Synthesis and biological screening of some novel amidocarbamate derivatives of ketoprofen

Article abstract of DOI:10.1016/j.ejmech.2010.04.008

A series of novel ketoprofen derivatives 4a-j bearing both amide and carbamate functionalities were prepared using benzotriazole. Selective reduction of ketoprofen produced hydroxy derivative 2, which reacts with one or 2 mol of 1-benzotriazole carboxylic acid chloride (1) gave benzotriazole derivatives 3a and 3b respectively. Antioxidative screenings revealed that the prepared compounds 3b and 4a-j possess excellent lipid peroxidation inhibition at 0.1 mM concentration. Two of the compounds 3b and 4g also showed high soybean lipoxygenase inhibition activity, where as the amidocarbamate derivatives of ketoprofen showed only weak reducing activity against 1,1-diphenyl-2- picrylhydrazyl radicals. No selective antiviral effects were noted for the tested compounds against a broad variety of DNA and RNA viruses.

Full text of DOI:10.1016/j.ejmech.2010.04.008

European Journal of Medicinal Chemistry 45 (2010) 3162e3168
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological screening of some novel amidocarbamate derivatives
of ketoprofen
,
b
Prasanta Kumar Sahoo a , Pritishova Behera
*
a Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Tadepalligudem, West Godavari District 534101, Andhra Pradesh, India
b K. G. R. L. College of Pharmacy, Bhimabharam, West Godavari, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel ketoprofen derivatives 4aej bearing both amide and carbamate functionalities were
prepared using benzotriazole. Selective reduction of ketoprofen produced hydroxy derivative 2, which
reacts with one or 2 mol of 1-benzotriazole carboxylic acid chloride (1) gave benzotriazole derivatives 3a
and 3b respectively. Antioxidative screenings revealed that the prepared compounds 3b and 4aej
possess excellent lipid peroxidation inhibition at 0.1 mM concentration. Two of the compounds 3b and
4g also showed high soybean lipoxygenase inhibition activity, where as the amidocarbamate derivatives
of ketoprofen showed only weak reducing activity against 1,1-diphenyl-2-picrylhydrazyl radicals. No
selective antiviral effects were noted for the tested compounds against a broad variety of DNA and RNA
viruses.
Received 15 September 2009
Received in revised form
30 March 2010
Accepted 7 April 2010
Available online 14 April 2010
Keywords:
Ketoprofen
Amidocarbamate
Antioxidant
Peroxidation
Lipoxygenase
Antiviral
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
2. Chemistry
Ketoprofen (Ket) is a non-steroidal anti-inflammatory drug
(NSAID) with pronounced analgesic and antipyretic properties.
Various ketoprofen derivatives have been synthesized in order to
minimize side-effects, prolong plasma half-life and increase solu-
bility [1,2]. Some amides have proved to be useful prodrugs, while
the others possess anti-inflammatory activity independent of the
parent compound. It has been demonstrated that amidation of
NSAIDs improves selectivity towards COX-2 [3], while modification
of the carboxylic group to hydroxamic acid leads to inhibition of
both cyclooxygenase and 5-lipoxygenase, these two enzymes are
crucial in inflammatory processes [4,5]. Glycine amides of keto-
profen and several other well-known NSAIDs are significantly less
irritating to gastric mucosa, while their anti-inflammatory activities
are comparable to their parent drugs [6]. Numerous studies suggest
that NSAIDs are promising anticancer drugs and may be associated
with reduced risk of colon, lung, liver and other types of cancers
[7,8]. In this paper, a series of novel amidocarbamate derivatives of
ketoprofen 4aej was prepared, characterized and screened for their
antioxidative, cytostatic and antiviral activities.
Benzotriazolides 3a, 3b were prepared from the reduced
ketoprofen derivative and 1-benzotriazole carboxylic acid
2
chloride (1), presented in Scheme 1 [9,10]. If the reaction was
performed with 1 equivalent of chloride 1 then product 3a with
free hydroxy group was obtained in 75% yield. When the reaction
was carried out with 2 equivalents of chloride 1, the main
product was benzotriazolide 3b, in which both carboxylic and
hydroxy groups were acylated. Minor amount of product 3a was
detected, even if the reaction was performed with the excess of
chloride 1. The reaction was carried out at room temperature in
order to avoid benzotriazolide 3a polycondensation. Compounds
4aej was prepared by the reaction of benzotriazolide 3b with
2 equivalents of an appropriate amine, in the presence of 5
equivalents of triethylamine (Scheme 1). All reactions were
performed in toluene, at room temperature, for 0.5e48 h. Trie-
thylamine formed a water soluble salt with benzotriazole, a by-
product of the reaction, which was readily extracted with water.
Structures of compounds 3a, 3b and 4aej were deduced from the
analysis of their IR, 1H and 13C NMR spectra and confirmed by the
elemental analysis. The chemical shifts were consistent with
the proposed structures of the novel compounds. The physico-
chemical data of the newly synthesized compounds were pre-
sented in Table 1.
* Corresponding author.
E-mail address: psahoo.phar@gmail.com (P.K. Sahoo).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.008

P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 3162e3168
CH3 OH CH3
3163
O
OH
CH3
OHH /Pd/C(en)
OH
Bt
BtCOCl
2
TEA, toluene
THF
O
O
O
Ket
2
3a
2 BtCOCl
2 TEA, toluene
O
O
N
N
N
Bt
O
CH3
R
O
CH3
Bt =
Bt 2 NH R / 5 TEA
R
2
toluene
O
O
3b
4a-j
4a
4b
4c
4d
4e
H
N
H
N
H
N
H
N
O
O
R
R
N
CH3
O
CH3
H
4f
4g
4h
4i
4j
H
N
N
H
N
H
N
N
Scheme 1. Synthesis of compounds 4aej.
3. Pharmacological results and discussion
3.1. Biological studies
more potent lipoxygenase inhibitors than the other
amidocarbamates.
3.1.3. Inhibition of linoleic acid lipid peroxidation
3.1.1. Antioxidant activity
Azo compounds generating free radicals through spontaneous
thermal decomposition are useful for free radical production
studies in vitro. The water soluble azo compound 2,20-azobis(2-
amidinopropane) dihydrochloride (AAPH) has been extensively
used as a clean and controllable source of thermally produced
alkylperoxyl free radicals. In this study AAPH was used as a free
radical initiator to follow oxidative changes of linoleic acid to
conjugated diene hydroperoxide. The results indicated that all the
compounds are excellent inhibitors of lipid peroxidation (LP)
(54.5e99.5%), significantly higher than ketoprofen (69.3%) at
0.1 mM concentration (Table 2). This inhibition was found to be
concentration dependent.
Regression analysis of the lipid peroxidation inhibition at
100 mM revealed that the overall molar refractivity (CMR) is the
main physicochemical parameter influencing the inhibition. The
linear CMR model suggests that the compounds with high CMR
value will be more active. No correlation for lipophilicity was found.
The interaction of the examined compounds with the stable free
radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) was studied. Interaction
with DPPH indicates radical scavenging ability in an iron-free system.
Interactions were monitored after 20 and 60 min at two concentrations
of DPPH (0.05 and 0.1 mM). Ketoprofen, the prototype compound,
benzotriazolide 3b as well as all the tested compounds presented very
low interaction values. The results are presented in Table 2.
3.1.2. Soybean lipoxygenase inhibition
Compounds were further evaluated for the inhibition of soybean
lipoxygenase (LOX) by the UV absorbance based enzyme assay [11].
Lipoxygenases oxidize certain fatty acids at specific positions to
hydroperoxides, precursors of leukotrienes, which contain
a conjugated triene structure, i.e. soybean lipoxygenase converts
linoleic to 13-hydroperoxylinoleic acid. Leukotrienes play an
important role as inflammatory and allergic mediators [12].
Inhibitors of LOX have attracted attention initially as potential
agents for the treatment of inflammatory and allergic diseases but
their therapeutic potential has now been expanded to certain types
of cancer and cardiovascular diseases [13]. Most of the LOX inhib-
itors are antioxidants or free radical scavengers, since lip-
oxygenation occurs via a carbon-centered radical [14]. According to
IC50 values compound 3b is the most active as compared to
logLP% ¼ 0:047ð0:018ÞCMR ꢀ 1:317ð0:234Þ
n ¼ 11; r ¼ 0:894; r2 ¼ 0:800; q2 ¼ 0:680;
s ¼ 0:045; F1;8 ¼ 31:555; a ¼ 0:01
3.1.4. Antiviral and cytostatic evaluation
Antiviral evaluation was performed on a series of DNA and RNA
viruses: herpes simplex virus type 1 (HSV-1), HSV-2 (G), vaccinia
compounds 4g, 4d and 4f (IC50 ¼ 21e95
mM). From Table 2 it is
obvious that aromatic and cycloalkyl derivatives 4g, 4d and 4f are

3164
P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 3162e3168
Table 1
Physicochemical properties of compounds 4aej.
R
17
3
O
O
CH3
9
16
13
R
11
8
1
4
2
10
15
14
O
12
5
7
6
4a - 4j
Compd.
No.
R
Molecular Molecular Elemental
formula weight analysis data
1H and 13C NMR (DMSO-d6,
d/ppm, J/Hz)
C
H
N
11.22 (s, 1H, NH carbamate), 10.65 (s, 1H, NH amide), 7.39e7.21 (m, 9H, arom.), 6.71
(s, 1H, 10), 3.58 and 3.51 (2s, 3H, 10),a 3.38 (q, 1H, 2, J ¼ 6.79), 1.31 (d, 3H, 3, J ¼ 7.02)
170.31 (17), 156.20 (1), 142.14, 141.10, 140.98 (4, 8, 11), 128.97, 128.27, 127.21,
126.96, 125.68, 125.42 (5e7, 9, 12e16), 77.15 (10), 64.03 and 63.49 (10),a 42.49 (2),
18.63 (3)
11.09 (s, 1H, NH carbamate), 10.54 (s, 1H, NH amide), 7.36e7.21 (m, 9H, arom.), 6.69
(s, 1H, 10), 3.81e3.66 (m, 3H, 2, 10) and 3.42e3.33 (m, 2H, 10),a 1.30 (d, 3H, 3,
J ¼ 6.99), 1.15e1.04 (m, 6H, 20) 170.37 (17), 156.30 (1), 142.25, 141.13, 141.07 (4, 8,
11), 128.95, 128.23, 127.18, 126.94, 125.62, 125.37 (6e8, 10, 13e17), 77.12 (10),
71.50 and 70.87 (10),a 42.48 (2), 18.61 (3), 13.87, 13.83 (20)a
O
1'
N
H
CH3
4a
4b
C19H22N2O5 358.34
C21H26N2O5 386.28
63.64 6.18 7.82
65.22 6.77 7.24
H
N
1'
2'
CH3
O
H
N
3'
6'
1'
11.21 (s, 1H, NH carbamate), 10.66 (s, 1H, NH amide), 7.38e7.21 (m, 19H, arom.),
6.72 (s, 1H, 10), 4.77 and 4.70 (2s, 4H, 10),a 3.41 (q, 1H, 3, J ¼ 6.96), 1.31 (d, 3H, 3,
J ¼ 6.86) 170.54 (17), 156.43 (1), 142.16, 141.08, 141.06 (4, 8, 11), 136.35, 136.27 (20),
129.37, 129.26, 128.74, 128.71 (30e70), 128.96, 128.25, 127.32, 126.96, 125.74,
125.42 (5e7, 9, 12e16), 77.92 and 77.09 (10),a 77.33 (10), 42.44 (2), 18.68 (3)
2'
4'
5'
O
4c
4d
4e
C31H30N2O5 510.64
C27H34N2O3 434.44
C29H38N2O3 462.54
72.91 5.92 5.42
74.62 7.88 6.44
75.26 8.27 6.05
7'
H
N
7.87 (d, 1H, NH carbamate, J ¼ 6.90), 7.44 (d, 1H, NH amide, J ¼ 6.90), 7.34e7.17 (m,
9H, arom.), 6.63 (s, 1H, 10), 3.98e3.87 and 3.81e3.73 (2m, 1H, 10),a 3.55 (q, 1H, 2,
J ¼ 6.96), 1.83e1.16 (m, 16H, 20e50), 1.28 (d, 3H, 3, J ¼ 7.00)
2'
1'
3'
172.86 (17), 155.26 (1), 143.12, 141.80, 141.65 (4, 8, 11), 128.81, 128.66, 127.94,
127.01, 126.91, 125.87, 125.09 (5e7, 9, 12e16), 76.39 (10), 52.61 and 50.65 (10),a
45.18 (2), 32.78 and 32.61 (20)a 32.69 (50), 23.95 and 23.91 (30),a 23.73 (40), 18.99 (3)
5'
4'
H
N
2'
7.75 (d, 1H, NH carbamate, J ¼ 7.77), 7.36e7.17 (m, 10H, arom., NH amide), 6.62 (s,
1H, 10), 3.55 (q, 1H, 2, J ¼ 6.94), 3.50e3.40 and 3.29e3.17 (2m, 1H, 10),a 1.74e1.52,
1.28e0.96 (2m, 20H, 20e60), 1.27 (d, 3H, 3, J ¼ 7.01) 172.42 (17), 154.96 (1), 143.16,
141.80, 141.66 (4, 8, 11), 128.78, 128.63, 127.91, 127.01, 126.92, 125.81, 125.10 (5e7,
9, 12e16), 76.37 (10), 50.01 and 47.85 (10),a 45.23 (2), 33.11 (20), 32.78 (60), 25.68 and
25.61 (30),a 25.06 (40) 24.97 (50), 18.95 (3)
1'
3'
4'
6'
5'
4'
7.86 (t, 1H, NH carbamate, J ¼ 5.48), 7.44e7.16 (m, 10H, arom., NH amide), 6.61 (s,
1H, 10), 3.59 (q, 1H, 2, J ¼ 6.88), 2.87e2.80 (m, 4H, 10), 1.72e1.53 (m, 10H, 20, 30, 70),
1.41e1.28, 1.20e1.04, 0.88e0.72 (3m, 12H, 40e60), 1.29 (d, 3H, 3, J ¼ 7.00) 173.42
(17), 155.93 (1), 143.10, 141.78, 141.59 (4, 8, 11), 128.80, 128.62, 127.96, 127.02,
125.85, 125.10 (5e7, 9, 12e16), 76.48 (10), 47.07 and 45.25 (10),a 45.45 (2), 38.13 and
37.86 (20),a 30.80 and 30.77 (30),a 30.71 (70), 26.49, 25.83 (40e60), 19.02 (3)
3'
2'
5'
H
4f
C31H42N2O3 490.54
75.85 8.64 5.68
6'
N
7'
1'
4'
8.49e8.44 (dd, 1H, NH carbamate, J ¼ 3.71, J ¼ 5.25), 8.04e8.00 (m, 1H, NH amide),
7.38e7.13 (m, 19H, arom.), 6.67 (s, 1H, 10), 4.24e4.19 (m, 4H, 10), 3.66 (q, 1H, 2,
J ¼ 6.83), 1.34 (d, 3H, 3, J ¼ 6.90)
3'
2'
5'
6'
H
N
4g
C31H30N2O3 478.52
77.78 6.31 5.85
173.54 (17), 156.10 (1), 142.92, 141.68, 141.53 (4, 8, 11), 140.07, 139.92 (20), 128.88,
128.77, 128.05, 127.25, 126.99, 126.09, 125.26 (5e7, 9, 12e16), 128.73, 128.69,
127.51, 127.40, 127.15 (30e70), 76.82 (10), 45.51 (2), 44.27 and 42.51 (10),a 19.07 (3)
7'
1'

P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 3162e3168
3165
Table 1 (continued)
Compd.
No.
R
Molecular Molecular Elemental
formula weight analysis data
1H and 13C NMR (DMSO-d6,
d/ppm, J/Hz)
C
H
N
8.01 (t, 1H, NH carbamate, J ¼ 5.05), 7.54 (t, 1H, NH amide, J ¼ 5.48), 7.34e7.08 (m,
19H, arom.), 6.64 (s, 1H, 10), 3.55 (q, 1H, 2, J ¼ 6.88), 3.28e3.20 (m, 4H, 10), 2.66e2.50
(m, 4H, 20), 1.28 (d, 3H, 3, J ¼ 7.16)
H
N
4'
7'
3'
1'
5'
2'
4h
4i
C33H34N2O3 506.48
6'
78.20 6.76 5.53
173.40 (17), 155.73 (1), 142.94, 141.71, 141.54 (4, 8, 11), 139.87, 139.70 (30), 128.83,
127.98, 127.12, 127.00, 125.97, 125.19 (5e7, 9, 12e16), 129.10, 128.75, 128.68,
126.53, 126.44 (40e80), 76.54 (10), 45.47 (2), 42.39 and 40.73 (10),a 35.79 and 35.49
(20),a 19.10 (3)
8'
1'
7.39e7.17 (m, 9H, arom.) 6.67 (s, 1H, 10), 3.87 (q, 1H, 2, J ¼ 6.62), 3.57e3.46,
3.30e3.14 and 3.03e2.95 (3m, 10, 40),a 1.89e1.62 (m, 8H, 20, 30), 1.27 (d, 3H, 3,
J ¼ 6.77) 171.51 (17), 153,46 (1), 142.55, 142.04, 141.81 (4, 8, 11), 129.15, 128.91,
127.99, 127.04, 126.79, 125.94, 125.10, (5e7, 9, 12e16), 76.85 (10), 46.45 and 46.18
(10),a 46.07 and 46.00 (40),a 43.71 (2), 25.98 and 25.72 (20),a 24.90 and 24.16 (30)a,
20.31 (3)
N
2'
C25H30N2O3 406.46
73.83 7.44 6.88
74.58 7.85 6.42
4'
3'
7.34e7.17 (m, 9H, arom.), 6.66 (d, 1H, 10, J ¼ 2.07), 4.05 (q, 1H, 2, J ¼ 6,65),
3.74e3.70, 3.59e3.43 and 3.21e3.01 (3m, 8H, 10, 50),a 1.59e1.31, 1.18e1.07,
0.62e0.48 (3m, 12H, 20e40), 1.24 (dd, 3H, 3, J ¼ 3.15, J ¼ 3.59) 171.04 (17), 153,87 (1),
143.37, 142.06, 141.64 (4, 8, 11), 129.25, 128.90, 127.99, 127.08, 126.87, 126.66,
125.36, 125.16, 124.64 (5e7, 9, 12e16), 77.28 (10), 46.27 and 44.89 (10),a 42.81
and 42.79 (50),a 41.85 (2), 25.76 and 25.73 (20),a 25.51 and 24.40 (30),a 24.24 (40),a
21.10 (3)
1'
4'
2'
3'
N
4j
C27H34N2O3 434.53
5'
a
The first signals belong to the carbamate 10, 20, 30, 40 or 50 atoms, while the second signals belong to amide 10, 20, 30, 40 or 50 atoms.
virus, vesicular stomatitis virus, parainfluenza-3, reovirus-1, Sind-
bis, Coxsackie B4, Punta Toro virus, vesicular stomatitis virus,
Coxsackie virus B4, respiratory syncytial virus, influenza A (H1N1;
H3N2), influenza B, HIV-1(IIIB) and HIV-2(ROD). No selective anti-
viral effects were noted for any of the tested compounds against
any of the viruses evaluated at subtoxic compound concentrations
(data not shown). Only 4g showed minor antiviral activity against
vesicular stomatitis virus, Coxsackie virus B4 and Punta Toro virus.
However, the activity was found at compound concentrations close
to their cytostatic activities, pointing to a toxic rather than a specific
antiviral effect. The compounds have also been evaluated for their
cytostatic activity against murine leukemia L1210, murine
mammary carcinoma FM3A and human T-lymphoblast CEM cell
cultures. The 50% inhibitory concentrations of the test compounds
substituents on the core structure for cytostatic activity, as long as
a bulky lipophilic (cyclic) entity has been present. Also, the pres-
ence of the amide groups might play an important role to even-
tually exert cytostatic potential.
4. Conclusions
A series of novel ketoprofen amidocarbamate derivatives 4aej
were prepared and screened for antioxidative, antiviral and cyto-
static activities. Antioxidative screenings revealed that the
prepared compounds possess excellent lipid peroxidation inhibi-
tion. Compounds 3b and 4g also showed high soybean lip-
oxygenase inhibition activity. No selective antiviral effects were
noted for the tested compounds against a broad variety of DNA and
RNA viruses. Most compounds showed a moderate cytostatic
activity.
ranked between 2.7
compound and the tumor cell line evaluated (Table 3). The majority
of the compounds show IC50 values around 10e25 M (i.e. 4c, 4d,
mM and 422 mM depending on the nature of the
m
4e, 4f, 4g, 4h and 4j), pointing to a relatively minor role of the R-
Table 2
Interaction with DPPH, in vitro inhibition of soybean lipoxygenase (LOX) and lipid peroxidation (LP).
Compd.
DPPH 20 mina (%) DPPH 0 mina (%) DPPH 20 minb (%) DPPH 60 minb(%) LOX Inhibitionb (%) LP Inhibitionc (%) LP Inhibitionb (%) clog Pd CMRd
3b
4a
4b
4c
4d
4e
4f
4g
4h
4i
2.9
n.a.e
2.5
3.4
3.2
2.0
1.7
4.2
3.6
5.7
8.6
3.7
4.1
3.7
5.6
2.8
3.1
3.1
5.3
8.5
6.5
10
2.5
1.8
2.3
n.a.
n.a.
n.a.
1.6
n.a.
n.a.
2.6
1.7
8.1
n.d.
93
5.4
3.2
1.1
n.a.
2.3
n.a.
5.2
2.4
2.2
2.7
4.3
7.2
n.d.
97
95.0f
26.9
22.3
40.8
69.6f
22.7
56.6f
83.8f
18.9
12.7
n.a.
15.0
2.5
n.a.
49.2
n.a.
5.4
98.0
61.0
54.5
99.3
95.2
96.1
99.5
98.4
99.0
77.2
97.4
69.3
n.d.
6.05
2.17
3.23
5.71
4.76
5.88
7.12
4.33
6.01
4.70
5.81
n.d.
14.16
9.71
10.64
14.74
12.76
13.69
14.62
14.43
15.36
11.84
12.76
n.d.
1.4
n.a.
22.5
16.4
25.5
n.d.
n.d.
n.d.
n.d.
4j
Ketoprofen 6.4
Caffeic acid n.d.
NDGA
Trolox
3.1
n.d.
83
n.d.g
600
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
81
n.d.
n.d.
n.d.
n.d.
63
n.d.
n.d.
n.d.
a
Concentrations of the tested compounds: 5 ꢁ 10ꢀ5 M.
Concentrations of the tested compounds: 1 ꢁ 10ꢀ4 M.
Concentrations of the tested compounds: 1 ꢁ 10ꢀ5 M.
Theoretically calculated values.
No activity under the reported experimental conditions.
IC50 value was also determined: 21 (3b), 86 (4d), 95 (4f), 32 (4g), 130 (Ket) mM.
b
c
d
e
f
g
Not determined.

3166
P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 3162e3168
Table 3
5.1.4. 2-(3-(Hydroxy(phenyl)methyl)phenyl)propanoic acid
benzotriazolide (3a)
Cytostatic activity of the test compounds in cell cultures.
Compd.
IC50
(
m
M)a
From the reaction of 2, chloride 1 (1.696 g, 10 mmol), and trie-
thylamine (1.393 mL, 10 mmol) in dry toluene (2 ꢁ 20 mL). 2.681 g
(75%) of 3a was obtained; mp 96e99 ꢂC; IR (KBr): nmax 3342, 3072,
3027, 3003, 2942, 2874, 1738, 1060, 1597, 1486, 1452, 1376, 959, 771,
L1210
FM3A
CEM
3b
4a
4b
4c
4d
4e
4f
4g
4h
4i
38 ꢃ 1
334 ꢃ 76
262 ꢃ 15
9.7 ꢃ 0.2
9.8 ꢃ 0.2
9.6 ꢃ 0.1
11 ꢃ 0
194 ꢃ 13
10 ꢃ 128
275 ꢃ 70
11 ꢃ 1
12 ꢃ 1
15 ꢃ 1
n.a.b
14 ꢃ 1
13 ꢃ 1
35 ꢃ 5
8.0 ꢃ 5.1
67 ꢃ 31
422 ꢃ 110
313 ꢃ 25
14 ꢃ 0
12 ꢃ 2
15
751, 746, 710 cmꢀ1; 1H NMR (DMSO-d6)
d 8.26e8.21 (m, 2H, arom.),
7.80e7.75 (m, 1H, arom.), 7.62e7.57 (m, 1H, arom.), 7.52e7.50 (m,
1H, arom.), 7.33e7.14 (m, 8H, arom.), 5.88 (d, 1H, 11, J ¼ 4.00 Hz),
5.65 (d, 1H, 10, J ¼ 3.83 Hz), 5.32 (q,1H, 2, J ¼ 6.89 Hz), 1.63 (d, 3H, 3,
n.a.
J ¼ 6.94 Hz); 13C NMR (DMSO-d6)
d 173.50 (1), 146.82, 145.88,
10 ꢃ 1
2.7 ꢃ 0.3
145.83, 140.01, 131.18 (4, 8, 12, 10, 60), 131.39, 129.08, 128.45, 127.14,
127.02, 126.66, 126.63, 126.58, 125.84, 120.53, 114.44 (5e7, 9, 13e17,
20e50), 74.49 (10), 45.12 (2), 19.07 (3). Anal. (C22H19N3O2) C, H, N.
8.2 ꢃ 1.0
41 ꢃ 3
14
42 ꢃ 0
13 ꢃ 6
4j
15 ꢃ 2
a
Compound concentration required to inhibit tumor cell proliferation by 50%.
No activity under the reported experimental conditions.
b
5.1.5. 2-(3-(N-Benzotriazolcarbonyloxy)(phenyl)(methyl)phenyl)
propanoic acid benzotriazolide (3b)
5. Experimental
From the reaction of 2, chloride 1 (5.080 g, 28 mmol) and trie-
thylamine (3.9 mL, 28 mmol) in dry toluene (2 ꢁ 50 mL) 3.52 g
(70%) of 3b was obtained; mp 124e127 ꢂC; IR (KBr): nmax 3091,
3032, 2980, 2937, 1764, 1732, 597, 1486, 1451, 1398, 1250, 1036, 951,
5.1. Chemistry
Melting points were determined on a Stuart Melting Point
Apparatus SMP3 (Lennox, Ireland) and were uncorrected. IR spectra
were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer
(SE Source, Canada). 1H and 13C NMR spectra were recorded on
a Varian Gemini 300 spectrometer (Lab X, Canada), operating at
300 and 75.5 MHz for the 1H and 13C nuclei, respectively. Samples
were measured in DMSO-d6 solutions at 20 ꢂC in 5-mm NMR tubes.
781, 760, 748, 708, 583 cmꢀ1 1H NMR (DMSO-d6)
; d 8.28e7.98
(m, 3H, arom.), 7.78e7.28 (m, 15H, arom.), 7.24 (s, 1H, 10), 5.38
(q, 1H, 2, J ¼ 6.84 Hz), 1.6600(d, 3H, 3, J ¼ 6.94 Hz); 13C NMR (DMSO-
d6)
d 173.28 (1), 147.91 (1 ), 145.80, 145.77, 140.72, 140.02, 139.17,
131.73 (4, 8, 11, 10, 60, 200, 700), 131.36, 131.11, 129.83, 129.20, 128.29,
127.36, 127.23, 126.97, 126.79, 126.55, 126.38, 120.70, 120.45, 114.39,
113.67 (5e7, 9, 12e16, 20e50, 300e600), 81.48 (10), 45.04 (2), 18.98 (3).
Anal. (C29H22N6O3) C, H, N.
Chemical shifts (d) were referred to TMS. Coupling constants (J) are
given in Hz. Elemental analysis was determined on CHN-LECO-932
(Choice Analytical, USA) and the elemental composition of the
compounds agreed within ꢃ0.4%. For thin-layer chromatography,
precoated Merck silica gel 60 F254 and solvent system cyclo-
hexane/ethyl acetate/methanol (3:1:0.5) were used. Spots were
visualized by short-wave UV light and iodine vapor. Column chro-
matography was performed on Merck silica gel 0.063e0.200 mm
with cyclohexane/ethyl acetate (1:2, 2:1 / 1:1, 1:1) as eluents.
Benzotriazole, triphosgene, triethylamine, cyclopentylamine,
cyclohexylamine, cyclohexanemethylamine, benzylamine, 2-
phenylethylamine, pyrrolidine, piperidine, O-methylhydroxyl-
amine hydrochloride, O-benzylhydroxylamine hydrochloride, eth-
ylenediamine, 10% Pd/C, DPPH, AAPH, nordihydroguaiaretic acid
(NDGA), sodium linoleate, soybean lipoxygenase, caffeic acid and
trolox were purchased from SigmaeAldrich. O-ethylhydroxylamine
hydrochloride was obtained from E. Merck, India, CDH, s.d. Fine
Chem. and Qualigens, India. Ketoprofen was obtained as a gift
sample from Ranbaxy Laboratories Ltd., India. All solvents were of
analytical grade and were dried prior to use.
5.1.6. Amidocarbamates 4aej. General procedure
A solution of benzotriazolide 3b (0.5 mmol), the appropriate
amine (1.1 mmol) and triethylamine (0.348 mL, 2.5 mmol) in
toluene (5 mL) was stirred at room temperature for 30 mine48 h.
The reaction mixture was extracted with brine (5 ꢁ 10 mL),
hydrochloric acid (w ¼ 1%, 1 ꢁ 5 mL) and washed with water till pH
7. After drying (anhydrous sodium sulphate) and evaporation of the
solvent, the crude product was obtained.
5.1.7. (3-(1-(Methoxycarbamoil)ethyl)phenyl)(phenyl)methyl
methoxycarbamate (4a)
To the product 3b, O-methylhydroxylamine hydrochloride
(0.092 g, 0.5 mmol) and triethylamine was added at room
temperature and stirred for 10 h. After purification by the column
chromatography (eluent cyclohexane/ethyl acetate 1:2) 0.143 g
(80%) of 4a was obtained; oil; IR (film): nmax 3455, 3217, 3065, 2978,
2938,1725, 1668,1606, 1489, 1454, 1254, 1115, 1043, 705 cmꢀ1. Anal.
(C19H22N2O5) C, H, N.
5.1.1. 1-Benzotriazole carboxylic acid chloride (BtCOCl, 1)
5.1.8. (3-(1-(Ethoxycarbamoil)ethyl)phenyl)(phenyl)methyl
ethoxycarbamate (4b)
BtCOCl was prepared from benzotriazole and triphosgene [9].
To the product 3b, O-ethylhydroxylamine hydrochloride
(0.107 g, 0.5 mmol) and triethylamine was added at room
temperature and stirred for 25 h. After purification by the column
chromatography (eluent cyclohexane/ethyl acetate 2:1 / 1:1)
0.140 g (73%) of 4b was obtained; oil; IR (film): nmax 3454, 3219,
3064, 2981, 2937, 2891, 1724, 1715, 1668, 1606, 1494, 1454, 1384,
1253, 1113, 1041, 704 cmꢀ1. Anal. (C21H26N2O5) C, H, N.
5.1.2. 2-(3-(Hydroxy(phenyl)methyl)phenyl)propanoic acid (2)
2-(3-(Hydroxy(phenyl)methyl)phenyl)propanoic acid (2) was
prepared by the catalytic hydrogenation of ketoprofen using H2/Pd/
C(en)/tetrahydrofurane [15].
5.1.3. Benzotriazolides 3a, b. General procedure
To a solution of 2 (2.561 g, 10 mmol) and triethylamine (10 or
28 mmol) in dry toluene (20 or 50 mL), a solution of chloride 1 (10 or
28 mmol) in dry toluene (20 or 50 mL) was added dropwise (15 min).
The reaction mixture was stirred at room temperature for 60 min
and extracted 4 times with water. The organic layer was dried over
anhydrous sodium sulphate, filtrated and evaporated. Thus obtained
crude products were purified by trituration with ether.
5.1.9. (3-(1-(Benzyloxycarbamoil)ethyl)phenyl)(phenyl)methyl
benzyloxycarbamate (4c)
To the product 3b, O-benzylhydroxylamine hydrochloride
(0.175 g, 0.5 mmol) and triethylamine was added at room
temperature and stirred for 48 h. After purification by the column
chromatography (eluent cyclohexane/ethyl acetate 1:1) 0.115 g

P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 3162e3168
3167
(45%) of 4c was obtained; mp 42e45 ꢂC; IR (KBr): nmax 3215, 3064,
5.1.17. Interaction with DPPH
3032, 2974, 2936,1722,1665,1605,1495,1454,1249,1107,1027, 749,
To a solution of DPPH (0.05 mM) in absolute ethanol an
equal volume of 0.1 or 0.05 mM ethanolic solution of the tested
compound was added [11]. After 20 and 60 min the absorbance
was recorded at 517 nm and compared to the appropriate
standard NDGA (Table 2). Ethanol was used as a control. Each in
vitro experiment was performed at least in triplicate and the
standard deviation of absorbance was less than 10% of the
mean.
699 cmꢀ1. Anal. (C31H30N2O5) C, H, N.
5.1.10. (3-(1-(Cyclopentylcarbamoil)ethyl)phenyl)(phenyl)methyl
cyclopentylcarbamate (4d)
To the product 3b, cyclopentylamine (0.109 mL, 0.5 mmol) and
triethylamine was added at room temperature and stirred for 1 h.
After trituration with ether 0.167 g (77%) of 4d was obtained; mp
132e135 ꢂC; IR (KBr): nmax 3305, 3269, 3065, 2962, 2870, 1700,
1651, 1606, 1545, 1452, 1249, 1040, 1017, 702 cmꢀ1
.
Anal.
5.1.18. Soybean lipoxygenase inhibition
(C27H34N2O3) C, H, N.
DMSO solution of the tested compound was incubated with
sodium linoleate (0.1 mM) and 0.2 mL of soybean lipoxygenase
solution (1/9 ꢁ 10ꢀ4 w/v in saline) at room temperature [11]. The
conversion of sodium linoleate to 13-hydroperoxylinoleic acid was
recorded at 234 nm and compared to the standard inhibitor caffeic
acid.
5.1.11. (3-(1-(Cyclohexylcarbamoil)ethyl)phenyl)(phenyl)methyl
cyclohexylcarbamate (4e)
To the product 3b, cyclopentylamine (0.126 mL, 0.5 mmol) and
triethylamine was added at room temperature and stirred for 5 h.
After trituration with ether 0.173 g (75%) of 4e was obtained; mp
139e142 ꢂC; IR (KBr): nmax 3303, 3265, 3066, 2933, 2854, 1695,
1650, 1603, 1547, 1450, 1235, 1042, 702 cmꢀ1. Anal. (C29H38N2O3) C,
H, N.
5.1.19. Inhibition of linoleic acid lipid peroxidation
Oxidation of linoleic acid to conjugated diene hydroperoxide
in an aqueous dispersion is monitored at 234 nm [16]. AAPH was
used as a free radical initiator. Ten microliters of the 16 mM
linoleic acid dispersion was added to the UV cuvette containing
0.93 mL of 0.05 M phosphate buffer, pH 7.4 prethermostated at
37 ꢂC. The oxidation reaction was initiated at 37 ꢂC under air by
5.1.12. (3-(1-(Cyclohexanemethylcarbamoil)ethyl)phenyl)(phenyl)
methyl cyclohexanemethylcarbamate (4f)
To the product 3b, cyclohexanemethylamine (0.143 mL,
0.5 mmol) and triethylamine was added at room temperature and
stirred for 30 min. After trituration with ether 0.184 g (75%) of 4f
was obtained; mp 128e129 ꢂC; IR (KBr): nmax 3340, 3278, 3064,
2922, 2851, 1707, 1654, 1604, 1551, 1449, 1249, 702 cmꢀ1. Anal.
(C31H42N2O3) C, H, N.
the addition of 50
carried out in the presence of compounds (10
m
L of 40 mM AAPH solution. Oxidation was
L, final concen-
m
tration 0.1 mM). In the assay with no antioxidant lipid oxidation
was measured in the presence of the same level of DMSO. The
rate of oxidation was monitored at 37 ꢂC by recording the
increase of absorption at 234 nm caused by conjugated diene
hydroperoxides. The results were compared to the standard
inhibitor trolox.
5.1.13. (3-(1-(Benzylcarbamoil)ethyl)phenyl)(phenyl)methyl
benzylcarbamate (4g)
To the product 3b, benzylamine (0.120 mL, 0.5 mmol) and
triethylamine was added at room temperature and stirred for
30 min. After trituration with ether 0.127 g (53%) of 4 g was
obtained; mp 107e109 ꢂC; IR (KBr): nmax 3316, 3267, 3087, 3063,
3032, 2932, 1684, 1641, 1606, 1551, 1519, 1454, 1248, 699 cmꢀ1. Anal.
(C31H30N2O3) C, H, N.
5.1.20. Antiviral and cytostatic activity assays
Murine leukemia L1210, murine mammary carcinoma FM3A
and human T-lymphocyte CEM cells were suspended at
300,000e500,000 cells/mL of culture medium, and 100
mL of a cell
suspension was added to 100 L of an appropriate dilution of the
m
test compounds in wells of 96-well microtiter plates. After incu-
bation at 37 ꢂC for two (L1210, FM3A) or three (CEM) days, the cell
number was determined using a Coulter counter. The IC50 was
defined as the compound concentration required to inhibit cell
proliferation by 50%.
The antiviral assays, other than the anti-HIV assays, were based
on inhibition of virus-induced cytopathicity in HEL [herpes simplex
virus type 1 (HSV-1), HSV-2 (G), vaccinia virus and vesicular
stomatitis virus], Vero (parainfluenza-3, reovirus-1, Sindbis, Cox-
sackie B4, and Punta Toro virus), HeLa (vesicular stomatitis virus,
Coxsackie virus B4, and respiratory syncytial virus) or MDCK
5.1.14. (3-(1-(Phenylethylcarbamoil)ethyl)phenyl)(phenyl)methyl
phenylethylcarbamate (4h)
To the product 3b, phenylethylamine (0.139 mL, 0.5 mmol) and
triethylamine was added at room temperature and stirred for
35 min. After trituration with ether 0.190 g (75%) of 4h was
obtained; oil; IR (KBr): nmax 3417, 3313, 3063, 3028, 2972, 2932,
2872, 1713, 1699, 1660, 1650, 1604, 1538, 1517, 1496, 1454, 1248,
1030, 749, 700 cmꢀ1. Anal. (C33H34N2O3) C, H, N.
5.1.15. (3-(1-Oxo-1-(pyrrolidin-1-yl)propan-2-yl)phenyl)(phenyl)
methyl pyrrolidine-1-carboxylate (4i)
(influenza
A (H1N1; H3N2); and influenza B) cell cultures.
To the product 3b, pyrrolidine (0.092 mL, 0.5 mmol) and trie-
thylamine was added at room temperature and stirred for 30 min.
After evaporation 0.152 g (75%) of 4i was obtained; oil; IR (film):
nmax 3061, 3030, 2973, 2875, 1700, 1643, 1634, 1588, 1454, 1416,
1126, 1096, 764, 709 cmꢀ1. Anal. (C25H30N2O3) C, H, N.
Confluent cell cultures in microtiter 96-well plates were inoculated
with 100 CCID50 of virus (CCID50 being the virus dose to infect 50%
of the cell cultures). After a 1 h virus adsorption period, residual
virus was removed, and the cell cultures were incubated in the
presence of varying concentrations (200, 40, 8
mM) of the test
compounds. Viral cytopathicity was recorded as soon as it reached
completion in the control virus-infected cell cultures that were not
treated with the test compounds. The methodology of the anti-HIV
assays was as follows: human CEM (w3 ꢁ 105 cells/mL) cells were
infected with 100 CCID50 of HIV-1(IIIB) or HIV-2(ROD)/mL and
5.1.16. (3-(1-Oxo-1-(piperidin-1-yl)propan-2-yl)phenyl)(phenyl)
methyl piperidine-1-carboxylate (4j)
To the product 3b, piperidine (0.109 mL, 0.5 mmol) and trie-
thylamine was added at room temperature and stirred for 45 min.
After trituration with ether 0.179 g (80%) of 4j was obtained; mp
106e109 ꢂC; IR (KBr): nmax 3050, 3028, 2971, 2940, 2852, 1694,
seeded in 200 mL wells of a microtiter plate containing appropriate
dilutions of the test compounds. After 4 days of incubation at 37 ꢂC,
HIV-induced CEM giant cell formation was examined
microscopically.
1628, 1587, 1469, 1426, 1258, 1236, 1148, 1083, 1026, 707, 699 cmꢀ1
.
Anal. (C27H34N2O3) C, H, N.

3168
P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 3162e3168
[6] V.R. Shanbhag, A.M. Crider, R. Gokhale, A. Harpalani, R.M. Dick, J. Pharm. Sci.
Acknowledgements
81 (1992) 149.
[7] N.R. Sivak-Sears, J.A. Schwartzbaum, R. Miike, M. Moghadassi, M. Wrensch,
Am. J. Epidemiol. 159 (2004) 1131.
The authors are thankful to the Management and Principal for
providing the research facilities to pursue this work.
[8] H. Xiao, C.S. Yang, Int. J. Cancer 123 (2008) 983.
[9] I. Butula, M.T.M. Jadrijevic, Croat. Chem. Acta 73 (2000) 569.
[10] B. Zorc, S. Antolic, I. Butula, Acta. Pharm. 43 (1993) 127.
[11] E. Pontiki, D. Hadjipavlou-Litina, Bioorg. Med. Chem. 15 (2007) 5819.
[12] H. Kühn, J. Belkner, R. Wiesner, A.R. Brash, J. Biol. Chem. 265 (1990)
18351.
[13] E. Pontiki, D. Hadjipavlou-Litina, Curr. Enz. Inh. 1 (2005) 309.
[14] K. Muller, Arch. Pharm. 327 (1994) 3.
[15] M. Allegretti, R. Bertini, M.C. Cesta, C. Bizzarri, R. Bitondo, V. Di Cioccio,
E. Galliera, V. Berdini, A. Topai, G. Zampella, V. Russo, N. Di Bello, G. Nano,
L. Nicolini, M. Locati, P. Fantucci, S. Florio, F. Colotta, J. Med. Chem. 48 (2005)
4312.
References
[1] F. Bonina, C. Puglia, N.A. Santagati, A. Saija, A. Tomaino, B. Tita, Pharmazie 57
(2002) 552.
[2] F. Bonina, N.A. Santagati, C. Puglia, Drug Dev. Ind. Pharm. 29 (2003) 181.
[3] A.S. Kalgutkar, B.C. Crews, S.W. Rowlinson, A.B. Marnett, K.R. Kozak, R.
P. Remmel, L.J. Marnett, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 925.
[4] D.L. Flynn, T. Capiris, W.J. Cetenko, D.T. Connor, R.D. Dyer, C.R. Kostlan, D.
E. Nies, D.J. Schrier, J.C. Sircar, J. Med. Chem. 33 (1990) 2070.
[5] E.M.F. Muri, M.J. Nieto, R.D. Sindelar, J.S. Williamson, Curr. Med. Chem. 9
(2002) 1631.
[16] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free.
Radic. Biol. Med. 26 (1999) 1231.