = 7.1 Hz), 4.25 (t, 2H, J = 6.3 Hz), 4.29–4.34 (m, 2H), 7.09 (d, 2H,
J = 8.1 Hz), 7.20 (d, 2H, J = 8.1 Hz), 7.38–7.42 (m, 1H), 8.26, 8.29
(dt, 1H, J = 1.8 Hz), 8.78, 8.80 (dd, 1H, J = 1.8 Hz), 9.21 (d, 1H,
J = 1.8 Hz). 13C NMR (CDCl3, 75 MHz): d 18.36, 22.35, 27.94,
30.15, 44.99, 45.09, 61.04, 61.87, 123.30, 125.98, 127.07, 129.35,
137.02, 137.58, 140.62, 150.88, 153.47, 165.05, 174.66. MS m/z:
370.2 [M + H]+. HRMS ESI m/z: [M + H]+ Calculated for
C22H28NO4: 370.2013, found: 370.2041.
2f: Colorless oil, 69%, Rf = 0.48 in 30% EtOAc/Pet.ether. 1H NMR
(300 MHz, CDCl3): d 1.95–2.06 (m, 2H), 2.36 (s, 3H), 3.76 (t, 2H,
J = 6.0 Hz), 4.46 (t, 2H, J = 6.1 Hz), 7.11, 7.13 (dd, 1H, J = 0.9 Hz),
7.33 (t, 1H, J = 7.5 Hz), 7.58 (t, 1H, J = 7.8 Hz), 8.01, 8.03 (dd, 1H,
J = 1.8 Hz). 13C NMR (75 MHz, CDCl3): d 21.03, 31.72, 59.01,
62.03, 123.23, 123.79, 126.05, 131.67, 133.96, 150.63, 164.88,
169.80. ESI–MS m/z: 239.1 [M + H]+.
C
23H21ClN2O4: 459.0873, found: 459.0885.
2c: Yellow oil, yield: 59%, Rf = 0.42 in 5% MeOH/CH2Cl2. 1H NMR
(300 MHz, CDCl3): d 1.79–1.91 (m, 2H), 2.40 (s, 3H), 3.53–3.68 (m,
2H), 3.70 (s, 3H), 3.85 (s, 3H), 4.28 (t, 2H, J = 6.0 Hz), 6.68, 6.71 (dd,
1H, J = 2.4 Hz), 6.88 (d, 1H, J = 9.3 Hz), 6.97 (d, 1H, J = 2.4 Hz), 7.49
(d, 2H, J = 8.4 Hz), 7.68 (d, 2H, J = 8.4 Hz). MS m/z: 416.1 [M + H]+,
438.1 [M + Na]+. HRMS ESI m/z: [M + Na]+ Calculated for C22H22-
ClNO5: 438.1079, found: 438.1118.
3f: Colorless oil, 81%, Rf = 0.51 in 60% EtOAc/Pet.ether. 1H NMR
(300 MHz, CDCl3): d 2.24–2.31 (m, 2H), 2.36 (s, 3H), 4.47 (t, 2H,
J = 6.3 Hz), 4.53 (t, 2H, J = 6.3 Hz), 7.11 (d, 1H, J = 8.1 Hz), 7.30–7.33
(m, 1H), 7.37, 7.40 (dd, 1H, J = 4.8 Hz), 7.57 (t, 1H, J = 7.8 Hz), 8.00,
8.02 (dd, 1H, J = 1.5 Hz), 8.29 (d, 1H, J = 8.1 Hz), 8.78, 8.80 (dd, 1H,
J = 1.5 Hz), 9.24 (d, 1H, J = 2.1 Hz). 13C NMR (75 MHz, CDCl3): d
21.01, 28.08, 61.71, 62.14, 122.97, 123.29, 123.83, 126.00, 131.59,
134.00, 137.05, 150.88, 153.49, 164.20, 165.14, 169.63. MS m/z:
344.1 [M + H]+, 366.1 [M + Na]+. HRMS ESI m/z: [M + Na]+ Calculated
for C18H17NO6: 366.0948, found: 366.0938.
3c: Yellow oil, yield: 83%, Rf = 0.37 in 5% MeOH/CH2Cl2. 1H NMR
(300 MHz, CDCl3): d 2.09–2.18 (m, 2H), 2.40 (s, 3H), 3.69 (s, 2H),
3.83 (s, 3H), 4.29 (t, 2H, J = 6.3 Hz), 4.39 (t, 2H, J = 6.3 Hz), 6.65,
6.68 (dd, 1H, J = 5.4, 2.4 Hz), 6.86 (d, 1H, J = 9.0 Hz), 6.96 (d, 1H,
J = 2.4 Hz), 7.38–7.43 (m, 1H), 7.47–7.50 (m, 2H), 7.66–7.70 (m,
2H), 8.26, 8.28 (dt, 1H, J = 1.95 Hz), 8.78, 8.80 (dd, 1H, J = 1.8 Hz),
9.21 (d, 1H, J = 1.5 Hz). 13C NMR (75 MHz, CDCl3): d 13.32, 27.97,
30.36, 31.66, 55.71, 59.16, 62.10, 101.27, 111.62, 114.98, 123.34,
125.90, 129.13, 130.81, 131.19, 133.85, 135.96, 137.04, 139.29,
150.84, 153.52, 156.04, 165.05, 171.24. MS m/z: 521.1 [M + H]+,
543.1 [M + Na]+. HRMS ESI m/z: [M + H]+ Calculated for C28H26-
ClN2O6: 521.1474, found: 521.1522.
3. Results and discussion
Our investigation begins with synthesis of hydroxy scaffold, 2
through the reaction of NSAIDs with the 1-bromo-3-hydroxypro-
pane (Scheme 1). The NSAIDs were reacted with the later in the
presence of caesium carbonate and dimethylformamide to afford

230
M. Gund et al. / European Journal of Pharmaceutical Sciences 49 (2013) 227–232
Table 1
the desired scaffold, 2 which in turn condensed with nicotinic acid
in the presence of TBTU to afford the desired prodrugs, 3.
Effect of amount of TBTU and diisopropyl ethyl amine loading in the esterification of
nicotinic acid with 2a.a
The coupling reagent TBTU, has been extensively used due to its
high efficiency, high stability, long shelf life, solubility in organic sol-
vents, ease of handling and availability. In the present study, TBTU is
applied for the esterification of nicotinic acid with the diverse
NSAID’s containing hydroxyl derivatives in good yields. The advan-
tages of this reaction includes low toxicity of by-products, easy han-
dling, simple work-up. The proposed mechanism of the reaction is
depicted below (Scheme 2). The reaction takes place through the
tautomeric forms of TBTU, of which the form I reacts with the car-
boxylate ion to produce the intermediate which then couples with
the hydroxyl derivative to form the desired ester product.
Entry
TBTU eq
DIPEA eq
Yieldb (%)
1
2
3
4
5
6
7
1.0
1.2
1.4
1.2
1.2
1.2
1.2
1.0
1.0
1.0
1.1
1.2
1.3
Nil
76
79
80
81
84
85
24
a
Reaction conditions: nicotinic acid (1.1 eq); alcohol, 2 (1.0 eq); diisopropylethyl
amine.
b
Isolated yield.
The optimization of the reaction was carried out by varying the
amount of TBTU reagent, diisopropyl ethylamine and the solvents
at room temperature (Tables 1 and 2). The experimental results
indicated that with an optimized amount of equimolar TBTU, diiso-
propyl amine is required for the reaction of 2 (1.0 eq) and nicotinic
acid (1.1 eq) and THF as a solvent is necessary for the effective con-
version. With this optimization result in hand various hydroxyl
derivatives of NSAID’s were condensed with nicotinic acid and
the results are reported in Table 3. The purified products (HPLC
purities more than 95%) were characterized by different spectral
techniques including 1H NMR, 13C NMR, IR and MS techniques.
Table 2
Effect of solvent in the esterification reaction of nicotinic acid and 2.a
Entry
Solvent
Yieldb (%)
1
2
3
4
5
Acetonitrile
Tetrahydrofuran
2-Methyl THF
Dichloromethane
N,N-dimethyl formamide
64
84
71
54
63
a
Reaction conditions: nicotinic acid (1.1 eq); alcohol, 2 (1.0 eq); diisopropylethyl
amine.
b
Isolated yield. TBTU; solvent (8 mL).
4.1. Screening for anti-inflammatory activity
examples of selective COX-1 inhibitors has been identified from
the literature, similarly conjugates of NSAID’s with nicotinic acid
have been known in literature (Perrone et al., 2010) and they retain
COX-inhibitory activity. The NSAID’s possess potent COX-1 inhibi-
tory activity and COX-1 is the main culprit in causing gastric ulcer-
ation (Nylander, 2011).
All the synthesized conjugates were screened for anti-inflamma-
tory activity at 30 lM concentration and the percentage inhibition
data is presented in Table 4. The anti-inflammatory effects of the
synthesized conjugates and that of standard NSAID’s used for conju-
gation were very similar. Thus our principle of mutual prodrug
worked as there is no reduction in biological activity. Also, we can
expect these ester containing conjugates to release the drugs over
a longer period and in a sustained manner, and thus, reducing the
doses and the intervals of the medication (Uddin et al., 2010). In
the traditional NSAID’s the aspirin and indomethacin are few
4.1.1. Detection of secreted cytokines
Human TNF-
a and IL-6 was quantified by an enzyme linked
immunosorbent assay (ELISA) as previously described. Briefly,
N
N
N
N
O
_
N
N
_
BF4
N
+
N
_
BF4
O
O
N
H
+
TBTU
Base
O
O
O
N
N
N
N
N
N
Nicotinic acid
O
OH
O
NSAID
2
O
N
N
N
_
O
NSAID
O
O
N
O
O
3
N
N
N
OH
N
N
N
BF4
+
O
N
N+
N
O-
_
+
N
BF4
N
Scheme 2. Mechanism of esterification mediated by TBTU reagent.

M. Gund et al. / European Journal of Pharmaceutical Sciences 49 (2013) 227–232
231
Table 3
Table 4
Esterification of nicotinic acid with 2.
Anti-inflammatory activities of the conjugates.
S. no.
1
Product, 3a–f
Yielda (%)
90
S.
no.
NSAID/
compound
IL-6
TNF-
IC50 % Inhibition @
30
a
% Inhibition @
IC50
N
30
l
M
lM
O
O
1
2
3
4
5
6
Naproxen
3a
Diclofenac
3b
Indomethacin
3c
Sulindac
3d
Ibuprofen
3e
21 2.1
35 3.2
35 3.2
44 4.1
21 3.5
37 3.9
36 2.1
42 3.1
21 1.1
26 2.1
>30 32 4.5
>30 39 1.9
>30 42 3.2
>30 48 1.9
>30 32 7.2
>30 40 2.1
>30 38 8.8
>30 44 7.1
>30 27 2.2
>30 29 3.7
>30 12 1.9
>30 10 2.1
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
O
O
O
2
3
89
92
Cl
N
NH
Cl
O
O
Aspirin
3f
12
10
1
2
O
O
Cl
acid-NSAID’s conjugates with the advantages of low toxicity of
by-products, easy handling and simple work-up procedures. All
the synthesized conjugates are equally or slight better potent
in vitro when compared with the respective parent drug.
O
N
N
CH3
O
H3C
O
O
Acknowledgement
O
O
4
88
The authors wish to express their gratitude to the VIT Univer-
sity for support and facilities.
CH3
S
O
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejps.2013.02.007.
N
CH3
O
F
O
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microwave
accelerated
palladium-catalyzed
amination
of
1-