Design and study of some novel ibuprofen derivatives with potential nootropic and neuroprotective properties

Article abstract of DOI:10.1016/j.bmc.2006.10.056

Six novel ibuprofen derivatives and related structures, incorporating a proline moiety and designed for neurodegenerative disorders, are studied. They possess anti-inflammatory properties and three of them inhibited lipoxygenase. One compound was found to

Full text of DOI:10.1016/j.bmc.2006.10.056

Bioorganic & Medicinal Chemistry 15 (2007) 951–961
Design and study of some novel ibuprofen derivatives with potential
nootropic and neuroprotective properties
Ioanna C. Siskou,a Eleni A. Rekka,a,* Angeliki P. Kourounakis,b Michael C. Chrysselis,a
Kariofyllis Tsiakitzisa and Panos N. Kourounakisa
aDepartment of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki, Thessaloniki 54124, Greece
bDepartment of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis Zografou,
Athens 15771, Greece
Received 21 June 2006; revised 6 October 2006; accepted 17 October 2006
Available online 28 October 2006
Abstract—Six novel ibuprofen derivatives and related structures, incorporating a proline moiety and designed for neurodegenerative
disorders, are studied. They possess anti-inflammatory properties and three of them inhibited lipoxygenase. One compound was
found to inhibit cyclooxygenase (COX)-2 production in spleenocytes from arthritic rats. The HS-containing compounds are potent
antioxidants and one of them protected against glutathione loss after cerebral ischemia/reperfusion. They demonstrated lipid-low-
ering ability and seem to acquire low gastrointestinal toxicity. Acetylcholinesterase inhibitory activity, found in two of these
compounds, may be an asset to their actions.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
quently, there are several targets for the medicinal
treatment of cognition disorders and the potential of
multifunctional therapeutic agents for the treatment
of complex neurodegenerative diseases has already
been considered.4
Neurodegenerative disorders of Alzheimer’s type (Se-
nile Dementia Alzheimer’s Type, SDAT), along with
cardiovascular diseases and cancer, are the top causes
of morbidity and they are estimated to affect more
than 37 million of people worldwide by the year
2020.1,2 Therefore, it appears interesting to design
and synthesise molecules for the treatment of SDAT.
Moreover, it is a pharmacochemical challenge, since
there is still enough uncertainty about the molecular
pathogenesis of nerve cell death3 and there has not
been a really effective intervention yet. However, there
is a series of pathobiochemical changes in the dement-
ed brain, which could be used as a starting point for
rational design of molecules for the particular condi-
tion. Thus, the appearance of neurofibrillary tangles
intracellularly, amyloid plaques extracellularly, a seri-
ous cholinergic deficit, oxidative stress, inflammation,
reduction of nerve growth factor, increased apolipo-
protein E4 and hypercholesterolemia are interrelated
factors contributing to cell death in SDAT.3 Conse-
In this paper, we describe the synthesis of derivatives
of ibuprofen, a known non-steroidal anti-inflammato-
ry drug which has also been found to affect amyloid
pathology in the brain5 and its long-term use has been
associated with reduced risk of neurodegeneration.6
Ibuprofen is connected, via an amide bond, with a
L-proline moiety, expected to contribute to the noo-
tropic properties, since structurally related amides or
esters of proline have some nootropic action and pro-
line dipeptides have been found to be about 2000
times more potent than piracetam.7,8 Proline carboxyl-
ic group has further been esterified with 2-methoxy-4-
methyl-phenol or 3-(3-hydroxypropyl)pyridine (com-
pounds 1 and 2, Scheme 1), expected to offer neuro-
trophic and/or neuroprotective activity, the former
by induction of nerve growth factor synthesis,9 the
latter independently of this factor.10,11 In addition,
the carboxylic group of the intermediate proline amide
has been converted to amide with cysteamine and
L-cysteine ethyl ester (compounds 3 and 4), known
antioxidants with low general toxicity.12
Keywords: Ibuprofen derivatives; Inflammation; Antioxidant activity;
Cerebral ischemia/reperfusion injury.
*
Corresponding author. Tel.: +30 2310 997614; fax: +30 2310
997622; e-mail: rekka@pharm.auth.gr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.056

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I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
OH
Cl
H
COOH
N
COR
N
O
N
O
COOH
O
O
(COCl)2
CH2Cl2
a, b, c or d
NaOH/H2O, 0o C
1-4
A
C
B
Cl
O
(C2H5)3N,
a or b
+
COR
COOH
N
N
COOH
(CH3)3SiCl
N
H
O
O
5, 6
D
Compounds 1, 5: R=
O
CH3
Compound 3: R=
Compound 4: R=
NHCH2CH2SH
CH3O
OCH2CH2CH2
NHCHCH2SH
COOCH2CH3
N
Compounds 2, 6: R=
Scheme 1. Synthesis of ibuprofen (A) and related derivatives. Reagents and conditions: (a) 2-methoxy-4-methyl-phenol, DCC/DMAP, room
temperature; (b) 3-(3-hydroxypropyl)-pyridine, DCC/DMAP, room temperature; (c) cysteamine, CDI, N2, 0 ꢁC; (d) L-cysteine ethyl ester, CDI, N2,
0 ꢁC.
Finally, in order to investigate the importance of the
ibuprofen moiety, the L-proline amide with isobutyric
acid has been prepared and esterified with 2-methoxy-
4-methyl-phenol or 3-(3-hydroxypropyl)pyridine (com-
pounds 5 and 6).
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 1–4 is shown in Scheme
1, starting from ibuprofen. As ibuprofen has a chiral
centre, two enantiomers exist. Pharmaceutical prepara-
tions contain ibuprofen racemate. In the organism,
ibuprofen undergoes unidirectional metabolic chiral
inversion from (R)- to (S)-antipode to an extent of
60%.6
The anti-inflammatory activity of the synthesised com-
pounds (1–6) was tested as reduction of paw oedema in-
duced by Freund’s complete adjuvant in mice and as
in vitro inhibition of lipoxygenase activity. The effect
of the administration of a selected compound (2) on
COX-2 production from spleenocytes from arthritic rats
was also studied. Their antioxidant potential was evalu-
ated by the offered inhibition of lipid peroxidation, using
rat hepatic microsomal membranes, and by their inter-
action with 2,2-diphenyl-1-picrylhydrazyl stable radical.
Ibuprofen [A, racemate or the (S)-isomer] was converted
to the acid chloride (B), and then reacted with L-proline
[(S)-pyrrolidine-2-carboxylic acid], to give the corre-
sponding amide (C). Compound C was further convert-
ed to the final esters (1 and 2) or amides (3 and 4) by
application of conventional methods under mild condi-
tions, with moderate to good yields.
The protective activity of 4, found to possess significant
anti-inflammatory and antioxidant capacity, against
oxidative insult to rat brain caused by ischemia–reperfu-
sion, was also tested. Furthermore, the effect of
compounds 3 and 4 on cholesterol and triglyceride
plasma levels of hyperlipidemic rats was studied. In view
of the potential long-term application of these
compounds and since ibuprofen, like most non-steroidal
anti-inflammatory drugs, has undesired effects mainly
from the gastrointestinal tract, the gastrointestinal
toxicity of compound 3 was examined. The ability of
compounds 1, 2, 4 and 6 to inhibit acetylcholinesterase
activity in vitro was evaluated.
When racemic ibuprofen was used, two diastereomers of
compounds 1 and 4 were isolated and only one diaste-
reomer of compounds 2 and 3 was practically obtained.
Diastereomers were separated with flash chromatogra-
phy and identified by comparison with the product
yielded from the (S)-isomer of ibuprofen. Diastereomer
II of 1 [1(SS)] and diastereomer I of 4 [4(RSR)] (see Sec-
tion 4.2) were used in the in vitro and in vivo
experiments.
L-Proline reacted with isobutyryl chloride in the
presence of chlorotrimethylsilane, to yield the amide
D, which was esterified to give compounds 5 and 6
(Scheme 1).
Finally, the polar surface area of compounds 1–6 was
calculated as an indicator of their ability to penetrate
the blood–brain barrier.

I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
953
1,2
2.2. Effect on inflammation induced by Freund’s complete
adjuvant (FCA)
1
1
0,8
0,6
0,4
0,2
0
The effect of compounds 1–6 and ibuprofen (racemate)
on paw oedema induced by FCA is given in Table 1.
FCA paw oedema is a model of acute inflammation in
which primarily histamine, serotonin and bradykinin
are involved, and secondarily the prostaglandin path-
way. The new compounds demonstrated anti-inflamma-
tory activity comparable to that of ibuprofen at the
same dose. The most active was compound 2 which, at
half the dose of ibuprofen, caused about the same oede-
ma inhibition (58%) with ibuprofen (53%).
2
3
5
4
Compounds 5 and 6, which do not contain the complete
ibuprofen structure, had no anti-inflammatory activity.
0
60
120
180
240
300
360
420
Time (sec)
2.3. In vitro effect on lipoxygenase activity
Figure 1. Lipoxygenase activity as affected by compound 1[SS] and
(S)-ibuprofen. 1, Control; 2, 0.1 mM; 3, 0.2 mM; 4, 0.3 mM; 5,
ibuprofen 0.3 mM.
In an attempt to investigate the mechanism of the anti-
inflammatory activity of compounds 1–6, their in vitro
effect on soybean lipoxygenase was examined. Lipoxy-
genases are cytosolic nonhaem iron dioxygenases
involved in the synthesis of leukotrienes, important
inflammatory mediators.13 Recent findings14 show that
the activation of brain lipoxygenases is an early event
in the pathogenesis of Alzheimer’s disease. Soybean lip-
oxygenases convert linoleic acid to 13-hydroperoxylino-
leic acid, producing a conjugated diene that absorbs at
234 nm. It is inhibited by non-steroidal anti-inflammato-
ry drugs in a way similar to that of the rat mast cell
lipoxygenase and is often used as a reliable tool for test-
ing lipoxygenase inhibitors.15 Compound 1 demonstrat-
ed about 78% inhibition at 0.3 mM, and this effect was
time- and concentration-dependent (Fig. 1). The IC50
value of compound 1, after 7 min of incubation, was
found to be 0.14 mM, using 250 U/mL of lipoxygenase.
inhibitor, albeit not an anti-inflammatory drug, had an
IC50 value of 1.3 lM under the same experimental con-
ditions.16 Thus, lipoxygenase inhibition may, at least
partially, contribute to the anti-inflammatory activity
of this compound. It is interesting to notice that com-
pound 5, structurally relevant to 1, but without the ibu-
profen moiety, retained a moderate inhibitory activity
(42% at 0.3 mM), indicating that lipoxygenase inhibition
by compound 1 is not entirely due to the presence of the
ibuprofen structure. At the same concentration, com-
pounds 3, 4 and 6 had little effect (results not shown).
Compound
2 was found practically inactive at
0.2 mM, while at higher concentrations it presented sol-
ubility problems during the experiment.
Nordihydroguaiaretic acid,
a potent lipoxygenase
Since there are not strict structural requirements for
lipoxygenase inhibition and a large number of chemicals
inhibit lipoxygenase activity, several mechanisms of ac-
tion have been proposed. However, there is no univer-
sally accepted approach to evaluate the relative
potency of different substances to cause lipoxygenase
inhibition.17 We could only suggest that the lipophilic
character of compounds may be involved in lipoxyge-
nase inhibition, possibly influencing their interaction
with the active site.
Table 1. Effect of ibuprofen (racemate) and the tested compounds on
FCA induced inflammation
Compound
Dose
(mmol/kg)
Percent oedema
increase
(means SEM)
Percent oedema
inhibition
Ibuprofen
0
103.5 25.5
(44.6 18.4)**
86.2 5.5
0
53
0
0.30
0
1
0.15
0.30
0
67.9 5.3**
49.6 8.6*
86.2 5.5
36.3 5.9**
16.4 2.0**
70.2 4.1
21
43
0
2.4. Determination of cyclooxygenase (COX)-2 producing
spleenocytes
2[SS]
0.15
0.30
0
58
81
0
Although a key event in the demented brain is the pres-
ence of neuroinflammation, the use of non-steroidal
anti-inflammatory drugs for the treatment of Alzhei-
mer’s disease patients is complex and still controver-
sial.18 COX-2 inhibitors have been used in clinical
trials for Alzheimer’s disease patients, but the results
reported so far have been negative.19 However, there is
evidence suggesting a role of COX-2 in synaptic plastic-
ity.20 Our results (Table 2) confirm the production of
COX-2 from spleenocytes of arthritic rats and its inhibi-
tion in the animals treated with compound 2, indicating
3[SS]
0.30
0
27.7 4.9**
102.2 1.5
68.8 8.1*
26.5 3.7**
83.5 6.1
61
0
4
0.30
0.60
0
33
74
0
5
6
0.30
0
79.3 13.5
83.5 6.1
77.0 9.7
1
0
0.30
1
* P < 0.01.
** P < 0.001 compared to controls (Student’s t-test).

954
I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
Table 2. Effect of compound 2 on COX-2 producing spleenocytes in
AID rats
1
0,8
0,6
0,4
0,2
0
A
Treatment
Percentage of spleenocytes
producing COX-2
(means SEM)
1
Day 6
Day 14
Absolute control
AID
AID + compound 2
0
0
2.50 0.25*
0.30 0.15*
0.25 0.05
0.00 0.00
2
* P < 0.001 compared to controls (Student’s t-test).
3
that this agent interferes with the production of COX-2
in chronic inflammatory conditions.
5
0
10
20
30
Time (min)
40
50
2.5. Antioxidant activity
1,5
B
2.5.1. In vitro lipid peroxidation. Oxidative stress, which
is induced in Alzheimer’s disease, can damage neurons
and glia by several mechanisms, increased lipid peroxi-
dation being one of them. Thus, the effect of compounds
1–6 on the Fe(II)/ascorbate-induced peroxidation of
hepatic microsomal membrane lipids was examined.
Compounds 1, 2, 5 and 6 had no effect under the condi-
tions of the experiment. On the contrary, compounds 3
and 4 inhibited very strongly the peroxidation reaction,
as this is demonstrated in Figure 2A and B. This effect
was time- and concentration-dependent. Apparently,
this property is due to the presence of the sulfhydryl
group in compounds 3 and 4, which may act via a
chain-breaking mechanism.21 However, applying the
above assay, cysteamine and L-cysteine ethyl ester were
found to inhibit lipid peroxidation only by 37% and
49%, respectively, at 1 mM,22 while (S)-ibuprofen,
L-proline and compounds C and D were inactive. The
most active was 3, with an IC50 value of 19 lM after
45 min of incubation. The IC50 of 4 was calculated to
be 75 lM under the same conditions. It could be sug-
gested that the difference in activity between 3 and 4 is
due to different physicochemical properties. Lipophilici-
ty of compounds is often considered as an important
factor contributing to their protection against lipid per-
oxidation, and this may partially explain the low activity
of cysteamine and L-cysteine ethyl ester. However, the
most active, compound 3, is less lipophilic than 4 (clogP
values calculated by the method of Leo–Hansch23 are
4.07 and 4.50 for 3 and 4, respectively). Yet, the calculat-
ed polar surface area of 3 is smaller than that of 4 (Table
3), possibly enabling its more effective access, retention
to and interaction with biological membranes, the site
of lipid peroxidation. Furthermore, the availability of
the SH group, due to different degrees of conformation-
al freedom, may be an additional important factor for
the differences in activity between 3 and 4, but also
between each of them and cysteamine or L-cysteine ethyl
ester, respectively.
1
1
3
0,5
4
5
0
0
10
20
30
40
50
Time (min)
Figure 2. Time course of lipid peroxidation, as affected by various
concentrations of compound 3[SS] (A) and compound 4[RSR] (B). 1,
Control; 2, 0.01 mM; 3, 0.1 mM; 4, 0.25 mM; 5, 0.5 mM.
Table 3. Lipophilicity (clogP) and polar surface area (TPSA) of
compounds
0
TPSA (A)
Compound
clogP
˚
1
2
3
4
5
6
5.667
5.185
4.066
4.501
2.200
1.710
55.84
59.50
49.41
75.71
55.84
59.50
interaction, while 3 and 4 interacted very efficiently
(Table 4), compound 3 being the most active. The above
results are in agreement with these on lipid peroxidation
Table 4. Percent interaction with DPPH (0.2 mM) after 60 min of
incubation
Compound (mM)
Percent interaction
2.5.2. Interaction with the stable 1,1-diphenyl-2-pic-
rylhydrazyl radical (DPPH). The radical scavenging
ability of compounds 1–6 was also determined as inter-
action with the hydrophobic, electron accepting radical
DPPH.24 Again, compounds 1, 2, 5 and 6 showed no
0.05
0.10
0.20
0.40
3[SS]
4[RSR]
Cysteamine
18
19
14
37
38
37
76
68
60
90
78
84

I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
955
and attributed to the presence of the sulfhydryl group. It
is known that the interaction of compounds with DPPH
is a measure of their reductive ability,21 which in this
case is due to the known reductive properties that a sulf-
hydryl group conveys.
damage, due to insufficiency of the glutathione system
in this vulnerable region of the demented brain.32
2.7. Effect on plasma cholesterol and triglyceride levels
Increased cholesterol concentration can trigger inflam-
matory reactions and is a causative factor for age-relat-
ed disorders such as atherosclerosis and Alzheimer’s
disease.33 Hypercholesterolemia is related to neurode-
generation34 and studies support that amyloid precursor
protein processing and brain cholesterol homeostasis are
connected,35 possibly because the low density lipopro-
tein (LDL) receptor-related protein is localised to mem-
brane regions that also contain secretases involved in the
production of Ab peptide.36 Additionally, we have
reported that several classical non-steroidal anti-inflam-
matory drugs possess lipid-lowering action.37 We have
also shown that the combination, in a single molecule,
of both anti-dyslipidemic and antioxidant properties is
an asset for anti-atheromatic action.38,39 Thus, com-
pounds 3 and 4 as well as the parent drug, ibuprofen,
were evaluated for anti-dyslipidemic activity in vivo,
and results are shown in Table 6. Both the synthesised
compounds decreased total cholesterol, LDL-cholester-
ol and triglyceride levels more than 50%, compared with
the control levels in the plasma of hyperlipidemic rats,
compound 4 being more active. Ibuprofen (racemate)
decreased these parameters by 32–41%.
2.6. Effect on brain oxidative stress caused by ischemia–
reperfusion
It has been established that the demented brain suffers
from serious oxidative stress,25 therefore, we found it
interesting to study the effect of compound 4 on oxida-
tive stress, challenged by ischemia–reperfusion in rat
brain and expressed as malondialdehyde production
and glutathione depletion. Ischemia–reperfusion injury
is caused by reactive oxygen species produced through
Fenton reaction, able to induce lipid peroxidation, as
well as by the ensuing inflammation.26 Compound 4
was selected because it combines significant anti-inflam-
matory and antioxidant properties with lipophilicity
which would enable its access to the CNS, as well as
expected lower general toxicity, compared with com-
pounds 1–3, as a derivative of a natural amino acid.
The ischemia–reperfusion injury was well developed un-
der the applied experimental conditions, since lipid per-
oxidation, expressed as malondialdehyde (MDA)
formation, was doubled, while glutathione (GSH) con-
centration was reduced by about 50%, compared with
the control values (Table 5). Treatment with 4 caused
a trend to decreased MDA formation towards normal
values; however, this effect was not statistically signifi-
cant. Nevertheless, the effect on glutathione depletion
was very significant, since glutathione content was about
90% of the control value. Depletion of total glutathione
is a marker for oxidative stress in ischemic brain. Ische-
mic outcome is aggravated by pharmacological glutathi-
one depletion but improved by administration of
glutathione mimetics or precursors.27 Our results dem-
onstrate that treatment with 4 protects from glutathione
depletion caused in the brain by ischemia–reperfusion.
This property is probably connected to the antioxidant
and reductive ability of 4, as well as with the anti-inflam-
matory activity of this compound.28–30 It has been
reported that disturbances of thiol homeostasis are
closely related to oxidative stress in the brain31 and that
a decrease of free thiol groups in proteins extracted from
hippocampus of patients with Alzheimer’s disease is
observed, attributed to increased oxidative protein
It can be summarised that compound 4, effective against
lipid peroxidation and protective against brain oxidative
damage, also possessed lipid-lowering ability. Compound
3, structurally very similar to 4, also demonstrated almost
all the above properties, with small variations. This may
Table 6. Plasma total cholesterol (TC), LDL-cholesterol (LDL-C) and
triglyceride (TG) levels after ibuprofen (racemate) and compounds
3[SS] and 4[RSR] treatment
Compound
Plasma values (mg/dl) SD (percent
reduction compared to hyperlipidemic
control group)
TC
LDL-C
56
TG
Hyperlipidemic
control group
Ibuprofen
3[SS]
180 12
3
386 30
111 8 (38)
54 4 (70)
29 2 (84)
38 2 (32)
25 1 (54)
20 1 (64)
228 16 (41)
70 5 (82)
43 3 (89)
4[RSR]
In all cases, P < 0.005 compared to control group (Student’s t-test).
Table 5. Protective effect of compound 4[RSR] against oxidative damage during cerebral ischemia/reperfusion
Treatment
MDA nmol/mg protein
(means SEM)
Percent MDA
increase
GSH lmol/g brain
(means SEM)
Percent GSH decrease
Control
Ischemia
Ischemia + 4[RSR]
0.80 0.13
1.73 0.31***
1.35 0.17** ,+
0
116
69
97.8 6.8
54.6 8.1***
90.3 10.0*,++
0
44
8
* P > 0.1 compared to control group.
** P < 0.05.
*** P < 0.001.
+ P > 0.1 compared to rats with ischemia (Student’s t-test).
++
P < 0.05.

956
I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
1,6
indicate an underlying common molecular mechanism,
possibly their antioxidant activity.
A
1
1,4
1,2
1
2.8. Gastrointestinal toxicity
2
Since ibuprofen, and most of the non-steroidal anti-in-
flammatory drugs currently in use, have serious gastro-
intestinal toxicity, intimately connected to their
molecular mode of action, we found it necessary to
examine one of these compounds, 3, on a model proto-
col of this activity which we have reported earlier.40,41
Furthermore, a prospective nootropic, an agent for
long-term application, should be free of this common
to the non-steroidal anti-inflammatory drugs’ side effect.
We selected compound 3, because it is one of the most
active ibuprofen derivatives, which, in addition, con-
tains a cysteamine residue. It has been known for several
years that cysteamine itself, at relatively high doses,
causes duodenal ulceration,42 possibly by altering the re-
dox status in duodenal mucosa.43 Thus, ibuprofen (race-
mate), cysteamine and compound 3 were given to rats at
1.6 mmol/kg, a dose equal to that at which ibuprofen
caused 50% mortality (Table 7). Compound 3 did not
cause any mortality or any of the signs of gastrointesti-
nal toxicity observed after ibuprofen or cysteamine
administration. This greatly reduced gastrointestinal
toxicity may be the result of the entire molecular struc-
ture, integrating antioxidant properties, since it is
known that oxidative stress is an important component
of gastrointestinal ulceration,44,45 and the elimination of
the free carboxylic group of ibuprofen through amide
bond formation.
3
0,8
0,6
0,4
0,2
0
4
0
60
120
180
240
300
360
420
Time (sec)
1,6
1,4
1,2
1
B
1
3
0,8
0,6
0,4
0,2
0
4
0
60
120
180
240
300
360
420
2.9. In vitro effect on acetylcholinesterase activity
Time (sec)
Figure 3. Acetylcholinesterase activity, as affected by (S)-ibuprofen,
compound 1[SS] (A) and compound 2[SS] (B). 1, Control; 2,
ibuprofen, 0.6 mM; 3, 0.3 mM; 4, 0.6 mM.
The compromised cholinergic system of the demented
brain has been well established.46 Accordingly, the
major current approaches to SDAT treatment aim to
an increase of acetylcholine in the brain by acetylcholin-
esterase inhibitors. These agents impede some of the
principal processes that contribute to neurodegeneration
via acetylcholine-mediated and non-acetylcholine-medi-
ated mechanisms.47 The inhibition of acetylcholinester-
ase may be irreversible, mainly via covalent bond
formation, or reversible, non-covalent binding. There-
fore, we investigated the in vitro effect of compounds
1, 2, 5 and 6 on acetylcholinesterase activity, using rat
brain homogenates and acetylthiocholine as substrate.48
Since the applied method is based on the colorimetric
determination of thiocholine produced through the cat-
alytic activity of acetylcholinesterase, by its reaction
with dithio-bisnitrobenzoic acid, this protocol could
not be used with the sulfhydryl-containing compounds
3 and 4.
The time course of acetylcholinesterase activity, as
affected by various concentrations of 1, 2 and (S)-ibu-
profen, is shown in Figure 3A and B. At the end of
the incubation period, 1, 2 and ibuprofen offered about
80%, 64% and 20% inhibition, respectively, at 0.6 mM,
while 5 and 6 were inactive. Although these are only pre-
liminary results and further investigation is needed, the
potential of these compounds to act as acetylcholinester-
ase inhibitors can be considered beneficial for their pro-
spective nootropic action and may contribute to the
mechanisms of action of reported7,8 structurally related
proline derivatives.
Table 7. Gastrointestinal toxicity (1.6 mmol/kg, ip) of ibuprofen
(racemate), cysteamine and compound 3 in rats
2.10. Evaluation of blood–brain barrier (BBB) penetration
Compound
Percent
Percent
Body weight Melena
incidence
mortality GI ulcers change
(g/100 g)
The compounds under investigation have been designed
to act in the CNS; consequently, a prerequisite for their
activity is their ability to pass the blood–brain barrier. A
method to predict the ability of compounds for BBB
penetration is the calculation of their molecular polar
Ibuprofen
Cysteamine
Compound 3[SS]
50
80
0
88
40
0
ꢀ13
ꢀ7
+
+
ꢀ
+3.5

I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
957
surface area (PSA), defined as the sum of the surface
areas of the polar atoms in a molecule. In a set of 125
different drugs, it has been shown that all those demon-
strating CNS activity could be found within a PSA
range not higher than 90 A . We calculated the topo-
logical polar surface area (TPSA) according to the liter-
ature50 and the results are given in Table 3, indicating
that all derivatives seem to be capable of entering CNS.
lane (TMS). Signals are designated as follows: s, singlet;
d, doublet; t, triplet; q, quadruplet; m, multiplet. Ele-
mental analyses were performed with a Perkin-Elmer
2400 CHN analyzer.
2
49
˚
4.2.1.
1-(2-(4-Isobutylphenyl)propanoyl)pyrrolidine-2-
carboxylic acid, C. A solution of ibuprofen (racemate
or the (S)-isomer, compound A) [48.5 mmol] in dichlo-
romethane [50 mL] was maintained at 0 ꢁC while oxalyl
chloride [145.5 mmol] was added dropwise. The reaction
mixture was stirred at 0 ꢁC for 30 min and then at room
temperature for 3 h. The volatile components were re-
moved by distillation under reduced pressure to give 2-
(4-isobutylphenyl)propanoyl chloride, B, as yellow oil.
Compound B [46.7 mmol] and a 4 N aqueous solution
of sodium hydroxide [12 mL] were then added alterna-
tively, at 0 ꢁC, to an aqueous solution of L-proline
[(S)-pyrrolidine-2-carboxylic acid], prepared by dissolv-
ing L-proline [46.2 mmol] in 2 N aqueous solution of
sodium hydroxide [26 mL]. After constant stirring at
0 ꢁC for 20 min and then at room temperature for 1 h,
the reaction mixture was washed with ethyl acetate
[30 mL], acidified with hydrochloric acid 1 N to pH 2,
extracted with chloroform [4· 60 mL] and dried
[Na2SO4]. The solvent was evaporated under reduced
pressure and the residue was recrystallised from ether
and petroleum ether, giving a white solid (compound
C), which was further used without separation of diaste-
reomers at this stage, due to their similar chromato-
graphic properties. Yield 90%, mp 89–93 ꢁC.
3. Conclusion
With the design of the described ibuprofen derivatives
we aimed to compounds that would acquire a series of
biological properties able to prevent or restore a number
of pathological changes implicated in SDAT and
appearing in the demented brain. This study has demon-
strated that, in general, the synthesised compounds pos-
sess a number of the desired properties. It has been
admitted that neurodegenerative disorders, such as Alz-
heimer’s disease, would require multiple drug therapy to
address the various pathological disturbances of the dis-
ease. Thus, it has been realised that non-steroidal anti-
inflammatory drugs, antioxidants, certain cholesterol-
lowering agents,3 drugs that specifically block lipoxyge-
nase activation14 would be promising agents as parts of
a multidrug treatment of Alzheimer’s disease. However,
the risk of a multidrug combination, with potentially
different pharmacokinetic properties of the individual
drugs, would be avoided by the development of multi-
functional compounds, which, in addition, would offer
a simplified treatment to the Alzheimer’s disease pa-
tients, often showing difficulties in compliance. The syn-
thesised type of compounds may add to this effort.
Starting from (S)-ibuprofen and L-proline, (S)-1((S)-2-
(4-isobutylphenyl)propanoyl)pyrrolidine-2-carboxylic
acid was obtained, Mp 112–114 ꢁC. IR, 1752,
1598 cmꢀ1
. d 0.83 (d, 6H,
1H NMR: (CDCl3),
CH(CH3)2), d 1.39 (d, 3H, CH3CHCO), d 1.76–1.94
(m, 4H, C-3 pyrr, C-4 pyrr), d 2.27–2.39 (m, 3H,
CH(CH3)2, CH2CH(CH3)2), d 3.17–3.46 (m, 2H, C-5
pyrr), d 3.69 (q, 1H, CH3CHCO), d 4.58 (t, 1H, C-2
pyrr) d 7.01–7.10 (dd, 4Harom), d 10.32 (s, 1H, OH).
Analysis for C18H25NO3 calculated: C 71.24%, H
8.31%, N 4.62%. Found: C 71.04%, H 8.53%, N 4.72%.
4. Experimental
4.1. Materials and animals
All commercially available chemicals (from Aldrich-
Chemie, Steinheim, Germany; Merck, Darmstadt, Ger-
many) were of the appropriate purity. 2-Thiobarbituric
acid, diagnostic kits for total cholesterol, LDL-choles-
terol and triglyceride determinations, anti-Rabbit-IgG
(A-9169) and anti-Goat-IgG (A-8919) were purchased
from Sigma Chemical Co. (St. Louis, MO, USA).
Anti-COX-2 (sc-1746) was from Santa Cruz Biotechnol-
ogy (Santa Cruz, USA). Ibuprofen racemate was kindly
donated by VIANEX Co. (Greece).
4.2.2. (R)-1-(Isobutyryl)pyrrolidine-2-carboxylic acid, D.
To a suspension of L-proline [40 mmol] in dichlorometh-
ane [280 mL], triethylamine [108 mmol] and chlorotri-
methylsilane [48 mmol] were added successively and
the mixture was stirred for 3 h at room temperature.
Then, isobutyryl chloride [20 mmol] was added at 0 ꢁC
and the mixture was stirred for 48 h at room tempera-
ture, concentrated under reduced pressure, the residue
was dissolved in aqueous K2CO3 solution [5%, 50 mL],
washed with diethyl ether [60 mL], cooled, acidified to
pH 2 with 6 M hydrochloric acid, extracted with ethyl
acetate [3· 40 mL] and dried [Na2SO4]. The solvent
was evaporated under reduced pressure and the residue
was recrystallised from ethyl acetate and petroleum
ether. Yield 46%, mp 118–120 ꢁC. IR, 3300–2500,
For the in vivo experiments, male Balb-C mice (20–30 g)
and Fischer-344 rats (150–220 g) were used.
4.2. Synthesis
Melting points (mp) were obtained on a MEL-TEMP II
(Laboratory Devices) apparatus and are uncorrected.
Infrared (IR) spectra were taken with a Perkin-Elmer
597 Infrared Spectrophotometer. Proton nuclear mag-
netic resonance (1H NMR) spectra were obtained on a
Brucker 400 MHz spectrometer and chemical shifts are
reported in parts per million (d) relative to tetramethylsi-
1718, 1598 cmꢀ1 1H NMR: (CDCl3), d 1.07 (d, 6H,
.
CH(CH3)2), d 1.90–2.29 (m, 4H, C-3pyrr, C-4pyrr), d
2.65 (m, 1H, CH(CH3)2), d 3.46–3.65 (m, 2H, C-5pyrr),
d 4.50 (t, 1H, C-2pyrr), d 10.76 (s, 1H, OH). Analysis for

958
I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
C9H15NO3 calculated: C 58.36%, H 8.16%, N 7.56%.
Found: C 58.59%, H 8.03%, N 7.83%.
pyrr, CH(CH3)2, phenyl-CH3, CH2CH(CH3)2), d 3.45
(m, 2H, C-5 pyrr), d 3.69–3.78 (m, 4H, CH3CHCO,
OCH3), d 4.72 (m, 1H, C-2 pyrr), d 6.67–6.75 (m, 2Har-
om), d 6.85–7.17 (m, 5Harom). Analysis for C26H33NO4:
diastereomer I [1(SR)], calculated: C 73.73%, H 7.86%,
N 3.31%. Found: C 73.59%, H 8.22%, N 3.84%. Diaste-
reomer II [1(SS)], calculated: C 73.73%, H 7.86%, N
3.31%. Found: C 73.43%, H 8.22%, N 3.41%.
4.2.3. Synthesis of esters and amides of compounds C and
D. Compounds 1–6 were synthesised by esterification or
amidation of C or esterification of D with the proper
alcohol or amine. Because of the use of racemic ibupro-
fen, L-proline [(S)-pyrrolidine-2-carboxylic acid], and
L-cysteine ethyl ester [(R)-ethyl 2-amino-3-mercaptopro-
panoate, for compound 4], two diastereomers of 1 and 4
were synthesised, which were separated and identified.
The applied synthetic method and purification
procedure for compounds 2 and 3, however, resulted
in practically only one product, identified to be the
(S)-3-(pyridin-3-yl)propyl 1-((S)-2-(4-isobutylphenyl)-
propanoyl)pyrrolidine-2-carboxylate diastereomer of 2
and the (S)-1-(S)-2-(4-isobutylphenyl)propanoyl-N-(2-
mercaptoethyl)pyrrolidine-2-carboxamide diastereomer
of 3.
4.2.6. 3-(Pyridin-3-yl)propyl 1-(2-(4-isobutylphenyl)pro-
panoyl)pyrrolidine-2-carboxylate, 2. It was isolated as
practically the only diastereomer, by flash chromatogra-
phy (petroleum ether/ethyl acetate 1:2), as a liquid. It
was identified as described for 1, and found to be (S)-
3-(pyridin-3-yl)propyl 1-(S)-(2-(4-isobutylphenyl)propa-
noyl)pyrrolidine-2-carboxylate [2(SS)]. Yield 39%. IR,
1739, 1645 cmꢀ1 1H NMR: (CDCl3), d 0.87 (d, 6H,
.
CH(CH3)2), d 1.39 (d, 3H, CH3CHCO), d 1.80–2.01
(m, 7H, CH2CH2CH2, CH(CH3)2, C-3 pyrr, C-4 pyrr),
d 2.37 (d, 2H, CH2CH(CH3)2), d 2.64 (m, 2H, CH2-pyr-
idyl), d 3.23 (m, 2H, C-5 pyrr), d 3.64–3.75 (m, 1H,
CH3CHCO), d 4.08–4.17 (m, 2H, OCH2), d 4.41 (t,
1H, C-2 pyrr), d 7.05 (d, 1Harom), d 7.15 (m, 3H, 2Har-
om, C-5 pyridyl), d 7.49 (d, 1H, C-4 pyridyl), d 8.42 (m,
2H, C-2 pyridyl, C-6 pyridyl). Analysis for C26H34N2O3
calculated: C 73.89%, H 8.11%, N 6.63%. Found: C
73.73%, H 8.37%, N 6.92%.
4.2.4. General procedure for the esters 1, 2, 5 and 6. A
solution of compound C [10 mmol], 2-methoxy-4-meth-
yl-phenol [11 mmol] (1) or 3-(3-hydroxypropyl)-pyridine
[11 mmol] (2), N,N0-dicyclohexylcarbodiimide [DCC,
11 mmol] and N,N-dimethylaminopyridine [DMAP,
1 mmol] in dichloromethane was maintained at room
temperature for 3 h. After filtration, the filtrate was
washed with water, then with a 5% aqueous solution
of acetic acid, again with water and dried [MgSO4].
The solvent was evaporated under reduced pressure
and the residue was dissolved in dry ethyl acetate, left
at 4 ꢁC for 24 h, filtered and evaporated under reduced
pressure.
4.2.7. 1-(2-(4-Isobutylphenyl)propanoyl)-N-(2-mercapto-
ethyl)pyrrolidine-2-carboxamide, 3. A solution of com-
pound
C
[13.2 mmol] in N,N-dimethylformamide
˚
[DMF, 4 mL] dried over molecular sieve 4 A was main-
tained at 0 ꢁC under nitrogen, while 1,10-carbonyldiimi-
dazole [CDI, 13.7 mmol] was added. When the vigorous
release of carbon dioxide stopped, the mixture was fur-
ther stirred at room temperature until carbon dioxide
was no longer released. Subsequently, cysteamine (2-
aminoethanethiol) hydrochloride [13.3 mmol] was add-
ed and the reaction mixture was stirred for 1 h. Then,
water [20 mL] was added; the mixture was maintained
at room temperature under nitrogen for 48 h, extracted
with chloroform [3· 30 mL] and dried [Na2SO4]. The
solvent was evaporated under reduced pressure and,
with this procedure, compound 3 was finally isolated
as the only diastereomer by flash chromatography
(petroleum ether/ethyl acetate 1:1) as a white solid. It
was identified as described for 1, and found to be (S)-
1-(S)-(2-(4-isobutylphenyl)propanoyl)pyrrrolidine-N-(2-
mercaptoethyl)pyrrolidine-2-carboxamide [3(SS)]. Yield
40%, mp 75–78 ꢁC. IR, 1628, 2363, 3282, 3432 cmꢀ1. 1H
NMR: (CDCl3), d 0.86 (d, 6H, CH(CH3)2), d 1.31 (t, 1H,
SH), d 1.44 (d, 3H, CH3CHCO), d 1.78–2.04 (m, 5H, C-
3 pyrr, C-4 pyrr, (CH3)2CH), d 2.33–2.64 (m, 4H,
CH2SH, (CH3)2CHCH2), d 3.23–3.75 (m, 5H, C-5 pyrr,
NHCH2, CH3CHCO), d 4.51–4.66 (m, 1H, C-2 pyrr), d
7.05–7.24 (m, 4Harom), d 7.47 (s, 1H, NH). Analysis for
C20H30N2O2S calculated: C 66.26%, H 8.35%, N 7.78%.
Found: C 66.69%, H 8.54%, N 7.46%.
The esters 5 and 6 of compound D were prepared from
D and 2-methoxy-4-methyl-phenol (5) or 3-(3-hydroxy-
propyl)-pyridine (6) according to the method described
for 1 and 2.
4.2.5. 2-Methoxy-4-methylphenyl 1-(2-(4-isobutylphe-
nyl)propanoyl)pyrrolidine-2-carboxylate, 1. The use of
ibuprofen racemate and L-proline resulted in two diaste-
reomers of compound 1, well separated in TLC. They
were isolated by flash chromatography (petroleum
ether/ethyl acetate 4:1) in a molecular ratio almost 1:1.
The less polar (diastereomer I) is a white solid with
mp 78–82 ꢁC. The other diastereomer (diastereomer II)
is a liquid. After the synthesis of compound 1, beginning
with (S)-ibuprofen, it was found that diastereomer I is
(S)-2-methoxy-4-methylphenyl 1((R)-2-(4-isobutylphe-
nyl)propanoyl)pyrrrolidine-2-carboxylate [1(SR)], and
diastereomer II is (S)-2-methoxy-4-methylphenyl 1((S)-
2-(4-isobutylphenyl)propanoyl)pyrrrolidine-2-carboxyl-
ate [1(SS)]. Yield for both diastereomers 37%. IR, 1769,
1659 cmꢀ1. 1H NMR: Diastereomer I [1(SR)] (CDCl3), d
0.87 (d, 6H, CH(CH3)2), d 1.42 (d, 3H, CH3CHCO), d
1.82 (m, 2H, C-4 pyrr), d 2.13 (m, 3H, C-3 pyrr,
CH(CH3)2), d 2.31 (s, 3H, phenyl-CH3), d 2.42 (d, 2H,
CH2CH(CH3)2), d 3.27 (m, 2H, C-5 pyrr), d 3.71 (m,
1H, CH3CHCO), d 3.78 (s, 3H, OCH3), d 4.68 (m, 1H,
C-2 pyrr), d 6.99–7.24 (m, 7Harom). Diastereomer II
[1(SS)] (CDCl3): d 0.82 (d, 6H, CH(CH3)2), d 1.39 (d,
3H, CH3CHCO), d 1.72–2.40 (m, 10H, C-3 pyrr, C-4
4.2.8. Ethyl 2-(1-(2-(4-isobutylphenyl)propanoyl)pyrroli-
A
dine-2-carboxamido)-3-mercaptopropanoate,
4.
solution of compound C [6.6 mmol] in dry N,N-dimeth-
ylformamide [4 mL] was maintained at 0 ꢁC under

I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
959
nitrogen, while CDI [6.8 mmol] was added. The mixture
was stirred at room temperature until carbon dioxide
was no longer released. Then, L-cysteine ethyl ester
[(R)-ethyl 2-amino-3-mercaptopropanoate] hydrochlo-
ride [6.7 mmol] was added and the reaction mixture
was maintained at room temperature for 24 h. The
mixture was washed with water twice, chloroform was
added and the organic phase was dried [CaCl2]. The
solvent was evaporated under reduced pressure and
the residue was flash chromatographed (petroleum
ether/ethyl acetate 3:1) for the separation of isomers.
Both diastereomers are colourless oils. After the
synthesis of compound 4, beginning with (S)-ibuprofen,
it was found that the less polar diastereomer I is (R)-ethyl
2-((S)-1-((R)-2-(4-isobutylphenyl)propanoyl)pyrrolidine-
2-carboxamido)3-mercaptopropanoate [4(RSR)] and
diastereomer II is (R)-ethyl 2-((S)-1-((S))-2-(4-isobutyl-
phenyl)- propanoyl)pyrrrolidine-2-carboxamido-3-mer-
captopropanoate [4(RSS)]. Yield 32% for diastereomer
I, and very poor (less than 10%) for diastereomer II.
intraperitoneally (ip) [0.15–0.60 mmol/kg body weight]
15 min before the FCA injection. Three hours later,
the hind paws were excised and weighted separately.
The produced oedema was estimated as paw weight
increase compared to the control animals that received
only the liquid vehicle.51
4.4. In vitro effect on lipoxygenase activity
The reaction mixture [total volume 3 mL] contained
100 lL of the test compounds [0.1–0.3 mM], dissolved
in 60% aqueous ethanol (sample), or 100 lL of the sol-
vent (reference) and 200 lL of soybean lipoxygenase
[250 U/mL] in 2.6 mL of Tris buffer (pH 9). The reaction
was initiated by the addition of 100 lL sodium linoleate
[0.1 mM] in the sample mixture, an equal volume of
buffer being added to the reference solution, and moni-
tored for 7 min at 28 ꢁC, by recording the absorbance of
a conjugated diene structure at 234 nm, due to the for-
mation of 13-hydroperoxylinoleic acid.52
1
IR, 1632, 1683, 1740, 2560 cmꢀ1. H NMR: Diastereo-
mer I [4(RSR)] (CDCl3), d 0.87 (d, 6H, CH(CH3)2), d
1.28 (t, 3H, OCH2CH3), d 1.43 (d, 3H, CH3CHCO),
d 1.67–2.26 (m, 6H, SH, C-3 pyrr, C-4 pyrr, (CH3)2CH),
d 2.41 (d, 2H, (CH3)2CHCH2), d 2.90–3.08 (m, 2H, C-5
pyrr), d 3.19–3.27 (m, 2H, CH2SH), d 3.76 (q, 1H,
CH3CHCO), d 4.21 (m, 2H, OCH2), d 4.54 (m, 1H,
C-2 pyrr), d 4.75–4.79 (m, 1H, NHCH), d 7.06 (d, 2Har-
om), d 7.16 (d, 2Harom), d 7.54 (d, 1H, NH). Analysis
for C23H34N2O4S 1.1H2O calculated: C 60.79%, H
8.03%, N 6.16%. Found: C 60.41%, H 7.98%, N 6.39%.
4.5. Effect on COX-2 production in rat spleenocytes
Adjuvant arthritis (AID) was induced in rats by id injec-
tion of FCA [0.1 mL] to the third distal of the tail, as
previously described.53 The following day and for 10
days, compound 2, suspended in water with few drops
of Tween 80, was given ip [0.15 mmol/kg body weight].
Animals were sacrificed on the sixth and the 14th day
post AID treatment, spleens were removed quickly,
immersion-fixed at 4 ꢁC for 24 h, dehydrated, embedded
in paraffin and cut into about 15 (5 lm thick) sections.
Tissues were then deparaffinised with toluene, rehydrat-
ed, treated with H2O2 [0.1% in methanol] for 30 min,
incubated in 5% foetal bovine serum (FBS) in phosphate
buffer saline (PBS) for 1 h, then incubated with the pri-
mary antibody for 1 h at a concentration of 10 lg/mL.
After washing, slices were incubated for 1 h with the
appropriate peroxidase-conjugated secondary antibody
(diluted 500 times in PBS). Finally, slices were incubated
for 10 min in a dianisidine solution [5 mg of dianisidine,
1 mL methanol, 9 mL PBS and 5 lL H2O2 0.1%]. Red–
orange areas, representing positive areas, were visualised
using the appropriate microscopic equipment.54
4.2.9. (R)-2-Methoxy-4-methylphenyl 1-(isobutyryl)pyr-
rolidine-2-carboxylate, 5. It was isolated by flash chro-
matography (petroleum ether/ethyl acetate 2:1) as a
1
yellow oil. Yield 60%. IR, 1769, 1650, 1508 cmꢀ1. H
NMR: (CDCl3), d 1.07 (d, 6H, CH(CH3)2), d 1.96–
2.37 (m, 7H, C-3pyrr, C-4pyrr, phenyl-CH3), d 2.56–
2.67 (m, 1H, CH(CH3)2), d 3.51–3.73 (m, 5H, C-5pyrr,
OCH3), d 4.65 (t, 1H, C-2pyrr), d 6.65–6.92 (m, 3Har-
om). Analysis for C17H23NO4 calculated: C 66.86%, H
7.59%, N 4.59%. Found: C 66.80%, H 7.65%, N 4.49%.
4.2.10. (R)-3-(Pyridin-3-yl)propyl 1-(isobutyryl)pyrroli-
dine-2-carboxylate,6. It was isolated by flash chromatog-
raphy (ethyl acetate) as yellow oil. Yield 32%. IR, 1743,
4.6. In vitro lipid peroxidation
1645, 1577 cmꢀ1 1H NMR: (CDCl3), d 1.04 (d, 6H,
.
CH(CH3)2), d 1.86–2.33 (m, 6H, C-3pyrr, C-4pyrr,
CH2-CH2CH2), d 2.58–2.65 (m, 3H, CH(CH3)2, pyr-
idyl-CH2), d 3.47–3.64 (m, 2H, C-5pyrr), d 4.04 (m,
2H, OCH2), d 4.40 (t, 1H, C-2pyrr), d 7.13 (d, 1H, C-
4pyridyl), d 7.45 (m, 1H, C-5pyridyl), d 8.37 (d, 2H,
C-2pyridyl, C-6pyridyl). Analysis for C17H24N2O3 0.4
H2O calculated: C 65.53%, H 8.02%, N 8.99%. Found:
C 65.34%, H 8.27%, N 9.00%.
Hepatic microsomal fraction from untreated rats was
prepared.38 The incubation mixture contained heat-in-
activated (90 ꢁC for 90 s) microsomal fraction, corre-
sponding to 2.5 mg protein/mL (final concentration)
or 4 mM fatty acid residues,55 ascorbic acid [0.2 mM]
in Tris–HCl/KCl buffer [50/150 mM] and the test com-
pounds dissolved in dimethylsulfoxide. The peroxida-
tion reaction was initiated by the addition of a
freshly prepared FeSO4 solution [10 lM] and the mix-
ture was incubated at 37 ꢁC. Aliquots [0.3 mL] were
taken at various time intervals for 45 min. Lipid perox-
idation was assessed spectrophotometrically (535
against 600 nm) by the determination of 2-thiobarbitu-
ric acid reactive material.39 All compounds as well as
dimethylsulfoxide were tested and found not to inter-
fere with the assay.
4.3. Effect on inflammation induced by Freund’s complete
adjuvant (FCA)
Oedema was induced by the intradermal (id) injection of
FCA [0.05 mL/paw] into the right hind paw of mice, the
left paw serving as control. The test compounds, sus-
pended in water with few drops of Tween 80, were given

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I. C. Siskou et al. / Bioorg. Med. Chem. 15 (2007) 951–961
4.7. Interaction with the stable radical DPPH
presence of the tested compounds (dissolved in 60% eth-
anol) and evaluating the reaction product of the liber-
ated thiocholine with DTNB at 412 nm.48 The used
solvent system was tested and found not to interfere
with the assay. Physostigmine sulfate, used as a stan-
dard, caused a 56% inhibition of acetylcholinesterase
at 0.15 lM, under the same experimental conditions.
The test compounds were dissolved in absolute ethanol
(analytical grade, iron content less than 10ꢀ5% w/v),
added to an equal volume of an ethanolic solution of
DPPH [final concentration 0.2 mM] at various concen-
trations [25–400 lM] and incubated at 22 2 ꢁC. Absor-
bance (517 nm) was recorded after 30 min.29
4.8. Effect on brain oxidative stress caused by ischemia–
reperfusion
References and notes
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