Synthesis and pharmacological evaluation of some 4-Oxo-quinoline-2- carboxylic acid derivatives as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1002/ardp.201000016

The synthesis and the pharmacological activity of a series of 1-aroyl derivatives of kynurenic acid methyl ester (4-oxo-quinolin-2-carboxy methyl (KYNA) esters), structurally related to NSAID indomethacin are described. The derivatives were screened in vi

Full text of DOI:10.1002/ardp.201000016

Arch. Pharm. Chem. Life Sci. 2010, 10, 561–569
561
Article
Synthesis and Pharmacological Evaluation of Some 4-Oxo-
quinoline-2-carboxylic Acid Derivatives as Anti-inflammatory
and Analgesic Agents
This manuscript is dedicated to Orazio Mazzoni
Orazio Mazzoni1, Giovanni Esposito1, Maria Vittoria Diurno1, Diego Brancaccio1,
Alfonso Carotenuto1, Paolo Grieco1, Ettore Novellino1 and Walter Filippelli2
1
`
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli ‘‘Federico II’’, Napoli, Italia
Istituto di Farmacologia e Tossicologia, Facolta di Medicina e Chirurgia, Seconda Universita di Napoli,
2
`
`
Napoli, Italia
The synthesis and the pharmacological activity of a series of 1-aroyl derivatives of kynurenic acid
methyl ester (4-oxo-quinolin-2-carboxy methyl (KYNA) esters), structurally related to NSAID indo-
methacin are described. The derivatives were screened in vivo for anti-inflammatory and analgesic
activities. Most of the compounds exhibited good anti-inflammatory and analgesic activities. An
automatic docking of the synthesized compounds was performed using X-ray structures of COX-1 and
COX-2. Docking results are in good accordance with the experimental biological data.
Keywords: Anti-inflammatory activity / Drug design / NSAIDs / Prostaglandin / Quinoline-2-carboxylic acid derivatives
Received: January 11, 2010; Accepted: February 26, 2010
DOI 10.1002/ardp.201000016
Introduction
[3, 4]. PGs possess both beneficial and harmful properties
in vivo. For instance, PGE2 plays an important role in inflam-
mation and hyperalgesia when produced at an inflammatory
site, whereas it exerts a cytoprotective action in the gastric
mucosa and promotes normal renal function in the kidney
[5–7]. Inhibition of COX by NSAIDs leads to a decrease in all
prostaglandin and thromboxane synthesis, which accounts
for the beneficial anti-inflammatory and analgesic effects of
NSAIDs as well as for their side effects.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among
the most widely used of all therapeutic agents. They possess
anti-inflammatory, analgesic, and antipyretic activities and
are useful in the treatment of acute and chronic inflamma-
tory disorders [1]. The mechanism of action of NSAIDs is
linked to the nonselective or selective inhibition of two
prostaglandin endoperoxide synthase or cyclooxygenase
(COX) isoforms, namely COX-1 and COX-2 [2]. COX catalyzes
the first two steps in the arachidonic acid cascade that leads
to several bioactive lipids including prostaglandins (PGs)
While COX-1 is a constitutive enzyme necessary for the
proper function of the kidney and stomach through the
synthesis of prostaglandins, COX-2 is an inducible form of
the enzyme that mediates the inflammatory process [8]. The
selective inhibition of COX-2 avoids the presence of gastro-
intestinal and renal side effects associated with the inhi-
bition of production of prostaglandins by COX-1 [9]. Thus,
COX-2 is a valid target whose selective inhibition is sought in
the development of anti-inflammatory therapies.
Correspondence: Prof. Maria V. Diurno, Dipartimento di Chimica
`
Farmaceutica e Tossicologica, Universita di Napoli ‘‘Federico II’’, Via D.
Montesano 49, 80131 Napoli, Italia.
E-mail: diurno@unina.it
Fax: þ39 081 678 630
The COX isozymes are bifunctional, heme-containing
enzymes with a cyclooxygenase and a peroxidase active site.
The cyclooxygenase activity bisoxygenates arachidonic acid
Abbreviations: consistent-valence force field (CVFF); indomethacin
(IMN); iodosuprofen (ISF); kynurenic acid (1,4-dihydro-4-oxo-quinoline-
2-carboxylic acid) (KYNA); prostaglandin (PG).
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to PGG2 and the peroxidase activity reduces PGG2 to PGH2
[10]. All NSAIDs, as well as selective COX-2 inhibitors, bind in
the COX active site but not the peroxidase site [11]. The
kinetics of COX inhibition reveals that most NSAIDs conform
to the two-step mechanism, typical of slow, tightly binding
inhibitors [12]. The first step involves the formation of a
rapidly reversible complex (EꢀI) which represents a compet-
itively inhibited enzyme [13]. The second step involves the
conversion of the (EꢀI) complex to the (EꢀIꢁ) complex, in which
the inhibitor is bound more tightly to the enzyme. This step
occurs within seconds to minutes and is thought to reflect an
enzyme conformational change [14–18]. An example of time-
dependent inhibitors is indomethacin [13]. Despite their
toxicities, NSAIDs remain one of the most commonly pre-
scribed groups of drugs and the new COX-2 inhibitors may
provide a new therapeutic option to patients suffering from
arthritis and other musculoskeletal diseases.
Figure 1. Main analogues of 1-aroyl derivatives of KYNA methyl
ester.
Additional inhibitor classes may also serve as tools in our
understanding of the molecular and structural basis for
selective COX-2 inhibition. For instance, the issue that
COX-2 selectivity is due to the time-dependent step in the
kinetic scheme is not easily explainable with existing
methods and re-interpretation of the kinetic events are begin-
ning to appear in the literature [14, 10, 18].
at room temperature afforded compounds 5–15 in good
yields (Scheme 1 and Table 1).
Biological activities
The quinolone derivatives 5–15 were investigated with
respect to their pharmacological activity by two screening
protocols widely used for the testing of NSAIDs. The carra-
geenan-induced rat paw edema was used to study the anti-
inflammatory activity [28], whereas the analgesic activity was
studied by means of the acetic acid writhing test in mice [29].
Indomethacin was used in all tests as reference drug. It was
suggested that the activity of anti-inflammatory agents eval-
uated by carrageenan edema is due in the first moment to the
inhibitor effects on the vasoactive mediators release. The
activity detected in the subsequent hours is mediated by
COX inhibition [30].
The currently available NSAIDs belong to different classes
[19]. Among the nonselective NSAIDs, we have indomethacin
that is tenfold more active than COX-1 while among selective
COX-2 inhibitors there are the pyrazolyl derivatives [20].
Furthermore, it was proposed that increased brain for-
mation of kynurenic acid (1,4-dihydro-4-oxo-quinoline-2-car-
boxylic acid (KYNA)) induced by NSAIDs, contributes to their
analgesic efficacy probably through an inhibitory action on
COX-1 [21]. In fact, an increased formation of brain kynurenic
acid (KYNA) has been suggested to contribute to the analgesic
action of diclofenac, a relatively non-selective COX-inhibitor
[22]. KYNA is a metabolite of tryptophan and is synthesized in
brain astrocytes.
The results obtained in the carrageenan test, reported in
Table 2 and summarized in Figs. 2 and 3, indicate that all the
compounds display significant anti-inflammatory activity
during the first hour, with the exception of 5 and 10.
Compound 5 was also the least active of the series during
the whole experiment. Compounds 11 and 15 were the most
active during the first hour (61 and 62%, respectively).
In the second hour, the best results were obtained for
compounds 6, 7, and 14 (62, 60, and 65% respectively), while
8, 11, 12, and 15 showed comparable levels of activity ranging
between 46 and 49%. These compounds shared the same
pharmacological profile at every hour of the experiment.
The maximum of activity at the third hour was given by 14,
with 71% and is also the maximum of the whole experiment.
Compound 14, with an activity ranging between 58 and 71%
during the four hours of testing, is the most active of the
whole series showing an indomethacin-like profile.
This paper reports the synthesis and the pharmacological
evaluation of 1-aroyl derivatives of KYNA methyl ester, a
nucleus sharing a common feature with the indole nucleus
of indomethacin and similarities with SC-558, a COX-2-selec-
tive pyrazolyl derivative (Fig. 1).
Results and discussion
Chemistry
Traditionally, synthesis of 2-[(4’-substituted-phenyl)-amino]-2-
butenedioates 1a–4a has been achieved using the method of
Conrad and Limpach [23, 24], involving the reaction between
anilines and acetylenedicarboxylate, followed by cyclization
in diphenylether at ꢂ2508C, affording compounds 1b–4b.
Aroylation of the nitrogen of quinolones in dichloromethane
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Quinoline-2-carboxylic Acid Derivatives
563
Scheme 1. Synthesis of KYNA methyl ester derivatives 5–15.
Table 1. Structures of KYNA methyl ester derivatives 5–15.
In the fourth hour, the best results were obtained for
compound 7, 9, 12, 14 (54, 54, 54, and 60%, respectively).
The analgesic activity in the acetic acid writhing test
reported in Table 3 very well paralleled the results of the
carrageenan test. In fact, compounds 12 and 14 showed the
highest analgesic activity.
Comp.
R
R’
Comp.
R
R’
5
6
7
8
9
10
H
H
H
CH3
CH3
CH3
H
CH3
Cl
H
CH3
F
11
12
13
14
15
CH3
OCH3
OCH3
I
I
Cl
CH3
Cl
CH3
Cl
Overall, biological data point to an inhibition of the COX
activity for most of the tested compounds.
Molecular modeling and docking
In order to investigate the hypothesized interaction between
compounds 5–15 and COX enzymes, an automatic docking of
these compound onto COX-1 and COX-2 was performed by
Table 2. Anti-inflammatory activity by the carrageenan rat paw edema test.
Compound§
Volume of edema (mL W S.E.) % related to control after:
0
1 h&
2 h&
3 h&
4 h&
Controls
Indomethacin#
5
6
7
1.6 ꢃ 0.1
1.4 ꢃ 0.1
1.4 ꢃ 0.1
1.4 ꢃ 0.1
1.3 ꢃ 0.1
1.5 ꢃ 0.1
1.3 ꢃ 0.1
1.5 ꢃ 0.1
1.6 ꢃ 0.1
1.5 ꢃ 0.1
1.4 ꢃ 0.1
1.5 ꢃ 0.1
1.6 ꢃ 0.1
2.1 ꢃ 0.1 (þ31)
1.6 ꢃ 0.1 (–54)
1.7 ꢃ 0.1 (–32)
1.6 ꢃ 0.1 (–55)
1.5 ꢃ 0.1 (–52)
1.7 ꢃ 0.1 (–58)
1.5 ꢃ 0.1 (–51)
1.8 ꢃ 0.1 (–35)
1.8 ꢃ 0.1 (–61)
1.7 ꢃ 0.1 (–58)
1.6 ꢃ 0.1 (–55)
1.7 ꢃ 0.1 (–58)
1.8 ꢃ 0.1 (–62)
2.2 ꢃ 0.1 (þ37)
1.6 ꢃ 0.1 (–62)
1.8 ꢃ 0.1 (–24)
1.6 ꢃ 0.1 (–62)
1.5 ꢃ 0.1 (–60)
1.8 ꢃ 0.1 (–46)
1.6 ꢃ 0.1 (–37)
1.8 ꢃ 0.1 (–46)
1.9 ꢃ 0.1 (–48)
1.8 ꢃ 0.1 (–46)
1.7 ꢃ 0.1 (–43)
1.7 ꢃ 0.1 (–65)
1.9 ꢃ 0.1 (–49)
2.3 ꢃ 0.1 (þ44)
1.6 ꢃ 0.1 (–68)
1.9 ꢃ 0.1 (–18)
1.7 ꢃ 0.1 (–52)
1.6 ꢃ 0.1 (–48)
1.8 ꢃ 0.1 (–54)
1.6 ꢃ 0.1 (–47)
1.9 ꢃ 0.1 (–38)
1.9 ꢃ 0.1 (–57)
1.8 ꢃ 0.1 (–55)
1.8 ꢃ 0.1 (–37)
1.7 ꢃ 0.1 (–71)
1.9 ꢃ 0.1 (–57)
2.4 ꢃ 0.1 (þ50)
1.6 ꢃ 0.1 (–72)
2.0 ꢃ 0.1 (–14)
1.8 ꢃ 0.1 (–44)
1.6 ꢃ 0.1 (–54)
1.9 ꢃ 0.1 (–46)
1.6 ꢃ 0.1 (–54)
1.9 ꢃ 0.1 (–46)
2.0 ꢃ 0.1 (–50)
1.9 ꢃ 0.1 (–54)
1.9 ꢃ 0.1 (–28)
1.8 ꢃ 0.1 (–60)
2.0 ꢃ 0.1 (–50)
8
9
10
11
12
13
14
15
§
5-15 100 mmol/kg per os;# 14 mmol/kg per os;& Time from the administration; values in parentheses indicate percentage of variation of
edema related to the control (see Experimental).
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Figure 2. Carrageenan-induced rat paw edema test for the compounds 5–15 related to controls and indomethacin as reference compound.
Figure 3. Percentage of variation of edema for all the synthesized compounds related to controls and indomethacin as reference compound.
Table 3. Analgesic activity by the acetic acid writing test in mice.
Table 4. Prediction of COX-1 and COX-2 binding energy and
inhibition constants for compounds 5–15.
Compound§
mean number of
writhes W S.E.
in 25 min
% Decrease of
mean number of
writhes in 25 min
after treatment
rel. to control
Compound
COX-1
COX-2
DG (kcal/Mol) Ki (nM) DG (kcal/Mol) Ki (nM)
5
6
7
8
9
10
11
12
13
14
15
IMN
ISF
–10.13
–8.91
–8.54
–10.23
–9.22
–8.53
–9.50
–8.83
–8.96
–9.79
–9.36
–
37.31
292.76
554.27
31.71
173.02
555.60
109.58
338.94
270.83
66.39
138.3
–
–9.33
–9.99
–10.25
–9.29
–10.34
–9.33
–10.63
–10.14
–10.40
–11.28
–11.50
–10.37
–
145.20
47.24
30.68
154.95
26.42
145.12
16.19
36.91
23.96
5.43
Control
Indomethacin#
5
6
7
45.8 W 4.3
22.9 W 4.2
26.5 ꢃ 3.2
28.7 ꢃ 4.1
26.4 ꢃ 3.7
30.3 ꢃ 2.8
28.7 ꢃ 5.1
31.3 ꢃ 3.6
27.0 ꢃ 4.5
24.0 ꢃ 3.5
30.1 ꢃ 2.8
25.1 ꢃ 2.6
29.2 ꢃ 4.7
–51.7%
–42.1%
–37.2%
–42.2%
–33.8%
–37.3%
–31.6%
–41.0%
–47.4%
–34.1%
–45.0%
–36.1%
8
9
10
11
12
13
14
15
3.74
25.21
–
–9.13
204.46
§
5–15 100 mmol/kg per os;# 14 mmol/kg per os.
because of their high resolution qualities (resolution 3.50
˚
and 2.90 A, respectively). Docking details are reported in
AutoDock (version 4.0) [25–27]. The crystal structures of COX-
1 in complex with p-(2’-iodo-5’-2 thenoyl)-hydrotropic acid
(iodosuprofen, ISF; PDB code 1PGE) and COX-2 in complex
with indomethacin (IMN; PDB code 4COX) were chosen
Section 4.
Calculated DG and Ki values for all the synthesized com-
pounds onto COX-1 and COX-2 are summarized in Table 4,
compared with IMN and ISF as reference compounds.
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Quinoline-2-carboxylic Acid Derivatives
565
Figure 5. Comparison between compound 14 and IMN onto COX-2
binding site.
by Val523, Trp387, Gly526, and Ala527. The second lipophilic
pocket is delimited by Leu352, Ser353, Met522, and Phe518 in
COX-2. Hydrogen bonds were present between the 4-carbonyl
oxygen of quinolone and Arg120 Ne and Tyr355 phenolic OH,
and between the amidic carbonyl oxygen and Ser530 OH.
Similar interaction was observed between IMN and COX-2
in the X-ray structure. In particular, the first pocket was
occupied by the aroyl group of IMN (Fig. 5), while the second
one interacted with the indole nucleus. Hydrogen bonds
were present between the IMN carboxyl oxygen and
Arg120 Ne and Tyr355 phenolic OH, and between amidic
carbonyl oxygen and Ser530 OH, as shown in Fig. 5.
The reduced number of hydrogen bonds within the COX-1
binding site can explain the lower calculated affinity values
of almost all the compounds towards COX-1 compared to
COX-2. Compounds 5 and 8 show relatively high affinities for
COX-1, probably because of steric hindrance of substituents
at position R’ with residues of the second lipophilic pocket of
this enzyme. Considering COX-1, the substituent position R’
in the aroyl system is in close van-der-Waals contact by
Leu359, while the substituent position R is close to Ile523.
Docking scores are generally in good accordance with
biological activities with the most active compound 14 hav-
ing a good affinity for COX-2 and COX-1, and the least anal-
gesic compound 10 showing the lowest affinity for COX-1 and
low affinity for COX-2.
Figure 4. Interaction between compound 14 and COX-1 (a) and
COX-2 (b).
Figure 4 represents the docking interactions of compound
14 onto COX-1 and COX-2. Considering COX-1 binding inter-
action, all compounds bind in
a similar disposition.
Particularly, the binding site is characterized by two lipo-
philic pockets. The first one, in which the aroyl group lies, is
delimited by Ala527, Leu352, and Leu531 of COX-1. The
second lipophilic pocket is delimited by Val349, Met113,
Val116, and Leu359 and interacts with the aromatic moiety
of the quinolone nucleus. A hydrogen bond between the 2-
carboxyl oxygen and Tyr355 phenolic OH is observed. These
interactions with COX-1 are similar to those observed in a
model of the sulindac bound to COX-1 [31]. In particular, the
first pocket was occupied by the phenyl-ethynyl group of
sulindac, while the second one interacted with the indene
group. Hydrogen bonds were present between the sulindac
carboxyl group and Arg120 Ne and Tyr355 phenolic OH, and
between the sulfoxide group and Ser530 OH.
Structure-activity relationships (SAR)
Even if the results do not allow us to discern any clear
structure-activity relationships, we can make some brief
remarks on the influence of substituents on the activities.
Considering the anti-inflammatory activity, a methyl group
on the R’ position yields the compounds with the highest
anti-inflammatory activity. The chemical nature of the sub-
stituent on the aromatic mojety of the quinolone nucleus
modulates these activities. Compounds 6, 9, and 12, with the
Considering COX-2, again two lipophilic pockets host the
ligands. The first one, in which the aroyl group lies, is formed
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hydrogen, methyl group, and methoxy group on the R po-
sition, respectively, showed an increasing activity. The iodine
atom, characteristic of compound 14, gives the best result.
Hence, a bulky, polarizable group is favored in this position.
On contrary, the presence of an electron-withdrawing sub-
stituent on the aroyl group leads to a decrease of activity
(compare 15 and 13 with 14). Compound 10, with a fluorine
group at the R’ position, was the least active.
Dimethyl 2-(phenyl-amino)-2-butendioate 1a
Oil. 1H-NMR (CDCl3): 9.60 (1H, bs), 7.19 (2H, t, J ¼ 8.0 Hz), 7.00 (1H,
t, J ¼ 7.5 Hz), 6.81 (2H, d, J ¼ 8.1 Hz), 5.31 (1H, s), 3.64 (3H, s), 3.60
(3H, s). Mass calcd.: 235.24; MS (ESIþ) m/z: 236.22 [M þ Hþ] (100).
Dimethyl 2-[(4’-methyl-phenyl)-amino]-2-butendioate 2a
Oil. 1H-NMR (CDCl3): 9.62 (1H, bs), 7.07 (2H, d, J ¼ 8.0 Hz), 6.80 (2H,
d, J ¼ 8.0 Hz), 5.33 (1H, s), 3.74 (3H, s), 3.69 (3H, s), 2.30 (3H, s). Mass
calcd.: 249.26; MS (ESIþ) m/z: 250.24 [M þ Hþ] (100).
With regard to analgesic activity, compounds 14 and 12
showed a reduction of writhes similar to indomethacin, with
a 45% and 47% reduction of pain, respectively, confirming
the SAR obtained above.
Dimethyl 2-[(4’-methoxy-phenyl)-amino]-2-butendioate 3a
Oil. 1H-NMR (CDCl3): 9.57 (1H, bs), 6.90 (2H, d, J ¼ 8.0 Hz), 6.81 (2H,
d, J ¼ 8.0 Hz), 5.30 (1H, s), 3.78 (3H, s), 3.73 (3H, s), 3.67 (3H, s). Mass
calcd.: 265.26; MS (ESIþ) m/z: 266.25 [M þ Hþ] (100).
Conclusion
Dimethyl 2-[(4’-iodo-phenyl)-amino]-2-butendioate 4a
Oil. 1H-NMR (CDCl3): 9.60 (1H, bs), 7.57 (2H, d, J ¼ 8.4 Hz), 6.65 (2H,
d, J ¼ 8.4 Hz), 5.46 (1H, s), 3.75 (3H, s), 3.73 (3H, s). Mass calcd.:
361.13; MS (ESIþ) m/z: 362.15 [M þ Hþ] (100).
In conclusion, we have succeeded in the creation of structurally
simple and novel quinolone derivates with interesting anti-
inflammatory and analgesic activities, probably through the
inhibition of the COX enzymes. Our models indicate that some
compounds could preferentially inhibit the COX-2 subtype. The
results presented in this paper should be useful for the develop-
ment of superior COX inhibitors with COX-subtype selectivity.
4-Oxo-1,4-dihydro-quinoline-2-carboxylic acid methyl ester
1b–4b
Phenyl ether (40 mL) was heated under stirring at 2408C. The
dimethyl butenedioates 1a–4a were slowly added and the result-
ing mixture was refluxed for 4 h. After the mixture was cooled at
room temperature, petroleum ether was added and the resulting
precipitate was collected by filtration, washed with petroleum
ether, and recrystallized from methanol to provide compounds
1b–4b as pale-colored solids with yields ranging between 70 to
77%.
Experimental
Chemistry
All commercial reagents and solvents were used without further
purification. Purity of the compounds was checked by analytical
TLC on F254 Merck’s precoated silica-gel plates, 0.25 mm (Merck,
Darmstadt, Germany) with fluorescent backing. The spots were
located by UV (254 and 366 nm) and/or with iodine. Silica gel 60,
230–400 mesh purchased from Merck was used for column
chromatography. Preparative thin-layer chromatography was
performed using silica gel from Merck, and the compounds were
extracted from silica gel by the following solvent system: CHCl3/
CH3CN (70:30). Electron impact (EI) mass spectra were obtained
on an API 2000 spectrometer (Applied Biosystems, Foster City,
CA, USA). 1H-NMR spectra were recorded on a Varian 500 MHz
spectrometer (Varian, Inc. Corporate Headquarters, Palo Alto,
CA, USA) with Me4Si as internal reference. Chemical shifts are
reported in (ppm) and coupling constants in Hertz (Hz). The
splitting patterns were designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet. Melting points were determined
using a Gallenkamp apparatus (Weiss-Gallenkamp, London, UK)
and remain uncorrected.
Methyl 4-oxo-1,4-dihydro-2-quinolinecarboxylate 1b [32]
M. p.: 224–2258C. 1H-NMR (CDCl3): 9.15 (1H, bs), 8.29 (1H, d, J ¼ 8.5
Hz), 7.91 (1H, d, J ¼ 8.5 Hz), 7,80 (1H, t, J ¼ 7.8 Hz), 7.50 (1H, t, J ¼
7.8 Hz), 6.94 (1H, s), 4.02 (3H, s). Mass calcd.: 203.19; MS (ESIþ) m/z:
204.21 [M þ Hþ] (100).
Methyl 6-methyl-4-oxo-1,4-dihydro-2-quinolinecarboxylate
2b
M. p.: 244–2458C. 1H-NMR (CDCl3): 9.02 (1H, bs), 8.15 (1H, s), 7.50
(1H, d, J ¼ 8.4 Hz), 7.34 (1H, d, J ¼ 8.4 Hz), 6.96 (1H, s), 4.03 (3H, s),
2.47 (3H, s). 13C-NMR: 179.64 (s), 163.78 (s), 137.21 (s), 135.95 (s),
134.93 (d), 127.45 (s), 126.62 (s), 125.83 (d), 117.97 (d), 111.65 (d),
53.94 (q), 21.31 (q). Mass calcd.: 217.22; MS (ESIþ) m/z: 218.20 [M þ
Hþ] (100).
Methyl 6-methoxy-4-oxo-1,4-dihydro-2-
quinolincarboxylate 3b [24]
M. p.: 241–2428C. 1H-NMR (CD3OD): 7.56 (1H, d, J ¼ 8.1 Hz), 7.38
(1H, d, J ¼ 2.8 Hz), 7.16 (1H, dd, J ¼ 8.1 Hz, 2.8 Hz), 6.60 (1H, s), 3.79
(3H, s), 3.67 (3H, s). Mass calcd. 233.22; MS (ESIþ) m/z: 234.23 [M þ
Hþ] (100).
2-[(4’-Substituted-phenyl)-amino]-2-butendioate 1a–4a
A mixture of acethylene acid dimethyl ester (9.30 mL, 46 mmol)
and appropriate aniline (4.20 mL, 46 mmol) in methanol was
heated at reflux temperature for 3 h.
The reaction mixture, cooled and evaporated in vacuo, afforded
a yellow pale oil that was used for cyclization without purifi-
cation. A pure sample of compounds for instrumental analysis
was obtained by thin layer chromatography using preparative
silica gel plates (0.5 mm) and a mixture of chloroform/aceto-
nitrile as eluant. Yields ranging between 70–80%.
Methyl 6-iodo-4-oxo-1,4-dihydro-2-quinolincarboxylate 4b
[33]
M. p.: 270–2718C. 1H-NMR (CD3OD): 8.61 (1H, d, J ¼ 2.3 Hz), 8.03
(1H, dd, J ¼ 8.5 Hz), 7,67 (1H, d, J ¼ 8.5 Hz), 7.00 (1H, s), 4.06 (1H, s).
Mass calcd.: 329.09; MS (ESIþ) m/z: 330.12 [M þ Hþ] (100).
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2010, 10, 561–569
Quinoline-2-carboxylic Acid Derivatives
567
139.79 (s), 133.46 (d), 130.79 (d), 130.73 (2C, d), 129.87 (2C, d), 125.89
(s), 124.01 (s), 120.18 (d), 113.90 (d), 53.43 (q), 22.26 (q), 22.06 (q). Mass
calcd.: 335.35; MS (ESIþ) m/z: 336.30 [M þ Hþ] (100).
1-(4’-Substituted benzoyl)-4-oxo-6-substituted-1,4-
dihydro-quinoline-2-carboxylic acid methyl esters 5–15
To the solution of 1b–4b (0.05 mol) in anhydrous CH2Cl2 (50 mL),
triethylamine (3 mL) and DMF (2 mL) were added together with
the appropriate benzoyl chloride (0.05 mol). The reaction mix-
ture, stirred 3 h at reflux temperature, cooled and evaporated in
vacuo, yielding the crude products that were recrystallized from
ethanol/ethyl acetate (2:1, v:v). Yields range between 70 to 90%.
Methyl 1-(4’-fluoro-benzoyl)-6-methyl-4-oxo-1,4-dihydro-
quinoline-2-carboxylate 10
Yield: 73%. M. p.: 150–1518C. 1H-NMR (CDCl3): 8.34 (2H, d, J ¼ 8.0
Hz), 8.25 (1H, d, J ¼ 8.7 Hz), 8.14 (1H, s), 7.74 (1H, s), 7.66 (1H, d, J ¼
8.75, 1.3 Hz), 7.27 (2H, d, J ¼ 8.1 Hz), 4.08 (3H, s), 2.55 (3H, s). 13C-
NMR: 165.59 (s), 163.16 (s), 154.88 (s), 148.31 (s), 147.99 (s), 139.93
(s), 139.82 (s), 133.44 (2C, d), 130.91 (2C, d), 128.85 (s), 123.87 (s),
120.01 (d), 116.59 (d), 113.87 (d), 53.48 (q), 22.29 (q). Mass calcd.:
339.32; MS (ESIþ) m/z: 340.28 [M þ Hþ] (100).
Methyl 1-benzoyl-4-oxo-1,4-dihydro-quinoline-2-
carboxylate 5
Yield: 80%. M. p.: 143–1448C. 1H-NMR (CDCl3): 8.37 (1H, d, J ¼ 8.5
Hz), 8.31 (2H, d, J ¼ 7.8 Hz), 8.21 (1H, s), 8.07 (1H, d, J ¼ 8.3 Hz),
7.83 (1H, t, J ¼ 7.5 Hz), 7.73 (1H, t, J ¼ 8.1 Hz), 7.67 (1H, t, J ¼ 7.9
Hz), 7.59 (2H, t, J ¼ 7.9 Hz), 4.09 (3H, s). 13C-NMR: 165.56 (s), 163.99
(s), 155.6 (s), 149.59 (s), 149.00 (s), 134.64 (d), 131.14 (d), 131.06 (d),
130.68 (2C, d), 129.20 (d), 129.16 (2C, d), 128.67 (s), 123.90 (s),
121.51 (d), 113.71 (d), 53.53 (q). Mass calcd.: 307.30; MS (ESIþ) m/z:
308.42 [M þ Hþ] (100).
Methyl 1-(4’-chloro-benzoyl)-6-methyl-4-oxo-1,4-dihydro-
quinoline-2-carboxylate 11
Yield: 75%. M. p.: 175–1768C. 1H-NMR (CDCl3): 8.25 (3H, dd, J ¼ 8.7,
8.4 Hz), 8.14 (1H, s), 7.74 (1H, s), 7.67 (1H, d, J ¼ 8.7 Hz), 7.59 (2H, d,
J ¼ 8.3 Hz), 4.09 (3H, s), 2.56 (3H, s). 13C-NMR: 165.62 (s), 163.32 (s),
154.81 (s), 148.31 (s), 147.99 (s), 141.33 (s), 139.97 (s), 133.57 (d),
132.03 (2C, d), 130.92 (d), 129.60 (2C, d), 128.74 (s), 123.80 (s),
119.98 (d), 113.85 (d), 53.50 (q), 22.30 (q). Mass calcd.: 355.77; MS
(ESIþ) m/z: 356.81 [M þ Hþ] (100).
Methyl 1-(4’-methyl-benzoyl)-4-oxo-1,4-dihydro-quinoline-
2-carboxylate 6
Yield: 60%. M. p.: 130–1318C. 1H-NMR (CDCl3): 8.35 (1H, d, J ¼ 8.4
Hz), 8.21 (1H, s), 8.19 (2H, d, J ¼ 8.4 Hz), 8.05 (1H, d, J ¼ 8.8 Hz),
7.84 (1H, t, J ¼ 7.8 Hz), 7.67 (1H, t, J ¼ 7.9 Hz), 7.40 (2H, d, J ¼ 7.7
Hz), 4.10 (3H, s), 2.51 (3H, s). 13C-NMR: 165.59 (s), 164.21 (s), 155.79
(s), 149.57 (s), 149.00 (s), 145.71 (s), 131.12 (d), 131.02 (d), 130.74
(2C, d), 129.88 (2C, d), 129.14 (s), 125.88 (s), 123.98 (s), 121.57 (d),
113.74 (d), 53.52 (q), 22.07 (q). Mass calcd.: 321.33; MS (ESIþ) m/z:
322.28 [M þ Hþ] (100).
Methyl 1-(4’-methyl-benzoyl)-6-methoxi-4-oxo-1,4-
dihydro-quinoline-2-carboxylate 12
Yield: 67%. M. p.: 182–1838C. 1H-NMR (CDCl3): 8.23 (1H, d, J ¼ 8.1
Hz), 8.20 (2H, d, J ¼ 8.0 Hz), 8.15 (1H, s), 7.46 (1H, dd, J ¼ 8.1, 2.5
Hz), 7.39 (2H, d, J ¼ 8.4 Hz), 7.21 (1H, d, J ¼ 2.5 Hz), 4.07 (3H, s),
3.90 (3H, s), 2.51 (3H, s). 13C-NMR: 165.75 (s), 164.15 (s), 160.08 (s),
154.37 (s), 146.18 (s), 145.77 (s), 132.76 (d), 130.73 (2C, d), 129.95
(2C, d), 127.68 (s), 125.90 (s), 125.44 (s), 124.13 (d), 121.00 (s),
114.37 (d), 98.93 (d), 55.92 (q), 53.51 (q), 22.14 (q). Mass calcd.:
351.35; MS (ESIþ) m/z: 352.30 [M þ Hþ] (100).
Methyl 1-(4’-chloro-benzoyl)-4-oxo-1,4-dihydro-quinoline-
2-carboyxlate 7
Yield: 62%. M. p.: 193–1948C. 1H-NMR (CDCl3): 8.38 (1H, d, J ¼ 8.4
Hz), 8.26 (2H, d, J ¼ 8.4 Hz), 8.20 (1H, s), 8.04 (1H, d, J ¼ 8.4 Hz),
7.85 (1H, t, J ¼ 7.7 Hz), 7.69 (1H, t, J ¼ 7.7 Hz), 7.58 (2H, d, J ¼ 8.8
Hz), 4.10 (3H, s). 13C-NMR: 165.58 (s), 163.00 (s), 155.46 (s), 149.59
(s), 149.00 (s), 141.39 (s), 132.01 (2C, d), 131.21 (d), 131.49 (d),
129.60 (2C, d), 129.31 (d), 127.09 (s), 123.74 (s), 121.35 (s), 113.66
(d), 94.97 (d), 53.57 (q). Mass calcd.: 341.75; MS (ESIþ) m/z: 342.70
[M þ Hþ] (100).
Methyl 1-(4’-chloro-benzoyl)-6-methoxy-4-oxo-1,4-
dihydro-quinoline-2-carboxylate 13
Yield: 68%. M. p.: 213–2148C. 1H-NMR (CDCl3): 8.26 (1H, d, J ¼ 9.4
Hz), 8.23 (2H, d. J ¼ 8.4 Hz), 8.12 (1H, s), 7.57 (2H, d, J ¼ 8.4 Hz), 7.45
(1H, dd, J ¼ 9.3, 2.6 Hz), 7.16 (1H, d, J ¼ 2.65 Hz), 4.06 (3H, s), 3.89
(3H, s). 13C-NMR: 165.62 (s), 163.23 (s), 160.16 (s), 154.03 (s), 146.18
(s), 145.76 (s), 141.33 (s), 132.78 (d), 131.95 (2C, d), 129.62 (2C, d),
127.12 (s), 125.18 (s), 124.10 (d), 114.22 (d), 98.87 (d), 55.89 (q), 53.44
(q). Mass calcd.: 371.77; MS (ESIþ) m/z: 372.72 [M þ Hþ] (100).
Methyl 1-benzoyl-6-methyl-4-oxo-1,4-dihydro-quinoline-2-
carboxylate 8
Yield: 83%. M. p.: 127–1288C. 1H-NMR (CDCl3): 8.33 (2H, d, J ¼ 7.65
Hz), 8.25 (1H, d, J ¼ 8.75 Hz), 8.15 (1H, s), 7.78 (1H, s), 7.74 (1H, t,
J ¼ 8.1 Hz), 7.66 (1H, dd, J ¼ 8.7, 1.3 Hz), 7.60 (2H, t, J ¼ 7.7 Hz), 4.08
(3H, s), 2.55 (3H, s). 13C-NMR: 165.66 (s), 164.12 (s), 155.05 (s),
148.30 (s), 147.99 (s), 139.85 (s), 134.59 (d), 133.50 (d), 130.86 (d),
130.70 (2C, d), 129.16 (2C, d), 128.74 (s), 123.95 (s), 120.14 (d),
113.89 (d), 53.46 (q), 22.28 (q). Mass calcd.: 321.33; MS (ESIþ) m/z:
322.30 [M þ Hþ] (100).
Methyl 1-(4’-methyl-benzoyl)-6-iodo-4-oxo-1,4-dihydro-
quinoline-2-carboxylate 14
Yield: 87%. M. p.: 209–2108C. 1H-NMR (CDCl3): 8.44 (1H, s), 8.19
(1H, s), 8.18 (2H, d, J ¼ 8.0 Hz), 8.06 (2H, dd), 7.40 (2H, d, J ¼ 8.0 Hz),
4.08 (3H, s), 2.50 (3H, s). 13C-NMR: 165.29 (s), 163.83 (s), 154.47 (s),
149.39 (s), 148.42 (s), 145.96 (s), 140.04 (d), 132.45 (d), 130.81 (2C,
d), 130.63 (d), 129.97 (2C, d), 125.59 (s), 125.40 (s), 114.42 (d), 95.81
(s), 53.60 (q), 22.10 (q). Mass calcd.: 447.22; MS (ESIþ) m/z: 448.15
[M þ Hþ] (100%).
Methyl 1-(4’-methyl-benzoyl)-6-methyl-4-oxo-1,4-dihydro-
quinoline-2-carboxylate 9
Yield: 78%. M. p.: 185–1868C. 1H-NMR (CDCl3): 8.25 (1H, d, J ¼ 8.7 Hz),
8.21 (2H, d, J ¼ 7.9 Hz), 8.14 (1H, s), 7.77 (1H, s), 7.66 (1H, d, J ¼ 8.5
Hz), 7.40 (2H, d, J ¼ 7.8 Hz), 4.08 (3H, s), 2.55 (3H, s), 2.51 (3H, s). 13C-
NMR: 165.67 (s), 164.15 (s), 155.13 (s), 148.26 (s), 147.94 (s), 145.68 (s),
Methyl 1-(4’-chloro-benzoyl)-6-iodo-4-oxo-1,4-dihydro-
quinoline-2-carboxylate 15
Yield: 85%. M. p.: 202–2038C. 1H-NMR (CDCl3): 8.39 (1H, s), 8.24
(2H, d, J ¼ 8.8 Hz), 8.18 (1H, s), 8.07 (H, d, J ¼ 8.7 Hz), 7.59 (2H, d, J ¼
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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568
O. Mazzoni et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 561–569
8.8 Hz), 7.50 (2H, d, J ¼ 8.7 Hz), 4.08 (3H, s). 13C-NMR: 165.22 (s),
163.03 (s), 154.12 (s), 149.39 (s), 148.44 (s), 141.62 (s), 140.19 (d),
132.51 (d), 130.08 (2C, d), 130.42 (d), 129.70 (2C, d), 129.59 (s),
126.78 (s), 125.17 (s), 114.36 (d), 53.66 (q). Mass calcd.: 467.64; MS
(ESIþ) m/z: 468.58 [M þ Hþ] (100).
coordinates were retrieved from the ProteinData Bank. From
the PDB file of the complex, the ligand and all the water mol-
ecules were removed. Missing hydrogens and CVFF (Consistent-
Valence Force Field) partial atomic charges were added using the
builder module of InsightII/Discover software package (Accelrys)
[34, 35]. A short minimization (steepest descent) using CVFF with
˚
a gradient convergence value of 0.05 kcal/mol A was performed
Biological activities
Anti-inflammatory activity
using the Discover module present in the InsightII program in
order to release any internal strain of the receptors. Then, with
the Autodock Tools package [36], the partial atomic charges were
calculated using Gasteiger–Marsili method [37] and nonpolar
hydrogens were merged.
For ligands, 3D structures were constructed using the module
Builder of the Insight program and then optimized by minimiz-
ing the steepest descent using CVFF with a gradient-convergence
value of 0.05 kcal/mol.
Anti-inflammatory activity was evaluated by the paw edema test
[28] using carrageenan (1%) on albino rats (outbred Wister) of
both sexes weighing 220–250 g; each group comprised ten
animals. (Rats were purchased from HARLAN Laboratories
S.r.l., Italy.) The compounds were administered by oral route
(gavage) 30 min before the carrageenan, at a dose of 100
mmol/kg (ꢂ30 mg/kg). The volume of the rat’s paw was measured
1, 2, and 4 h after the administration of carrageenan, and
indomethacin was used as reference compound (14 mmol/kg, 2
mg/kg). The reported values were the average of five determi-
nations ꢃ S.E. The percentage of anti-inflammatory activity was
calculated by the formula:
Docking protocol
The grid maps representing the COX-1 and COX-2 in the actual
docking process were calculated with Autogrid (part of the
Autodock package). The grids (one for each atom type in the
ligand, plus one for the electrostatic interactions) were chosen to
be sufficiently large to include not only the active site but also
significant portions of the surrounding surface. The edges of the
% anti-inflammatory activity ¼ ð1ꢄ dt=dcÞ ꢅ 100
where dt ¼ % of paw-volume variation in the drug treated-
groups; dc ¼ % of paw-volume variation in the control groups.
The significance was calculated by Student’s test for coupled
values (Table 3).
(1 )
˚
˚
grid were 40 ꢅ 40 ꢅ 40 A with a grid spacing of 0.375 A (roughly
a quarter of a length of a carbon-carbon single bond).
The grid was centered on the ligand’s binding site of the
original crystal structures of COX-1 complexed with ISF and
COX-2 complexed with indomethacin, respectively.
Analgesic activity
Automated docking studies were carried out using Autodock
version 4.0 [25]. Of the three different search algorithm offered
by Autodock 4.0, the Lamarckian genetic algorithm (LGA) was
applied to model the interaction/binding between the COX-1,
COX-2, and ligands. The docking process was performed in two
steps. In the first short steps, consisting of 100 LGA runs, the
initial position of the ligand was random. The population was
100, the maximum number of generations was 27 000, and the
maximum number of energy evaluations was limited to 250 000.
The best ligand orientation in the first step, based on the score
criteria, was used as the input position for the second docking
step, were the number of energy evaluations was 2.5 ꢅ 106. The
second step provided the most probable ligand geometries and
orientations in the binding pocket. The resultant ligand orien-
tations and conformations were score-based on the docking and
binding energies (the cut-off value for the energies was 2 kcal/
mol). Multiple docking runs can increase the performance of
docking programs [38], as was shown specifically in the case of
Autodock [39]. To meet aspect of calculation time and data size
on one hand, and convergence criteria and statistical relevance
on the other hand, 50 independent docking runs were per-
formed for each docking case. Cluster analysis was performed
on the docked results using a root-mean-square (RMS) tolerance
Analgesic activity was determined by means of acetic acid
(writhing test) [29] carried out on albino mice outbred Swiss
(Mus musculus; HARLAN Laboratories, Italy). Each group
was composed of ten animals of both sexes; body weight (18–
25 g). The compounds were administered orally at a dose
of 100 mmol/kg and indomethacin was used as reference
compound (2 mg/kg).
The animals were injected intraperitoneally with 0.25 mL/
mouse of 0.5% aqueous acetic acid solution and writhes were
counted during the subsequent 25 min. An oral dose of a test
compound was administered 30 min before the injection. The
mean number of writhes for each experimental group ꢃ S.E. and
percent-decrease compared with the control group (five mice not
treated with the test compounds) were calculated. Results are
given in Table 3.
Molecular modeling
Molecular modeling and graphics manipulations were per-
formed using the InsightII/Discover software package
(Accelrys, San Diego, CA, USA) running on a SGI Octane 2 work-
station and on a Dell Precision T3400, an Intel Core 2 Quad
Q6600 machine running Linux Fedora 7.0 as operating system.
Automated docking studies were carried out using AutoDock
(version 4.0) [25–27]. Molecular graphics images of the complex
were produced using the UCSF Chimera package [40].
˚
of 0.5 A.
The LC-MS and NMR spectral data were provided by Centro di
Ricerca Interdipartimentale di Analisi Strumentale, Universita` degli
Studi di Napoli ‘‘Federico II’’. The assistance of the staff is gratefully
appreciated.
Preparation of enzymes and ligand molecules
Crystal structures of COX-1 and COX-2 complexed with a non-
selective inhibitor were used. Particularly the co-crystallized
complex of ISF onto COX-1 (PDB entry code 1PGE) and co-crystal-
lized complex of indomethacin onto COX-2 (PDB entry code
4COX) were chosen due to their high resolution. The spatial
The authors have declared no conflict of interest.
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www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 10, 561–569
Quinoline-2-carboxylic Acid Derivatives
569
[21] L. Schwieler, S. Erhardt, C. Erhardt, G. Engberg, J. Neural
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www.archpharm.com