The novel amidocarbamate derivatives of ketoprofen: Synthesis and biological activity

Article abstract of DOI:10.1007/s00044-010-9309-2

A series of novel ketoprofen derivatives 4a-j bearing both amide and carbamate functionalities were prepared using the benzotriazole method of carboxylic and hydroxy group activation. Selective reduction of ketoprofen produced hydroxy derivative 2, which

Full text of DOI:10.1007/s00044-010-9309-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:210–219
DOI 10.1007/s00044-010-9309-2
ORIGINAL RESEARCH
The novel amidocarbamate derivatives of ketoprofen: synthesis
and biological activity
•
Zrinka Rajic Dimitra Hadjipavlou-Litina
•
´
Eleni Pontiki Jan Balzarini Branka Zorc
•
•
Received: 16 July 2009 / Accepted: 13 January 2010 / Published online: 3 February 2010
ꢀ Springer Science+Business Media, LLC 2010
Abstract A series of novel ketoprofen derivatives 4a–
j bearing both amide and carbamate functionalities were
prepared using the benzotriazole method of carboxylic and
hydroxy group activation. Selective reduction of ketopro-
fen produced hydroxy derivative 2, which in the reaction
with one or two moles of 1-benzotriazole carboxylic acid
chloride (1) gave benzotriazole derivatives 3a and 3b,
respectively. Compounds 3a and 3b with various amines
afforded amidocarbamates 4a–j. Antioxidative screenings
revealed that the prepared compounds 3b and 4a–j possess
excellent lipid peroxidation inhibition at 0.1 mM concen-
tration, higher than 95% for the derivatives bearing aro-
matic, cycloalkyl or heterocyclic substituents. Two of the
compounds, 3b and 4g, also show high soybean lipoxy-
genase inhibition activity (95 and 83.5%, respectively). On
the other hand, the amidocarbamate derivatives of keto-
profen show 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. Most compounds
were endowed with a moderate (IC50: 10–25 lM) cyto-
static activity.
Keywords Ketoprofen amide ꢀ Carbamate ꢀ Antioxidant ꢀ
Peroxidation ꢀ Lipoxygenase ꢀ Antiviral activity ꢀ
Cytostatic activity
Introduction
Ketoprofen (Ket) is a non-steroidal anti-inflammatory drug
(NSAID) with pronounced analgesic and antipyretic
properties. Numerous ketoprofen derivatives have been
synthesized in order to minimize side-effects, prolong
plasma half-life and increase solubility (Bonina et al.,
2002a, b, 2003). 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 selec-
tivity towards COX-2 (Kalgutkar et al., 2000), while
modification of the carboxylic group to hydroxamic acid
leads to inhibition of both cyclooxygenase and 5-lipoxy-
genase, two enzymes crucial in inflammatory processes
(Flynn et al., 1990; Muri et al., 2002). Glycine amides of
ketoprofen and several other well-known NSAIDs are
significantly less irritating to gastric mucosa, while their
anti-inflammatory activities are comparable to their parent
drugs (Shanbhag et al., 1992; Singh et al., 1990). Keto-
profen glycinate methyl ester has higher anti-inflammatory
and analgesic activity than the parent drug (Dhaneshwar
and Chaturvedi, 1994). Ketoprofenamides with heterocy-
clic residues (2-thiazolinyl, 4-methylpyridyl, 3-hydroxy-
pyridyl, pyridyl, 1,5-dimethyl-2-phenylpyrazolonyl or
thiazolyl) also possess significant analgesic and anti-
inflammatory activities (Spickett et al., 1976), while
ketoprofen 2-hydroxyethylamide and ketoprofen esters
with bis-(hydroxyalkylthio)-alkanes are useful in the
treatment and prevention of atherosclerosis (Lafon, 1977).
´
Z. Rajic ꢀ B. Zorc (&)
Department of Medicinal Chemistry, Faculty of Pharmacy and
ˇ ´
Biochemistry, University of Zagreb, A. Kovacica 1,
10000 Zagreb, Croatia
e-mail: bzbz@pharma.hr
D. Hadjipavlou-Litina ꢀ E. Pontiki
School of Pharmacy, Aristotles University of Thessaloniki,
541 24 Thessaloniki, Greece
J. Balzarini
Rega Institute for Medical Research, Katholieke Universiteit
Leuven, 3000 Leuven, Belgium
123

Med Chem Res (2011) 20:210–219
211
Numerous studies suggest that NSAIDs are promising
anticancer drugs as well and may be associated with
reduced risk of colon, lung, liver and other types of cancers
(Thun et al., 2002; Sivak-Sears et al., 2004).
reaction was run at room temperature in order to avoid
benzotriazolide 3a polycondensation.
Compounds 4a–j were prepared by the reaction of
benzotriazolide 3b with two equivalents of an appropriate
amine, in the presence of five equivalents of triethylamine
(Scheme 1). All reactions were performed in toluene, at
room temperature, for 0.5–48 h. Triethylamine formed a
water soluble salt with benzotriazole, a by-product of the
reaction, which was readily extracted with water.
Structures of compounds 3a,b and 4a–j 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 (Fig. 1, Table 1).
In our previous research potency of amides and hy-
droxamic acid derivatives of ketoprofen and related
NSAIDs as cytostatic and antioxidant agents was screened
´
(Zovko et al., 2003; Marjanovic et al., 2007; Wittine et al.,
2009). Our articles and the extensive literature data
describe the effect of carboxylic group derivatization. To
our knowledge, a modification of both carboxylic and
carbonyl functionalities in ketoprofen molecule has not
been studied. In this article, a series of novel derivatives of
ketoprofen bearing amide and carbamate moieties were
prepared, characterized and screened for their antioxida-
tive, cytostatic and antiviral activities.
Biological studies
Antioxidant activity
Results and discussion
The interaction of the examined compounds with the stable
free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) was
studied. Interaction with DPPH indicates radical scaveng-
ing ability in an iron-free system. Interactions were mon-
itored 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
shown in Table 2.
Chemistry
Benzotriazolides 3a,b were prepared from the reduced
ketoprofen derivative 2 and 1-benzotriazole carboxylic
acid chloride (1), following our previously developed
´
procedure (Scheme 1) (Butula and Jadrijevic Mladar
ˇ
Takac, 2000; Zorc et al., 1993). If the reaction was per-
formed with one equivalent of chloride 1, product 3a with
free hydroxy group was obtained in 75% yield. When the
reaction was carried out with two 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 as well, even if the
reaction was performed with the excess of chloride 1. The
Soybean lipoxygenase inhibition
Compounds were further evaluated for the inhibition of
soybean lipoxygenase (LOX) by the UV absorbance
O
CH3
OH
CH3
OH
CH3
Scheme 1 Synthesis of
compounds 4a–j
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
R
NH2R / TEA
toluene
O
O
3b
4a-j
4a
4b
4c
4h
4d
4e
H
N
H
H
N
N
H
O
O
N
R
N
CH3
O
CH3
H
4f
4g
4i
4j
H
N
N
H
H
N
R
N
N
123

212
Med Chem Res (2011) 20:210–219
3''
4''
Fig. 1 Atom enumeration of
compounds 3a and 3b
2''
N
5''
N
7''
6''
N
1''
3
3
N
N
N
N
N
N
OH
CH3
O
16
O
CH3
1'
1'
2'
2'
9
9
16
13
11
11
8
1
8
1
4
4
2
2
10
10
15
14
15
14
6'
6'
3'
3'
O
O
12
7
12
7
5
5
5'
5'
4'
4'
6
6
13
3a
3b
Table 1 1H- and 13C-NMR data for compounds 4a–j
R
17
3
O
O
CH3
16
13
9
R
11
8
1
2
4
10
15
14
O
12
5
7
6
4a-j
Compd. R
1H- and 13C-NMR (DMSO-d6, d/ppm, J/Hz)
4a
11.22 (s, 1H, NH carbamate), 10.65 (s, 1H, NH amide), 7.39–7.21 (m, 9H, arom.), 6.71 (s, 1H, 10), 3.58a
O
1'
CH3
and 3.51b (2s, 3H, 10), 3.38 (q, 1H, 2, J = 6.79), 1.31 (d, 3H, 3, J = 7.02)
N
H
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
(5–7, 9, 12–16), 77.15 (10), 64.03a and 63.49b (10), 42.49 (2), 18.63 (3)
4b
4c
11.09 (s, 1H, NH carbamate), 10.54 (s, 1H, NH amide), 7.36–7.21 (m, 9H, arom.), 6.69 (s, 1H, 10),
3.81–3.66a (m, 3H, 2, 10) and 3.42–3.33b (m, 2H, 10), 1.30 (d, 3H, 3, J = 6.99), 1.15–1.04 (m, 6H, 20)
H
1'
N
2'
CH3
O
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
(6–8, 10, 13–17), 77.12 (10), 71.50a and 70.87b (10), 42.48 (2), 18.61 (3), 13.87a, 13.83b (20)
11.21 (s, 1H, NH carbamate), 10.66 (s, 1H, NH amide), 7.38–7.21 (m, 19H, arom.), 6.72 (s, 1H, 10), 4.77a
and 4.70b (2s, 4H, 10), 3.41 (q, 1H, 3, J = 6.96), 1.31 (d, 3H, 3, J = 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 (30–70), 128.96, 128.25, 127.32, 126.96, 125.74, 125.42 (5–7, 9, 12–16), 77.92a and 77.09b (10),
77.33 (10), 42.44 (2), 18.68 (3)
H
N
3'
6'
1'
2'
4'
5'
O
7'
4d
4e
7.87 (d, 1H, NH carbamate, J = 6.90), 7.44 (d, 1H, NH amide, J = 6.90), 7.34–7.17 (m, 9H, arom.), 6.63
(s, 1H, 10), 3.98–3.87a and 3.81–3.73b (2m, 1H, 10), 3.55 (q, 1H, 2, J = 6.96), 1.83–1.16 (m, 16H, 20–50),
1.28 (d, 3H, 3, J = 7.00)
H
N
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 (5–7, 9, 12–16), 76.39 (10), 52.61a and 50.65b (10), 45.18 (2), 32.78a and 32.61b (20) 32.69 (50),
23.95a and 23.91b (30), 23.73 (40), 18.99 (3)
5'
1'
4'
7.75 (d, 1H, NH carbamate, J = 7.77), 7.36–7.17 (m, 10H, arom., NH amide), 6.62 (s, 1H, 10), 3.55 (q,
1H, 2, J = 6.94), 3.50–3.40a and 3.29–3.17b (2m, 1H, 10), 1.74–1.52, 1.28–0.96 (2m, 20H, 20–60), 1.27
(d, 3H, 3, J = 7.01)
H
N
2'
3'
4'
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 (5–7, 9, 12–16), 76.37 (10), 50.01a and 47.85b (10), 45.23 (2), 33.11 (20), 32.78 (60), 25.68a and
25.61b (30), 25.06 (40) 24.97 (50), 18.95 (3)
6'
5'
123

Med Chem Res (2011) 20:210–219
213
Table 1 continued
Compd. R
1H- and 13C-NMR (DMSO-d6, d/ppm, J/Hz)
4f
4'
7.86 (t, 1H, NH carbamate, J = 5.48), 7.44-7.16 (m, 10H, arom., NH amide), 6.61 (s, 1H, 10), 3.59 (q,
1H, 2, J = 6.88), 2.87-2.80 (m, 4H, 10), 1.72-1.53 (m, 10H, 20, 30, 70), 1.41-1.28, 1.20-1.04, 0.88-0.72
(3m, 12H, 40–60), 1.29 (d, 3H, 3, J = 7.00)
3'
2'
5'
6'
H
N
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 (5–7, 9, 12–16), 76.48 (10), 47.07a and 45.25b (10), 45.45 (2), 38.13a and 37.86b (20), 30.80a and
30.77b (30), 30.71 (70), 26.49, 25.83 (40–60), 19.02 (3)
7'
4'
1'
1'
4g
4h
8.49–8.44 (dd, 1H, NH carbamate, J = 3.71, J = 5.25), 8.04-8.00 (m, 1H, NH amide), 7.38–7.13 (m,
19H, arom.), 6.67 (s, 1H, 10), 4.24–4.19 (m, 4H, 10), 3.66 (q, 1H, 2, J = 6.83), 1.34 (d, 3H, 3,
J = 6.90)
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 (5–7, 9, 12–16), 128.73, 128.69, 127.51, 127.40, 127.15 (30–70), 76.82
(10), 45.51 (2), 44.27a and 42.51b (10), 19.07 (3)
3'
2'
5'
6'
H
N
7'
8.01 (t, 1H, NH carbamate, J = 5.05), 7.54 (t, 1H, NH amide, J = 5.48), 7.34–7.08 (m, 19H, arom.),
6.64 (s, 1H, 10), 3.55 (q, 1H, 2, J = 6.88), 3.28–3.20 (m, 4H, 10), 2.66–2.50 (m, 4H, 20), 1.28 (d, 3H, 3,
J = 7.16)
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 (5–7, 9, 12–16), 129.10, 128.75, 128.68, 126.53, 126.44 (40–80), 76.54 (10),
45.47 (2), 42.39a and 40.73b (10), 35.79a and 35.49b (20), 19.10 (3)
H
4'
7'
N
3'
1'
5'
2'
8'
6'
4i
4j
7.39–7.17 (m, 9H, arom.) 6.67 (s, 1H, 10), 3.87 (q, 1H, 2, J = 6.62), 3.57–3.46, 3.30–3.14a
and 3.03–2.95b (3m, 10, 40), 1.89–1.62 (m, 8H, 20, 30), 1.27 (d, 3H, 3, J = 6.77)
1'
N
2'
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, (5–7, 9, 12–16), 76.85 (10), 46.45a and 46.18b (10), 46.07a and 46.00b (40), 43.71 (2),
25.98a and 25.72b (20), 24.90a and 24.16b (30), 20.31 (3)
7.34–7.17 (m, 9H, arom.), 6.66 (d, 1H, 10, J = 2.07), 4.05 (q, 1H, 2, J = 6,65), 3.74–3.70, 3.59–3.43a
and 3.21–3.01b (3m, 8H, 10, 50), 1.59–1.31, 1.18–1.07, 0.62–0.48 (3m, 12H, 20–40), 1.24 (dd, 3H, 3,
J = 3.15, J = 3.59)
4'
3'
1'
2'
3'
N
5'
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 (5–7, 9, 12–16), 77.28 (10), 46.27a and 44.89b (10), 42.81a and 42.79b
(50), 41.85 (2), 25.76a and 25.73b (20), 25.51a and 24.40b (30), 24.24 (40), 21.10 (3)
4'
a
Signals of the carbamate atoms
Signals of the amide atoms
b
based enzyme assay (Pontiki and Hadjipavlou-Litina,
2007). Lipoxygenases oxidize certain fatty acids at spe-
cific positions to hydroperoxides, precursors of leukotri-
enes, which contain a conjugated triene structure, i.e.
soybean lipoxygenase converts linoleic to 13-hydroper-
oxylinoleic acid. Leukotrienes play an important role as
mediators of a variety of inflammatory and allergic pro-
cesses (Ku¨hn et al., 1990). 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 (Pontiki and
Hadjipavlou-Litina, 2005). Most of the LOX inhibitors
are antioxidants or free radical scavengers, since lipoxy-
genation occurs via a carbon-centred radical (Muller,
1994). Perusal of IC50 values shows that compound 3b is
the most active by far, followed by compounds 4g, 4d
and 4f (IC50 = 21–95 lM). From Table 2 it is obvious
that aromatic and cycloalkyl derivatives 4g, 4d and 4f are
more potent lipoxygenase inhibitors than the other
amidocarbamates.
Inhibition of linoleic acid lipid peroxidation
Azo compounds generating free radicals through sponta-
neous thermal decomposition are useful for free radical
production studies in vitro. The water soluble azo com-
pound 2,20-azobis(2-amidinopropane) dihydrochloride
(AAPH) has been extensively used as a clean and con-
trollable source of thermally produced alkylperoxyl free
radicals. In our studies, 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) (54.5–99.5%), significantly higher than
123

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Med Chem Res (2011) 20:210–219
Table 2 Interaction with DPPH, in vitro inhibition of soybean lipoxygenase (LOX) and lipid peroxidation (LP)
Compd.
DPPH
DPPH
DPPH
DPPH
LOX
LP inhibitionc LP inhibitionb c log Pd CMRd
20 mina (%) 60 mina (%) 20 minb (%) 60 minb (%) inhibitionb (%) (%)
(%)
3b
4a
4b
4c
4d
4e
4f
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
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
n.a.
49.2
n.a.
5.4
10.64
14.74
12.76
13.69
14.62
14.43
15.36
11.84
12.76
n.d.
1.4
4g
4h
4i
n.a.
22.5
16.4
25.5
n.d.
n.d.
n.d.
n.d.
4j
Ketoprofen 6.4
Caffeic acid n.d.
3.1
n.d.
83
n.d.g
600
n.d.
n.d.
NDGA
Trolox
81
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
63
n.d.
n.d.
a
b
c
Concentrations of the tested compounds: 5 9 10-5 M; 1 9 10-4 M; 1 9 10-5
M
d
Theoretically calculated values
e
No activity under the reported experimental conditions
f
IC50 value was also determined: 21 (3b), 86 (4d), 95 (4f), 32 (4g), 130 (Ket) lM
g
Not determined
ketoprofen (69.3%) at 0.1 mM concentration (Table 2).
This inhibition was found to be concentration dependent.
Regression analysis of the LP inhibition at 100 lM
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.
leukaemia L1210, murine mammary carcinoma FM3A and
human T-lymphoblast CEM cell cultures. The 50% inhibi-
tory concentrations of the test compounds ranked between
2.7 and 422 lM depending on the nature of the compound
and the tumour cell line evaluated (Table 3). The majority of
the compounds show IC50 values around 10–25 lM (i.e. 4c,
4d, 4e, 4f, 4g, 4h and 4j), pointing to a relatively minor role
of the R-substituents on the core structure for cytostatic
log LP% ¼ 0:047ð0:018Þ CMR ꢁ 1:317ð0:234Þ
Table 3 Cytostatic activity of the test compounds in cell cultures
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
Antiviral and cytostatic evaluation
Compd.
IC50 (lM)a
L1210
FM3A
CEM
3b
4a
4b
4c
4d
4e
4f
4g
4h
4i
38
1
194 13
10 128
275 70
67 31
334 76
262 15
9.7 0.2
9.8 0.2
9.6 0.1
422 110
313 25
Antiviral evaluation was performed on a broad series of
DNA and RNA viruses (as listed under the ‘‘Experimental’’
section). No antiviral 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 in HeLa cell cultures (EC50: 3 lM), Sindbis virus
(EC50: 11 lM) and Punta Toro virus in Vero cell cultures
(EC50: 11 lM). However, the activity was found at com-
pound concentrations close to their cytostatic activities
(CC50: 2.7–14 lM) (Table 3), pointing to a toxic rather than
a specific antiviral effect. The compounds have also been
evaluated for their cytostatic activity against murine
11
1
1
1
14
0
2
12
12
15
n.a.b
15
11
10
0
1
n.a.
14
1
1
5
2.7 0.3
14
8.2 1.0
13
41
15
3
2
35
42
13
0
6
4j
8.0 5.1
a
Compound concentration required to inhibit tumour cell prolifera-
tion by 50%
b
No activity under the reported experimental conditions
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Med Chem Res (2011) 20:210–219
215
activity, as long as a bulky lipophilic (cyclic) entity has been
present. Also, the presence of the amide groups might play
an important role to eventually exert cytostatic potential.
using H2/Pd/C(en)/tetrahydrofurane (Hattori et al., 2001),
according to the modified published procedure (Allegretti
et al., 2003; 2005).
2-(3-(Hydroxy(phenyl)methyl)phenyl)propanoic acid
benzotriazolide (3a)
Conclusions
A series of novel ketoprofen amidocarbamate derivatives
4a–j were prepared and screened for antioxidative, anti-
viral and cytostatic activities. Antioxidative screenings
revealed that the prepared compounds possess excellent LP
inhibition. Compounds 3b and 4g also showed high soy-
bean lipoxygenase inhibition activity. No selective antivi-
ral effects were noted for the tested compounds against a
broad variety of DNA and RNA viruses. Most compounds
were endowed with a moderate cytostatic activity.
To a solution of 2 (2.561 g, 10 mmol) and triethylamine
(1.4 ml, 10 mmol) in dry toluene (20 ml), a solution of
chloride 1 (1.696 g, 10 mmol) in dry toluene (20 ml) was
added dropwise (0.25 h). The reaction mixture was stirred
at room temperature for 1 h and washed four times with
water. The organic layer was dried over anhydrous sodium
sulphate, filtrated and evaporated. Thus, obtained crude
product was purified by the trituration with ether. Yield:
2.681 g (75%); mp 96–99ꢁC; IR (KBr): mmax 3342, 3072,
3027, 3003, 2942, 2874, 1738, 1060, 1597, 1486, 1452,
1376, 959, 771, 751, 746, 710 cm-1; 1H-NMR (DMSO-d6)
d 8.26–8.21 (m, 2H, arom.), 7.80–7.75 (m, 1H, arom.),
7.62–7.57 (m, 1H, arom.), 7.52–7.50 (m, 1H, arom.),
7.33–7.14 (m, 8H, arom.), 5.88 (d, 1H, 11, J = 4.00 Hz),
5.65 (d, 1H, 10, J = 3.83 Hz), 5.32 (q, 1H, 2,
J = 6.89 Hz), 1.63 (d, 3H, 3, J = 6.94 Hz); 13C-NMR
(DMSO-d6) d 173.50 (1), 146.82, 145.88, 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
(5–7, 9, 13–17, 20–50), 74.49 (10), 45.12 (2), 19.07 (3).
Atom enumeration is given in Fig. 1. Anal. Calcd. for
C22H19N3O2: C, 73.93; H, 5.36; N, 11.76. Found: C, 73.64;
H 5.39; N, 11.39.
Experimental
Melting points were determined on a Stuart Melting Point
Apparatus SMP3 and were uncorrected. IR spectra were
recorded on a FTIR Perkin Elmer Paragon 500 spectrom-
1
eter. H- and 13C-NMR spectra were recorded on a Varian
Gemini 300 spectrometer, operating at 300 and 75.5 MHz
for the 1H and 13C nuclei, respectively. Samples were
measured in DMSO-d6 solutions at 20ꢁC in 5-mm NMR
tubes. Chemical shifts (d) were referred to TMS. Coupling
constants (J) are given in Hz. Elemental analysis was
determined on CHN-LECO-932. For thin-layer chroma-
tography, precoated Merck silica gel 60 F254 and solvent
system cyclohexane/ethyl acetate/methanol (3:1:0.5) were
used. Spots were visualized by short-wave UV light and
iodine vapour. Column chromatography was performed on
Merck silica gel 0.063–0.200 mm with cyclohexane/ethyl
acetate (1:2, 2:1 ? 1:1, 1:1) as eluents. O-ethylhydroxyl-
amine hydrochloride was obtained from Fluka and keto-
profen from PLIVA. All chemicals were purchased from
Sigma-Aldrich. All solvents were of analytical grade purity
and were dried prior to use.
2-(3-(N-benzotriazolcarbonyloxy)(phenyl)methyl)
phenyl)propanoic acid benzotriazolide (3b)
To a solution of 2 (2.561 g, 10 mmol) and triethylamine
(3.9 ml, 28 mmol) in dry toluene (50 ml), a solution of
chloride 1 (5.080 g, 28 mmol) in dry toluene (50 ml) was
added dropwise (0.25 h). The reaction mixture was stirred
at room temperature for 1 h and washed four times with
water. The organic layer was dried over anhydrous sodium
sulphate, filtrated and evaporated. Thus, obtained crude
products was purified by the trituration with ether. Yield:
3.52 g (70%); mp 124–127ꢁC; IR (KBr): mmax 3091, 3032,
2980, 2937, 1764, 1732, 597, 1486, 1451, 1398, 1250,
1-Benzotriazole carboxylic acid chloride (BtCOCl, 1)
Solution of benzotriazole (1.191 g, 10 mmol) and tri-
phosgene (2.523 g, 8.5 mmol) in dry toluene was refluxed
for 3 h. The reaction mixture was evaporated under
reduced pressure. The crude product was used in the fol-
lowing reactions without further purification (Butula and
1036, 951, 781, 760, 748, 708, 583 cm-1 1H-NMR
;
(DMSO-d6) d 8.28–7.98 (m, 3H, arom.), 7.78–7.28 (m,
15H, arom.), 7.24 (s, 1H, 10), 5.38 (q, 1H, 2, J = 6.84 Hz),
1.66 (d, 3H, 3, J = 6.94 Hz); 13C-NMR (DMSO-d6) d
173.28 (1), 147.91 (100), 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 (5–7, 9,
12–16, 20–50, 300–600), 81.48 (10), 45.04 (2), 18.98 (3).
´ ˇ
Jadrijevic Mladar Takac, 2000).
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
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Med Chem Res (2011) 20:210–219
Atom enumeration is given in Fig. 1. Anal. Calcd. for
C29H22N6O3: C, 69.31; H, 4.41; N, 16.72. Found: C, 69.39;
H 4.63; N, 16.99.
evaporated under reduced pressure. The crude product was
purified by the column chromatography (eluent cyclohex-
ane/ethyl acetate 1:1). Yield: 0.115 g (45%); mp 42–45ꢁC;
IR (KBr): mmax 3215, 3064, 3032, 2974, 2936, 1722, 1665,
1605, 1495, 1454, 1249, 1107, 1027, 749, 699 cm-1. Anal.
Calcd. for C31H30N2O5: C, 72.92; H, 5.92; N, 5.49. Found:
C, 73.00; H 6.04; N, 5.77.
(3-(1-(Methoxycarbamoyl)ethyl)phenyl)(phenyl)methyl
methoxycarbamate (4a)
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
O-methylhydroxylamine hydrochloride (0.092 g, 1.1 mmol)
and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was
stirred at room temperature for 10 h. The reaction mixture
was extracted with brine (5 9 10 ml), 1% hydrochloric acid
(1 9 5 ml) and washed with water till pH 7. The organic
layer was dried over anhydrous sodium sulphate and
evaporated under reduced pressure. Thus, obtained the
crude product was purified by the column chromatography
(eluent cyclohexane/ethyl acetate 1:2). Yield: 0.143 g
(80%); oil; IR (film): mmax 3455, 3217, 3065, 2978, 2938,
1725, 1668, 1606, 1489, 1454, 1254, 1115, 1043,
705 cm-1. Anal. Calcd. for C19H22N2O5: C, 63.67; H, 6.19;
N, 7.82. Found: C, 63.75; H 6.03; N, 7.48.
(3-(1-(Cyclopentylcarbamoyl)ethyl)phenyl)(phenyl)methyl
cyclopentylcarbamate (4d)
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
cyclopentylamine (0.109 ml, 1.1 mmol) and triethylamine
(0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room
temperature for 1 h. The reaction mixture was extracted
with brine (5 9 10 ml), 1% hydrochloric acid (1 9 5 ml)
and washed with water till pH 7. The organic layer was
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The crude product was triturated
with ether several times. Yield: 0.167 g (77%); mp
132–135ꢁC; IR (KBr): mmax 3305, 3269, 3065, 2962, 2870,
1700, 1651, 1606, 1545, 1452, 1249, 1040, 1017,
702 cm-1. Anal. Calcd. for C27H34N2O3: C, 74.62; H,
7.89; N, 6.45. Found: C, 74.39; H 7.76; N, 6.28.
(3-(1-(Ethoxycarbamoyl)ethyl)phenyl)(phenyl)methyl
ethoxycarbamate (4b)
(3-(1-(Cyclohexylcarbamoyl)ethyl)phenyl)(phenyl)methyl
cyclohexylcarbamate (4e)
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
O-ethylhydroxylamine hydrochloride (0.107 g, 1.1 mmol)
and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml)
was stirred at room temperature for 25 h. The reaction
mixture was extracted with brine (5 9 10 ml), 1% hydro-
chloric acid (1 9 5 ml) and washed with water till pH 7.
The organic layer was dried over anhydrous sodium
sulphate and evaporated under reduced pressure. Thus,
obtained the crude product was purified by the column
chromatography (eluent cyclohexane/ethyl acetate 2:1 ?
1:1). Yield: 0.140 g (73%); oil; IR (film): mmax 3454, 3219,
3064, 2981, 2937, 2891, 1724, 1715, 1668, 1606, 1494,
1454, 1384, 1253, 1113, 1041, 704 cm-1. Anal. Calcd. for
C21H26N2O5: C, 65.27; H, 6.78; N, 7.25. Found: C, 65.34;
H 6.43; N, 7.29.
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
cyclohexylamine (0.126 ml, 1.1 mmol) and triethylamine
(0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room
temperature for 5 h. The reaction mixture was extracted
with brine (5 9 10 ml), 1% hydrochloric acid (1 9 5 ml)
and washed with water till pH 7. The organic layer was
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The crude product was triturated
with ether several times. Yield: 0.173 g (75%); mp
139–142ꢁC; IR (KBr): mmax 3303, 3265, 3066, 2933, 2854,
1695, 1650, 1603, 1547, 1450, 1235, 1042, 702 cm-1
.
Anal. Calcd. for C29H38N2O3: C, 75.29; H, 8.28; N, 6.06.
Found: C, 75.55; H 8.01; N, 6.16.
(3-(1-(Cyclohexanemethylcarbamoyl)ethyl)phenyl)
(phenyl)methyl cyclohexanemethylcarbamate (4f)
(3-(1-(Benzyloxycarbamoyl)ethyl)phenyl)(phenyl)methyl
benzyloxycarbamate (4c)
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
cyclohexanemethylamine (0.143 ml, 1.1 mmol) and tri-
ethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was
stirred at room temperature for 0.5 h. The reaction mixture
was extracted with brine (5 9 10 ml), 1% hydrochloric
acid (1 9 5 ml) and washed with water till pH 7. The
organic layer was dried over anhydrous sodium sulphate
and evaporated under reduced pressure. The crude product
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
O-benzylhydroxylamine hydrochloride (0.175 g, 1.1 mmol)
and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was
stirred at room temperature for 48 h. The reaction mixture
was extracted with brine (5 9 10 ml), 1% hydrochloric acid
(1 9 5 ml) and washed with water till pH 7. The organic
layer was dried over anhydrous sodium sulphate and
123

Med Chem Res (2011) 20:210–219
217
was triturated with ether several times. Yield: 0.184 g
(75%); mp 128–129ꢁC; IR (KBr): mmax 3340, 3278, 3064,
2922, 2851, 1707, 1654, 1604, 1551, 1449, 1249,
702 cm-1. Anal. Calcd. for C31H42N2O3: C, 75.88; H,
8.63; N, 5.71. Found: C, 75.58; H 8.22; N, 5.99.
the column chromatography (eluent cyclohexane/ethyl
acetate 1:1). Yield: 0.152 g (75%); oil; IR (film): mmax
3061, 3030, 2973, 2875, 1700, 1643, 1634, 1588, 1454,
1416, 1126, 1096, 764, 709 cm-1. Anal. Calcd. for
C25H30N2O3: C, 73.86; H, 7.44; N, 6.89. Found: C, 73.57;
H 7.67; N, 6.80.
(3-(1-(Benzylcarbamoyl)ethyl)phenyl)(phenyl)methyl
benzylcarbamate (4g)
(3-(1-Oxo-1-(piperidin-1-yl)propan-2-
yl)phenyl)(phenyl)methyl piperidine-1-carboxylate (4j)
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
benzylamine (0.120 ml, 1.1 mmol) and triethylamine
(0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room
temperature for 0.5 h. The reaction mixture was extracted
with brine (5 9 10 ml), 1% hydrochloric acid (1 9 5 ml)
and washed with water till pH 7. The organic layer was
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The crude product was triturated
with ether several times. Yield: 0.127 g (53%); mp
107–109ꢁC; IR (KBr): mmax 3316, 3267, 3087, 3063, 3032,
2932, 1684, 1641, 1606, 1551, 1519, 1454, 1248,
699 cm-1. Anal. Calcd. for C31H30N2O3: C, 77.80; H,
6.32; N, 5.85. Found: C, 77.66; H 6.48; N, 6.01.
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
piperidine (0.109 ml, 1.1 mmol) and triethylamine
(0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room
temperature for 0.75 h. The reaction mixture was extracted
with brine (5 9 10 ml), 1% hydrochloric acid (1 9 5 ml)
and washed with water till pH 7. The organic layer was
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. After trituration with ether affor-
ded the pure product. Yield: 0.179 g (80%); mp
106–109ꢁC; IR (KBr): mmax 3050, 3028, 2971, 2940, 2852,
1694, 1628, 1587, 1469, 1426, 1258, 1236, 1148, 1083,
1026, 707, 699 cm-1. Anal. Calcd. for C27H34N2O3: C,
74.62; H, 7.89; N, 6.45. Found: C, 74.36; H 7.63; N, 6.55.
(3-(1-(Phenylethylcarbamoyl)ethyl)phenyl)(phenyl)methyl
phenylethylcarbamate (4h)
Interaction with DPPH
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 (Pontiki and Hadjipavlou-Li-
tina, 2007). 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 tripli-
cate and the standard deviation of absorbance was\10% of
the mean.
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
phenylethylamine (0.139 ml, 1.1 mmol) and triethylamine
(0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room
temperature for 0.6 h. The reaction mixture was extracted
with brine (5 9 10 ml), 1% hydrochloric acid (1 9 5 ml)
and washed with water till pH 7. The organic layer was
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The pure product was obtained
after trituration with ether. Yield: 0.190 g (75%); oil; IR
(KBr): mmax 3417, 3313, 3063, 3028, 2972, 2932, 2872,
1713, 1699, 1660, 1650, 1604, 1538, 1517, 1496, 1454,
1248, 1030, 749, 700 cm-1. Anal. Calcd. for C33H34N2O3:
C, 78.23; H, 6.76; N, 5.53. Found: C, 78.47; H 6.44; N,
5.70.
Soybean lipoxygenase inhibition
DMSO solution of the tested compound was incubated with
sodium linoleate (0.1 mM) and 0.2 ml of soybean lipoxy-
genase solution (1/9 9 10-4 w/v in saline) at room tem-
perature (Pontiki and Hadjipavlou-Litina, 2007). The
conversion of sodium linoleate to 13-hydroperoxylinoleic
acid was recorded at 234 nm and compared to the standard
inhibitor caffeic acid, according to the procedure previ-
ously reported.
(3-(1-Oxo-1-(pyrrolidin-1-yl)propan-2-
yl)phenyl)(phenyl)methyl pyrrolidine-1-carboxylate (4i)
A solution of benzotriazolide 3b (0.251 g, 0.5 mmol),
pyrrolidine (0.092 ml, 1.1 mmol) and triethylamine
(0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room
temperature for 0.5 h. The reaction mixture was extracted
with brine (5 9 10 ml), 1% hydrochloric acid (1 9 5 ml)
and washed with water till pH 7. The organic layer was
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The crude product was purified by
Inhibition of linoleic acid lipid peroxidation
Oxidation of linoleic acid to conjugated diene hydroper-
oxide in an aqueous dispersion is monitored at 234 nm (Re
et al., 1999). AAPH was used as a free radical initiator. Ten
microliters of the 16 mM linoleic acid dispersion was
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Med Chem Res (2011) 20:210–219
added to the UV cuvette containing 0.93 ml of 0.05 M
phosphate buffer, pH 7.4 prethermostated at 37ꢁC. The
oxidation reaction was initiated at 37ꢁC under air by the
addition of 50 ll of 40 mM AAPH solution. Oxidation was
carried out in the presence of compounds (10 ll, final
concentration 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
37ꢁC by recording the increase of absorption at 234 nm
caused by conjugated diene hydroperoxides. The results
were compared to the standard inhibitor trolox.
of the test compounds. After 4 days of incubation at 37ꢁC,
HIV-induced CEM giant cell formation was examined
microscopically.
Acknowledgements Support for this study was provided by the
Ministry of Science, Education and Sports of the Republic of Croatia
(Project 006-0000000-3216) and by the Concerted Actions (GOA No.
05/19) of the K. U. Leuven. We thank Mrs. Leen Ingels, Leentje
Persoons, Vicky Broeckx and Frieda De Meyer for excellent technical
assistance and Dr C. Hansch and Biobyte Corp. 201 West 4th Str.,
Suite 204, Claremont CA California, 91711, USA for free access to
the C-QSAR program.
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