Compounds against inflammation and oxidative insult as potential agents for neurodegenerative disorders

Article abstract of DOI:10.1007/s00044-011-9726-x

Amides of proline, a feature encountered in nootropics, via the carboxylic group of ibuprofen, indomethacin, ketoprofen and naproxen were prepared. Proline carboxylic group was amidated or esterified with potential antioxidant or neuroprotective compounds. Proline was replaced by 4-hydroxyproline, 2-pipecolic acid or omitted, for investigating the contribution of structure to activity. Anti-inflammatory activity was determined, and selected compounds were examined for anti-dyslipidemic action, protection against brain ischaemia/reperfusion and brain penetration. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9726-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2280–2291
DOI 10.1007/s00044-011-9726-x
ORIGINAL RESEARCH
Compounds against inflammation and oxidative insult as potential
agents for neurodegenerative disorders
•
Christos M. Doulgkeris Ioanna C. Siskou Nikoletta Xanthopoulou
•
•
•
Vassiliki Lagouri Constantina Kravaritou Phaedra Eleftheriou
•
•
•
Panos N. Kourounakis Eleni A. Rekka
Received: 6 June 2010 / Accepted: 21 June 2011 / Published online: 28 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Amides of proline, a feature encountered in
nootropics, via the carboxylic group of ibuprofen, indo-
methacin, ketoprofen and naproxen were prepared. Proline
carboxylic group was amidated or esterified with potential
antioxidant or neuroprotective compounds. Proline was
replaced by 4-hydroxyproline, 2-pipecolic acid or omitted,
for investigating the contribution of structure to activity.
Anti-inflammatory activity was determined, and selected
compounds were examined for anti-dyslipidemic action,
protection against brain ischaemia/reperfusion and brain
penetration.
often observed during AD, and inflammation products are
found in the central nervous system of AD patients
(Eikelenboom and Veerhuis, 1996; Streit et al., 2004). Brain
capillaries themselves are also affected in the course of AD
(De Vries et al., 1997). In addition, cerebral ischaemia seems
to play an important role in the pathogenesis of AD (Qi et al.,
2007). Finally, there is a significant role of oxidative stress in
the development of AD (Moreira et al., 2008). Reactive
oxygen species released in the central nervous system after
an ischaemic event may influence blood brain barrier per-
meability. It is already known that the function of blood brain
barrier is impaired in AD patients (De Vries et al., 1997).
Thus, despite the fact that there is still enough uncer-
tainty about the molecular mechanism in AD pathogenesis,
and although a really effective intervention has not evolved
yet, the above pathobiochemical changes in the demented
brain can be used as a starting point for rational design of
plurifunctional molecules for the medicinal treatment of
cognition disorders.
Keywords Non steroidal anti-inflammatory drugs ꢀ
Proline conjugates ꢀ Inflammation ꢀ Brain penetration
Introduction
Alzheimer’s disease (AD) is a chronic neurodegenerative
condition that affects an increasing number of individuals
as life expectancy is rising. Neurofibrillary tangles and
senile plaques (Crentsil, 2004), as well as a serious cho-
linergic deficit in the brain are main pathological features
of Alzheimer-type dementia (Palmer, 2002). Inflammatory
reactions, surrounding the cerebral microvasculature, are
In previous investigations, we have reported the design
and study of a number of properly modified non steroidal
anti-inflammatory drugs (NSAIDs) designed to prevent or
restore some pathological changes appearing in AD, via a
combination of biological properties that the new molecules
would acquire (Siskou et al., 2007; Doulgkeris et al., 2006).
In this study, an effort is made to complete and extend those
studies with the synthesis and biological evaluation of some
more compounds, to identify favourable structural charac-
teristics. Thus, amides of proline, a feature often encountered
in nootropic agents (Ostrovskaya et al., 2007), with the
carboxylic group of known NSAIDs, i.e., ibuprofen, indo-
methacin, ketoprofen and naproxen were prepared. Proline
carboxylic group was esterified with 2-methoxy-4-methyl
phenol and 3-(3-hydroxypropyl)pyridine, expected to offer
neuroprotective or neurotrophic activity, or with
C. M. Doulgkeris ꢀ I. C. Siskou ꢀ N. Xanthopoulou ꢀ
V. Lagouri ꢀ C. Kravaritou ꢀ P. Eleftheriou ꢀ
P. N. Kourounakis ꢀ E. A. Rekka (&)
Department of Pharmaceutical Chemistry, School of Pharmacy,
Aristotelian University of Thessaloniki,
Thessaloniki 54124, Greece
e-mail: rekka@pharm.auth.gr
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Med Chem Res (2012) 21:2280–2291
2281
2-nitroxyethanol, that can liberate nitric monoxide. Amida-
tion of the proline carboxylic group with the thiol-containing
cysteamine and cysteine ethyl ester, that would present
antioxidant properties, was also performed. For further
investigation about the contribution of structural features to
activity, esters of phenylpropanol and ethylenediol were
prepared. Finally, the proline moiety was replaced by
4-hydroxy-proline, 2-pipecolic acid, or it was excluded and
the NSAIDs were directly converted to esters or amides
using some of the above mentioned alcohols or amines.
The synthesized compounds were tested for anti-
inflammatory and antioxidant activity. An effective inhib-
itor of the in vitro lipid peroxidation was also tested in
vivo, by evaluation of lipid peroxidation after rat brain
ischaemia and reperfusion. Finally, since the designed site
of action of these compounds is the central nervous system,
a HPLC method was developed using brain homogenates
of rats treated with a number of the most promising com-
pounds, for the determination of blood–brain barrier
penetration.
Table 1 Structures of the synthesized compounds 1–8
O
C
O R1
N
D
No.
1
D
R1
CH3
(CH2)3
CH3
CH3
O
H3C
CH3
–CH2CH2Br
2
3
O
H3C
CH3
O
O
H3CO
CH3
CH3
N
Cl
O
H3C
CH3
O
4
5
O
Results and discussion
CH3
CH3
N
O
CH3
Chemistry
CH
3 O
O
H3C
H3C
O
The synthesized compounds are given in Tables 1, 2 and 3.
Their synthesis has been performed by methods we have
applied previously (Siskou et al., 2007; Doulgkeris et al.,
2006; Galanakis et al., 2004). During the purification of the
nitric acid derivative 7 by flash chromatography, a second
compound was isolated and identified as the hydrolysis
product 8.
CH3
(CH2)3
6
7
O
O
CH3
–CH2CH2ONO2
–CH2CH2OH
CH3
CH3
O
H3C
H3C
In an effort to react the pipecolic acid amide of indo-
methacin with cysteine ethyl ester, compound 14 was
obtained as the main reaction product. Amide bond
hydrolysis of indomethacin has been known to occur in
both acid (Garcia et al., 2006) and alkaline environment
(Suleiman and Najib, 1990) even at 25°C.
CH3
8
O
during inflammation can be indicated. This antioxidant
action can be mediated via different mechanisms, via NO
liberation in the former case, the reductive effect of the
–SH group in the latter. We have shown the anti-inflam-
matory activity of nitric oxide donors before (Ziakas et al.,
2006) examining the anti-inflammatory potential of various
tolfenamic acid nitric ester derivatives. The positive effect
of the thiol group is further verified by the ketoprofen
derivative 10 (52% inhibition), compared to ketoprofen
(42%).
Acute inflammation
All tested compounds present anti-inflammatory activity,
yet most of them are weaker than or equal to the parent
NSAIDs (Tables 4, 5). However, this study reveals some
important structural characteristics, which possibly are
implicated with this activity. Thus, the nitric ester 7
induces 62% inhibition, better than ibuprofen, but the
hydrolysis product 8 only 24%. Given the fact that ibu-
profen amide with proline, when amidated with cyste-
amine, produces 61% inhibition of oedema at the same
experiment (Siskou et al., 2007), the role of the action of
these compounds against reactive oxygen species formed
Another point that deserves further investigation is the
presence of the 3-pyridyl-propyl group, which seems to
contribute to the anti-inflammatory action in several cases.
Thus, a simple esterification with 3-pyridyl-propanol
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Med Chem Res (2012) 21:2280–2291
Table 2 Structures of the
synthesized compounds 9–14
No.
9
D
n
R2
R3
R2
O
H3CO
1
H
–CH(COOC2H5)CH2SH
n
O
CH3
N
O
C NH R3
Cl
N
D
H3C
O
10
11
1
1
H
H
–CH(COOC2H5)CH2SH
–CH2CH2SH
O
O
H3C
O
CH3
O
H3C
H3C
12
13
14
1
1
2
H
OH
H
–CH2CH2SH
–CH2CH2SH
O
CH3
O
O
O
–CH(COOC2H5)CH2SH
H3C O
CH3
N
H
increases the anti-inflammatory activity of ketoprofen (17)
and ibuprofen (16). We have found (Siskou et al., 2007)
that the activity of the ibuprofen derivative is greatly
increased (81%) when a proline moiety is incorporated
between ibuprofen and 3-pyridyl-propanol. On the con-
trary, when the pyridyl group is replaced by phenyl, a great
drop in activity is observed (1).
same experimental conditions, and found to inhibit COX-1
by 52 and 86% and COX-2 by 88 and 78%, respectively.
It is known that small molecules incorporating a proline
moiety esterified with 2-pyridyl-propanol cross the blood–
brain barrier and possess neurotrophic action (Steiner et al.,
1997). By this study, some additional properties of such
derivatives are demonstrated, with possible further
applications.
Lipoxygenase and cycloxygenase activities
In vitro lipid peroxidation
The effect of a number of the synthesized compounds on
the in vitro lipoxygenase activity is shown in Table 6. A
similar to the in vivo anti-inflammatory effect of the
3-pyridyl-propanol derivatives is also observed with
lipoxygenase inhibition. Thus, the three most active
lipoxygenase inhibitors are compounds 6, 16 and 19 (IC50
46, 45 and 28 lM, respectively). Compound 17, although
moderately inhibiting lipoxygenase, strongly inhibits
COX-1 and COX-2 (87 and 69%, respectively). Further-
more, the naproxen ester with 3-pyridyl-propanol (19)
significantly inhibits both COX isoforms (COX-1: 71%;
COX-2: 50%), whilst this activity almost disappears with
the naproxen derivative 5 (16% inhibition for COX-1, no
effect on COX-2), as well as with the indomethacin ana-
logue 3 (16 and 18% for COX isoforms). Naproxen and
ketoprofen were included as reference compounds at the
Most of the compounds tested had a minor effect on the
peroxidation of rat microsomal membrane lipids, e.g., the
nitric ester 7 inhibited the peroxidation reaction by 14% at
1 mM. As expected, the highest activity was observed with
the thiol-containing compounds 10 and 20, with IC50 value
(after 45 min of incubation) of 440 and 650 lM, respec-
tively, being very much in accordance with the reported
(Doulgkeris et al., 2006) value of 390 lM for compound 9.
However, compound 14, derived after amide bond hydro-
lysis of the indomethacin moiety, was found to be a very
potent antioxidant, with IC50 value of 8 lM, under the
same experimental conditions. It is known that melatonin,
being structurally related to 14, as well as other indole
derivatives show some antioxidant activity in vitro. This
effect can be attributed to the donation of hydrogen by the
indole NH group (Marshall et al., 1996).
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Med Chem Res (2012) 21:2280–2291
2283
Table 3 Structures of the
synthesized compounds 15–20
No.
15
D
R4
O
CH3
CH3
O
O
C
R4
D
CH3
CH3
CH3
O
H3C
H3C
CH3
(CH2)3
O
O
16
17
18
N
O
H3C
(CH2)3
O
N
O
CH3
CH3
O
O
O
H3C
H3C
CH3
O
CH3
(CH2)3
O
19
20
O
N
O
H3C
–NHCH2CH2SH
O
O
Table 4 Effect of the tested compounds on FCA-induced mice paw
oedema
Brain oxidative stress caused by ischaemia–reperfusion
The effect of the indomethacin derivative 9 on oxidative
stress induced in vivo by reperfusion of ischaemic rat
brain is determined. We selected this compound for
several reasons: it has been found (Doulgkeris et al.,
2006) to inhibit considerably in vitro lipid peroxidation,
to reduce chronic inflammation induced by Freund’s
complete adjuvant (FCA) by 77%, whilst it did not
produce any gastrointestinal irritation, compared to
equimolar dose of indomethacin, which demonstrated
serious gastrointestinal toxicity. Furthermore, a similar
compound, integrating ibuprofen in the place of indo-
methacin, has been found (Siskou et al., 2007) to protect
the brain from oxidative insult induced by ischaemia–
reperfusion. Finally, both ibuprofen and indomethacin
have been found able to cross the blood–brain barrier
(Parepally et al., 2006).
Compound
Dose
(mmol/kg)
Percent oedema
increase
(mean SEM)
Percent
oedema
inhibition
1
3
0
103.5 25.5
73.0 7.0**
80.2 7.7
0
31
0
0.30
0
0.15
0.30
0
40.6 3.5**
36.6 4.5 **
74.1 8.7
49
54
0
7
8
9
0.30
0
24.8 3.5**
103.5 25.5
74.7 9.7*
96.8 5.6
62
0
0.30
0
24
0
0.15
0.30
0
72.7 3.0**
54.6 1.7**
83.5 6.1
25
44
0
15
Ischaemia is found to increase significantly (P \ 0.05
compared to controls) oxidative stress in the brain, expressed
as malondialdehyde formation (3.7 0.2, against control
2.0 0.7 nmol/mg brain protein) and treatment with 9
caused a statistically significant (P \ 0.01 compared to
ischaemia; not significant compared to controls) reduction of
malondialdehyde formation (3.0 0.2 nmol/mg brain pro-
tein). These results can be considered interesting, since,
besides providing evidence that this compound can enter the
0.30
0
45.4 7.6**
74.1 10.7
29.5 6.3**
103.5 25.5
49.6 18.6**
81.7 4.9
43
0
16
0.30
0
59
0
S-Ibuprofen
Indomethacin
0.30
0
53
0
0.15
26.3 2.8**
68
* P \ 0.01, ** P \ 0.001 compared to controls (Student’s t test)
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Med Chem Res (2012) 21:2280–2291
Gastrointestinal toxicity
Table 5 Effect of the tested compounds on carrageenan-induced rat
paw oedema
Nonsteroidal anti-inflammatory drugs induce gastric muco-
sal lesions in part by the activation of inflammatory cells and
the production of proinflammatory cytokines. In particular,
indomethacin causes serious gastric ulcers through various
processes, including generation of reactive oxygen species,
initiation of lipid peroxidation, infiltration of leucocytes,
induction of apoptosis, in addition to inhibition of prosta-
glandin synthesis (Bech et al., 2000). These effects are dose
dependent and in many casessevere enough to limit their use.
In this study, we examined the appearance of gastrointestinal
toxicity after the administration of the indomethacin deriv-
ative 3, one of the most potent synthesized anti-inflammatory
agents, and compound 17, a ketoprofen derivative with high
anti-inflammatory action and a potent inhibitor of both
cyclooxygenase isoforms. At doses equimolar to those that
the parent drugs induced 50% mortality, both compounds
had mild effects on the gastrointestinal tract (Table 7),
compound 3 being less toxic. Since these compounds have
no antioxidant activity and do not seem to possess any tissue
protective properties, we could only attribute their low gas-
trointestinal toxicity to the masking of the carboxylic group.
The parent non steroidal anti-inflammatory drug may grad-
ually be liberated after hydrolysis. The higher gastrointesti-
nal toxicity of the ketoprofen derivative 17, compared to the
indomethacin analogue, could be explained by the easier
hydrolysis of an ester than an amide bond. Additional gastric
irritation by 17 may be due to the fact that it has been found to
be a quite strong COX-1 inhibitor, whilst 3 has only weak
cyclooxygenase inhibitory action.
Compound
Dose
(mmol/kg)
Percent oedema
increase
(means SEM)
Percent
oedema
inhibition
4
0
38.4 2.6
32.7 3.0NS
59.8 12.0
40.0 3.9*
37.7 9.7
28.6 7.1*
45.1 5.3
21.7 3.4**
37.7 9.7
14.8 7.6*
37.7 9.7
22.7 3.7*
37.7 9.7
27.4 4.4*
45.2 5.6
27.3 3.7*
45.2 5.6
26.2 1.2*
58.5 4.3
28.7 4.2*
0
13
0
0.30
0
5
0.30
0
20
0
6
0.30
0
24
0
10
0.30
0
52
0
17
0.30
0
61
0
18
0.30
0
40
0
19
0.30
0
27
0
20
0.30
0
39
0
S-Ketoprofen
S-Naproxen
0.30
0
42
0
0.30
51
NS not significant (Student’s t test)
* P \ 0.01, ** P \ 0.001 compared to controls
Table 6 In vitro evaluation of
Compound
LOX inhibition
IC50 (lM)
lipoxygenase (LOX) inhibition
Measurement of compounds in brain and blood
1
60
60
4
Although, we have computational and indirect experi-
mental evidence that the compounds of the above general
structure can reach the brain, we decided to get a direct
proof, by experimentally determining them in rat brain by
HPLC. We start this examination by a preliminary
assessment of four compounds (Table 8) previously
reported to possess remarkable antioxidant, anti-inflam-
matory activities and low gastrointestinal toxicity (Siskou
et al., 2007; Doulgkeris et al., 2006).
6
46
7
240
320
170
65
8
10
15
16
45
17
145
90
18
19
28
Furthermore, the ibuprofen derivative ethyl 2-(1-(2-(4-
isobutylphenyl)propanoyl)pyrrolidine-2-carboxamido)-3-
mercaptopropanoate (compound ii, Table 8) has been
found to protect rat brain from oxidative stress and gluta-
thione depletion after ischaemia–reperfusion injury (Siskou
et al., 2007).
Ibuprofen
Ketoprofen
Naproxen
200
220
220
brain, it is known that ischaemia–reperfusion injury is
mainly due to radical attack (Rodrigo et al., 2005), and fur-
ther that ischaemic neuronal changes are implicated in
dementia (Pluta and Amek, 2008).
All compounds are found able to enter the brain relatively
soon, and to remain there at measurable concentrations for
more than 2 h. The ibuprofen derivatives i and ii seem to
follow a different pattern of the presence in the brain,
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Med Chem Res (2012) 21:2280–2291
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Table 7 Gastrointestinal toxicity of two new compounds, indomethacin and ketoprofen
Compound
Dose
(lmol/kg)
Mortality
(%)a
Perforating
ulcers (%)b
Body weight
changec
Incidence of
melena (%)d
3
84
84
0
50
33
50
0
80
50
85
?11
-15
-13
-24
0
100
60
Indomethacin
17
200
200
Ketoprofen
100
a
Dead per total 9 100
b
Percent of animals developing perforating intestinal ulcers
c
In g/100 g body weight. Standard deviation of the weight change is always within 10% of the average value
Percent of animals developing melena defecation
d
Table 8 Determination of compounds in rat brain
R2
O
C
X
NH
CH SH
N
D
Compound
D
30 min
60 min
120 min
a
%
a
%
a
%
R2
X
lg/g brain ( SEM)
lg/g brain ( SEM)
lg/g brain ( SEM)
i. Ibuprofen
ii. Ibuprofen
iii. Naproxen
iv. Naproxen
a
H
H
9.78 1.28
1.59 0.61
7.66 1.93
26.55 3.62
4.9
0.6
10.22 2.56
40.77 4.55
1.94 0.26
5.70 0.86
5.2
15.6
0.9
35.17 4.95
27.84 2.77
1.53 0.05
0.71 0.12
17.8
10.7
0.7
H
COOCH2CH3
H
H
H
3.3
OH
11.0
2.4
0.3
Percentage brain penetration
compared with the naproxen derivatives iii and iv: the con-
centrations of the former are gradually increased with time,
whereas the latter reaches the brain within the first 30-min
post injection and a decrease is observed in the next
90 min. Although it has been observed, in retrospective
clinical studies, that chronic treatment with non steroidal
anti-inflammatory drugs may protect against Alzheimer’s
disease, there are not enough studies concerning their con-
centration in the brain. Both ibuprofen and naproxen have
been reported to have access to the brain, however, it has
been claimed (Mannila et al., 2005) that ibuprofen brain
penetration is insufficient for its use in Alzheimer’s disease.
Better results were obtained with ibuprofen derivatives
having higher lipophilicity, such as ibuprofen glycerides
(Deguchi et al., 2000). Regarding the above, we consider the
performed molecular modifications a significant improve-
ment for better brain penetration.
found that the ratio of brain to blood concentration is 0.04
2-h post injection, which drops to 0.004 1 h later, possibly
due to redistribution.
Conclusion
Disorders, such as Alzheimer’s disease, could be treated
effectively with compounds designed to act at different
causes and stages of their pathogenesis. The described
molecules could become useful for developing agents that
may slow the progression or delay the onset of neurode-
generative diseases.
Experimental
We also determined the blood concentrations of 1(2-(6-
methoxynaphthalen-2-yl)propanoyl)-4-hydroxypyrrolidine-
2-carboxylic acid 2-mercaptoethylamide (compound iv,
Table 8), which has the lowest lipophilicity of the four, by
the same method and at the same time intervals. It was
Materials and methods
All commercially available reagents (from Aldrich-Chemie,
Steinheim, Germany; Merck, Darmstadt, Germany) were of
the appropriate purity. 2-Thiobarbituric acid, soybean
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Med Chem Res (2012) 21:2280–2291
1-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetyl)pyrrolidine-2-carboxylic acid, B (Doulgkeris
et al., 2006)
lipoxidase and diagnostic kits for cycloxygenase 1 and 2
determinations were purchased from Cayman Chemical
Company (Ann Arbor, Michigan, USA).
Mp 120–124°C (from ether and petroleum ether), yield
87%.
Synthesis
Melting points were obtained on a MEL-TEMP II (Labo-
ratory Devices) apparatus and are uncorrected. Infrared
(IR) spectra were taken with a FTIR-810M (Shimadzu)
Fourier Transform Spectrophotometer. Proton nuclear
magnetic resonance (1H NMR) spectra were obtained on a
Brucker 400 MHz spectrometer and chemical shifts are
reported in parts per million (d) relative to tetramethysi-
lane. Signals are designated as follows: s, singlet; d, dou-
blet; t, triplet; q, quadruplet and m, multiplet. Elemental
analyses were performed with a Perkin-Elmer 2400 CHN
analyzer and are within 0.4% of the calculated values.
1-(2-(3-Benzoylphenyl)propanoyl)pyrrolidine-2-carboxylic
acid, C
Oil, yield 85%. IR (neat) 1606, 1670, 1756 cm-1. 1H NMR
(CDCl3) d 1.50 (d, 3H, CH3CH; J = 9.0 Hz), d 1.90–2.30
(m, 4H, C-3 pyrr., C-4 pyrr.), d 3.30–3.40 (m, 2H, C-5
pyrr.), d 3.90 (q, 1H CH3CHCO), d 4.40 (t, 1H, C-2 pyrr.),
d 7.40–7.70 (m, 9H arom.).
1-(2-(6-Methoxynaphthalen-2-yl)propanoyl)pyrrolidine-2-
carboxylic acid, D
General method for the preparation of proline/pipecolic
acid amides with the NSAIDs
Mp 158–160°C (from ether and petroleum ether), yield
90%. IR (KBr) 1632, 1735 cm-1. 1H NMR (CDCl3) d 1.50
(d, 3H, CH3CH; J = 8.9 Hz), d 1.80–2.30 (m, 4H, C-3
pyrr., C-4 pyrr.), d 3.20–3.60 (m, 2H, C-5 pyrr.), d 3.70 (m,
1H, CHCH3), d 3.90 (s, 3H, CH3O), d 4.65 (t, 1H, C-2
pyrr.), d 7.10 (s, 2H arom., C-1 naphth., C-5 naphth.), d
7.40 (d, 1H arom., C-7 naphth.; J = 11.4 Hz), d 7.65–7.80
(m, 3H arom.).
The corresponding NSAID, i.e., (S)-ibuprofen ((S)-2-(4-
isobutylphenyl)propanoic acid), indomethacin (1-(4-chlo-
robenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid),
(S)-ketoprofen ((S)-2-(3-benzoylphenyl)propanoic acid) or
(S)-naproxen ((S)-(6-methoxy-2-naphthyl)propanoic acid)
(48.5 mmol) was dissolved in dichloromethane (50 ml)
and maintained at 0°C. Oxalyl chloride (145.5 mmol) was
added dropwise, the mixture was kept at 0°C for 30 min
and then at room temperature for 3–5 h. The volatile
components were distilled off under reduced pressure to
give the chlorides as yellow oils or solids (indomethacin
acid chloride, Mp 124–126°C, naproxen acid chloride, Mp
92–94°C). The chloride (46.7 mmol) and a 4 N aqueous
solution of sodium hydroxide (12 ml) were then added
alternatively, at 0°C, to an aqueous solution of L-proline
((S)-pyrrolidine-2-carboxylic acid) or trans-4-hydroxy-L-
proline ((2S, 4R)-4-hydroxypyrrolidine-2-carboxylic acid)
or L-pipecolic acid ((S)-2-piperidine carboxylic acid),
prepared by dissolving the amino acids (46.2 mmol) in 2 N
aqueous solution of sodium hydroxide (26 ml). The mix-
ture was stirred at 0°C for 20 min, then at room tempera-
ture for 1–6 h, washed with ethyl acetate (3 9 40 ml),
acidified with hydrochloric acid 1 N to pH 2, extracted
with chloroform (4 9 60 ml) and dried (Na2SO4). The
solvent was evaporated under reduced pressure and the
residue was purified and identified.
1(2-(6-Methoxynaphthalen-2-yl)propanoyl)-4-
hydroxypyrrolidine-2-carboxylic acid, E
Mp 152–156°C (from ethyl alcohol and water), yield 73%.
IR (KBr) 1632, 1726, 3368 cm-1. 1H NMR (CDCl3) d 1.60
(d, 3H, CH3CH; J = 9.0 Hz), d 2.10–2.45 (m 2H, C-3
pyrr.), d 3.15–3.65 (m, 2H, C-5 pyrr.), d 3.80 (m, 1H, C-4
pyrr.), d 3.90 (s, 3H, CH3O), d 4.35–4.45 (m, 1H C-2
pyrr.), d 4.75–4.85 (m, 1H, CH3CH), d 7.10 (s, 1H arom.,
C-5 naphth.), d 7.15 (s, 1H arom., C-1 naphth.), d 7.4 (d,
1Harom., C-7 naphth.; J = 11.4 Hz), d 7.60–7.70 (m, 3H
arom.).
1-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetyl)-piperidine-2-carboxylic acid, F
Mp 161–163°C (from cold chloroform), yield 73%. IR
(KBr) 1625, 1680, 1718 cm-1. 1H NMR (CDCl3) d 1.2–1.4
(m. 2H, C-3 piper.), d 1.50–1.70 (m, 4H, C-4, C-5 piper.), d
2.00–2.30 (m, 2H, C-6 piper.), d 2.35–2.40 (s, 3H, indolyl-
CH3), d 3.60 (s, 2H, CH2CO), d 3.70 (s, 3H, OCH3), d 5.20
(s, 1H, C-2 piper.), d 6.70–7.40 (m, 4H arom., C-6 indolyl,
C-7 indolyl, C-3 4-chlorobenzoyl and C-5 4-chloroben-
zoyl), d 7.60–8.00 (m, 3H arom., C-4 indolyl, C-2 4-chlo-
robenzoyl, C-6 4-chlorobenzoyl).
1-(2-(4-Isobutylphenyl)propanoyl)pyrrolidine-2-carboxylic
acid, A (Siskou et al., 2007)
Mp 112–114°C (from ether and petroleum ether), yield
90%.
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Med Chem Res (2012) 21:2280–2291
2287
General method for the preparation of esters of the proline
derivatives
CH2O), d 7.03 (d, 2H arom.; J = 12.0 Hz), d 7.13 (d, 2H
arom.; J = 12.0 Hz). Anal. (C20H28BrNO3), C, H, N.
A solution of the proline derivatives (A–D) (10 mmol), the
corresponding alcohol or phenol (11 mmol), N,N0-dicy-
clohexylcarbodiimide (DCC, 11 mmol) and N,N-dimeth-
ylaminopyridine (DMAP, 1 mmol) in dichloromethane
(15–20 ml) was maintained at room temperature for
5–10 h. After filtration, the filtrate was washed with water,
dried (MgSO4), dichloromethane was evaporated under
reduced pressure, the residue was dissolved in dry ethyl
acetate, kept at 4°C for 24 h, filtered and concentrated
under reduced pressure. The final esters were purified with
flash chromatography and identified.
2-Methoxy-4-methylphenyl 1-(2-(1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl) acetyl)pyrrolidine-2-
carboxylate, 3
Yellow solid purified by flash chromatography (petroleum
ether/ethyl acetate 1/1), mp 76–78°C, yield 41%. IR (KBr)
1630, 1645, 1765 cm-1. 1H NMR (CDCl3) d 1.90–2.20 (m,
4H, C-3 pyrr. C-4 pyrr.), d 2.20–2.50 (m, 6H, phenyl-CH3,
indolyl-CH3), d 3.50–3.65 (m, 2H, C-5 pyrr.), d 3.70 (s,
2H, CH2CO), d 3.75 (s, 6H, phenyl-OCH3, indolyl-OCH3),
d 4.80 (t, 1H, C-2 pyrr.), d 6.60–7.00 (m, 6H, indolyl-3H
arom., 2-methoxy-4-methylphenyl-3H arom.), d 7.40 (d,
2H arom., C-3 4-chlorobenzoyl, C-5 4-chlorobenzoyl;
J = 6.5 Hz), d 7.65 (d, 2H arom., C-2 4-chlorobenzoyl,
C-6 4-chlorobenzoyl; J = 9.0 Hz). Anal. (C32H31ClN2O6),
C, H, N.
Compound 2 (see below) was further converted to the
corresponding nitric ester, compound 7: compound 2
(10 mmol) was dissolved in acetonitrile (20 ml) and
brought to 80°C. A solution of silver nitrate (15 mmol) in
acetonitrile was added and the mixture was refluxed for 8 h.
The produced silver bromide was filtered off, the filtrate
was concentrated under reduced pressure, dichloromethane
was added (30 ml), the mixture was washed with water
(2 9 20 ml), dried (CaCl2), the solvent was evaporated and
the residue was purified with flash chromatography.
During the preparation and purification procedure, a
second, less polar compound was isolated and identified,
compound 8.
2-Methoxy-4-methylphenyl 1-(2-(3-
benzoylphenyl)propanoyl)pyrrolidine-2-carboxylate, 4
Colourless oil purified by flash chromatography (petroleum
ether/ethyl acetate 1/1), yield 40%. IR (neat) 1606, 1653,
.
1774 cm-1 1H NMR (CDCl3) d 1.45 (d, 3H, CH3CH;
J = 9.0 Hz), d 1.90–2.20 (m, 4H, C-3 pyrr. C-4 pyrr.), d
2.40 (s, 3H, phenyl-CH3), d 3.30 (m, 2H, C-5 pyrr.), d 3.70
(s, 3H, CH3O), d 3.90 (q, 1H, CH3CHCO), d 4.50 (t, 1H
C-2 pyrr.), d 6.50–7.00 (m, 3H, 2-methoxy-4-methyl-
phenyl-3H arom.), d 7.40–7.70 (m, 9H arom.). Anal.
(C29H29NO5), C, H, N.
3-(Phenyl)propyl 1-(2-(4-isobutylphenyl)propanoyl)
pyrrolidine-2-carboxylate, 1
Yellow oil, purified by flash chromatography (petroleum
ether/ethyl acetate 4/1), yield 74%. IR (neat) 1645,
1
1739 cm-1. H NMR (CDCl3) d 0.83 (d, 6H, CH(CH3)2;
2-Methoxy-4-methylphenyl 1-(2-(6-methoxynaphthalen-2-
yl)propanoyl)pyrrolidine-2-carboxylate, 5
J = 8.0 Hz), d 1.37 (d, 3H, CH3CHCO; J = 8.0 Hz), d
1.75–2.11 (m, 7H, C-3 pyrr., C-4 pyrr., CH2CH2CH2,
CH(CH3)2), d 2.37 (d, 2H, CH2CH(CH3)2; J = 8.0 Hz), d
2.58 (t, 2H, phenyl-CH2), d 3.17–3.45 (m, 2H, C-5 pyrr.), d
3.67 (q, 1H, CH3CHCO), d 4.07 (t, 2H, CH2O), d 4.48 (t,
1H, C-2 pyrr.), d 7.00–7.22 (m, 9H arom.). Anal.
(C27H35NO3), C, H, N.
White solid purified by flash chromatography (petroleum
ether/ethyl acetate 4/3), mp 148–150°C, yield 51%. IR
1
(KBr) 1645, 1770 cm-1. H NMR (CDCl3) d 1.50–1.58
(m, 3H, CH3CHCON), d 1.90–1.95 (m, 2H, C-4 pyrr.), d
2.00–2.30 (m, 2H, C-3 pyrr.), d 2.35 (s, 3H, CH3-phenyl), d
3.40–3.50 (m, 2H, C-5 pyrr.), d 3.85 (s, 6H, CH3O-phenyl,
CH3O-naphth.), d 4.50 (t, 1H C-2 pyrr.), d 4.75–4.80 (q,
1H, CH3CHCON.), d 6.70–7.70 (m, 9H arom.). Anal.
(C27H29NO5), C, H, N.
2-Bromoethyl 1-(2-(4-isobutylphenyl)propanoyl)
pyrrolidine-2-carboxylate, 2
Colourless oil purified by flash chromatography (petroleum
ether/ethyl acetate 4/1), yield 85%. IR (neat) 1645,
3-(Pyridin-3-yl)propyl 1-(2-(6-methoxynaphthalen-2-
yl)propanoyl)pyrrolidine-2-carboxylate, 6
1
1743 cm-1. H NMR (CDCl3) d 0.83 (d, 6H, CH(CH3)2;
J = 8.0 Hz), d 1.35 (d, 3H, CH3CHO; J = 8.0 Hz), d
1.77–2.12 (m, 5H, C-3 pyrr., C-4 pyrr., CH(CH3)2), d 2.38
(d, 2H, CH2CH(CH3)2; J = 8.0 Hz), d 3.19 (m, 2H, C-5
pyrr.), d 3.29–3.54 (m, 2H, CH2Br), d 3.66 (q, 1H,
CH3CHCO), d 4.08 (t, 1H, C-2 pyrr.), d 4.30–4.39 (m, 2H,
Purified by flash chromatography (ethyl acetate/acetonitrile
10/1) as viscous oil, yield 50%. IR (Nujol) 1611,
1750 cm-1 1H NMR (CDCl3) d 1.54 (d, 3H, CH3CH;
.
J = 9.0 Hz), d 1.86 (m, 2H, CH2CH2CH2), d 1.90–2.16
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Med Chem Res (2012) 21:2280–2291
(m, 4H, C-3 pyrr. C-4 pyrr.), d 2.30 (t, 2H, pyridyl-CH2), d
3.30–3.55 (m, 2H, C-5 pyrr.), d 3.75 (s, 3H, CH3O), d 3.89
(q, 1H, CH3CHCO), d 4.11 (t, 2H COOCH2), d 4.55 (m,
1H C-2 pyrr.), d 7.05 (d, 1H arom., C-7 naphth.;
J = 11.4 Hz), d 7.20–7.70 (m, 6H arom., C-1 naphth., C-3
naphth., C-8 naphth., C-9 naphth., C-2 pyridyl, C-6 pyri-
dyl), d 8.37 (m, 3H arom., C-4 naphth. C-2 pyridyl, C-6
pyridyl). Anal. (C27H30N2O4 9 0.5 CH3CN), C, H, N.
Then, water (20 ml) was added, the mixture was extracted
with chloroform (3 9 30 ml) and dried (MgSO4). The
solvent was evaporated under reduced pressure and the
residue was purified and identified.
Ethyl 2-(1-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
1H-indol-3-yl) acetyl)pyrrolidine-2-carboxamido)-3-
mercaptopropanoate, 9 (Doulgkeris et al., 2006).
Purified by flash chromatography (petroleum ether/ethyl
2-Nitroxyethyl 1-(2-(4-isobutylphenyl)propanoyl)
pyrrolidine-2-carboxylate, 7
acetate 1/2) as yellow solid, mp 109–111°C, yield 30%.
Ethyl 2-(1-(2-(3-benzoylphenyl)propanoyl)pyrrolidine-2-
carboxamido)-3-mercaptopropanoate, 10
Colourless oil, purified by flash chromatography (petro-
leum ether/ethyl acetate 4/1 and then 3/1), yield 35%. IR
1
(neat) 1641, 1748 cm-1. H NMR (CDCl3) d 0.86 (d, 6H,
Colourless oil, purified by flash chromatography (petro-
leum ether/ethyl acetate 3/2 and then 1/1), yield 30%. IR
.
(neat) 1606, 1636, 1662, 1717, 2555 cm-1 1H NMR
CH(CH3)2; J = 8.0 Hz), d 1.38 (d, 3H, CH3CHCO;
J = 8.0 Hz), d 1.75–1.92 (m, 4H, C-3 pyrr., C-4 pyrr.), d
2.15 (m, 1H, CH(CH3)2), d 2.41 (d, 2H, CH2CH(CH3)2;
J = 8.0 Hz), d 3.20–3.48 (m, 2H, C-5 pyrr.), d 3.69 (q, 1H,
CH3CHCO), d 4.34 (t, 2H, CH2O), d 4.44–4.63 (m, 3H,
C-2 pyrr., CH2ONO2), d 7.06–7.16 (m, 4H arom.). Anal.
(C20H28N2O6), C, H, N.
(CDCl3) d 1.25 (t, 3H, CH3CH2O), d 1.50 (d, 3H, CH3CH;
J = 8.5 Hz), d 1.55 (t, 1H, SH), d 1.90–2.30 (m, 4H, C-3
pyrr. C-4 pyrr.), d 2.90–3.10 (m, 2H, CH2SH), d 3.20–3.40
(m, 2H, C-5 pyrr.), d 3.70–3.80 (m, 1H, CH3CH), d
3.90–4.00 (q, 2H, CH3CH2O), d 4.15–4.30 (m, 1H C-2
pyrr.), d 4.72 (t, 1H, CHCH2SH), d 7.40–7.80 (m, 9H
arom.). Anal. (C26H30N2O5S), C, H, N.
2-Hydroxyethyl 1-(2-(4-isobutylphenyl)propanoyl)
pyrrolidine-2-carboxylate, 8
1-(2-(3-Benzoylphenyl)propanoyl)-N-(2-
mercaptoethyl)pyrrolidine-2-carboxamide, 11
Colourless oil, purified by flash chromatography (petro-
leum ether/ethyl acetate 4/1), yield 25%. IR (neat) 1632,
1743, 3432 cm-1 1H NMR (CDCl3) d 0.85 (d, 6H,
.
Colourless oil, purified by flash chromatography (petro-
leum ether/ethyl acetate 3/2 and then 1/1), yield 35%. IR
CH(CH3)2; J = 8.0 Hz), d 1.37 (d, 3H, CH3CHCO;
J = 8.0 Hz), d 1.78–2.17 (m, 6H, C-3 pyrr., C-4 pyrr.,
1
(neat) 1606, 1640, 1662, 2555 cm-1. H NMR (CDCl3) d
CH(CH3)2,OH),
d
2.40 (d, 2H, CH2CH(CH3)2;
1.35 (t, 1H, SH), d 1.50 (d, 3H, CH3CH; J = 9.0 Hz), d
1.80–2.20 (m, 4H, C-3 pyrr. C-4 pyrr.), d 2.30–2.70 (m,
2H, CH2SH), d 3.30–3.80 (m, 4H, C-5 pyrr., CH2CH2SH),
d 3.90–4.00 (q, 1H, CHCH3), d 4.50 (m, 1H, C-2 pyrr.), d
7.30–7.90 (m, 9H arom). Anal. (C23H26N2O3S 9 0.3
CH3COOCH2CH3), C, H, N.
J = 8.0 Hz), d 3.20–3.26 (m, 2H, C-5 pyrr.), d 3.43–3.53
(q, 1H, CH3CHCO), d 3.62 (m, 2H, CH2OH), d 4.16–4.31
(m, 2H, CH2O), d 4.44 (m, 1H, C-2 pyrr.), d 7.07 (d, 2H
arom.; J = 12.0 Hz) d 7.15 (d, 2H arom.; J = 12.0 Hz).
Anal. (C20H29NO4), C, H, N.
General method for the preparation of amides
of the proline/pipecolic acid derivatives
1-(2-(6-Methoxynaphthalen-2-yl)propanoyl)-N-(2-
mercaptoethyl)pyrrolidine-2-carboxamide, 12
(Doulgkeris et al., 2006)
A solution of compounds B–F (10 mmol) in dry N,N-
dimethylformamide (10 ml) was maintained at 0°C under
nitrogen and 1,10-carbonyldiimidazole (CDI, 10.4 mmol)
was added. When the vigorous release of carbon dioxide
subsided, the mixture was further stirred at room temper-
ature until no more gas was released. Then, L-cysteine ethyl
ester [(R)-ethyl 2-amino-3-mercaptopropanoate] hydro-
chloride (10.1 mmol) was added to compounds B, C and F,
or cysteamine (2-aminoethanethiol) hydrochloride
(10.1 mmol) was added to compounds C, D and E, and the
reaction mixture was stirred at room temperature for 24 h.
Purified by flash chromatography (petroleum ether/ethyl
acetate 1/3) as white solid, mp 151–152°C, yield 42%.
1-(2-(6-Methoxynaphthalen-2-yl)propanoyl)-N-(2-
mercaptoethyl)-4-hydroxypyrrolidine-2-carboxamide,
13 (Doulgkeris et al., 2006)
Purified by flash chromatography (ethyl acetate/ethanol
95/5) as white solid, mp 140–141°C, yield 29%.
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2289
Ethyl 2-(1-(2-(5-methoxy-2-methyl-1H-indol-3-
yl)acetyl)piperidine-2-carboxamido)-3-
mercaptopropanoate, 14
2-Methoxy-4-methylphenyl
2-(6-methoxynaphthalen-2-
yl)propanoate, 18 White solid, purified by flash chroma-
tography (petroleum ether/ethyl acetate 4/1), mp 84–85°C,
yield 48%. 1H NMR (CDCl3) d 1.68 (d, 3H, CH3CH;
J = 8.0 Hz), d 2.30 (s, 3H, phenyl-CH3), d 3.65 (s, 3H,
phenyl-OCH3), d 3.91 (s, 3H, naphthyl-OCH3), d 4.12 (q,
After the reaction of compound F with L-cysteine ethyl ester
according to the general method, compound 14 was isolated
by flash chromatography (petroleum ether/ethyl acetate 1/1)
as the main reaction product. Yellow solid, mp 54–57°C,
yield 49%. IR (KBr). 1624, 1670, 1739, 2556 cm-1. 1H NMR
(CDCl3) d 1.30 (t, 3H, CH3CH2), d 1.40–1.70 (m, 7H, C-3
piper. C-4 piper., C-5 piper., SH), d 2.35 (s, 3H, indolyl-CH3),
d 2.80–3.30 (m, 6H, CH2CO C-6 piper., CH2SH), d 3.80 (s,
3H, CH3O), d 4.10–4.15 (m, 3H, CH3CH2O) d 4.70–4.90 (m,
2H, C-2 piper., CHCO), d 6.60 (s, 1H arom., C-4 indolyl), d
6.80–7.10 (m, 2H arom., C-6 indolyl, C-7 indolyl), d 7.90 (m,
1H, NHCO). Anal. (C23H31N3O5S), C, H, N.
1H, CH3CH), d 6.66–7.75 (m, 9H arom.). Anal.
(C22H22O4, 90.8 H2O), C, H, N.
3-(Pyridin-3-yl)propyl 2-(6-methoxynaphthalen-2-yl)pro-
panoate, 19 White solid, purified by flash chromatogra-
phy (petroleum ether/ethyl acetate 1/4), mp 55–56°C, yield
52%. 1H NMR (CDCl3) d 1.52 (d, 3H, CH3CH;
J = 8.0 Hz), d 1.82–1.85 (m, 2H, CH2CH2CH2), d 2.45 (t,
2H, pyridyl-CH2), d 3.80 (q, 1H, CH3CH), d 4.01 (s, 3H,
CH3O), d 4.05 (m, 2H, CH2O), d 7.05–7.35 (m, 8H arom.,
C10H6, C-4 pyridyl, C-5 pyridyl), d 8.30 (s, 1H arom., C-2
pyridyl), d 8.40 (d, 1H arom. C-6 pyridyl; J = 9.0 Hz).
Anal. (C22H25NO3), C, H, N.
Preparation of esters and amide of NSAIDs According to
the described general methods, the following derivatives of
NSAIDs were synthesized. Their IR spectra had a peak at
N-(2-Mercaptoethyl)-2-(3-benzoylphenyl)-2-propanamide,
20 Colourless oil, prepared, according to the general
method, from S-ketoprofen and cysteamine, and purified by
flash chromatography (petroleum ether/ethyl acetate 2/1),
yield 40%. 1H NMR (CDCl3) d 1.15 (t, 1H, SH), d 1.55 (d,
3H, CH3CH; J = 8.0 Hz), d 2.65 (m, 2H, CH2SH), d 3.65
(m, 2H, CH2CH2SH), d 3.8 (q, 1H CHCH3), d 7.30–7.70
(m, 9H arom.). Anal. (C18H19N2S), C, H, N.
1740–1760 cm-1
.
2-Methoxy-4-methylphenyl 2-(4-isobutylphenyl)propano-
ate, 15 White solid, purified by flash chromatography
(petroleum ether/ethyl acetate 5/1), mp 27–29°C, yield
51%. 1H NMR (CDCl3) d 0.92 (d, 6H, CH(CH3)2;
J = 8.0 Hz) d 1.61 (d, 3H, CH3CHCO; J = 8.0 Hz), d
1.88 (m, 1H, CH(CH3)2), d 2.32 (s, 3H, phenyl-CH3), d
2.48 (d, 2H, CH2CH(CH3)2; J = 8.0 Hz), d 3.69 (s, 3H,
CH3O), d 3.98 (q, 1H, CH3CHCO), d 6.67–6.83 (m, 3H
arom., C-3 phenyl, C-5 phenyl, C-6 phenyl), d 7.14 (d, 2H
arom.; J = 12.0 Hz), d 7.34 (d, 2H arom.; J = 12.0 Hz).
Anal. (C21H26O3), C, H, N.
Effect on acute inflammation For the in vivo experiments,
male Fischer-344 rats (200–280 g) or Balb-C mice
(20–30 g) were used. Animals were kept and treated
according to the European Communities Council Directive
of 24 November 1986 (86/609/EEC). They were housed in
controlled rooms, humidity 50–60%, temperature 23°C,
with a 12 h light/dark cycle, and free access to standard
laboratory chow and tap water.
3-(Pyridin-3-yl)propyl
2-(4-isobutylphenyl)propanoate,
16 Yellow oil, purified by flash chromatography (petro-
leum ether/ethyl acetate 3/1), yield 60%. 1H NMR (CDCl3) d
0.87 (d, 6H, CH(CH3)2), d 1.50 (d, 3H, CH3CHCO;
FCA Acute inflammation was induced by the id injection
of FCA (0.05 ml/paw) into the right hind paw of mice, the
left paw serving as control. The test compounds, suspended
in water with few drops of Tween 80, were given ip
(0.15–0.30 mmol/kg body weight) 5 min before the FCA
injection. Three hours later, the hind paws were excised
and weighted separately. The produced oedema was esti-
mated as paw weight increase compared to the control
animals that received only the liquid vehicle (Hadjipetrou-
Kourounakis et al., 1992).
J = 8.0 Hz),
d
1.77–1.96 (m, 3H, CH(CH3)2,
CH2CH2CH2), d 2.43–2.56 (m, 4H, CH2CH(CH3)2, pyridyl-
CH2), d 3.71 (q, 1H, CH3CHCO), d 4.01–4.14 (m, 2H,
CH2O), d 7.09–7.25 (m, 5H arom., C6H4, C-5 pyridyl), d 7.34
(d, 1H arom., C-4 pyridyl), d 8.34–8.45 (m, 2H arom. C-2
pyridyl, C-6 pyridyl). Anal. (C21H27NO2), C, H, N.
3-(Pyridin-3-yl)propyl
2-(3-benzoylphenyl)propanoate,
17 Yellow oil, purified by flash chromatography (petro-
leum ether/ethyl acetate 3/7), yield 60%. 1H NMR (CDCl3)
d 1.54 (d, 3H, CH3CH), d 1.90 (m, 2H, CH2CH2CH2), d
2.56 (t, 2H, pyridyl-CH2), d 3.80(q, 1H, CH3CH), d 4.09 (t,
2H, CH2O), d 7.16–7.78 (m, 11H arom., C6H5, C6H4, C-4
pyridyl, C-5 pyridyl), d 8.35 (s, 1H arom., C-2 pyridyl), d
8.42 (d, 1H arom. C-6 pyridyl; J = 9.0 Hz). Anal.
(C24H23NO3), C, H, N.
Carrageenan paw oedema An aqueous solution of carra-
geenan was prepared (1% w/v) and 0.1 ml of this was
injected into the right hind paw of male rats, the left paw
serving as control. The tested compounds (dissolved or
suspended in water with a few drops of Tween 80) were
given ip (0.3 mmol/kg of body weight) 5 min before the
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Med Chem Res (2012) 21:2280–2291
carrageenan injection. After 3 h, the hind paws were
excised and weighed separately. The produced oedema was
estimated as paw weight increase (Hadjipetrou-Kourouna-
kis et al., 1992).
induce incomplete cerebral ischaemia. Animals were sub-
jected to 45 min of carotid occlusion, then the clips were
removed and brain was reperfused for 90 min. Animals
were subsequently sacrificed, brains were removed quickly,
homogenised (1 g in 10 ml cold isotonic KCl solution) for
1 min and stored at -80°C for further evaluation. Lipid
peroxidation of rat brain homogenates was determined
fluorometrically as 2-thiobarbituric acid reactive substance.
The degree of lipid peroxidation was expressed as mal-
ondialdehyde formation (nmol/mg brain protein), using
malondialdehyde bis(dimethyl acetal) as reference.
In vitro effect on lipoxygenase activity The reaction
mixture (total volume 3 ml) contained 100 ll of the test
compounds at various concentrations, dissolved in 60%
aqueous ethanol (sample), or 100 ll of the solvent (refer-
ence) 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 monitored for 7 min at 28°C, by
recording the absorbance of a conjugated diene structure at
234 nm, due to the formation of 13-hydroperoxylinoleic
acid (Siskou et al., 2007).
Gastrointestinal toxicity Compounds 3, 17 as well as
indomethacin and ketoprofen were administered sc to rats
once daily for 4 days. Perforating gastrointestinal ulcers,
melena defecation, body weight change and mortality were
recorded 24 h after the last injection (Doulgkeris et al.,
2006).
In vitro evaluation of cyclooxygenase activity (isoforms 1
and 2) The effect of compounds 3, 5, 17 and 19 on COX-
1 and COX-2 activity was measured using ovine COX-1
and human recombinant COX-2 enzymes included in the
‘COX Inhibitor Screening Assay’ kit provided by Cayman.
The assay directly measures PGF2a produced by SnCl2
reduction of COX-derived PGH2. The prostanoid product is
quantified via enzyme immunoassay using a broadly spe-
cific antibody that binds to all the major prostaglandin
compounds. The compounds were added to the reaction
mixture at a final concentration of 200 lM. Arachidonic
acid concentration, 0.1 lM; COX, 10-9 IU/ml for both
isoforms (Ziakas et al., 2006).
Measurement of compounds in brain and blood Rats
were divided into three groups and the test compounds
(0.6 mmol/kg) were administered ip. Brains were quickly
removed 30, 60 and 120-min post injection and homoge-
nized in 1.15% potassium chloride solution.
In another set of experiments, compound iv (Table 8)
was administered as described above and blood was taken
from the aorta, under light ether anaesthesia, 30, 60 and
120-min post injection. Blood was centrifuged (30009g,
15 min) and compound iv was determined in the plasma.
The compounds were isolated from the brain samples by
protein precipitation and solid phase extraction. Brain
homogenates (300 ll) were acidified with 2 M hydro-
chloric acid (300 ll) vortexed for 5 min, acetonitrile
(4.0 ml) was added, mixed for 2 min and centrifuged for
10 min (75009g). The supernatants were diluted with
ultrapure water (2.0 ml) and applied to the preconditioned
and equilibrated C18 solid phase extraction cartridges
(Discovery DSC-18; Supelco, Bellefonte, PA, USA)
(Mannila et al., 2005). The cartridges were first washed
with methanol (2.0 ml) and then with water (2.0 ml). The
samples were eluted with acetonitrile (3.0 ml), evaporated
to dryness under a nitrogen stream at 50°C and reconsti-
tuted in acetonitrile (0.5 ml).
In vitro lipid peroxidation Hepatic microsomal fraction
from untreated rats was prepared. The incubation mixture
contained heat inactivated (90°C for 90 s) microsomal
fraction, corresponding to 2.5 mg protein/ml (final con-
centration) or 4 mM fatty acid residues, ascorbic acid
(0.2 mM) in Tris–HCl/KCl buffer (50 mM/150 mM) and
the test compounds dissolved in dimethyl sulphoxide. The
peroxidation reaction was initiated by the addition of a
freshly prepared FeSO4 solution (10 lM) and the mixture
was incubated at 37°C. Aliquots (0.3 ml) were taken at
various time intervals for 45 min. Lipid peroxidation was
assessed spectrophotometrically (535 against 600 nm) by
the determination of 2-thiobarbituric acid reactive material.
All compounds as well as dimethyl sulphoxide were tested
and found not to interfere with the assay (Siskou et al.,
2007).
The concentrations of the examined compounds in brain
samples were measured by a Dionex isocratic RP-HPLC
system with UV-detection at 230 nm. The HPLC system
consisted of a D-6000A interface, L-6200A intelligent
pump, L-4500 diode array detector, L-7350 column oven,
D-7000 chromatography data station software (version 3.1)
and a Zorbax SB-C18 (4.6 9 150 mm, 5 lm) analytical
column (Agilent Technologies Inc., Little Falls, Wilming-
ton, DE, USA). Loop injection volume was 10 and 30 ll
for plasma and brain samples, respectively. The mobile
Effect on brain oxidative stress caused by ischaemia–
reperfusion Compound 9 (0.4 mmol/kg body weight)
was administered ip once per day for 4 days and once more
2 h before the initiation of ischaemia. The common carotid
arteries were exposed and clamped with aneurysm clips to
123

Med Chem Res (2012) 21:2280–2291
2291
opioid agonist which also possesses antioxidant activity. Ann
NY Acad Sci 650:19–24
phase was composed of 20 mM phosphate buffer solution
(pH 2.5) and acetonitrile in volume ratios of 55:45 and
46:54 for plasma and brain samples, respectively. The
calibration curves of the plasma and the brain methods
were linear over a range of 0.2–50 and 0.2–6 lg/ml,
respectively. The precision of the plasma and the brain
sample methods were studied at a concentration level of the
lower limit of quantification, and expressed as coefficients
of variation (CV%); 4.6% (n = 5) and 8.9% (n = 4) for
spiked samples containing 5 and 0.2 lg/ml, respectively.
The recovery was over 84% in all methods studied.
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