Synthesis and biological evaluation of N-antipyrine-4-substituted amino-3-chloromaleimide derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.07.040

This paper describes the synthesis of new cyclic imides obtained by reaction with N-antipyrine-3,4-dichloromaleimides and different aromatic amines. The analgesic activity of the synthesized compounds was initially investigated against the writhing test in mice, followed by analysis of the most promising compounds in this model and in the formalin-induced model. The results indicate that the compounds containing the electron-withdrawing substituents in the para position of the substitute ring exerted more potent analgesic activity in mice, being much more potent than the prototype N-antipyrine-3,4-dichloromaleimide and some reference drugs. Some compounds exhibited activity against human opportunistic and pathogenic fungi, with MIC values of between 40 and 100 μg/mL (91.74 and 236.96 μM), and it was verified that only a few compounds presented potential for cytotoxic activity.

Full text of DOI:10.1016/j.ejmech.2010.07.040

European Journal of Medicinal Chemistry 45 (2010) 4761e4768
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of N-antipyrine-4-substituted
amino-3-chloromaleimide derivatives
Fernanda Mahle a, Tatiana da Rosa Guimarães a, Aleandra Vergilina Meira a, Rogério Corrêa a,
Rosana Cé Bella Cruz a, Alexandre Bella Cruz a, Ricardo José Nunes b, Valdir Cechinel-Filho a,
,
Fátima de Campos-Buzzi a
*
a Núcleo de Investigações Químico-Farmacêuticas (NIQFAR)/CCS, Universidade do Vale do Itajaí (UNIVALI), Caixa Postal 360, CEP 88302-202, Itajaí, Santa Catarina, Brazil
b Departamento de Química, Curso de Pós-Graduação em Química, Universidade Federal de Santa Catarina, UFSC, Florianópolis, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the synthesis of new cyclic imides obtained by reaction with N-antipyrine-3,4-
dichloromaleimides and different aromatic amines. The analgesic activity of the synthesized compounds
was initially investigated against the writhing test in mice, followed by analysis of the most promising
compounds in this model and in the formalin-induced model. The results indicate that the compounds
containing the electron-withdrawing substituents in the para position of the substitute ring exerted
more potent analgesic activity in mice, being much more potent than the prototype N-antipyrine-3,4-
dichloromaleimide and some reference drugs. Some compounds exhibited activity against human
Received 11 February 2010
Received in revised form
16 July 2010
Accepted 21 July 2010
Available online 24 July 2010
Keywords:
Cyclic imides
Antipyrine
Antinociceptive activity
Antimicrobial activity
opportunistic and pathogenic fungi, with MIC values of between 40 and 100
mg/mL (91.74 and
236.96 M), and it was verified that only a few compounds presented potential for cytotoxic activity.
m
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
We have recently observed considerable evidence that the
derivative (3) produces anti-hypernociception in mice at the
peripheral, spinal and supraspinal sites, and that interaction with
group I metabotropic glutamate receptors (mGluRs) and N-methyl-
During our previous studies to achieve compounds with anal-
gesic potential, it was verified that a new cyclic imide derivative (1)
from 4-aminoantipyrine caused potent analgesic effects when
analyzed against the writhing test in mice [1], but it also showed
the toxicity signal.
The fragment 4-aminoantipyrine presents a certain similarity
with metamizole (2), a well-known analgesic, anti-inflammatory
and antipyretic agent, whose efficacy is unquestionable. Current
studies suggest that the risk of agranulocytosis was exaggerated in
the 1970’s, and the risks from adverse reactions of metamizole are
similar, or even lower, than for other analgesic/antipyretic drugs
available on the market [2].
Based on these studies, a series of new cyclic imides obtained
from 4-aminoantipyrine (1) were synthesized, particularly mal-
eimide, succinimide, naphtalimide and glutarimide derivatives, and
their analgesic potential evaluated in mice. The most promising
compound, a 3,4-dichloromaleimide derivative (3), was analyzed
using other models of nociception, and it was observed that this
compound exerted potent analgesic activity in mice, being more
active than some reference drugs [3] (Fig. 1).
D-aspartate (NMDA) receptors contributes to the mechanisms
underlying its effect [4].
In order to obtain new therapeutic compounds which are
structurally related to cyclic imides, and considering the high
activity antinociceptive of derivative 3, we have now synthesized
and evaluated other analogues of this compound, keeping the
imide fragments and antipyrine and therefore replacing one of the
chlorines with different substituents.
In addition, given that some cyclic imides have antimicrobial
effects, the compounds were screened by agar dilution, as an
antibacterial and antifungal agent against a panel of human path-
ogens, to determine the minimum inhibitory concentration (MIC).
Their cytotoxicity potential was also determined.
2. Results and discussion
2.1. Chemistry
Twenty-one compounds, being nineteen new compounds
and two previously evaluated on mitochondrial effects by
our group [5], were obtained from N-antipyrine-3,4-
* Corresponding author. Tel.: þ55 (47) 3341 7855; fax: þ55 (47) 3341 7601.
E-mail address: fcamposbuzzi@univali.br (F. de Campos-Buzzi).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.040

4762
F. Mahle et al. / European Journal of Medicinal Chemistry 45 (2010) 4761e4768
CH3
CH3
H3C
H3C
N
H3C
N
N
N
N
O
N
O
N
N
H3C
H3C
Cl
O
O
O
N
CH2SO3-Na+
O
O
Cl
(1)
(2)
(3)
Fig. 1. Structures of N-antipyrinemaleimide (1), metamizole (2) and N-antipyrine-3,4-dichloromaleimide (3).
dichloromaleimide (3). It was obtained by the reaction of 3,4-
dichloromaleic anhydride with aminophenazone (4-amino-
antipyrine) in ether, and subsequently dehydrated by treatment
with acetic acid/reflux to give the cyclic compound, as described
previously [3]. This compound was used as a prototype and reacted
with specific amines at a proportion of 1:2 under stirring, at room
temperature. The substitution of only one chlorine atom probably
occurs by a mechanism of additioneelimination, involving a tetra-
hedral intermediary, which loses a chlorine atom to form the
desired product (Scheme 1). The substitution of the second chlorine
atom by nucleophiles was not observed in the conditions studied.
This can be attributed to the canonic structures, where the resonant
structures indicate that a nucleophilic attack on the other olefinic
carbon is unlikely [6].
Although none of the replacements was more effective than
compound 3 (inhibition of 99.0%), used as a prototype, the
compounds with a halogen in the para position (5, 9, 10 and 20)
were the most effective of the derivatives, and were therefore
selected for more detailed studies in this test, to evaluate their
potencies. As shown in Table 2, they exhibited notable activity,
being about 33e284-fold more potent than the reference drugs
ASA (acetyl salicylic acid) and MET (metamizole), and about 3e19-
fold more potent than compound 3 itself.
Since compounds 5, 9, 10 and 20 were the most active in the
writhing test, these were selected for another model of pain: the
formalin-induced pain model (Table 3). In this model ASA, an
analgesic and anti-inflammatory drug, which is frequently used in
therapeutic treatments, was inactive in preventing the first phase of
formalin-induced (neurogenic) pain, and derivatives 5 and 10
presented signs of activity at a dosage of 10 mg/kg. In the second
The yields and melting points of the compounds are listed in
Table 1. The chemical identification data (1H NMR, 13C NMR, IR and
CHN) confirmed the structures of compounds 3e25.
phase, ASA presented an ID50 value of 123.0 (77e209)
derivative 20 presented inhibition of 49% with 20.5
m
mol/kg, and
mol/kg
m
(10 mg/kg), being about 6-fold more active than ASA.
2.2. Evaluation of antinociceptive activity
The evaluation in the formalin test presents a distinctive biphasic
nociception response termed the early and late phases. Drugs that
act primarily on the central nervous system inhibit both phases
equally, while peripherally acting drugs inhibit only the late phase.
The early phase is probably a direct result of the stimulation of
nociceptors in the paw, and reflects centrally mediated pain, while
the late phase is due to inflammation with a release of serotonin,
histamine, bradykinin and prostaglandins, and at least to some
degree, to the sensitization of the central nociceptive neurons [7,8].
Besides the aniline derivatives, some other substituents were
introduced and demonstrated interesting results. Compound 21, with
the broken conjugation between unsubstituted benzene and amine,
showed less activity than compound 4 (aniline derivative unsub-
stituted). However, compound 22, with two carbon atoms (phenethyl
derivative unsubstituted), showed betteractivity than compound 4. It
is recognized that phenethyl derivatives increase narcotic analgesic
potency in morphine analogues [9]. Such compounds with different
substituents are being prepared, and the results will be shown later.
Also, piperidine derivative 23 and piperazine derivative 24 demon-
strated more activity than aniline derivative 4.
In previous studies, compound 3 was evaluated using the
acetic acid-induced writhing model, presenting an ID50 value of
10.8 (4.8e24.8)
mmol/kg, being about 12-fold more potent than
the standard drugs used as references in the same model [3].
The aim of this work was to obtain new and more active
derivatives. All the new compounds were initially evaluated
using the writhing test in mice, after intraperitoneal adminis-
tration of a 10 mg/kg dose in a preliminary test. The results
summarized in Table 1 reveal that the derivative compounds,
with the exception of compounds 12 and 25, showed a higher
or similar antinociceptive effect when compared with non-
steroidal drugs.
Among all the studied compounds, the most active were aniline
derivatives with electron-withdrawing substituents in the para
position. When compared with those of the meta position, it can be
suggested that the electronic and steric effects contribute to the
antinociception activity of these compounds. These hypotheses
may be evidenced in the comparison between compounds 5 and 15,
which presented inhibitions of 75% and 39%, both with substitution
of the chlorine atom in the para and meta positions, respectively.
The contribution of electronegative substituents such as halogens,
which have a balance between inductive electron-withdrawal and
2.3. Evaluation of antimicrobial activities
p
e
p
conjugation, is extended to the more effective compounds in
The antimicrobial activity was determined by the agar dilution
method. Our results indicate that some of the maleimide
this series.
H3C
O
H3C
H3C
CH3
O
O
CH3
CH3
Cl
N
CH CH OH or CH Cl
2
CH COOH
3
Reflux
3
2
2
Cl
N
Cl
N
Stirred
N
NHR2
+
+
N
+
HCl
O
H2N
N
N
N
25 - 80 °C
R
Cl
O
Cl
O
O
O
O
O
R = alkyl- or arylamine
Scheme 1. The synthesis of N-antipyrine-4-substituted amino-3-chloromaleimide derivatives.

F. Mahle et al. / European Journal of Medicinal Chemistry 45 (2010) 4761e4768
Table 1 (continued)
4763
Table 1
Structure and physical data and antinociceptive activity of N-antipyrine-3-4-
dichloromaleimide derivatives and non-steroidal analgesic drugs (ASA and MET)
given intraperitoneally at 10 mg/kg in mice in the writhing test.
Reaction
time (h)
Yield
(%)
Inhibition
(%)b
Compounds
Code
mp (ꢀC)a
Cl
NH
15
9
82.7 223.0e225.0 38.9 ꢁ 4.4**
H3C
NH
16
17
7
8
52.0 136.5e138.7 49.7 ꢁ 6.6**
56.0 139.5e142.1 56.7 ꢁ 3.1**
Compounds
Code Reaction
time (h)
Yield mp (ꢀC)a
(%)
Inhibition
(%)b
H CO
3
NH
NH
4
5
3
8
63.0 266.3e268.4 71.6 ꢁ 3.9**
57.2 254.3e256.0 97.4 ꢁ 0.4**
H3C
NH
NH
18
19
7
8
61.0 137.8e139.9 68.4 ꢁ 4.0**
Cl
Cl
CH3
NH
H CO
3
NH
6
7
9
6
60.9 253.0e254.8 75.0 ꢁ 2.0**
93.0 178.0e180.4 33.6 ꢁ 3.7*
Cl
90.0 140.0e141.9 72.0 ꢁ 2.0**
NH
OCH
3
H3C
O N
2
NH
NH
20
21
22
9
8
1
82.0 121.9e123.2 98.5 ꢁ 0.4**
64.0 123.0e126.0 35.4 ꢁ 3.6**
54.8 157.7e159.0 81.8 ꢁ 1.2**
Cl
8
9
6
6
6
95.0 208.8e209.5 31.2 ꢁ 3.8*
70.0 243.8e245.2 97.5 ꢁ 0.6**
80.0 263.8e265.0 93.2 ꢁ 1.5**
H CO
3
NH
NH
Br
NH
NH
10
F
NH
N
23
24
10
10
45.0 176.4e178.7 84.6 ꢁ 1.3**
70.0 132.2e133.0 77.7 ꢁ 2.8**
11
12
7
65.0 226.8e229.1 49.5 ꢁ 2.9**
60.0 118.2e120.0 29.5 ꢁ 2.9*
H3C
N
N
O N
2
NH
N
10
H
N
NH
25
36
38.0 123.3e125.1 23.3 ꢁ 2.8
13
14
8
8
60.0 222.7e224.1 63.6 ꢁ 3.3**
68.0 224.8e226.0 33.9 ꢁ 1.4*
HOOC
HOOC
Clꢂ
3
ASA
MET
99.0 ꢁ 0.3**c
35.0 ꢁ 2.0*c
33.0 ꢁ 3.5*c
NH
a
Melting points were uncorrected.
b
Each group represents the mean of six experiments *p < 0.05 and **p < 0.01
compared with the corresponding control values.
c
Campos et al., 2002.

4764
F. Mahle et al. / European Journal of Medicinal Chemistry 45 (2010) 4761e4768
Table 2
Table 4
Effects of compounds 3, 5, 9, 10, 20, ASA and MET on acetic acid-induced abdominal
Antifungal activity of some compounds against pathogenic fungi.
constrictions in mice.
Compound
Minimum inhibitory concentration (mg/mL)
Compound
ID50
(
m
mol/kg, i.p.)a
Inhibition (%)b
A. fuma
C. neob
M. gypc
Rhizopusd
T. mene
T. rubf
3
5
9
10
20
ASA
MET
10.8 (4.8e24.4)
4.05 (2.26e7.25)
0.57 (0.37e0.92)
0.73 (0.52e1.03)
1.29 (0.96e1.74)
133 (73e243)
99.0 ꢁ 1.0**
97.4 ꢁ 0.4**
97.5 ꢁ 0.6**
96.7 ꢁ 0.7**
98.5 ꢁ 0.4**
83 ꢁ 1.4**
04
06
9
10
11
21
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
40
>100
100
>100
100
100
>100
6
80
80
100
>100
>100
>100
15
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
100
162 (88e296)
54 ꢁ 1.4**
Ketoconazole
7
5
8
3
Each group represents the mean ꢁ SEM of six to eight animals. Compounds,
acetyl salicylic (ASA) and metamizole (MET) were given intraperitoneally at
0.1e100 mg/kg.
a
Aspergillus fumigatus (A. fum).
Cryptococcus neoformans (C. neo).
Microsporum gypseum (M. gyp).
Rhizopus sp (Rhizopus).
Trichophyton mentagrophytes (T. men).
Trichophyton rubrum (T. rub).
b
c
d
e
f
a
95% confidence limit.
b
**p < 0.01 compared with the corresponding control value.
compounds tested exhibited activity, the fungi being selectively
inhibited by compounds 4, 6, 9, 10, 11 and 21 (Table 4).
toxicity of some drugs [17]. However, compounds 15 and 16 also
presented moderate toxicity (LC50 ¼ 86.23 and 90.54, respectively,
Analysis of the structures of the compounds did not enable
a parameter of correlation with the antifungal activity to be
established. However, compound 21 presented activity against
some fungi. The main difference observed between this compound
and the other active compounds (4, 6, 9, 10 and 11) appeared to be
related to the distance of a carbon between the imide ring and the
aromatic ring, suggesting that the broken conjugation between
the imide ring and the substituent group of the amino group, and
consequently the hydrophobic factor, may be contributing to the
antifungal activity. A similar effect has already been observed in
previous studies [10,11].
equivalent to 194.52 and 214.11
mM), and compound 19
(LC50 ¼ 248.68
mg/mL, equivalent to 532.64 mM) exhibited very low
toxicity. For these compounds, it was not possible to establish
a correlation with the steric, electronic and hydrophobic effects.
The compounds that presented cytotoxicity also presented
substitution in the meta position of the aromatic ring. However,
other compounds of this same series with substituents in this
position were inactive. It is emphasized out of all active
compounds, only compound 20 presented potential for toxicity.
Moreover, future studies could be carried out with compounds 15,
16 and 20 to evaluate the possible antitumoral activity, as it has
been found that this test is predictive of this activity [18,19].
Although previous studies have demonstrated that some cyclic
imides present antibacterial activity [12e16], none of the tested
compounds showed activity against bacteria at doses lower than
100 mg/mL. In other studies, activity was generally found at higher
concentrations, as previously observed by Prado et al. [5]. For
3. Conclusion
example, for the same compound 3 tested here, these authors
In summary, the synthetic method enabled the preparation of 21
new N-antipyrine-4-substituted amino-3-chloromaleimide deriva-
tives, with satisfactory to very good yields (37e95%). Four of these
molecules (5, 9, 10 and 20) presented lower efficacy, but higher
potency than prototype 3 and the positive control drugs (acetylsa-
licylic acid and metamizole), being all promising candidates for
future analgesic drugs. The structure-activity analysis shows that
the aromatic ring with electronegative substituent in the para
position contributed to improving the analgesic action. The mech-
anism(s) of action by which these compounds exert analgesic
activity still remain undetermined, and further studies are required
toelucidatethem. Also, the results indicatethatsomecompounds (4,
6, 9, 10, 11 and 21), exhibited activity against pathogenic fungi, with
found a MIC value of 250
mg/mL for antibacterial activity against
Staphylococcus aureus. The maximum concentration value (100
mg/
mL), was used as criteria because most of the clinically useful
antibiotics are active against the tested strains at a level of at least
10
mg/mL. A compound which is not active until doses of at least
100
mg/mL is unlikely to be a serious candidate for clinical use.
2.4. Evaluation of cytotoxic activity
Through the brine shrimp lethality test, it was verified that the
majority of the compounds tested were virtually non-cytotoxic
(LC50 > 1000
presented potential for toxicity; compound 20 showed moderate
g/mL (192.81 M), and
mg/mL). Only four maleimides (15, 16, 19 and 20)
MIC values of between 40 and 100
mg/mL (91.74 and 236.96 mM).
Finally, through the cytotoxicity assay, it was verified that only
compounds 15, 16, 19 and 20 presented potential for toxicity.
toxicity to brine shrimp with LC50 ¼ 94.15
m
m
seems to be related the presence of the electronic and hydrophobic
nitro group, which is classified as being largely responsible for the
4. Experimental
Table 3
4.1. General procedures for the synthesis of N-antipyrine-3,4
dichloromaleimide (3)
Antinociceptive effects of compounds 9, 10, 20 and ASA against the first (0e5 min)
and second (15e30 min) phases of formalin-induced licking.
Compound
Formalin test e Inhibition (%)
Compound 3 was obtained directly by the reaction of 3,4-
dichloromaleic anhydride with 4-aminoantipyrine, followed by
dehydrated in acetic acid under reflux, as previously described [3].
First phase
Second phase
5
9
10
20
ASA
28.2 ꢁ 9.5*
11.4 ꢁ 4.5
28.9 ꢁ 5.9**
19.6 ꢁ 1.6
16.0 ꢁ 2.0
29.3 ꢁ 9.5
22.5 ꢁ 8.3
4.5 ꢁ 9.0
4.2. General procedures for the synthesis of compounds 4e25
49.0 ꢁ 9.2**
45.0 ꢁ 5.0*
Compounds (4e25) were obtained by the reaction of the N-anti-
pyrine-3,4-dichloromaleimide (0.057 mmol) [3], with appropriate
Each group represents the mean ꢁ SEM of six to eight animals. *p < 0.05 and
**p < 0.01 compared with the corresponding control value.

F. Mahle et al. / European Journal of Medicinal Chemistry 45 (2010) 4761e4768
4765
amines (0.057 mmol) in ethanol (15 mL), which were stirred over-
night at room temperature. The product was filtered, but in cases
where there was no precipitate, it was extracted with dichlor-
ometane. The combined organic layers were dried with Na2SO4,
filtered and evaporated. The product was purified by recrystallization
with hexane and dichloromethane, or through chromatographic
silica column gel, using chloroform:methanol as eluent.
109.3 (]CeCl); 123.8 and 132.3(2CeCl, Ar); 115.9e138.3 (11 C, Ar);
127.0 (]CeN); 136.5 (]CeCH3); 158.0 (C]O, imide); 161.3 (C]O,
imide); 162.9 (C]O, pyrazolone). Anal. Calcd. for C21H15N4O3Cl3
(477.78 g/mol): C, 52.80; H, 3.16; N, 11.73. Found: C, 52.40; H, 3.49;
N, 11.32%.
4.3.5. N-antipyrine-3-chloro-4-[4-methylaniline maleimide] (7)
Molecular formula (MW): C22H19N4O4Cl2 (421.72 g/mol); Yield
4.3. Physicochemical data of the synthesized compounds
(%): 93%; mp (ꢀC): 178.0e180.4 ꢀC; IR (KBr) (cmꢂ1): 1781.5; 1726.4
n
(
n
eC]O, imide); 1648.1 (
1H NMR (CDCl3)
(ppm): 2.21 (s, 3H, CH3); 2.35 (s, 3H, NeCH3); 3.20
(q, 2H, CH2); 7.12e7.40 (m, 5H, ar); 7.69 (s,1H, NH); 13C NMR (CDCl3)
(ppm): 11.4 (CH3); 20.5 (CH3); 35.1 (NeCH3); 106.5 (]CeN); 109.3
n eC]O, antipyrine); 3200.1 (n eNHe);
Melting points were determined with a Microquimica AP-300
apparatus (Florianópolis, Brazil) and were uncorrected. IR spectra
were recorded with a Bomem 100 (Quebec, Canada) on KBr disks.
Elemental analysis was determined with a Perkin Elmer 2400
(Norwalk, USA). Percentages of C and H were in agreement with
the product formula (within ꢁ0.4% of theoretical values). The 13C
and 1H NMR spectra were recorded on a Brucker 200 MHz
(Karlsruhe, Germany). The compounds were dissolved in
deuterated solvents from commercial sources, with TMS as
internal standard. The purity of the synthesized substances was
monitored by thin-layer cromatography using Sigma (St. Louis,
d
d
(]CeCl); 118.0e144.2 (12 C, Ar); 127.0 (]CeN); 136.5 (]CeCH3);
158.0 (C]O, imide); 161.3 (C]O, imide); 162.9 (C]O, pyrazolone).
Anal. Calcd. for C22H19N4O4Cl2 (421.72 g/mol): C, 62.49; H, 4.53; N,
13.25. Found: C, 62.67; H, 4.82; N, 12.98%.
4.3.6. N-antipyrine-3-chloro-4-[4-methoxyaniline maleimide] (8)
Molecular formula (MW): C22H19N4O4Cl2 (438.72 g/mol); Yield
(%): 95%; mp (ꢀC): 208.8e209.5 ꢀC; IR (KBr) (cmꢂ1): 1789.1; 1723.0
n
USA) silica pre-coated plastic plates of 200
m
m in thickness, with
(
n
eC]O, imide); 1639.8 (
1H NMR (CDCl3)
(ppm): 2.23 (s, 3H, CH3); 3.24 (s, 3H, NeCH3); 3.18
(q, 2H, CH2); 3.81 (q, 2H, CH2),7.40 (m,5H, ar); 8.09 (s, 1H, NH); 13
NMR (CDCl3) (ppm): 11.4 (CH3); 35.1 (NeCH3); 55.5 (OCH3); 106.5
n eC]O, antipyrine); 3200.1 (n eNHe);
various solvent systems of different polarities. Spots were visu-
alized by short-wave UV light and iodine vapor. The spectral data
(IR, 1H and 13C NMR), shown below, were in accordance with the
structures.
d
C
d
(]CeN); 109.3 (]CeCl); 118.0e139.5 (11C, Ar); 127.0 (]CeN);
136.5 (]CeCH3); 157.60 (CeOCH3, Ar); 158.0 (C]O, imide); 161.3
(C]O, imide); 162.9 (C]O, pyrazolone). Anal. Calcd. for
C22H19N4O4Cl2 (438.72 g/mol): C, 60.21; H, 4.36; N, 12.77. Found: C,
60.32; H, 4.89; N, 12.41%.
4.3.1. N-antipyrine-3,4 dichloromaleimide (3)
Yield (%): 58.16; mp (ꢀC): 208.2e209.2 ꢀC; IR (KBr)
n
(cmꢂ1):
eC]O, antipyrine); 1H NMR
(ppm): 2.18 (s, 3H, CH3); 3.21 (s, 3H, NeCH3); 7.34e7.50
(m, 5H, Ar); 13C NMR (CDCl3)
(ppm): 10.8 (CH3); 35.2 (NeCH3);
1796; 1742 (n eC]O, imide), 1642 (n
(CDCl3)
d
d
4.3.7. N-antipyrine-3-chloro-4-[4-bromoaniline maleimide] (9)
100.1 (]CeN); 125.1e134.2 (5 C, Ar); 127.7 (2 ]CeCl); 152.6 (]
CeCH3); 160.4 (2 C]O, imide); 161.8 (C]O, pyrazolone). Anal.
Calcd. for C15H11N3O3Cl2 (352.15 g/mol): C, 51.16; H, 3.15; N, 11.93.
Found: C, 51.32; H, 3.22; N, 11.81%.
Yield (%): 70%; mp (ꢀC): 243.8e245.2 ꢀC; IR (KBr)
n
(cmꢂ1): 1781;
eNHe);
(ppm): 2.18 (s, 3H, CH3); 3.20 (s, 3H, NeCH3);
6.97e7.49 (m, 9H, Ar); 8.47 (s, 1H, NH); 13C NMR (CDCl3)
(ppm):
1726 (
n eC]O, imide); 1652 (n eC]O, antipyrine); 3615 (n
1H NMR (CDCl3)
d
d
10.9 (CH3); 35.1 (NeCH3); 95.3 (]CeN); 100.7 (]CeCl); 118.7
(CeBr, Ar); 125.2e152.4 (11C, Ar); 135.2 (]CeN); 137.4 (]CeCH3);
160.7 (C]O, imide); 164.7 (C]O, imide); 166.1 (C]O, pyrazolone).
Anal. Calcd. for C21H16Br Cl N4O3(487.73 g/mol): C, 51.71; H, 3.31; N,
11.49. Found: C, 51.62; H, 3.56; N, 11.33%.
4.3.2. N-antipyrine-3-chloro-4-[aniline maleimide] (4)
Yield (%): 63%; mp (ꢀC): 266.3e268.4 ꢀC; IR (KBr)
n
(cmꢂ1):
eC]O, antipyrine);
(ppm): 2.22 (s, 3H, CH3); 3.22
(s, 3H, NeCH3); 7.31e7.51 (m,5H, ar); 13C NMR (CDCl3)
(ppm): 11.4
1781.8; 1722.6 (
n eC]O, imide); 1640.0 (n
3204.54 (
n
eNH); 1H NMR (CDCl3)
d
d
(CH3); 35.1 (NeCH3); 106.5 (]CeN); 109.3 (]CeCl); 116.5e143.2
(12C, Ar); 127.0 (]CeN); 136.5 (]CeCH3); 158.0 (C]O, imide);
161.3 (C]O, imide); 162.9 (C]O, pyrazolone). Anal. Calcd. for
C21H17N4O3Cl (408.7 g/mol); C, 61.69; H, 4.19; N, 13.70. Found: C,
61.02; H, 4.31; N, 13.43%.
4.3.8. N-antipyrine-3-chloro-4-[4-fluoroaniline maleimide] (10)
Yield (%): 80%; mp (ꢀC): 263.8e265.0 ꢀC; IR (KBr)
n
(cmꢂ1): 1783;
eNHe);
(ppm): 2.20 (s, 3H, CH3); 3.21 (s, 3H, NeCH3);
6.99e7.46 (m, 9H, Ar); 7.77 (s, 1H, NH); 13C NMR (CDCl3)
(ppm):
1725 (
n eC]O, imide); 1658 (n eC]O, antipyrine); 3619 (n
1H NMR (CDCl3)
d
d
11.0 (CH3); 35.3 (NeCH3); 94.3 (]CeN); 101.1 (]CeCl); 115.3 e
115.7 (CH, AreF); 125.9 e 126.0 (CH, AreF); 125.2e134.0 (6C, Ar);
131.7 (]CeN); 137.7 (]CeCH3); 152.6 (CeNH, Ar); 158.4 e 163.5
(CeF, Ar); 160.8 (C]O, imide); 164.8 (C]O, imide); 166.1 (C]O,
pyrazolone). Anal. Calcd. for C21H16F Cl N4O3 (426.82 g/mol): C,
59.09; H, 3.78; N, 13.13. Found: C, 59.63; H, 3.92; N, 12.98%.
4.3.3. N-antipyrine-3-chloro-4-[4-chloroaniline maleimide] (5)
Yield (%): 57.2%; mp (ꢀC): 254.3e256.0 ꢀC; IR (KBr)
n
(cmꢂ1):
1780.1; 1727.3 (
NMR (CDCl3)
7.10e7.40 (m,9H, ar); 8.09 (s, 1H, NH); 13C NMR (CDCl3)
n
eC]O, imide); 1649.4 (
(ppm): 2.21 (s, 3H, CH3); 3.22 (s, 3H, NeCH3);
(ppm):
n
eC]O, antipyrine); 1H
d
d
11.4 (CH3); 35.1 (NeCH3); 106.5 (]CeN); 109.3 (]CeCl); 122.5
(CeCl, Ar); 116.7e143.1 (11C, Ar); 127.0 (]CeN); 136.5 (]CeCH3);
158.0 (C]O, imide); 161.3 (C]O, imide); 162.9 (C]O, pyrazolone).
Anal. Calcd. for C21H16N4O3Cl2 (474.21 g/mol): C, 56.90; H, 3.64; N,
12.64. Found: C, 56.41; H, 3.99; N, 12.01%.
4.3.9. N-antipyrine-3-chloro-4-[4-ethylaniline maleimide] (11)
Yield (%): 65%; mp (ꢀC): 226.8e229.1 ꢀC; IR (KBr)
n
(cmꢂ1): 1750;
eNHe);
(ppm): 1.17 (t, 3H, CH3), 2.15 (s, 3H, CH3), 2.58 (q,
2H, CH2); 3.16 (s, 3H, NeCH3), 7.0e7.40 (m, 9H, Ar); 8.80 (s,1H, NH);
13C NMR (CDCl3)
(ppm): 11.0 (CH3); 15.7 (CH3eEt); 28.6 (CH2);
1727 (
n eC]O, imide), 1659 (n eC]O, antipyrine); 3176 (n
1H NMR (CDCl3)
d
4.3.4. N-antipyrine-3-chloro-4-[3,4dichloroaniline maleimide] (6)
d
Yield (%): 75%; mp (ꢀC): 253.0e254.8 ꢀC; IR (KBr)
n
(cmꢂ1):
35.3 (NeCH3); 94.0 (]CeN); 101.1 (]CeCl); 114.6 (]CeNH);
124.2e142.7 (11C, Ar); 137.8 (]CeCH3); 152.54 (CeEt, Ar), 160.6
(C]O, imide); 165.3 (C]O, imide); 166.6 (C]O, pyrazolone). Anal.
Calcd. for C23H21Cl N4O3 (436.89 g/mol): C, 63.23; H, 4.84; N, 12.82.
Found: C, 63.51; H, 4.98; N, 12.65%.
1781.5; 1722.7 (
3195.7 (
eNHe); 1H NMR (CDCl3)
3H, NeCH3); 6.75e7.70 (m,8H, ar); 13.9 (s, 1H, NH); 13C
NMR (CDCl3) (ppm): 11.4 (CH3); 35.1 (NeCH3); 106.5 (]CeN);
n
eC]O, imide), 1648.6 (n eC]O, antipyrine);
n
d
(ppm): 2.10 (s, 3H, CH3); 2.95 (s,
d

4766
F. Mahle et al. / European Journal of Medicinal Chemistry 45 (2010) 4761e4768
4.3.10. N-antipyrine-3-chloro-4-[3-nitroaniline maleimide] (12)
Yield (%): 60%; mp (ꢀC): 118.2e120.0 ꢀC; IR (KBr) (cmꢂ1): 1750;
1727 ( eC]O, imide), 1663 ( eC]O, antipyrine); 3290 ( eNHe);
1H NMR (CDCl3)
(ppm): 2.26 (s, 3H, CH3); 3.23 (s, 3H, NeCH3);
7.36e8.04 (m, 9H, Ar); 10.32 (s, 1H, NH); 13C NMR (CDCl3)
(ppm):
10.2 (CH3); 35.1 (NeCH3); 95.9 (]CeN); 98.9 (]CeCl); 117.6e138.1
(11 CH, Ar); 138.2 (]CeCH3); 147.5 (]CeN); 154.2 (CeNO2, Ar);
160.6 (C]O, imide); 164.5 (C]O, imide); 166.7 (C]O, pyrazolone).
Anal. Calcd. for C21H16Cl N5O5 (453.83 g/mol): C, 55.58; H, 3.55; N,
15.43. Found: C, 55.76; H, 3.95; N, 15.06%.
(CDCl3) d (ppm): 10.9 (CH3); 35.3 (NeCH3); 55.3 (OCH3); 94.7 (]
n
CeN); 101.2 (]CeCl); 109.2e152.8 (11C, Ar); 134.1 (]CeNH); 137.3
(]CeCH3); 159.8 (CeOCH3, Ar); 160.8 (C]O, imide); 165.0 (C]O,
imide); 166.3 (C]O, pyrazolone). Anal. Calcd. for C22H19Cl N4O4
(438.86 g/mol); C, 60.21; H, 4.36; N, 12.77. Found: C, 60.34; H, 4.54;
N, 12.59%.
n
n
n
d
d
4.3.16. N-antipyrine-3-chloro-4-[3,5-dimethylaniline maleimide]
(18)
Molecular formula (MW): C23H21Cl N4O3 (436.89 g/mol); Yield
(%): 61%; mp (ꢀC): 137.8e139.9 ꢀC; IR (KBr) (cmꢂ1): 1781; 1725 (
n n
4.3.11. N-antipyrine-3-chloro-4-[4-carboxyaniline maleimide] (13)
eC]O, imide), 1661 (
NMR (CDCl3) (ppm): 2.21 (s, 3H, CH3); 3.20 (s, 3H, NeCH3); 2.32 (s,
6H, CH3); 6.80e4.76 (m, 8H, Ar); 13C NMR (CDCl3)
(ppm): 11.0
n eC]O, antipyrine); 3300 (n
eNHe); 1H
Yield (%): 60%; mp (ꢀC): 222.7e224.1 ꢀC; IR (KBr)
n
(cmꢂ1): 1750;
d
1728 (
1H NMR (CDCl3)
6.30e6.85 (m, 9H, Ar); 8.60 (s,1H, NH); 8.69 (s,1H, COOH); 13C NMR
(CDCl3) (ppm): 10.3 (CH3); 35.1 (NeCH3); 93.6 (]CeN); 99.2 (]
n
eC]O, imide), 1653 (
n
eC]O, antipyrine); 3316 (
n
eNHe);
d
d
(ppm): 2.30 (s, 3H, CH3); 3.08 (s, 3H, CH3);
(CH3); 35.3 (NeCH3); 21.3 (2 CH3); 94.2 (]CeN); 101.4 (]CeCl);
121.4e152.9 (11C, Ar); 134.21 (]CeN); 137.4 (]CeCH3); 160.8 (C]
O, imide); 165.1 (C]O, imide); 166.3 (C]O, pyrazolone). Anal.
Calcd. for C23H21Cl N4O3 (436.89 g/mol): C, 63.23; H, 4.84; N, 12.82.
Found: C, 63.31; H, 4.98; N, 12.67%.
d
CeCl); 122.7e134.3 (11C, Ar); 137.0 (]CeCH3); 141.0 (]CeN);
154.2 (CeCOOH, Ar); 160.6 (C]O, imide); 164.6 (C]O, imide);
165.9 (C]O, pyrazolone), 166.9 (C]O, COOH). Anal. Calcd. for
C22H17Cl N4O5 (452.84 g/mol): C, 58.35; H, 3.78; N, 12.37. Found: C,
58.27; H, 3.97; N, 12.62%.
4.3.17. N-antipyrine 3-chloro-4-[3,5-dimethoxyaniline maleimide]
(19)
Yield (%): 90%; mp (ꢀC): 140.0e141.9 ꢀC; IR (KBr)
eC]O, imide), 1653 ( eC]O, antipyrine); 3360 (
1H NMR (CDCl3)
(ppm): 2.19 (s, 3H, CH3); 3.19 (s, 3H, NeCH3); 3.77
(s, 6H, OCH3); 6.32 (s, CH, Ar); 7.25e7.45 (m, 9H, Ar); 7.74 (s, 1H,
NH); 13C NMR (CDCl3)
(ppm): 10.9 (CH3); 35.2 (NeCH3); 55.5 (2
n
(cmꢂ1): 1780;
eNHe);
4.3.12. N-antipyrine-3-chloro-4-[3-carboxyaniline maleimide] (14)
1726 (
n
n
n
Yield (%): 68%; mp (ꢀC): 224.8e226.0 ꢀC; IR (KBr)
n
(cmꢂ1): 1780;
d
1719 (
2531 (
(s, 3H, NeCH3), 7.37e7.79 (m, 9H, Ar); 10.15 (s, 1H, COOH); 13C NMR
(CDCl3) (ppm): 10.2 (CH3); 35.1 (NeCH3); 93.6 (]CeN); 99.1 (]
n
eC]O, imide), 1653 (
n
eC]O, antipyrine); 3309 (
n eNHe),
n
eCOOHe); 1H NMR (CDCl3)
d
(ppm): 2.26 (s, 3H,CH3); 3.23
d
OCH3); 95.0 (]CeN); 98.5e137.4 (10C, Ar); 101.0 (]CeCl); 137.2
(]C-CH3); 152.6 (C]O, imide); 160.6 (2 CeOCH3); 165.0 (C]O,
imide); 166.2 (C]O, pyrazolone). Anal. Calcd. for C23H21Cl N4O5
(466.88 g/mol): C, 58.91; H, 4.51; N, 11.95. Found: C, 58.96; H, 4.76;
N, 11.82%.
d
CeCl); 124.4e154.3 (11C, Ar); 128.5 (]CeNH); 130.9 (CeCOOH,
Ar); 138.5 (]CeCH3, Ar); 160.6 (C]O, imide); 164.6 (C]O, imide);
165.9 (C]O, pyrazolone); 166.8 (C]O, COOH). Anal. Calcd. for
C22H17Cl N4O5 (452.84 g/mol): C, 58.35; H, 3.78; N, 12.37. Found: C,
58.29; H, 3.95; N, 12.61%.
4.3.18. N-antipyrine-3-chloro-4-[4-chloro-3-nitroaniline
maleimide] (20)
4.3.13. N-antipyrine-3-chloro-4-[3-chloroaniline maleimide] (15)
Yield (%): 82%; mp (ꢀC): 121.9e123.2 ꢀC; IR (KBr)
n
(cmꢂ1):
eC]O, antipyrine); 3439 (
(ppm): 2.23 (s, 3H, CH3); 3.21 (s, 3H,
NeCH3); 7.35e7.87 (m, 10H, Ar); 10.32 (s, 1H, NH); 13C NMR
(CDCl3) (ppm): 10.9 (CH3); 35.8 (NeCH3); 97.8 (]CeN); 99.5 (]
Yield (%): 82.75%; mp (ꢀC): 223.0e226.0 ꢀC; IR (KBr)
n
(cmꢂ1):
1798; 1727 (
n
eC]O, imide), 1658 (
n
n
1779; 1726 (
eNHe); 1H NMR (CDCl3)
NeCH3), 7.0e7.45 (m, 9H, Ar); 8.58 (s, 1H, NH); 13C NMR (CDCl3)
(ppm): 10.9 (CH3); 35.1 (NeCH3); 96.1 (]CeN); 100.9 (]CeCl);
n
eC]O, imide), 1645 (
n
eC]O, antipyrine); 3950 (
n
eNHe); 1H NMR (CDCl3)
d
d
(ppm): 2.15 (s, 3H, CH3), 3.19 (s, 3H,
d
d
CeCl); 120.4e147.6 (10 C, Ar); 127.8 (]CeN); 132.1 (CeCl, Ar);
138.6 (]CeCH3); 154.8 (CeNO2, Ar); 161.2 (C]O, imide); 165.1
(C]O, imide); 166.2 (C]O, pyrazolone). Anal. Calcd. for C21H16Cl2
N5O5 (488.28 g/mol): C, 51.66; H, 3.10; N, 14.34. Found: C, 51.82;
H, 3.31; N, 14.13%.
121.6e152.6 (11C, Ar); 127.6 (]CeNH); 137.1 (CeCl, Ar); 137.3 (]
CeCH3); 160.8 (C]O,imide); 164.7 (C]O, imide); 165.9 (C]O,
pyrazolone). Anal. Calcd. for C21H16Cl2N4O3 (443.28 g/mol): C,
56.90; H, 3.64; N, 12.64. Found: C, 57.03; H, 3.87; N, 12.41%.
4.3.14. N-antipyrine-3-chloro-4-[3-methylaniline maleimide] (16)
4.3.19. N-antipyrine 3-chloro-4-[benzylamine maleimide] (21)
Molecular formula (MW): C22H19Cl N4O3 (422.86 g/mol); Yield
Yield (%): 64%; mp (ꢀC): 123.0e126.0 ꢀC; IR (KBr)
n
(cmꢂ1): 1750;
eNHe);
(ppm): 2.19 (s, 3H, CH3); 3.19 (s, 3H, NeCH3); 4.84
(d, 2H, CH2); 7.34e7.48 (m,10H, Ar); 5.80 (s, 1H, NH); 13C NMR
(CDCl3) (ppm): 11.2 (CH3); 35.6 (NeeCH3); 47.4 (CH2); 125.1 (]
(%): 52%; mp (ꢀC): 136.5e138.7 ꢀC; IR (KBr)
n
(cmꢂ1): 1782; 1725 (
n
1726 (
n eC]O, imide); 1662 (n eC]O, antipyrine); 3285 (n
eC]O, imide),1658 (
(CDCl3) (ppm): 2.20 (s, 3H, CH3), 2.35 (s, 3H, CH3ear), 3.19 (s, 3H,
NeCH3), 6.99e7.48 (m, 9H, Ar), 7.71 (s, 1H, NH); 13C NMR (CDCl3)
(ppm): 10.9 (CH3); 21.4 (CH3eAr); 35.3 (NeCH3); 94.3 (]CeN);
n
eC]O, antipyrine); 3617 (
n
eNHe); 1H NMR
1H NMR (CDCl3)
d
d
d
d
CeN); 125.1 (]CeCl); 126.7 (]CeNH); 127.7e137.1 (12 C, Ar);
140.8 (]CeCH3); 153.1 (C]O, imide); 164.7 (C]O, imide); 166.4
(C]O, pyrazolone). Anal. Calcd. for C22H19Cl N5O5 (422.86 g/mol):
C, 62.49; H, 4.53; N, 13.25. Found: C, 62.57; H, 4.67; N, 13.08%.
101.2 (]CeCl); 120.8e138.6 (11C, Ar); 134.2 (]CeNH); 135.6
(CeN, Ar); 137.4 (]CeCH3); 152.9 (CeCH3, Ar); 160.8 (C]O,
imide); 165.0 (C]O, imide); 166.3 (C]O, pyrazolone). Anal. Calcd.
for C22H19Cl N4O3 (422.86 g/mol): C, 62.49; H, 4.53; N,13.25. Found:
C, 62.53; H, 4.78; N, 13.11%.
4.3.20. N-antipyrine 3-chloro-4-[phenethylamine maleimide] (22)
Yield (%): 54.8%; mp (ꢀC): 157.7e159.0 ꢀC; IR (KBr)
n
(cmꢂ1):
4.3.15. N-antipyrine-3-chloro-4-[3-methoxyaniline maleimide]
1725; 1661 (
eNHe); 1H NMR (CDCl3)
CH2); 3.07 (s, 3H, NeCH3); 3.34 (t, 2H, CH2); 7.21e7.58 (m, 10 H, Ar);
9.71 (s, 1H, NH); 13C NMR (CDCl3)
(ppm): 10.9 (CH3); 34.5
(NeCH3); 35.8 (CH2); 41.1 (CH2); 105.9 (]CeCl); 123.7e152.7 (12C,
Ar); 126.2 (]CeN); 126.4 (]CeNH); 139.1 (]CeCH3); 160.2 (C]O,
n
eC]O, imide); 1623 (
n eC]O, antipyrine); 3297 (n
(17)
d
(ppm): 2.21 (s, 3H, CH3); 2.76 (t, 2H,
Yield (%): 56%; mp (ꢀC): 139.5e142.1 ꢀC; IR (KBr)
eC]O, imide), 1658 ( eC]O, antipyrine); 3639 (
1H NMR (CDCl3)
(ppm): 2.20 (s, 3H,CH3); 3.20 (s, 3H, NeCH3), 3.81
(s, 3H, OCH3), 6.74e7.49 (m, 9H, Ar); 7.87 (s, 1H, NH); 13C NMR
n
(cmꢂ1): 1781;
eNHe);
1726 (
n
n
n
d
d

F. Mahle et al. / European Journal of Medicinal Chemistry 45 (2010) 4761e4768
4767
imide); 160.4 (C]O, imide); 161.3 (C]O, pyrazolone). Anal. Calcd.
for C23H21Cl N4O3 (436.90 g/mol): C, 63.23; H, 4.84; N,12.82. Found:
C, 63.31; H, 4.99; N, 12.73%.
at 20e22 ꢀC. After the challenge, each animal was placed in
a separate glass funnel, and the number of abdominal constrictions,
together with stretching of the hind limbs, was cumulatively
counted over a period of 20 min. For compounds 5, 9, 10 and 20, the
abdominal constriction response was analyzed, following
pretreatment via the intraperitoneal route in smaller doses
(0.1e10 mg/kg) and for standard drugs at higher doses (10e100 mg/
4.3.21. N-antipyrine 3-chloro-4-[piperidine maleimide] (23)
Yield (%): 45%; mp (ꢀC): 176.4e178.7 ꢀC; IR (KBr)
n
(cmꢂ1): 1724
eNHe); 1H
(ppm): 1.71 (m, 6H, CH2); 2.18 (s, 3H, CH3); 3.18 (s,
3H, NeCH3); 3.91 (m, 4H, CH2); 7.27e7.48 (m, 5H, Ar); 13C NMR
(CDCl3) (ppm): 11.0 (CH3); 24.1 (CH2); 26.7 (CH2); 35.4 (NeCH3);
(n
eC]O, imide), 1655 (n eC]O, antipyrine); 3397 (n
NMR (CDCl3)
d
kg) for evaluation of ID50.
d
4.4.3. Formalin test
49.7 (CH2); 94.1 (]CeCl); 102.0 (]CeN); 124.7e142.1 (6C, Ar);
134.4 (]CeN); 153.1 (]CeCH3); 160.9 (C]O, imide); 164.5 (C]O,
imide); 165.2 (C]O, pyrazolone). Anal. Calcd. for C20H21Cl N4O3
(400.85 g/mol): C, 59.92; H, 5.28; N, 13.98. Found: C, 60.01; H, 5.42;
N, 13.74%.
The observation chamber was a glass cylinder of 20 cm in
diameter, with a mirror at a 45ꢀ angle to allow clear observation
of the animal’s paws. The mice were treated with vehicle solu-
tion (i.p.), compounds 5, 9, 10 and 20 and ASA (10 mg/kg, i.p.),
30 min before formalin injection. Each animal was placed in the
chamber for 5 min before treatment, in order to acclimatize to
the new environment. The formalin test was carried out as
described by Hunskaar et al. [23], with minor modifications.
Twenty microliters of a 2.5% formalin solution (0.92% formalde-
hyde) in 0.9% saline solution were injected intraplantarly into the
right hind paw. The animal was then returned to the chamber,
and the amount of time spent licking the injected paw was
considered indicative of pain. Two distinct phases of intensive
licking activity were identified: an early acute phase and a late or
tonic phase (0e5 and 15e30 min after formalin injection,
respectively).
4.3.22. N-antipyrine 3-chloro-4-[(pyridin-2-yl)piperazine
maleimide] (24)
Yield (%): 70%, mp (ꢀC): 132.2e133.0 ꢀC; IR (KBr)
1657 (n eC]O, imide); 1625 (n
n
(cm-1): 1714;
eC]O, antipyrine); 1H NMR (CDCl3)
(ppm): 2.19 (s, 3H, CH3); 3.19 (s, 3H, NeCH3); 3.70 (dd, 2H, CH2);
4.12 (dd, 2H, CH2); 6.66e8.22 (m, 9H, Ar); 13C NMR (CDCl3)
(ppm):
d
d
11.0 (CH3); 35.4 (NeCH3); 45.6 (CH2); 47.7 (CH2); 95.9 (]CeN);
101.7 (]CeCl); 107.3e158.6 (11C, Ar); 134.4 (CeN); 153.1 (]
CeCH3); 160.9 (C]O, imide); 164.5 (C]O, imide); 165.0 (C]O,
pyrazolone). Anal. Calcd. for C24H23Cl N6O3 (478.94 g/mol): C, 60.19;
H, 4.84; N, 17.55. Found: C, 60.32; H, 4.97; N, 17.35%.
4.4.4. Statistical analysis
4.3.23. N-antipyrine 3-chloro-4-[1H-pyrrol-2-yl maleimide] (25)
The results are presented as mean ꢁ S.E.M., except for the mean
ID50 values (i.e. the dose of drugs or compounds that reduces the
antinociceptive response by 50% relative to the control value)
which are reported as geometric means accompanied by their
respective 95% confidence limits. The statistical significance
between the groups was determined by analysis of variance, fol-
lowed by Dunnett’s multiple comparison test. P-values of less than
0.05 were considered indicative of significance. The ID50 values
were determined by graphical interpolation from the individual
experiments.
Molecular formula (MW): C19H15ClN4O3 (382.80 g/mol); Yield
(%): 36.8%; mp (ꢀC): 123.3e125.1 ꢀC; IR (KBr)
n n
(cm-1): 3399 (
eNHe), 1718; 1713 (
NMR (CDCl3) (ppm): 2.19 (s, 3H, CH3); 3.21 (s, 3H, NeCH3);
6.42e7.50 (m, 8H, Ar); 10.49 (s, 1H, NH); 13C NMR (CDCl3)
(ppm):
n eC]O, imide), 1656 (n
eC]O, antipyrine); 1H
d
d
10.9 (CH3); 35.3 (NeCH3); 101.0 (]CeN); 112.1 (CH, pyrrole); 117.9
(CH, pyrrole); 118.5 (CH, pyrrole); 121.4 (CeCl); 124.9e129.3 (6C,
Ar); 134.1 (]CeCH3); 152.8 (Cepyrrole); 160.7 (C]O, imide); 164.8
(C]O, imide); 168.6 (C]O, pyrazolone). Anal. Calcd. for
C19H15ClN4O3 (382.80 g/mol): C, 59.61; H, 3.95; N, 14.64. Found: C,
59.84; H, 4.11; N, 14.39%.
4.4.5. Drugs
The following drugs were used: ASA, acetaminophen, meta-
mizole (all from Sigma Chemical) and acetic acid (E. Merck). All the
compounds were dissolved in Tween 80 (E. Merck), plus 0.9% of
NaCl solution. The final concentration of Tween 80 did not exceed
5% and did not cause any effect per se.
4.4. Biological assay
4.4.1. Animals
Swiss mice (25e35 g) were obtained from the Animal House of
the University of Vale do Itajaí (Itajaí, Brazil). The animals were
housed in automatically controlled temperature conditions
(23 ꢁ 2 ꢀC and 12 h lightedark cycles). They were given access to
water and Nuvital chow ad libitum, and remained in the appro-
priate laboratory at UNIVALI until several hours before the exper-
iments. The allocation of animals into the different groups was
randomized, and the experiments were carried out under
blind conditions. Since some suffering could result from the
experiments, the guidelines of the IASP Committee on Ethical
Issues were followed [20].
4.5. Antimicrobial analysis
4.5.1. Microorganisms
For the antimicrobial evaluation, standardized strains of the
following bacteria were used, obtained from the American Type
Culture Collection (ATCC) Rockville, MD, USA, and CEREMIC (C),
Centro de Referencia Micológica, Facultad de Ciencias Bioquímicas y
Farmacéuticas, Rosario, Argentina, and Control Lab (CL), Rio de
Janeiro, Brazil: Bacillus cereus ATCC 14579, Bacillus subtilis ATCC
23858, Enterobacter cloacae ATCC 35030, Escherichia coli ATCC 11775,
Proteus mirabilis ATCC 25933, Pseudomonas aeruginosa ATCC 27853,
Salmonella typhimurium ATCC 14028, S. aureus ATCC 6538P, SSta-
phylococcus saprophyticus ATCC 35552 and Streptococcus agalactiae
ATCC 13813, and fungi: Aspergillus flavus ATCC 9170, Aspergillus
fumigatus ATCC 26934, Aspergillus niger ATCC 9092, Rhizopus sp CL
35, Microsporum canis C112, Microsporum gypseum C115, Trichophy-
ton mentagrophytes ATCC 9972, Trichophyton rubrum C137, Crypto-
coccus neoformans ATCC 32264, Candida albicans ATCC 10231 and
Candida krusei ATCC 6582.
4.4.2. Acetic acid-induced writhing
The abdominal constriction response caused by intraperitoneal
injection of diluted acetic acid (0.6%) was determined according to
the procedures described previously, with minor modifications
[21,22]. The animals were pretreated with 10 mg/kg of N-antipy-
rine-3,4-dichloromaleimide derivatives (4e25) or standard drugs
via the intraperitoneal route, 30 min before acetic acid injection.
The control animals received a similar volume of vehicle (Tween 80
and 0.9% NaCl) (10 ml/kg, i.p.). All the experiments were carried out

4768
F. Mahle et al. / European Journal of Medicinal Chemistry 45 (2010) 4761e4768
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The bacteria used were cultivated on Mueller-Hinton agar (MHA
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adjusted to give a final concentration of 1.5 ꢃ 108 cell/mL, stan-
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l
¼ 530 nm) [24]. The
fungi were cultivated on Sabouraud dextrose agar (SDA e Difco).
For the filamentous fungi, suspensions were obtained according to
the reported procedures, adjusted to between 1.0 ꢃ 106 and
5.0 ꢃ 106 spores/mL by microscopic enumeration with a cell
counting hemocytometer (Neubauer chamber) [25], and the yeasts
were prepared according to Pfaller et al. [26], adjusting the
suspension to give a final concentration of between 1.0 ꢃ 106 and
5.0 ꢃ 106 cell/mL, also standardized with 0.5 on the McFarland scale
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¼ 530 nm).
4.5.3. Quantitative antimicrobial evaluation
The minimum inhibitory concentration (MIC) was determined
for the organisms by the agar dilution method, carried out on slants
(1 mL). Stock solutions of each compound were diluted in dime-
thylsulfoxide (DMSO) to give serial two-fold dilutions that were
added to each medium (MHA for bacteria and SDA for fungi),
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123e132.
resulting in concentrations ranging from 10 to 100
wards, a volume of 1 L of inoculum suspension, prepared previ-
mg/mL. After-
m
ously, was added to each slant, with the exception of the sterile
control. The final concentration of DMSO in the assay did not
exceed 2%. A drug-free solution was used as blank control and the
antifungal agent ketoconazole (Sigma) was included in the assay as
positive control. Each assay was repeated three times. The slants
were incubated at 35 ꢀC for the bacteria and yeasts and 28e30 ꢀC
for the hialohyphomycete and dermatophyte strains. MIC for each
compound was defined as the lowest concentration that produces
no visible microbial growth after the incubation time.
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the compounds. Dried brine shrimp eggs (1 g/L) (Maramar, Arraial
do Cabo, RJ, Brazil) were placed in artificial salt water consisting of
23 g NaCl, 11 g MgCl2$6H2O, 4 g Na2SO4, 1.3 g CaCl2$2H2O, 0.7 g KCl
in 1000 mL distilled water. The pH was adjusted to 9.0 using Na2CO3
[27], and incubated at 25 ꢀC with strong aeration, under a contin-
uous light regime (2000 Lux).
Approximately 12 h after hatching, the phototropic nauplii were
collected, and ten brine shrimp were transferred to each well using
adequate pipettes. Ten brine shrimp were transferred to each well
using pipettes. Each test consisted of exposing groups of 10 larvae
aged 12 h to various concentrations of the compound. The toxicity
was determined after 24 h of exposure. The numbers of survivors
were counted and the percentage of deaths calculated. Larvae were
considered dead if they did not exhibit any internal or external
movement over several seconds of observation.
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Acknowledgements
The authors are grateful to CNPq, FAPESC and ProPPEC/UNIVALI
for financial support.
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