N-Benzyl-2-chloroindole-3-carboxylic acids as potential anti-inflammatory agents. Synthesis and screening for the effects on human neutrophil functions and on COX1/COX2 activity

Article abstract of DOI:10.1016/j.ejmech.2004.06.008

The synthesis of N-benzyl-2-chloroindole-3-carboxylic acids related to indomethacin is reported. These compounds were tested on in vitro human neutrophil activation. Some of them, more soluble in water, were tested to define the influence on prostaglandin biosynthesis via inhibition of cyclooxygenases (COX1 and COX2) by a chemiluminescent method suitable for fast screening. Several derivatives showed inhibitory effects and in some cases were more active than the parent compound.

Full text of DOI:10.1016/j.ejmech.2004.06.008

European Journal of Medicinal Chemistry 39 (2004) 785–791
www.elsevier.com/locate/ejmech
Original article
N-Benzyl-2-chloroindole-3-carboxylic acids as potential
anti-inflammatory agents. Synthesis and screening for the effects
on human neutrophil functions and on COX1/COX2 activity
Aldo Andreani a,*, Massimiliano Granaiola a, Alberto Leoni a, Alessandra Locatelli a,
Rita Morigi a, Mirella Rambaldi a, Aldo Roda a, Massimo Guardigli a,
Serena Traniello b, Susanna Spisani b
a Dipartimento di Scienze Farmaceutiche, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Via L.Borsari 46, 44100 Ferrara, Italy
Received 9 January 2004; received in revised form 28 May 2004; accepted 9 June 2004
Available online 10 August 2004
Abstract
The synthesis of N-benzyl-2-chloroindole-3-carboxylic acids related to indomethacin is reported. These compounds were tested on in vitro
human neutrophil activation. Some of them, more soluble in water, were tested to define the influence on prostaglandin biosynthesis via
inhibition of cyclooxygenases (COX1 and COX2) by a chemiluminescent method suitable for fast screening. Several derivatives showed
inhibitory effects and in some cases were more active than the parent compound.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Indole; Inflammation; Neutrophil; COX1/COX2
1. Introduction
In pursuing our search on the anti-inflammatory activity of
heterocyclic compounds [14–18], we planned the synthesis
of 2-chloro-1-(4-chlorobenzyl)-5-methoxyindole-3-carbo-
xylic acid 3c (Scheme 1) which has a steric hindrance analo-
gous to indomethacin with a shorter acidic chain (the ap-
proach of chain shortening has been applied for example in
the case of diclofenac and its fenamic analog). The substitu-
ents at the indole ring are for example the methoxy group
(see indomethacin) and the acethyl group (see acetylsalicylic
acid where it is well known that the acetyl group binds a
serine residue in COX).
Indomethacin was first reported by Shen et al. [19]. The
paper stimulated the search in this field and a lot of deriva-
tives have been prepared, including rigid analogs [20] and
benzyl derivatives [21,22].
The encouraging effects shown by compound 3c on neu-
trophil functions prompted us to prepare a small series of
analogues in order to study the effect of different substituents
at the position 4–6 of the indole and at the position 4 of the
benzyl ring. The present work investigates the in vitro effects
of new compounds and indomethacin on human neutrophil
activation induced by different stimuli. Chemotaxis, super-
Nonsteroidal anti-inflammatory drugs (NSAIDs) show in
vitro effects on neutrophil functions [1–3]. Some NSAIDs
inhibit neutrophil activation by inflammatory stimuli, such as
C5a, LTB4, or formyl-methionine-leucine-phenylalanine
(FMLP), by interfering with early events of signal transduc-
tion [4–7]. Indomethacin inhibits neutrophil chemotaxis and
leukotriene B4 generation [8]. Moreover indomethacin, sali-
cylic acid and acetylsalicylic acid were found to inhibit
superoxide generation by human neutrophils exposed to
phorbol 12-myristate 13-acetate (PMA) in a whole-cell sys-
tem [9] while prednisolone and NSAIDs inhibited elastase
release from neutrophils exposed to FMLP [10,11]. For a
recent review on neutrophils in the inflammatory process see
Ref. [12]. The trend of measuring the effects on neutrophil
functions for the evaluation of new potential anti-
inflammatory agents is confirmed by a recent paper [13].
* Corresponding author. Tel.: +39-51-2099714; fax: +39-51-2099734.
E-mail address: aldo.andreani@unibo.it (A. Andreani).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.06.008

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A. Andreani et al. / European Journal of Medicinal Chemistry 39 (2004) 785–791
Scheme 1. (R–R3 see Table 1).
–
oxide anion (O2 ) generation and lysozyme degranulation
were evaluated after cell pulse with different concentrations
of the drugs.
order to obtain the corresponding phenols 4c–f. Aim of the
subsequent step was the synthesis of the corresponding
5-acetyloxy derivatives but under the planned experimental
conditions we were able to obtain only the expected com-
pounds 5c,d,f. Under the same experimental conditions com-
pound 4e led to complex reaction mixtures in which little, if
any, of the desired product was present. The spectroscopic
data of the new compounds (see Table 2) are in agreement
with the assigned structures and with the data reported on a
In order to better define the biological profile of the newly
synthesized compounds we also evaluated the inhibitory ac-
tivity in vitro on two COX isoenzymes, COX1 and COX2.
Among the numerous assays developed for this purpose over
the years, we decided to measure the inhibition using a
chemiluminescent (CL) method which allows to measure the
rate of the peroxidase reaction, i.e., the second step of the
PGH synthase-catalyzed reaction, in which the intermediate
product prostaglandin G2 is converted into the final product
prostaglandin H2 [23], by using luminol (a well-known CL
substrate) as the reducing cosubstrate [24]. It has been previ-
ously demonstrated that the cyclooxygenation reaction is the
rate limiting step for the generation of the CL signal, thus the
easily detectable CL emission produced by the oxidation of
luminol could be regarded as a real-time index of cyclooxy-
genase acitivity [24]. This method allowed us to determine
the IC50 value of the newly synthesized compounds in a very
short time and to consume small amounts of samples and
reagents.
1
previous papers [28]. The H-NMR spectra of compounds
2–5 show the methylene of the benzyl group in the range
5.32–5.67 ppm; in all compounds 4, the indole 5-hydroxy
group is between 9.10 and 9.25 ppm i.e. clearly separated
from the OH of the 4-hydroxyphenyl group present in two of
them (9.40–9.44 ppm).
3. Biological results
One of the targets of the present research was to determine
whether the tested compounds 3–5 affect some important
neutrophil functions. We analyzed the in vitro effect (see
Refs. [17,18]) of the new compounds on a wide range of
neutrophil functionality triggered by different stimuli acting
either through specific cell surface receptors (e.g. FMLP) or
by post-receptor signaling (e.g. casein and PMA). It is gen-
erally accepted that receptor and nonreceptor agonists utilize
different transduction mechanisms to activate chemotaxis
and free radical production [29,30]. In order to assess pos-
sible cytotoxic effects of the tested compounds, the cell
viability was measured. Release of the cytoplasmic enzyme
lactate dehydrogenase (LDH) was used as an indicator of cell
viability. In none of the above described experiments the
percentage of total LDH release was >3% (data not shown).
In the first step of our study, we tested whether compounds
2. Chemistry
The starting 2-chloroaldehydes 1 (Scheme 1) have been
previously reported in the literature [25,26]. N-Alkylation
was conducted in dimethylformamide in the presence of
sodium hydride [27]. The oxidation of the resulting
N-benzyl-2-chloroindole-3-carboxaldehydes 2a–f (Table 1)
to the corresponding carboxylic acids 3a–f was performed
with potassium permanganate in a mixture of acetone–water.
The acids bearing a methoxy group at the 5 position (3c–f)
were treated with boron tribromide in methylene chloride in

A. Andreani et al. / European Journal of Medicinal Chemistry 39 (2004) 785–791
787
Table 1
Compounds 2–5
Compound
R
R1
R2
R3
Formula
MW
m.p. (°C) or
literature
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
4c
4d
4e
4f
5c
5d
5f
H
Cl
H
H
H
H
H
Cl
H
H
H
H
–
H
H
Cl
C16H11Cl2NO
C16H10Cl3NO
C17H13Cl2NO2
C18H15Cl2NO2
C18H16ClNO3
C19H18ClNO3
C16H11Cl2NO2
C16H10Cl3NO2
C17H13Cl2NO3
C18H15Cl2NO3
C18H16ClNO4
C19H18ClNO4
C16H11Cl2NO3
C17H13Cl2NO3
C16H12ClNO4
C17H14ClNO4
C18H13Cl2NO4
C19H15Cl2NO4
C21H18ClNO6
304.2
338.6
334.2
348.2
329.8
343.8
320.2
354.6
350.2
364.2
345.8
359.8
336.2
350.2
317.7
331.7
378.2
392.2
415.8
[27]
H
H
Cl
180–184
OCH3
H
Cl
[16]
OCH3
CH3
H
Cl
175–180 dec
134–136
OCH3
OCH3
OCH3
Cl
OCH3
CH3
H
130–133
H
[27]
H
H
Cl
160–163 dec
215–218 dec
240–241 dec
211–214 dec
229–231 dec
237–239
OCH3
H
Cl
OCH3
CH3
H
Cl
OCH3
OCH3
OCH3
Cl
OCH3
CH3
H
–
–
–
–
–
–
–
–
CH3
H
Cl
210–212 dec
200–205 dec
203–206 dec
223–225
–
OH
OH
Cl
–
CH3
H
–
–
CH3
CH3
Cl
261–264 dec
233–236
–
OAc
3–5 and indomethacin were chemotaxins for human neutro-
phils, secretagogue agents or were able per se to trigger
superoxide anion production. None of the molecules was
able to mediate these activities. In the second step, we tested
a possible influence of the compounds on neutrophil func-
tions. In order to study the initial event of inflammation, we
analyzed the influence of the prepared compounds and in-
domethacin on random locomotion in vitro. All compounds
showed slight effects: however, these were not statistically
(Table 4) were in reasonable agreement with those reported
in the literature, in particular as concerned the COX1/COX2
selectivity and the relative potency of these compounds
[31,32]. Cyclooxygenase inhibitory activity was measured
only for compounds 3a,b, 4c–f, 5c,d,f which showed a good
solubility in DMSO/water 1:10 (v/v). The IC50 values found
for these compounds were in the range 10–4 to 10–6
M
(Table 4). Most of the compounds were more active than
ibuprofen and some of them (4d and 4f) showed an inhibitory
effect close to that of indomethacin. As indomethacin, both
the derivatives were approximately equally effective on both
isoenzymes. The IC50 values obtained for compounds 4d and
4f in COX2 inhibition experiments (IC50 = 10–6 M) were an
order of magnitude higher than that of the COX2 selective
inhibitor rofecoxib (IC50 = 10–7 M).
significant (P > 0.05) at the concentrations tested (10–11
,
10–9, 10–7 and 10–5 M) (data not shown). Conversely, when
the same compounds were tested for their ability to influence
directed migration, inhibition of FMLP-induced chemotaxis
(Table 3) was observed in the presence of compounds 3a,d–f,
4d–f and 5f. Most of the compounds were able to affect
FMLP-induced respiratory burst. The inhibition became sta-
tistically significant (P < 0.05) at a drug concentration of 10–9
M, reaching the maximal inhibition at the highest concentra-
tions (P < 0.01). Compounds 4c,d,f, 5c,d reached about 60%
inhibition at 10–5 M. Only few compounds (3f, 4e,f and 5f)
elicited a PMA-induced response (inhibition = 30% at 10–5
M). Only compounds 3e,f were active in PMA-stimulated
lysozyme release (inhibition = 35% at 10–5 M). None of the
tested derivatives affect either casein-activated chemotaxis or
FMLP-induced lysozyme secretion (data not shown). No
activity by any of the molecules was found at 10–11 M (data
not shown). Ineffective derivatives are not presented in the
Table 3.
4. Conclusion
According to our results, compounds 3–5 are not toxic
(measurement of cell viability, for experimental see Refs.
–
[17,18]). In particular, chemotaxis and O2 production trig-
gered by the optimal active concentration of the agonist
FMLP, seems more sensible to these derivatives. Some of
them display interesting properties as inhibitors of human
neutrophil functions and retain much of the inhibitory power
of indomethacin. In some cases they are even more active
(see 3d, 4c,d,f and 5c,d in FMLP-triggered O2– generation)
and are able to inhibit neutrophil activity at concentrations as
low as 10–9 M and for this reason may be considered as
potential anti-inflammatory agents. They could block recruit-
ment of neutrophils into inflammatory lesions and control
neutrophil degranulation, showing a protective effect against
the direct injury to the target tissue derived from proteolityc
The second target of the biological activity tests was the
study of COX1 and COX2 inhibition. The performance of the
CL method was determined by measuring the IC50 values of
known cyclooxygenase inhibitors (indomethacin, ibuprofen
and rofecoxib). The method proved to be reliable in term of
reproducibility and the obtained data for known inhibitors

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A. Andreani et al. / European Journal of Medicinal Chemistry 39 (2004) 785–791
Table 2
IR and 1H-NMR of compounds 2–5
Compound
IR a: mmax, cm-1
1H-NMR b: d (ppm); J (Hz) in DMSO-d6; ph, phenyl; ind, indole
2b
1650, 1035, 770, 725
1640, 1500, 1035, 715
1645, 1510, 1245, 1140
5.65 (2H, s, CH2), 7.15 (2H, d, ph, J = 8.4), 7.31 (1H, t, ind, J = 8.1), 7.39 (3H: 2H, d, ph, J = 8.4 + 1H, d,
ind, J = 8.1), 7.70 (1H, d, ind, J = 8.1), 10.67 (1H, s, CHO)
2d
2e
2.23 (3H, s, CH3), 3.84 (3H, s, OCH3), 5.57 (2H, s, CH2), 7.16 (2H, d, ph, J = 6.6), 7.42 (2H, d, ph, J = 6.6),
7.49 (1H, s, ind), 7.59 (1H, s, ind), 10.00 (1H, s, CHO)
3.70 (3H, s, OCH3), 3.79 (3H, s, OCH3), 5.49 (2H, s, CH2), 6.89 (2H, d, ph, J = 8.7), 6.94 (1H, dd, ind6,
J = 9.1, J = 2.8), 7.16 (2H, d, ph, J = 8.7), 7.59 (1H, d, ind7, J = 9.1), 7.61 (1H, d, ind4, J = 2.8), 10.00 (1H,
s, CHO)
2f
1650, 1510, 1250, 1030
1670, 1230, 1165, 760
1680, 1520, 1280, 1225
1665, 1505, 1210, 1180
1650, 1610, 1500, 1170
1680, 1510, 1210, 1175
3150, 1660, 1510, 1165
3450, 1650, 1520, 1225
3230, 1675, 1515, 1235
3300, 1660, 1500, 1250,
1730, 1650, 1490, 1200
1740, 1650, 1500, 1200
1740, 1680, 1640, 1185
2.20 (3H, s, CH3), 3.67 (3H, s, OCH3), 3.80 (3H, s, OCH3), 5.43 (2H, s, CH2), 6.86 (2H, d, ph, J = 8.4), 7.11
(2H, d, ph, J = 8.4), 7.48 (1H, s, ind), 7.54 (1H, s, ind), 9.96 (1H, s, CHO)
5.59 (2H, s, CH2), 7.13 (2H, d, ph, J = 8.5), 7.26(2H, d, ind, J = 4.6), 7.41 (2H, d, ph, J = 8.5), 7.62 (1H, t,
ind, J = 4.6)
3b
3c
3d
3e
3f
3.78 (3H, s, OCH3), 5.56 (2H, s, CH2), 6.90 (1H, dd, ind6, J = 9.0, J = 2.5), 7.11 (2H, d, ph, J = 8.5), 7.39
(2H, d, ph, J = 8.5), 7.51 (1H, d, ind7, J = 9.0), 7.55 (1H, d, ind4, J = 2.5)
2.22 (3H, s, CH3), 3.82 (3H, s, OCH3), 5.53 (2H, s, CH2), 7.09 (2H, d, ph, J = 8.5), 7.40 (3H: 2H, d, ph,
J = 8.5 + 1H, s, ind), 7.51 (1H, s, ind)
3.66 (3H, s, OCH3), 3.75 (3H, s, OCH3), 5.43 (2H, s, CH2), 6.84 (2H, d, ph, J = 8.8), 6.86 (1H, dd, ind6,
J = 9.1, J = 2.7) 7.06 (2H, d, ph, J = 8.8), 7.49 (1H, d, ind7, J = 9.1), 7.50 (1H, d, ind4, J = 2.7)
2.22 (3H, s, CH3), 3.69 (3H, s, OCH3), 3.81 (3H, s, OCH3), 5.43 (2H, s, CH2), 6.87 (2H, d, ph, J = 8.6), 7.06
(2H, d, ph, J = 8.6), 7.42 (1H, s, ind), 7.49 (1H, s, ind)
4c
4d
4e
4f
5.51 (2H, s, CH2), 6.73 (1H, dd, ind6, J = 8.8, J = 2.4), 7.12 (2H, d, ph, J = 8.5), 7.38 (1H, d, ind, J = 8.8),
7.40 (2H, d, ph, J = 8.5), 7.48 (1H, d, ind, J = 2.4), 9.20 (1H, s, OH)
2.21 (3H, s, CH3), 5.51 (2H, s, CH2), 7.12 (2H, d, ph, J = 8.5), 7.32 (1H, s, ind), 7.43 (2H, d, ph, J = 8.5),
7.52 (1H, s, ind), 9.25 (1H, s, OH)
5.33 (2H, s, CH2), 6.67 (2H, d, ph, J = 7.9), 6.70 (1H, dd, ind6, J = 8.8, J = 1.6), 6.97 (2H, d, ph, J = 7.9),
7.36 (1H, d, ind7, J = 8.8), 7.41 (1H, d, ind4, J = 1.6), 9.10 (1H, s, OH-5), 9.40 (1H, s, OH-para)
2.18 (3H, s, CH3), 5.32 (2H, s, CH2), 6.69 (2H, d, ph, J = 8.5), 6.97 (2H, d, ph, J = 8.5), 7.28 (1H, s, ind),
7.46 (1H, s, ind), 9.16 (1H, s, OH-5), 9.44 (1H, s, OH-para)
5c
5d
5f
2.29 (3H, s, CH3), 5.61 (2H, s, CH2), 7.04 (1H, dd, ind6, J = 8.8, J = 2.2), 7.15 (2H, d, ph, J = 8.4), 7.41 (2H,
d, ph, J = 8.4), 7.64 (1H, d, ind7, J = 8.8), 7.76 (1H, d, ind4, J = 2.2)
2.19 (3H, s, CH3), 2.33 (3H, s, CH3), 5.58 (2H, s, CH2), 7.15 (2H, d, ph, J = 7.7), 7.42 (2H, d, ph, J = 7.7),
7.56 (1H, s, ind), 7.69 (1H, s, ind)
2.16 (3H, s, CH3), 2.22 (3H, s, CH3), 2.30 (3H, s, CH3), 5.54 (2H, s, CH2), 7.07 (2H, d, ph, J = 8.7), 7.14
(2H, d, ph, J = 8.7), 7.54 (1H, s, ind), 7.65 (1H, s, ind)
a The COOH group is broad (3400–2300 cm–1).
b The COOH group is a singlet in the range 12.5–13 ppm.
enzyme release. In some cases (see for example 5d in
Table 3) the pharmacological response was peculiar and
difficult to explain.
1H-NMR spectra were recorded in (CD3)2SO on a Varian
Gemini (300 MHz); the chemical shift (referenced to solvent
signal) is expressed in d (ppm) and J in Hz (see Table 2).
We demonstrated that, thanks to the use of 96-well micro-
titer plates and the high detectability and fast kinetics of the
CL signal, the CL COX activity assay facilitated the rapid
screening of the biological activity of newly synthesized
compounds and the identification of the most promising
molecules with a reduced consumption of reagents.
5.1.1. General procedure for the synthesis
of the N-benzyl-2-chloroindole-3-carboxaldehydes 2a–f
The appropriate 2-chloroindolaldehyde 1 (10 mmol) was
dissolved in 20 ml of DMF and the stirred solution was
treated with small portions of NaH (15 mmol). The reaction
mixture was stirred at room temperature for 10 min, treated
with the appropriate benzyl chloride (60 mmol) and main-
tained at 90 °C for 1–3 h according to a TLC test. The
reaction mixture was then poured into ice and acidified with
2 N HCl. The crude N-benzyl-2-chloroindole-3-carbo-
xaldehydes 2a–f thus obtained was collected by filtration and
crystallized from ethanol with a yield of 80–90%.
5. Experimental
5.1. Chemistry
The melting points are uncorrected. Analyses (C, H, N)
were within 0.4% of the theoretical values. TLC was per-
formed on Bakerflex plates (Silica gel IB2-F): the eluent was
a mixture of petroleum ether/acetone in various proportions.
The same eluent was used for column chromatography with
Kieselgel 60 (Merck) as the stationary phase. The IR spectra
were recorded in nujol on a Nicolet Avatar 320 ESP. The
5.1.2. General procedure for the synthesis
of the N-benzyl-2-chloroindole-3-carboxylic acids 3a–f
The appropriate N-benzyl-2-chloroindole-3-carboxal-
dehyde (2a–f, 5 mmol) was dissolved in 100 ml of acetone
and treated with a solution of KMnO4 (15 mmol) in water

A. Andreani et al. / European Journal of Medicinal Chemistry 39 (2004) 785–791
789
Table 3
Inhibitory activity (%) of compounds 3–5 and indomethacin on human neutrophil functions
Compound
Concentration (M)
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
FMLP-induced chemotaxis a
FMLP-triggered O2– generation a
3a
n.s. b
52
31
33
32
35
35
30
31
31
41
44
51
n.s.
29
31
n.s.
n.s.
n.s.
44
53
55
51
58
62
n.s.
n.s.
28
41
44
49
61
60
59
58
53
52
28
32
47
22
22
36
3b
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
25
3c
3d
n.s.
n.s.
28
3e
29
31
3f
25
33
35
4c
n.s.
n.s.
n.s.
25
4d
44
35
4e
29
33
35
4f
26
28
30
5c
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
25
5d
5f
27
33
Indomethacin
49
52
59
a Cells were incubated for 10 min before the assay with the compounds at the concentration of 10–11 (n.s.), 10–9, 10–7, 10–5 M. Each value is the average of the
inhibitory activity from 8–10 separate experiments carried out in triplicate. SEM are within 10%. Statistical significance is reported in the text.
b n.s.=not significant.
(40 ml). The reaction mixture was stirred at room tempera-
ture for 5–16 h (according to a TLC test), discolored with
10% H2O2 and filtered. Acetone was evaporated under re-
duced pressure and the resulting solution, filtered again if
necessary, was acidified with 2 N HCl. The precipitate was
collected by filtration and crystallized from ethanol with a
yield of 60–70%.
stirred overnight at room temperature, treated with 33%
NaOH (pH 9) and stirred for additional 10 min. The aqueous
phase was separated and acidified with 2 N HCl at 0 °C: the
precipitate thus obtained was collected by filtration with a
yield of 50–60% and was not subjected to further purifica-
tion.
5.1.4. General procedure for the synthesis of the 5-acetyl
derivatives 5c,d,f
The appropriate 5-hydroxy derivative (4c–f, 2 mmol) was
dissolved in 15 ml of acetic anhydride and two drops of 98%
H2SO4. The mixture was stirred at 50–60 °C for 15 min and
water (30 ml) was added. The aqueous phase was extracted
5.1.3. General procedure for the synthesis of the 5-hydroxy
derivatives 4c–f
The appropriate 5-methoxy derivative (3c–f, 3 mmol) was
dissolved in 40 ml of anhydrous CH2Cl2 and treated under
nitrogen at 0–5 °C with BBr3 (7 mmol). The mixture was

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A. Andreani et al. / European Journal of Medicinal Chemistry 39 (2004) 785–791
lysozyme release was 45 4% or 52 5%/3 × 106 cells/min
from 15 separate experiments induced, respectively, by PMA
or FMLP. Stock solutions in dimethylsulfoxide (DMSO)
were diluted before use in Krebs–Ringer-phosphate buffer:
10–2 M for the tested compounds and FMLP; 1 mg/ml for
PMA. DMSO used did not interfere with any of the biologi-
cal assays performed. stock solution. The nonparametric
Wilcoxon test was used in the statistical evaluation of differ-
ences between groups (level of significance P ≤ 0.05).
Table 4
COX1 and COX2 inhibition IC50 values measured for compounds 3a,b,
4c–f, 5c,d,f and three reference drugs, using the CL method a
Compound
IC50: µmol/l SEM
COX1 inhibition
100 30
COX2 inhibition
350 200
Ibuprofen
Indomethacin
0.4 0.1
0.20 0.06
0.08 0.02
230 120
Rofecoxib
60 12
3a
3b
4c
4d
4e
4f
5c
5d
5f
95 15
70 15
12
6
4.8 0.4
1.7 0.3
5.3. COX1/COX2 inhibition
0.48 0.06
3.2 0.6
0.65 0.15
30
6
0.6 0.1
1.2 0.5
4.6 2.6
22 10
Ovine COX1 and COX2 were purchased from Cayman
Chemical, Ann Arbor, MI. Porcine hematin, luminol (5-
amino-2,3-dihydro-1,4-phthalazinedione sodium salt) and
arachidonic acid, as well as the reference inhibitor ibuprofen,
were from Sigma Chemical Co. (St. Louis, MO). Rofecoxib
was obtained from Merck & Co., Inc. (Rahway, NJ). Chemi-
luminescence measurements were performed in 96-well
white polystyrene microtiter plates (White Microstrip Break-
able, Labsystems Oy, Helsinki, Finland) using a microtiter
plate luminometer (Luminoskan Ascent, Labsystems Oy)
equipped with an on-board incubator and a reagent dispenser.
7.5 3.0
3.3 0.3
1.2 0.2
1.7 0.7
a IC50 values were calculated from inhibition graphs containing data
collected in three independent experiments.
with CH2Cl2 (3 × 20 ml) and the crude product thus obtained
(5c,d,f) was purified by column chromatography with a yield
of 20–25%. Under the above experimental conditions, com-
pound 4e led to complex reaction mixtures.
5.2. Effects on human neutrophil functions
5.3.1. Evaluation of cyclooxygenase inhibition
The CL cyclooxygenase activity was measured following
the procedure reported in the literature [24], with slight
modifications. Briefly, cyclooxygenases (COX1 or COX2,
50 U/ml in 0.1 M phosphate buffer, pH 6.5, with 1 mg/ml
gelatine), hematin (6 µM in 0.1 M phosphate buffer, pH 6.5)
and luminol (500 µM in 0.1 M TRIS buffer, pH 6.5) were
added to the microtiter plate wells to obtain a reaction mix-
ture containing 1.0 U of cyclooxygenase, 350 µM luminol
and 1 µM hematin in a 150 µl final volume. Then, 10 µl of
scalar dilutions of the inhibitors in DMSO/water 1:10 (v/v)
were added, and the solution was incubated in the microtiter
plate luminometer at 35 °C for 30 min. The enzymatic reac-
tion was started by adding to each well 200 µl of a 35 µM
arachidonic acid solution in water (corresponding to a final
arachidonic acid concentration of 20 µM), and the CL emis-
sion was measured at 1-s intervals up to 60 s after initiation of
the reaction with substrate. The cyclooxygenase activity was
evaluated by interpolating the integrated CL signal (which is
proportional to the amount of product formed) on a calibra-
tion curve obtained by measuring in the same analytical
session samples containing different amounts of cyclooxyge-
nases. The inhibition graph for each inhibitor was con-
structed from data collected in three independent experi-
ments. Data were analyzed by using the software package
Prism 3.0 (Graph Pad, San Diego, CA) and the results were
expressed as IC50 SEM (mol/l).
The detailed procedures have been described in a previous
papers [17]. Cells were purified employing the standard
techniques of dextran sedimentation of heparinized blood,
followed by centrifugation on Ficoll–Paque and hypotonic
lysis of contaminating red cells. Random locomotion and
chemotaxis were evaluated with a 48-well microchemotaxis
chamber, by estimating the distance in micrometers which
the leading front of the cell migrated. The chemotactic fac-
tors used were FMLP (10–8 M in KRPG-A) or casein
(2 mg/ml in KRPG-A). The actual control of chemotaxis was
95 4 µm or 83 2 µm SEM, from 15 separate experiments
induced, respectively, by FMLP or casein. The superoxide
anion production was measured by the superoxide
dismutase-inhibitable reduction of ferricytochrome modified
for microplate-based assays. The stimulants employed were
PMA (100 ng/ml in KRPG) or FMLP (10–6 M in KRPG).
Neutrophils were preincubated with cytochalasin B 5 µg/ml
for 5 min prior to activation by FMLP. The actual control of
O2– generation was, 35 2 nmoles or 30 2 nmoles/1 × 106
cells/5 min from 15 separate experiments induced, respec-
tively, by PMA or FMLP. The release of lysozyme was
quantified nephelometrically by the lysis rate of cell-wall
suspension of Micrococcus lysodeikticus. The enzyme was
expressed as a net percentage of the total enzyme content
released by 0.1% Triton X-100. The total enzyme activity
was 85 1 µg/1 × 107 cells/min. Spontaneous release was less
than 10%. The degranulating agents used were PMA
(100 ng/ml) or FMLP (10–6 M), in KRPG. Cells were prein-
cubated with cytochalasin B 5 µg/ml for 15 min prior to
activation by FMLP. Assays were currently done in triplicate
for each experimental condition. The actual control of
Acknowledgments
This work was supported by a grant from MIUR. We are
grateful to ”Banca del Sangue di Ferrara” for supplying fresh

A. Andreani et al. / European Journal of Medicinal Chemistry 39 (2004) 785–791
791
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