Synthesis and analgesic profile of conformationally constrained N-acylhydrazone analogues: Discovery of novel N-arylideneamino quinazolin-4(3H)-one compounds derived from natural safrole

Article abstract of DOI:10.1016/j.bmc.2009.08.009

In this work we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and platelet anti-aggregating properties of new 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-one derivatives (3a-j), designed as conformationally constrained analogues of analgesic 1,3-benzodioxolyl-N-acylhydrazones (1) previously developed at LASSBio. Target compounds were synthesized in very good yields exploiting abundant Brazilian natural product safrole (2) as starting material. The pharmacological assays lead us to identify compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) as new analgesic prototypes, presenting an antinociceptive profile more potent and effective than dipyrone and indomethacin used, respectively, as standards in AcOH-induced abdominal constrictions assay and in the formalin test. These results confirmed the success in the exploitation of conformation restriction strategy for identification of novel cyclic N-acylhydrazone analogues with optimized analgesic profile.

Full text of DOI:10.1016/j.bmc.2009.08.009

Bioorganic & Medicinal Chemistry 17 (2009) 6517–6525
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and analgesic profile of conformationally constrained
N-acylhydrazone analogues: Discovery of novel N-arylideneamino
quinazolin-4(3H)-one compounds derived from natural safrole
Rodolfo C. Maia a,b, Leandro L. Silva a,c, Eduardo F. Mazzeu a, Milla M. Fumian a,c, Claudia M. de Rezende b,
Antonio C. Doriguetto d, Rodrigo S. Corrêa d,e, Ana Luisa P. Miranda a,c, Eliezer J. Barreiro a,b,c
,
Carlos Alberto Manssour Fraga a,b,c,
*
a LASSBio—Laboratório de Avaliação e Síntese de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, PO Box 68023, ZIP 21941902 Rio de Janeiro,
RJ, Brazil
b Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
d Laboratório de Cristalografia, Departamento de Ciências Exatas, Universidade Federal de Alfenas (UNIFAL), ZIP 37130-000 Alfenas, MG, Brazil
e Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and platelet
anti-aggregating properties of new 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-
4(3H)-one derivatives (3a–j), designed as conformationally constrained analogues of analgesic 1,3-ben-
zodioxolyl-N-acylhydrazones (1) previously developed at LASSBio. Target compounds were synthesized
in very good yields exploiting abundant Brazilian natural product safrole (2) as starting material. The
pharmacological assays lead us to identify compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) as
new analgesic prototypes, presenting an antinociceptive profile more potent and effective than dipyrone
and indomethacin used, respectively, as standards in AcOH-induced abdominal constrictions assay and in
the formalin test. These results confirmed the success in the exploitation of conformation restriction
strategy for identification of novel cyclic N-acylhydrazone analogues with optimized analgesic profile.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 8 June 2009
Revised 3 August 2009
Accepted 5 August 2009
Available online 9 August 2009
Keywords:
N-Acylhydrazone
Quinazolin-4(3H)-one
Analgesic
Conformational restriction
Safrole
1,3-Benzodioxole
1. Introduction
Bio-1029 (1d) presented also potent analgesic activity, that are,
8.1 and 5.9 lmol/kg, respectively, in the same pharmacological
model (Fig. 1).
The N-acylhydrazone (NAH) subunit has been described1,2 as
the pharmacophoric framework of several anti-inflammatory,3
analgesic,4–6 antiplatelet,7–9 cardioactive,10,11 and trypanocyde
derivatives,12 among others. Its importance and recurrent appear-
ance in bioactive substances from different therapeutic classes has
indicating the privileged status of the NAH moiety.13
Trying to achieve the optimization of the analgesic profile
presented by the N-acylhydrazone prototypes (1a–d), we
planned the novel series of quinazolin-4(3H)-one derivatives
(3a–j) using the strategy of conformational restriction15 as tool
for molecular modification and design (Fig. 1). We first promoted
a cyclization between the position 6 of the 1,3-benzodioxole ring
and the amidic nitrogen of the NAH function of (1), restricting
the rotation of bonds A and B. On the other hand, we have also
introduced a methyl group in the position 2 of the new quinaz-
olin-4(3H)-one ring in order to restrict the free rotation of the
single bond C, favoring the stabilization of the s-cis conformer
(Fig. 1) by ca. 40 kcal/mol.16 So, in addition to the quinazolin-
4(3H)-one derivatives (3a–d), analogues to the corresponding
N-acylhydrazones (1a–d), we have exploited the bioisosterism
concept17 for designing the structurally related carba-analogue
derivative (3e) and the isosteric imine-attached heterocyclic
derivatives (3f–j) (Fig. 1).
The construction of NAH derivatives4 (1) bearing the 1,3-ben-
zodioxole ring derived from natural safrole14 (2) lead us to found
out novel orally active prototypes presenting remarkable analge-
sic profile when evaluated as inhibitor of AcOH-induced constric-
tions in mice, represented by phenyl derivative LASSBio-123 (1a)
and N,N-dimethylaminophenyl derivative LASSBio-125 (1b),
which showed ID50 of 6.9 and 95.5 lmol/kg, respectively. On the
other hand, the thiophene-containing isoster LASSBio-294 (1c)
and their corresponding imine-attached methyl analogue LASS-
* Corresponding author. Tel.: +55 21 25626503; fax: +55 21 25626644.
E-mail address: cmfraga@pharma.ufrj.br (C.A.M. Fraga).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.009

6518
R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
Figure 1. Design concept of new 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-one derivatives (3a–j).
The key quinazolin-4(3H)-one intermediate (9) was obtained in
90% yield by treatment of an ethanolic solution of the ester (8) with
hydrazine hydrate at reflux.23 The ‘one-pot’ formation of desired
six-member heterocyclic ring of (9) was unambiguously confirmed
by X-ray analysis (Fig. 2). The molecule is almost flat considering
the non-H atoms (rms deviation = 0.0290 Å). The largest deviation
from the least-squares plane through the all non-hydrogenous
atoms occurs for atom N1 [displacement = 0.076(1) Å]. The posi-
tional parameters of the two H atoms connected to the amine N
atom were not constrained during the refinements performed here
permitting to determine the amine geometry. As expected for –
NH2 groups, our experimental data show that the amine H atoms
are in an off-plane position. It was also shown that the position
of the amine H atoms is a result of crystal packing forces or inter-
molecular bonding motifs. That means, intermolecular hydrogen
bonds as described below. In the solid state, the compound (9)
exhibits a strong hydrogen bond involving N1–H1bꢀ ꢀ ꢀO1, in which
2. Results and discussion
2.1. Chemistry
The synthetic route used to construct the target compounds
(3a–j) is depicted in Scheme 1. The starting material was piperonal
(4) obtained in ca. 75% overall yield from isomerization and oxida-
tive cleavage of the double bond of safrole (2),18,19 an abundant
Brazilian natural product.14 Regioselective nitration of position 6
of the 1,3-bezondioxole ring of (4),20 by using concentrated nitric
acid, afforded the nitro-aldehyde derivative (5) in 95% yield, which
was converted to the corresponding methyl ester (6) through the
exploitation of oxidative Yamada’s procedure.21 Next, after the
chemoselective reduction of nitro group of (6) by treatment with
Feo and NH4Cl in ethanol at reflux,22 the obtained amino-ester
derivative (7) was acylated by using acetic anhydride in ethanol
under reflux,23 yielding compound (8) in 85% yield.
Scheme 1. Synthesis of 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-one derivatives (3a–j). Reagents and conditions: (a) concd HNO3, 20–25 °C,
30 min, 95%; (b) I2, KOH, MeOH, 0 °C, 1.5 h, 84%; (c) Fe, NH4Cl, EtOH:H2O, reflux, 2 h, 75%; (d) Ac2O, EtOH, reflux, 1 h, 85%; (e) NH2NH2ꢀH2O 98%, EtOH, reflux, 1.5 h, 90%; (f)
ArCHO EtOH, HClcat., rt, 0.5–1 h or ArCOCH3, EtOH, HClcat., 50–60 °C, 96 h.

R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
6519
demonstrating that (E)-diastereomer of N-acylhydrazone deriva-
tives presented a fragment in the mass spectrum referent to the
McLafferty rearrangement25 with high relative abundance, in con-
trast to the modest relative abundance (ca. 1%) observed for the
corresponding (Z)-diastereomer, due to the unfavorable orienta-
tion between the carbonyl oxygen atom and the
c-hydrogen
(Fig. 3). Considering that 3-arylideneamino quinazolin-4(3H)-
onequinazolin-4(3H)-one derivatives (3) have the N-acylhydraz-
one moiety internalized in the azaheterocyclic ring we assumed
that they presented (E) configuration because of the high relative
abundance evidenced for the peak with m/z 204.1, resultant from
McLafferty rearrangement of representative isosteric derivatives
(3a), (3c), (3i), and (3j) (Fig. 3).
Figure 2. The structure of key 3-amino-6,7-methylenedioxy-quinazolin-4(3H)-one
intermediate (9), showing the atom-numbering scheme. Displacement ellipsoids
are drawn at the 50% probability level and the hydrogen atoms are represented as
spheres of arbitrary radius.
2.2. Pharmacology
The evaluation of the analgesic profile of all quinazolin-4(3H)-
onequinazolin-4(3H)-one derivatives (3a–j) was performed using
the classical acetic acid-induced mice abdominal constrictions as-
forms a centrosymmetric dimer. Another classical hydrogen bond
that also contributes for packing stabilization occurs between
N1–H1aꢀ ꢀ ꢀN3. In this structure the non-classical hydrogen bonds
[C8–H8ꢀ ꢀ ꢀO2, C6–H6aꢀ ꢀ ꢀO1, and C6–H6bꢀ ꢀ ꢀO1] link the molecules
of (9) to form sheets parallel to the (ꢁ3 0 4) plane. In addition, a
say,26 in the screening concentration of 100
lmol/kg po and using
dipyrone as standard drug. The results are displayed in Table 2.
Almost all 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-
quinazolin-4(3H)-onequinazolin-4(3H)-one derivatives (3) inhib-
ited significantly the constrictions induced by acetic acid in a range
from 16.9% to 45.3% (Table 2). Among them, the most active anal-
gesic compounds were LASSBio-1240 (3b) and LASSBio-1272 (3d),
respectively, with 45.3% and 43.0% of inhibition of induced
constrictions.
The presence of a basic nitrogen in the aromatic ring attached to
the imine subunit showed to be an important feature for the anal-
gesic activity, as could be evidenced by comparison of the analgesic
profile of 4-dimethylaminophenyl derivative (3b, 43% inhibition)
with those displayed by its carbaisostere (3e, 32% inhibition) and
additionally, corroborating this observation, phenyl derivative
(3a) is less active than the 4-pyridinyl isostere (3h). In this context,
observing the pharmacological behavior of derivatives (3f–h) from
isomeric pyridinyl series and 4-dimethylaminophenyl derivative
(3b), we can notice that the analgesic activity increases as the basic
nitrogen is more distant from the quinazolin-4(3H)-one moiety, so
the order of activity presented is, 4-dimethylaminophenyl (3b) > 4-
pyridinyl (3h) > 3-pyridinyl (3g) > 2-pyridinyl (3h).
p-stacking interaction plays an important role in the assembly. In
this hydrophobic contact, the rings A of (9) are stacked along to
the c axis, forming an infinite one-dimensional chain. All informa-
tion concerning the intra- and intermolecular geometry is given in
the Supplementary data.
Finally, the desired quinazolin-4(3H)-one compounds ( 3a–c,
3e–j) were obtained, in good yields (81–98%), by condensing inter-
mediate 9 with the corresponding aromatic aldehydes (ArCHO) in
ethanol at room temperature, using hydrochloric acid as catalyst.4
On the other hand, compound 3d was obtained through the con-
densation of 9 with 2-acetylthiophene under the same conditions
except for the higher temperatures (50–60 °C) and longer time of
reaction (96 h) needed to promote the full conversion of the start-
ing material into the desired product (Table 1).
The next step of this work was to determine the relative config-
uration of the imine double bond in the novel quinazolin-4(3H)-
onequinazolin-4(3H)-one derivatives (3a–j), in order to assure
the diastereomeric ratio, essential to the complete understanding
of the biological results. The analysis of the 1H NMR spectra of
3a–j, allowed us to detect the presence of only one signal corre-
spondent to the imine hydrogen (Table 1), demonstrating the dia-
stereoselective profile of the last step of our synthetic route. The
characterization of the relative configuration of target quinazoli-
non-4-one derivatives (3) exploited mass spectrometry as auxiliary
tool, guided by the previous results from Pereira and co-workers24
On the other hand, the change of the imine-attached hydrogen
by a methyl group demonstrated to be very favorable for the anal-
gesic activity, since thienylidene derivative (3c) inhibited in 10.7%
the AcOH-induced abdominal constrictions while the correspond-
ing methyl analogue (3d) presented remarkable antinociceptive
profile, that is, 45.3% inhibition in the same bioassay.
Table 1
Yields and physical properties of the 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one derivatives (3a–j)
Compound
Molecular formulaa
Molecular weight
Yieldb (%)
Melting pointc (°C)
dd (ppm) N@CH
3a
3b
3c
3d
3e
3f
3g
3h
3i
C17H13N3O3
C19H18N4O3
C15H11N3O3S
C16H13N3O3S
307.10
350.14
313.05
327.07
349.14
308.09
308.09
308.09
297.07
313.05
87
81
92
89
98
88
97
94
99
94
240–242
226–227
238–239
255–256
114–116
216–218
264–265
>270
9.01
8.61
9.23
—
8.83
9.21
9.29
9.46
8.90
8.93
C20H19N3O3
C16H12N4O3
C16H12N4O3
C16H12N4O3
C15H11N3O4
C15H11N3O3S
225–226
225–227
3j
a
b
c
The analytical results for C, H, N, S were within 0.4% of calculated values.
Isolated yield from the condensation of 3-amino-6,7-methylenedioxy-quinazolin-4(3H)-one intermediate (9) with the corresponding carbonyl derivative.
Not corrected.
d
Data obtained from 1H NMR at 200 MHz, using CDCl3 as solvent.

6520
R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
Figure 4. Effect of compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) against
formalin-induced pain in mice after ip administration of a dose of 100 mol/kg.
l
Each column represents the mean SEM. of 6–12 experimental values. *P <0.05, **P
<0.01, ***P <0.001 Two-way ANOVA Bonferroni post-test.
(Fig. 5).28 However, none of these two derivatives was able to in-
crease the latency time of treated animals in this experimental pro-
tocol (Fig. 5).
Figure 3. McLafferty rearrangement peak in the mass spectra of (E)-3-(arylidenea-
mino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one
derivatives (3).
On the other hand, when we performed a modified version of
the classical hot-plate method,29 suitable for measuring inflamma-
tory nociception (hyperalgesia) through the previous treatment of
the animals with carrageenan, the analgesic actions of quinazolin-
For the two most active quinazolin-4(3H)-one compounds, that
are, (3b) and (3d), was determined a dose–response curve to verify
the potency and efficacy of these derivatives. LASSBio-1272 (3d)
presented a maximum effect of 49.8 2.16% inhibition of AcOH-in-
duced constrictions while LASSBio-1240 (3b) showed a maximum
effect of 41.2 1.94%. Relative to the potency, LASSBio-1240 (3b)
and LASSBio-1272 (3d) presented an ED50 = 5.4 and 3.5
respectively, both higher than dipyrone (ED50 = 144.5
used as standard analgesic drug (Table 2).
l
mol/kg,
lmol/kg)
Compounds (3b) and (3d) also had their antinociceptive activity
evaluated in the formalin test27 (Fig. 4), being both able to be active
in both phases of this pharmacological model after ip administra-
tion of a dose of 100 lmol/kg. However, they inhibited the noci-
ceptive response more expressively in the inflammatory phase
than in the neurogenic phase. Both derivatives showed to be more
active than indomethacin, used as standard drug, in the two phases
of this model.
The analgesic actions on neurogenic phase of formalin test lead
us to investigate an eventual CNS-acting profile of compounds
LASSBio-1240 (3b) and LASSBio-1272 (3d) in the hot-plate test
Figure 5. Effect of compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) in the
hot-plate test in mice, at 100
seven experimental values.
lmol/kg, ip. Each point represents the mean SEM of
Table 2
Analgesic profile of 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one derivatives (3a–j) and dipyrone in AcOH-induced abdominal
constriction test in mice
Compound
Dosea
(l
mol/kg)
Number of constrictionsb
nc
Inhibitiond (%)
ED50
—
144.5
—
5.4
—
3.5
—
—
—
—
—
(lmol/kg)
Vehicle
Dipyrone
—
64.40 1.25
41.22 3.06
53.07 1.05
36.67 1.65
57.51 2.07
35.23 1.81
43.22 1.66
52.44 2.04
41.70 1.73
39.60 1.98
53.52 1.16
42.05 1.57
10
10
10
9
8
9
9
9
10
8
10
9
—
100
100
100
100
100
100
100
100
100
100
100
36.0*
17.6*
43.0*
10.7
3a
3b
3c
3d
3e
3f
3g
3h
3i
45.3*
32.9*
18.5*
35.2*
38.5*
16.9*
34.7*
3j
—
a
b
c
All compounds were administered po.
Results expressed in terms of mean SEM.
n = number of animals.
% of inhibition obtained by comparison with vehicle control group.
P <0.01 One-way ANOVA (Dunnett’s Multiple Comparison Test).
d
*

R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
6521
at the same concentration utilized for (3b) and (3d), that is,
100 mol/kg (ip).
l
Moreover, the new quinazolin-4(3H)-onequinazolin-4(3H)-one
derivatives (3a–j) were also screened in order to evaluate their ef-
fects on in vitro rabbit platelet aggregation31 induced by arachi-
donic acid (AA, 100 lM). None of the compounds was able to
inhibit significantly the aggregation induced by the arachidonic
acid. These results indicated, in association with the absence of
activity observed in the classical carrageenan-induced rat paw ede-
ma assay, that the compounds LASSBio-1240 (3b) and LASSBio-
1272 (3d) do not exert their pharmacological effects through the
inhibition of cyclooxigenase enzymes.
3. Conclusions
As concluding remarks, we can establish that the synthetic
route used to access the new functionalized quinazolin-4(3H)-
onequinazolin-4(3H)-one derivatives (3a–j) described herein was
efficient, furnishing the titled compounds in high overall yields
(30–37%) and high diastereoselectivity, as they were obtained in
diastereopure (E)-form.
Figure 6. Effect of compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) against
carrageen-induced hyperalgesia in rats, at 100 mol/kg, ip. Each point represents
the mean SEM of 8–10 experimental values. ***P <0.001 Two-way ANOVA
l
Bonferroni post-test.
The pharmacological assays lead us to identify compounds
LASSBio-1240 (3b) and LASSBio-1272 (3d) as new analgesic proto-
types, presenting an antinociceptive profile more potent and effec-
tive than the standards used, dipyrone, in AcOH-induced
abdominal constrictions assay, and indomethacin, in the formalin
test. These results confirmed the success in the exploitation of con-
formation restriction strategy for identification of novel cyclic N-
acylhydrazone analogues with optimized analgesic profile. Com-
plementary to this work, we are performing in the laboratory a
deeper pharmacological study in order to elucidate the mechanism
of action of prototypes LASSBio-1240 (3b) and LASSBio-1272 (3d),
which will be described in due course.
4(3H)-onequinazolin-4(3H)-one derivatives LASSBio-1240 (3b)
and LASSBio-1272 (3d) could be evidenced (Fig. 6).
In spite to be less effective than standard drug indomethacin,
the compounds (3b) and (3d) showed to be able to reduce in ca.
50% the
D latency time in comparison with vehicle group (Fig. 6),
indicating their ability to control the hyperalgesic stimuli in
inflammatory pain models, in agreement with the results obtained
from formalin-induced murine hypernociception model.
We therefore turned our attention to the next step in the phar-
macological evaluation of the derivatives LASSBio-1240 (3b) and
LASSBio-1272 (3d) which consisted in the investigation of their
anti-inflammatory profile by using classical carrageenan-induced
rat paw edema (CIRPE) test (Fig. 7).30 The results obtained from
this model showed that the new quinazolin-4(3H)-onequinazo-
lin-4(3H)-one compounds did not possess a significant anti-inflam-
matory activity when compared with standard indomethacin,
which was able to inhibit the edema formation in ca. 61% (Fig. 7)
4. Experimental protocols
4.1. Chemistry
Melting points were determined with a Quimis Q340M13 appa-
ratus and are uncorrected. Proton magnetic resonance (1H NMR),
unless otherwise stated, was determined in deuterated chloroform
containing ca. 1% tetramethylsilane as an internal standard with
Bruker AC 200 spectrometer at 200 MHz. Splitting patterns are as
follows: s, singlet; d, doublet; t, triplet; q, quartet; qt, quintet;
dd, double doublet; br, broad; m, multiplet. Carbon magnetic reso-
nance (13C NMR) was determined in the same spectrometer de-
scribed above at 50 MHz, using CF3CO2D as solvent. Infrared (IR)
spectra were obtained with a Bomem FTLA2000 spectrophotome-
ter by using potassium bromide plates. The mass spectra (MS)
were obtained by electron impact (70 eV) with a GC/VG Micromass
12 spectrometer.
The progress of all reactions was monitored by thin-layer chro-
matography performed on aluminum sheets precoated with Silica
Gel 60 (HF-254, Merck) to a thickness of 0.25 mm. The developed
chromatograms were viewed under ultraviolet light at 254 nm.
4.1.1. 6-Nitro-1,3-benzodioxole-5-carbaldehyde (5)
A mixture of 0.7 g (4.7 mmol) of tritured piperonal (4) and
2.3 mL of concentrated nitric acid was kept at 20–25 °C, in a water
bath, until the complete dissolution of starting material. After
30 min, a mixture of ice/water was added to the reaction media,
promoting an intense precipitation of a yellow solid, which was
collected by filtration, washed with 140 mL of distilled water and
recrystallized with ethanol/water. Yield 0.85 g (93%); mp 91–
92 °C (lit. 93–94°C20); 1H NMR (200 MHz, DMSO-d6) d: 6.33 (s,
Figure 7. Effect of compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) against
carrageen-induced rat paw edema, at 100 lmol/kg, ip. Each point column the
mean SEM of 8–10 experimental values. ***P <0.001 One-way ANOVA ‘Dunnett’s
Multiple Comparison Test’.

6522
R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
2H, O–CH2–O), 7.32 (s, 1H, H4–Ar), 7.74 (s, 1H, H7–Ar), 10.08 (s, 1H,
–CHO); 13C NMR (50 MHz, DMSO-d6) d: 104.8 (O–CH2–O), 105.4
(C7–Ar), 107.4 (C4–Ar), 127.9 (C5–Ar), 146.1 (C6–Ar), 151.7 (C3–
NMR (200 MHz, DMSO-d6) d: 2.49 (s, 3H, CH3), 6.15 (s, 2H, O–
CH2–O), 7.00 (s, 1H, H5–Ar), 7.34 (s, 1H, H8–Ar); 13C NMR
(50 MHz, DMSO-d6) d: 19.3 (CH3), 99.2 (O–CH2–O), 103.8 (C5–Ar),
104.1 (C8–Ar), 113.4 (C4a–Ar), 136.0 (C1a–Ar), 148.9 (C6–Ar),
Ar), 152.3 (C1–Ar), 188.6 (C@O); IR (
mmax, KBr): 1682, 1518, 1368,
1336, 1126, 1119, 1032, 1021 cmꢁ1
.
155.0 (C7–Ar), 158.5 (C2–Ar), 159.5 (O@C4–Ar); IR (mmax, KBr):
3289, 3196, 1667, 1630, 1582, 1471, 1264, 1250, 1034 cmꢁ1
.
4.1.2. Methyl 6-nitro-1,3-benzodioxole-5-carboxylate (6)
To a solution of nitro-aldehyde derivative (5) (1.2 g, 6.1 mmol)
in absolute methanol (7.9 mL) cooled at 0 °C were successively
added methanolic solutions (each 7.9 mL) of iodine (2.0 g,
18.3 mmol) and KOH (1.0 g, 18.3 mmol) at 0 °C. After stirring for
1.5 h at 0 °C, small amounts of saturated NaHSO3 solution were
added until the disappearance of the brown color. Next, the meth-
anol was almost totally evaporated under reduced pressure. To the
residue was added water and ice, and the desired nitro-ester (6)
was obtained by filtration and recrystallized in ethanol/water as
a light yellow solid. Yield 1.15 g (84%); mp 102–103 °C (lit. 102–
103 °C6); 1H NMR (200 MHz, CDCl3,) d: 3.88 (s, 3H, OCH3), 6.17
(s, 2H, O–CH2–O), 7.03 (s, 1H, H4–Ar), 7.37 (s, 1H, H7–Ar); 13C
NMR (50 MHz, CDCl3) d: 53.4 (OCH3), 103.7 (O–CH2–O), 105.0
(C7–Ar), 108.5 (C4–Ar), 123.8 (C5–Ar), 143.2 (C6–Ar), 149.7 (C3–
4.1.6. General procedure for preparation of 3-(arylide-
neamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-
onequinazolin-4(3H)-one derivatives (3a–c) and (3e–j)
To a solution of 1 mmol of 3-amino-quinazolin-4(3H)-onequi-
nazolin-4(3H)-one derivative (9) in absolute ethanol (10 mL) con-
taining two drops of 37% hydrochloric acid was added 1.1 mmol
of corresponding aromatic aldehyde derivative. The mixture was
stirred at room temperature for 1 h and then, it was poured into
cold water, neutralized with 10% aqueous sodium bicarbonate
solution, and the precipitate formed was filtered out and recrystal-
lized from ethanol–water.
4.1.6.1. 3-(Benzylideneamino)-2-methyl-6,7-methylenedioxy-qui-
nazolin-4(3H)-onequinazolin-4(3H)-one derivative (3a). The
derivative (3a) was obtained, as a white solid, by condensation of
(9) with benzaldehyde. Yield 87%; mp 240–242 °C. Elemental Anal.
(CHNS) Calcd: C, 66.44; H, 4.26; N, 13.67. Found: C, 66.38; H, 4.21;
N, 13.57; 1H NMR (200 MHz, CDCl3) d: 2.72 (s, 3H, CH3), 6.14 (s,
Ar), 151.4 (C1–Ar), 165.8 (C@O); IR (
mmax, KBr): 1717, 1547, 1509,
1488, 1362, 1272, 1108, 1038 cmꢁ1
.
4.1.3. Methyl 6-amino-1,3-benzodioxole-5-carboxylate (7)
To a solution of nitro-ester (6) (1.0 g, 4.4 mmol) in 75 mL of an
ethanol/water mixture (2:1), were successively added metallic iron
(1.39 g, 24.9 mmol) and ammonium chloride (0.14 g, 2.7 mmol).
Next, the reaction mixture was refluxed for 1 h, filtered under Cel-
iteÒ and the obtained solution was concentrated under reduced
pressure. To the residue was added ice, and the resulting precipi-
tate was filtered out furnishing the desired amino-ester derivative
(7) as a light brown solid. Yield 0.66 g (75%); mp 175–176 °C (lit.
175–176 °C3); 1H NMR (200 MHz, CDCl3) d: 3.83 (s, 3H, OCH3),
5.60 (br, 2H, NH2), 5.88 (s, 2H, O–CH2–O), 6.16 (s, 1H, H4–Ar),
7.25 (s, 1H, H7–Ar); 13C NMR (50 MHz, CDCl3) d: 51.3 (OCH3),
96.7 (O–CH2–O), 101.2 (C7–Ar), 102.4 (C4–Ar), 108.5 (C5–Ar),
0
2H, O–CH2–O), 7.18 (m, 1H, H4 –Ph), 7.27 (s, 1H, H5–Ar), 7.56 (m,
0
0
0
2H, H3 and H5 –Ph), 7.58 (s, 1H, H8–Ar), 7.91 (d, 2H, J = 4.6 Hz, H2
and H6 –Ph), 9.01 (s, 1H, N@CH–Ar); 13C NMR (50 MHz, CF3CO2D)
d: 20.4 (CH3), 100.2 (O–CH2–O), 106.6 (C5–Ar), 106.9 (C8–Ar), 116.1
0
0
0
0
0
0
(C4a–Ar), 131.6 (C3 and C5 –Ph), 132.1 (C2 and C6 –Ph), 133.3 (C4 –
0
Ph), 136.4 (C1a–Ar), 137.5 (C1 –Ar), 153.4 (N@CH–Ar), 159.3 (C6–
Ar), 159.7 (C7–Ar), 160.0 (C2–Ar), 177.9 (O@C4–Ar); IR (mmax, KBr):
3048, 2910, 1663, 1590, 1471, 1038 cmꢁ1; MS (70 eV) m/z (relative
abundance): 307.1 (5.8%), 204.1 (100%), 163.1 (5.9%), 103.1 (34.5%).
4.1.6.2. 3-[(4-Dimethylaminobenzylidene)amino]-2-methyl-6,7-
methylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one
derivative (3b). The derivative (3b) was obtained, as a light
yellow solid, by condensation of (9) with 4-dimethylaminobenzal-
dehyde. Yield 81%; mp 226–227 °C. Elemental Anal. (CHNS) Calcd:
C, 65.13; H, 5.18; N, 15.99. Found: C, 65.18; H, 5.21; N, 16.07; 1H
NMR (200 MHz, CDCl3) d: 2.63 (s, 3H, CH3), 3.09 (s, 6H, N–
139.1 (C6–Ar), 148.8 (C3–Ar), 152.8 (C1–Ar), 168.2 (C@O); IR (mmax
,
KBr): 3449, 3350, 1671, 1616, 1479, 1401, 1235 cmꢁ1
.
4.1.4. Methyl 6-acetylamino-1,3-benzodioxole-5-carboxylate
(8)
0
A solution of amino-ester derivative (7) (1 g, 5.12 mmol) in
absolute ethanol (25 mL) and acetic anhydride (10 mL, 12.7 mmol)
was refluxed for 1 h and then concentrated under reduced pres-
sure. To the obtained residue was added a water/ice mixture and
the desired acetamide derivative (8) was collected by filtration as
a white solid. Yield 1.03 g (85%); mp 182–184 °C; 1H NMR
(200 MHz, CDCl3) d: 2.19 (s, 3H, OCH3), 3.86 (s, 3H, NHCOCH3),
5.99 (s, 2H, O–CH2–O), 7.38 (s, 1H, H4–Ar), 8.29 (s, 1H, H7–Ar);
13C NMR (50 MHz, CDCl3) d: 25.4 (COCH3), 52.1 (NHCOCH3),
101.4 (O–CH2–O), 102.0 (C4–Ar), 107.5 (C7–Ar), 108.8 (C5–Ar),
139.2 (C6–Ar), 142.6 (C3–Ar), 152.6 (C1–Ar), 168.3 (NHCOCH3),
(CH3)2), 6.11 (s, 2H, O–CH2–O), 6.74 (d, 2H, J = 8.0 Hz, H2 - and
0
H6 –Ph), 7.12 (s, 1H, H5–Ar), 7.58 (s, 1H, H8–Ar), 7.76 (d, 2H,
J = 8.0 Hz, H3 and H5 –Ph), 8.61 (s, 1H, N@CH–Ar); 13C NMR
(50 MHz, CF3CO2D) d: 18.1 (CH3), 47.1 (N(CH3)2), 97.9 (O–CH2–
0
0
0
O), 104.5 (C5–Ar), 104.9 (C8–Ar), 113.7 (C4a–Ar), 121.1 (C3 - and
0
0
0
0
0
C5 -Ph), 132.0 (C2 - and C6 -Ph), 133.0 (C4 -Ph), 133.7 (C1 -Ph),
145.7 (N@CH–Ar), 151.1 (C1a–Ar), 156.2 (C6–Ar), 157.3 (C7–Ar),
158.0 (C2–Ar), 171.2 (O@C4–Ar); IR (
mmax, KBr): 3044, 2917, 1661,
1614, 1594, 1479, 1365, 1234, 1184, 1033 cmꢁ1
.
4.1.6.3. 3-[(2-Thienylmethylidene)amino]-2-methyl-6,7-methyl-
enedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one derivative
(3c). The derivative (3c) was obtained, as a white solid, by con-
densation of (9) with 2-thiophenecarboxaldehyde. Yield 92%; mp
238–239 °C. Elemental Anal. (CHNS) Calcd: C, 57.50, H, 3.54; N,
13.41; S, 10.23. Found: C, 57.58; H, 3.51; N, 13.37; S, 10.17; 1H
NMR (200 MHz, CDCl3) d: 2.65 (s, 3H, CH3), 6.11 (s, 2H, O–CH2–
168.9 (CO2CH3); IR (
mmax, KBr): 3273, 2960, 1698, 1674, 1630,
1536, 1495, 1446, 1374, 1248, 1181, 1129, 1039 cmꢁ1
.
4.1.5. 3-Amino-2-methyl-6,7-methylenedioxyquinazolin-
4(3H)-onequinazolin-4(3H)-one (9)
A solution of ortho-acetamide-ester (8) (1 g, 4 mmol) in 30 mL
of ethanol containing 5.8 mL (30 equiv) of hydrazine monohydrate
98%, was maintained under reflux for 1.5 h. Then, this solution was
concentrated under reduced pressure, followed by addition of a
water/ice mixture (1:1) to the obtained residue, which promotes
extensive precipitation. The solid was collected by filtration and
recrystallized in ethanol/chloroform to furnish the desired deriva-
tive (9) as white needles. Yield 0.74 g (90%); mp 269–270 °C; 1H
0
O), 7.08 (s, 1H, H5–Ar), 7.17 (m, 1H, H4 –Ar), 7.56 (m, 3H, H8–Ar,
H3 -2-thienyl and H5 -2-thienyl), 9.23 (s, 1H, N@CH–Ar); 13C NMR
(50 MHz, CF3CO2D) d: 20.4 (CH3), 100.2 (O–CH2–O), 106.9 (C5–
0
0
0
Ar), 107.1 (C8–Ar), 116.0 (C4a–Ar), 131.1 (C3 -2-thienyl), 131.5
(C4 -2-thienyl), 136.1 (C1a–Ar), 138.4 (C5 -2-thienyl), 141.4 (C2 -2-
thienyl), 153.4 (N@CH–Ar), 159.5 (C6–Ar), 159.7 (C7–Ar), 160.2
(C2–Ar), 170.4 (O@C4–Ar); IR (mmax, KBr): 3109, 3038, 2904, 1661,
0
0
0

R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
6523
1583, 1475, 1039 cmꢁ1. MS (70 eV) m/z (relative abundance):
313.1 (4.6%), 204.1 (100%), 163.1 (17%), 103.1 (34.5%).
(C1a–Ar), 153.9 (N@CH–Ar), 158.3 (C6–Ar), 160.0 (C7–Ar), 162.1
(C2–Ar), 166.7 (O@C4–Ar); IR ( max, KBr): 3126, 3010, 1668, 1622,
1476, 1401, 1136 cmꢁ1
m
.
4.1.6.4. 3-[(4-Isopropylbenzylidene)amino]-2-methyl-6,7-meth-
ylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one deriva-
tive (3e). The derivative (3e) was obtained, as a light yellow solid,
by condensation of (9) with 4-isopropylbenzaldehyde. Yield 98%;
mp 114–116 °C. Elemental Anal. (CHNS) Calcd: C, 68.75; H, 5.48;
N, 12.03. Found: C, 68.85; H, 5.51; N, 12.97; 1H NMR (200 MHz,
CDCl3) d: 1.15 (d, 6H, J = 9.0 Hz, CH(CH3)2), 2.57 (s, 3H, CH3), 2.87
(m, 1H, CH(CH3)2), 6.03 (s, 2H, O–CH2–O), 7.04 (s, 1H, H5–Ar),
4.1.6.8. 3-[(2-Furylidene)amino]-2-methyl-6,7-methylenedioxy-
quinazolin-4(3H)-one derivative (3i). The derivative (3i) was ob-
tained, as a light yellow solid, by condensation of (9) with 2-furfur-
aldehyde. Yield 99%; mp 225–226 °C. Elemental Anal. (CHNS)
Calcd: C, 60.61; H, 3.73; N, 14.14. Found: C, 60.45; H, 3.80; N,
14.07; 1H NMR (200 MHz, CDCl3) d: 2.62 (s, 3H, CH3), 6.11 (s, 2H,
0
O–CH2–O), 6.61 (dd, 1H, J = 1.7 and 3.5 Hz, H4 -2-furyl), 7.02 (s,
0
0
0
7.27 (d, 2H, J = 8.0 Hz, H3 - and H5 -Ph), 7.47 (s, 1H, H8–Ar), 7.73
1H, H5–Ar), 7.05 (d, 1H, J = 3.5 Hz, H5 -2-furyl), 7.56 (s, 1H, H8–
(d, 2H, J = 8.0 Hz, H2 - and H6 -Ph), 8.83 (s, 1H, N@CH–Ar); 13C
NMR (50 MHz, CF3CO2D) d: 17.9 (CH(CH3)2), 21.9 (CH3), 34.6
(CH(CH3)2), 97.7 (O–CH2–O), 104.4 (C5–Ar), 104.6 (C8–Ar), 113.6
Ar), 7.68 (d, 1H, J = 1.7 Hz, H3 -2-furyl), 8.90 (s, 1H, N@CH–Ar);
0
0
0
13C NMR (50 MHz, CF3CO2D) d: 20.5 (CH3), 100.3 (O–CH2–O),
0
0
106.9 (C5–Ar), 107.2 (C8–Ar), 115.9 (C4 -2-furyl), 116.2 (C3 -2-furyl),
0
0
0
0
0
0
(C4a–Ar), 127.3 (C4 -Ph), 127.5 (C3 - and C5 -Ph), 130.0 (C2 - and
129.1 (C4a–Ar), 136.4 (C1a–Ar), 148.0 (C5 -2-furyl), 153.3 (C2 -2-fur-
0
0
C6 -Ph), 131.4 (C1 -Ph), 134.0 (N@CH–Ar), 150.9 (C1a–Ar), 157.2
(C6–Ar), 157.5 (C7–Ar), 158.5 (C2–Ar), 175.7 (O@C4–Ar); IR (mmax
KBr): 3034, 2961, 2908, 1659, 1581, 1472, 1327, 1233, 1037 cmꢁ1
yl), 153.5 (N@CH–Ar), 159.2 (C6–Ar), 159.8 (C7–Ar), 160.3 (C2–Ar),
,
.
165.4 (O@C4–Ar); IR (mmax, KBr): 3142, 3042, 1664, 1620, 1588,
1475, 1405, 1307, 1233, 1036 cmꢁ1. MS (70 eV) m/z (relative abun-
dance): 297.1 (7.7%), 204.1 (100%), 163.1 (10.5%), 120.1 (23.3%).
4.1.6.5. 3-[(2-Pyridinylmethylidene)amino]-2-methyl-6,7-meth-
ylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one deriva-
tive (3f). The derivative (3f) was obtained as a white solid by
condensation of (9) with 2-pyridinecarboxaldehyde. Yield 88%;
mp 216–218 °C. Elemental Anal. (CHNS) Calcd: C, 62.33; H, 3.92;
N, 18.17. Found: C, 62.45; H, 3.85; N, 18.03; 1H NMR (200 MHz,
CDCl3) d: 2.63 (s, 3H, CH3), 6.11 (s, 2H, O–CH2–O), 7.02 (s, 1H,
H5–Ar), 7.43 (t, 1H, J = 5.0 Hz, H5’-2-Py), 7.59 (s, 1H, H8–Ar), 7.84
4.1.6.9. 3-[(3-Thienylidene)amino]-2-methyl-6,7-methylenedi-
oxy-quinazolin-4(3H)-one derivative (3j). The derivative (3j)
was obtained, as a white solid, by condensation of (9) with 3-thio-
phenecarboxaldehyde. Yield 94%; mp 225–227 °C. Elemental Anal.
(CHNS) Calcd: C, 57.50; H, 3.54; N, 13.41; S, 10.23. Found: C, 57.37;
H, 3.62; N, 13.47; S, 10.27; 1H NMR (200 MHz, CDCl3) d: 2.55 (s, 3H,
CH3), 6.04 (s, 2H, O–CH2–O), 6.99 (s, 1H, H5–Ar), 7.35 (dd, 1H,
0
0
0
(t, 1H, J = 7.6 Hz, H4 -2-Py), 8.17 (d, 1H, J = 7.6 Hz, H6 -2-Py), 8.75
J = 2.0 and 4.0 Hz, H4 -3-thienyl), 7.44 (s, 1H, H8–Ar), 7.62 (d, 1H,
(d, 1H, J = 5.0 Hz, H3 -2-Py), 9.21 (s, 1H, N@CH–Ar); 13C NMR
(50 MHz, CF3CO2D) d: 21.1 (CH3), 100.8 (O–CH2–O), 107.2 (C5–
J = 4.0 Hz, H5 -3-thienyl), 7.79 (d, 1H, J = 2.0 Hz, H2 -3-thienyl),
0
0
0
8.93 (s, 1H, N@CH–Ar); 13C NMR (50 MHz, CF3CO2D) d: 17.9
0
0
Ar), 108.0 (C8–Ar), 116.5 (C4a–Ar), 129.3 (C4 and C5 -2-Py), 135.7
(CH3), 97.8 (O–CH2–O), 104.4 (C5–Ar), 104.6 (C8–Ar), 113.6 (C4a–
0
0
0
0
0
0
(C6 -2-Py), 145.2 (C1 -2-Py and C1a-Ar), 152.0 (C3 -2-Py), 153.9
Ar), 124.2 (C4 -3-thienyl), 128.2 (C5 -3-thienyl), 133.2 (C2 -3-thie-
0
(N@CH–Ar), 158.3 (C2–Ar), 160.0 (C6–Ar), 162.1 (C7–Ar), 166.7
nyl), 134.0 (C3 -3-thienyl), 137.8 (N@CH–Ar), 151.0 (C1a–Ar),
(O@C4–Ar); IR (
mmax, KBr): 3135, 3006, 1665, 1625, 1472, 1401,
156.9 (C6–Ar), 157.3 (C7–Ar), 157.5 (C2–Ar), 169.2 (O@C4–Ar); IR
1041 cmꢁ1
.
(mmax, KBr): 3074, 3037, 2920, 1663, 1585, 1476, 1238,
1044 cmꢁ1. MS (70 eV) m/z (relative abundance): 313.1 (5.9%),
4.1.6.6. 3-[(3-Pyridinylmethylidene)amino]-2-methyl-6,7-meth-
ylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one deriva-
tive (3g). The derivative (3g) was obtained, as a white solid, by
condensation of (9) with 3-pyridinecarboxaldehyde. Yield 97%;
mp 264–265 °C. Elemental Anal. (CHNS) Calcd: C, 62.33; H, 3.92;
N, 18.17. Found: C, 62.25; H, 4.00; N, 18.19; 1H NMR (200 MHz,
CDCl3) d: 2.65 (s, 3H, CH3), 6.12 (s, 2H, O–CH2–O), 7.03 (s, 1H,
204.1 (100%), 163.1 (8.9%), 109.1 (21.6%).
4.1.7. Preparation of 3-[2-thienylethylidene)amino]-2-methyl-
6,7-methylenedioxy-quinazolin-4(3H)-one derivatives (3d)
To a solution of 1 mmol of 3-amino-quinazolin-4(3H)-one
derivative (9) in absolute ethanol (10 mL) containing two drops
of 37% hydrochloric acid was added 1.1 mmol of 2-acetyl-thio-
phenecarboxaldehyde. The mixture was stirred at 50–60 °C for
96 h and then, it was poured into cold water, neutralized with
10% aqueous sodium bicarbonate solution, and the precipitate
formed was filtered out and recrystallized from ethanol–water.
Yield 89%; mp 255–256 °C. Elemental Anal. (CHNS) Calcd: C,
58.70; H, 4.00; N, 12.84; S, 9.80. Found: C, 58.55; H, 4.10; N,
12.77; S, 9.65; 1H NMR (200 MHz, CDCl3) d: 2.27 (s, 3H, CH3–
(Ar)C@N), 2.47 (s, 3H, CH3), 6.11 (s, 2H, O–CH2–O), 7.06 (s, 1H,
0
H5–Ar), 7.45 (dd, 1H, J = 4.0 and 8.0 Hz, H5 -3-Py), 7.57 (s, 1H,
0
H8–Ar), 8.25 (d, 1H, J = 8.0 Hz, H6 -3-Py), 8.75 (d, 1H, J = 4.0 Hz,
H4 -3-Py), 9.01 (s, 1H, H2 -3-Py), 9.29 (s, 1H, N@CH–Ar); 13C NMR
(50 MHz, CF3CO2D) d: 21.0 (CH3), 100.7 (O–CH2–O), 107.2
0
0
0
(C5–Ar), 107.7 (C8–Ar), 116.3 (C4a–Ar), 131.0 (C6 -3-Py), 135.1
0
0
0
(C5 -3-Py), 135.9 (C1a–Ar), 144.7 (C1 -3-Py), 146.8 (C4 -3-Py), 149.1
0
(C2 -3-Py), 153.8 (N@CH–Ar), 158.3 (C6–Ar), 160.0 (C7–Ar), 161.5
(C2–Ar), 167.1 (O@C4–Ar); IR (
mmax, KBr): 3130, 3045, 1665, 1589,
1475, 1403, 1037 cmꢁ1
.
H5–Ar), 7.15 (t, 1H, J = 5.0 Hz, H4 -2-thienyl), 7.55 (d, 1H,
0
0
J = 5.0 Hz, H3 -2-thienyl), 7.58 (s, 1H, H8–Ar), 7.63 (d, 1H,
0
4.1.6.7. 3-[(4-Pyridinylmethylidene)amino]-2-methyl-6,7-meth-
ylenedioxy-quinazolin-4(3H)-onequinazolin-4(3H)-one deriva-
tive (3h). The derivative (3h) was obtained, as a white solid, by
condensation of (9) with 4-pyridinecarboxaldehyde. Yield 94%;
mp >270 °C. Elemental Anal. (CHNS) Calcd: C, 62.33; H, 3.92; N,
18.17. Found: C, 62.05; H, 4.05; N, 18.37; 1H NMR (200 MHz,
CDCl3) d: 2.68 (s, 3H, CH3), 6.13 (s, 2H, O–CH2–O), 7.05 (s, 1H,
J = 5.0 Hz, H5 -2-thienyl); 13C NMR (50 MHz, CF3CO2D) d: 17.4
(N@C(Ar)–CH3), 18.1 (Ar–CH3), 97.9 (O–CH2–O), 104.3 (C5–Ar),
104.6 (C8–Ar), 112.8 (C4a–Ar), 128.9 (C3 -2-thienyl), 134.4 (C4 -2-
thienyl), 136.7 (C1a–Ar), 137.7 (C5 -2-thienyl), 141.8 (C2 -2-thienyl),
151.0 (N@CH–Ar), 151.6 (C6–Ar), 157.4 (C7–Ar), 157.8 (C2–Ar),
0
0
0
0
159.9 (O@C4–Ar); IR (
mmax, KBr): 2928, 1672, 1581, 1475, 1411,
1262, 1028, 705 cmꢁ1
.
0
H5–Ar), 7.57 (s, 1H, H8–Ar), 7.77 (d, 2H, J = 6.0 Hz, H2 - and
0
0
0
H6 -Ar), 8.80 (d, 2H, J = 6.0 Hz, H3 - and H5 -Ar), 9.46 (s, 1H,
N@CH–Ar N); 13C NMR (50 MHz, CF3CO2D) d: 21.1 (CH3), 100.8
(O–CH2–O), 107.2 (C5–Ar), 108.0 (C8–Ar), 116.5 (C4a–Ar), 129.3
4.1.8. Single crystal X-ray diffraction
After the synthesis and purification procedures, a well-shaped
single crystal of (9) was obtained by recrystallization from etha-
nol/chloroform (1:1 v/v) solution at room temperature. Intensity
0
0
0
0
0
(C2 and C6 -4-Py), 135.7 (C1 -4-Py), 145.2 (C3 and C5 -4-Py), 152.0

6524
R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
data were measured with the crystal at room temperature (298 K)
and with graphite monochromated Mo radiation
(100 lmol/kg) was used as standard drug under same conditions.
Ka
Acetic acid solution was administered intraperitoneally 1 h after
administration of quinazolin-4(3H)-one compounds (3a–j). Ten
minutes following intraperitoneally acetic acid injection the num-
ber of constrictions per animal was recorded for 20 min. Control
animals received an equal volume of vehicle. Analgesic activity
was expressed as percentage of inhibition of constrictions when
compared with the vehicle control group (Table 2). The dose–re-
sponse curve was constructed varying the molar concentration of
(k = 0.71073 Å), using the Enraf-Nonius Kappa-CCD diffractometer.
32
The cell refinements were performed using the software COLLECT
33
and SCALEPACK
,
and the final cell parameters were obtained on all
reflections. Data reduction was carried out using the software DEN-
33
ZO-SMN and SCALEPACK
.
Since the absorption coefficient is insignifi-
cant for (9), no absorption correction was applied. The structure
was solved using the software SHELXS-97,34 where all the non-
hydrogen atoms were readily found from the electronic density
map constructed by Fourier synthesis. The model obtained was re-
compounds (3b) and (3j) from 0.3 to 100 lmol/kg. Results are ex-
pressed as the mean SEM of n animals per group. The data were
statistically analyzed by the One-way ANOVA test for a significance
level of P <0.01.
fined by full-matrix least-squares on F2 using the software SHELXL
-
97.34 The refinement was carried on with anisotropic thermal
parameters for all the non-hydrogen atoms. With regard to the
hydrogen atoms of (9), the C–H hydrogen atoms were positioned
stereochemically and were refined with fixed individual displace-
4.2.1.2. Formalin-induced pain in mice. The formalin test was
carried out as described by Hunskaar and Hole.27 Albino swiss mic-
es of both sexes weighting 18–25 g fasted for a period of 8 h were
ment parameters [Uiso(H) = 1.2Ueq (C2 ) or 1.5Ueq (Cs3p)] using a rid-
sp
ing model with aromatic C–H bond length of 0.93 Å, methyl C–H
one of 0.96 Å, and methylene C–H one of 0.97 Å. The hydrogen
atoms linked to nitrogen of the amine group were located by differ-
ence Fourier synthesis and were set as isotropic. Crystal, collection
and structure refinement data are summarized in Table 3. Tables
injected subplantarly with 20
The compounds were administrated intraperitoneally (100
l
L of 2.5% formalin in one hind paw.
mol/
l
kg in arabic gum 5% as vehicle) 60 min before formalin injection.
The time the mice spent licking or biting the injected paw or leg
was recorded. Two distinct periods of intensive licking activity
were identified and scored separately unless otherwise stated.
The first period (earlier or neurogenic phase) was recorded 0–
5 min after formalin injection and the second period (later or
inflammatory phase) was recorded 15–30 min after injection.
35
were generated by WINGX and the structure representations by
37
ORTEP-336 and MERCURY
.
Atomic coordinates, bond lengths, angles and thermal parame-
ters have been deposited at the Cambridge Crystallographic Data
Center, deposition number CCDC 734003.
4.2.1.3. Hot-plate test. The central analgesic activity was deter-
mined in vivo by the hot-plate test according to Kuraishi et al.28
Swiss mice of both sexes were used, maintained with water ad libi-
tum and fasted for a period of 8 h.
Animals were placed on heated plate at 55 0.1 °C and their re-
sponses to thermal stimulation (licking or withdraw of hind paw)
were timed. Two control measures were done (in the absence of test
drugs) in intervals of 30 min to determine the control latency mean
time and the cut-off time (maximum time of permanence of the ani-
mal in the plate), calculated as three times the control mean value.
The response time for each mouse is registered at 30 min intervals
after compounds administration for a total of 150 min. The quinaz-
olin-4(3H)-one derivatives LASSBio-1240 (3b) and LASSBio-1272
4.2. Pharmacology
4.2.1. Analgesic activity
4.2.1.1. Acetic acid-induced abdominal constrictions assay.26
The abdominal constrictions assay induced by acetic acid (0.6%;
0.1 mL/10 g) was performed using albino mice of both sexes (18–
23 g). Compounds were administered orally (100 lmol/kg) as a
suspension in 5% Arabic gum in saline (vehicle). Dipyrone
Table 3
Crystal data and structure refinement for 3-amino-6,7-methylenedioxy-quinazolin-
4(3H)-one intermediate (9)
(3d) were administrated intraperitoneally (100 lmol/kg, in tween
1% in saline as vehicle).
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
C10H9N3O3
219.20
298(2)
0.71073
Monoclinic
P21/c
4.2.1.4. Modified hot-plate test (hyperalgesia).29
Wistar rats were placed individually on a hot plate with the tem-
perature adjusted to 51 0.1 °C. The latency of the withdrawal
response of the left hind paw was determined at 0, 30, 60, 120,
180, and 240 min post-challenge. The left paw was stimulated with
carrageenan (1 mg/paw) and the right with saline (0.9% NaCl), both
in a final volume of 100 lL, injected intraplantarly (ipl). The time of
maximum permanence permitted on the hot surface was 20 s. The
quinazolin-4(3H)-one derivatives LASSBio-1240 (3b) and LASSBio-
1272 (3d) were administrated intraperitoneally (100 lmol/kg, in
1% tween 80 in saline as vehicle) 60 min before carrageenan injec-
tion. Hyperalgesia to heat was defined as a decrease in withdrawal
latency and calculated as follows: d paw withdrawal latency
(s) = (left paw withdrawal latency in time 0)—(left paw withdrawal
latency in others times).
Unit cell dimensions
a (Å)
b (Å)
b (°)
10.8231(3)
12.6064(4)
103.983(2)
7.1213(2)
942.83(5)
4
1.544
0.117
c (Å)
Volume (Å3)
Z
Dcalc (Mg/m3)
Absorption coefficient (mmꢁ1
F(0 0 0)
)
456
Crystal size (mm3)
0.30 ꢂ 0.15 ꢂ 0.10
h Range for data collection (°)
Index ranges
Reflections collected
Independent reflections [R(int)]
Completeness to h = 25.0°
Max. and min. transmission
Refinement method
3.5–25.0
ꢁ12 6 h 6 12, ꢁ14 6 k 6 14, ꢁ8 6 l 6 8
3221
1651 [0.0110]
99.2%
4.2.2. Anti-inflammatory activity
0.9931 and 0.9638
Full-matrix least-squares on F2
1651/0/154
The anti-inflammatory activity was determined in vivo using
the carrageenan-induced rat paw edema test according to Ferre-
ira.30 Albino rats of both sexes (150–200 g) were used in this pro-
tocol. Compounds were administered in the dose of choice as a
suspension in 1% tween 80 in saline (vehicle). Control animals re-
ceived equal volume of the vehicle. One hour after, the animals
Data/restraints/parameters
Goodness-of-fit on F2
1.070
Final R indices [I > 2
R indices (all data)
r(I)]
R1 = 0.0399, wR2 = 0.1076
R1 = 0.0454, wR2 = 0.1126
0.180 and ꢁ0.173
Largest diff. peak and hole (e Åꢁ3
)

R. C. Maia et al. / Bioorg. Med. Chem. 17 (2009) 6517–6525
6525
3. Tributino, J. L. M.; Duarte, C. D.; Corrêa, R. S.; Dorigetto, A. C.; Ellena, J.; Romeiro,
N. C.; Castro, N. G.; Miranda, A. L. P.; Barreiro, E. J.; Fraga, C. A. M. Biorg. Med.
Chem. 2009, 17, 1125.
4. Lima, P. C.; Lima, L. M.; Silva, K. C. M.; Léda, P. H. O.; Miranda, A. L. P.; Fraga, C.
A. M.; Barreiro, E. J. Eur. J. Med. Chem. 2000, 35, 187.
5. Bezerra-Neto, H. J. C.; Lacerda, D. I.; Miranda, A. L. P.; Alves, H. M.; Barreiro, E. J.;
Fraga, C. A. M. Bioorg. Med. Chem. 2006, 14, 7924.
6. Duarte, C. D.; Tributino, J. L. M.; Lacerda, D. I.; Martins, M. V.; Alexandre-
Moreira, M. S.; Dutra, F.; Bechara, E. J. H.; De-Paula, F. S.; Goulart, M. O. F.;
Ferreira, J.; Calixto, J. B.; Nunes, M. P.; Bertho, A. L.; Miranda, A. L. P.; Barreiro, E.
J.; Fraga, C. A. M. Bioorg. Med. Chem. 2007, 15, 2421.
7. Cunha, A. C.; Figueiredo, J. M.; Tributino, J. L. M.; Miranda, A. L. P.; Castro, H. C.;
Zingali, R. B.; Fraga, C. A. M.; de Souza, M. C. B. V.; Ferreira, V. F.; Barreiro, E. J.
Bioorg. Med. Chem. 2003, 11, 2051.
8. Silva, G. A.; Costa, L. M. M.; Brito, F. C. F.; Miranda, A. L. P.; Barreiro, E. J.; Fraga,
C. A. M. Bioorg. Med. Chem. 2004, 12, 3149.
9. Lima, L. M.; Frattani, F. S.; dos Santos, J. L.; Castro, H. C.; Fraga, C. A. M.; Zingali,
R. B.; Barreiro, E. J. Eur. J. Med. Chem. 2008, 43, 348–356.
10. Gonzalez-Serratos, H.; Chang, R.; Pereira, E. F.; Castro, N. G.; Aracava, Y.; Melo,
P. A.; Lima, P. C.; Fraga, C. A. M.; Barreiro, E. J.; Albuquerque, E. X. J. Pharmacol.
Exp. Ther. 2001, 299, 558.
were then injected with either 0.1 mL of 1% carrageenan solution in
saline (0.1 mg/paw) or sterile saline (NaCl 0.9%), into the subplan-
tar surface of one of the hind paws, respectively. The paw volumes
were measured using a glass plethysmometer coupled to a peri-
staltic pump, 3 h after the subplantar injection. The edema was cal-
culated as the volume difference between the carrageenan and
saline-treated paw. Anti-inflammatory activity was expressed as
percentage of inhibition of the edema when compared with vehicle
control group.
4.2.3. Platelet anti-aggregating activity
4.2.3.1. Preparation of rabbit and human platelet rich
plasma. Rabbit blood was collected from the central ear artery
of rabbits weighing 2.5–3.0 kg. Blood samples were collected into
3.8% trisodium citrate (9:1 v/v). Platelet-rich plasma (PRP) was pre-
pared by centrifugation at 1800 rpm for 10 min at room tempera-
ture. The platelet poor plasma (PPP) was prepared by
centrifugation of the pellet at 3600 rpm for 10 min at room tem-
11. Silva, A. G.; Zapata-Sudo, G.; Kummerle, A. E.; Fraga, C. A. M.; Barreiro, E. J.;
Sudo, R. T. Bioorg. Med. Chem. 2005, 13, 3431.
12. Romeiro, N. C.; Aguirre, G.; Hernández, P.; González, M.; Cerecetto, H.; Aldana,
I.; Pérez-Silanes, S.; Monge, A.; Barreiro, E. J.; Lima, L. M. Bioorg. Med. Chem.
2009, 17, 641.
perature. Platelet count was adjusted to 5 ꢂ 108 mLꢁ1
.
13. Duarte, C. D.; Barreiro, E. J.; Fraga, C. A. M. Mini-Rev. Med. Chem. 2007, 7, 1108.
14. Barreiro, E. J.; Fraga, C. A. M. Quím. Nova 1999, 22, 744.
15. Wermuth, C. G. The Practice of Medicinal Chemistry, 2nd ed.; Academic Press:
New York, 2003. Chapter 6.
16. The energetic barrier was determined using the semi-empirical method AM1 in
the PC Spartan Pro program (Wavefunction Inc., Irvine, CA, 2000). It was
calculated by studying the energetic demand involved in the rotation of the
dihedral C–N–N@C in the novel quinazolin-4(3H)-one derivatives (3), when it
was turned by 15 to 15 degrees.
4.2.3.2. Platelet aggregation assay. Platelet aggregation was
monitored by the turbidimetric method of Born and Cross31 in a
Chrono-Log aggregometer. PRP (400
for 1 min with continuous stirring at 900 rpm. Aggregation of
PRP was induced by arachidonic acid (200 M). Test compounds
and the vehicle (0.5% DMSO, 2 L) were added to the PRP samples
lL) was incubated at 37 °C
l
l
5 min before addition of the aggregating agent. The DMSO used as
vehicle did not have either pro- or antiplatelet aggregation activity.
The platelet aggregation was expressed as percentage of aggrega-
tion for AA.
17. Lima, L. M.; Barreiro, E. J. Curr. Med. Chem. 2005, 12, 23.
18. Barreiro, E. J.; Lima, M. E. F. J. Pharm. Sci. 1992, 81, 1219.
19. Barreiro, E. J.; Costa, P. R. R.; Coelho, F. A. S. J. Chem. Res. 1985, 220.
20. Ekeley, J. B.; Klemme, M. J. Am. Chem. Soc. 1928, 50, 2711.
21. Yamada, S.; Morizono, D.; Yamamoto, K. Tetrahedron Lett. 1992, 33, 4329.
22. Lages, A. S.; Silva, K. C. M.; Miranda, A. L. P.; Fraga, C. A. M.; Barreiro, E. J. Bioorg.
Med. Chem. Lett. 1998, 8, 183.
Acknowledgements
23. (a) Peet, N. P.; Sunder, S.; Cregge, R. J. J. Org. Chem. 1976, 41, 2733; (b) Dallacker,
F. Monasth. Chem. 1959, 90, 846.
24. Pereira, A. S.; Violante, F. A.; Aquino Neto, F. R.; Cardoso, J. N.; Fraga, C. A. M.;
Barreiro, E. J. Anal. Lett. 1998, 31, 719.
25. McLafferty, F. W. Anal. Chem. 1956, 28, 306.
26. Coolier, H. O. J.; Dinneen, L. C.; Johnson, C. A.; Schneider, C. Br. J. Pharmacol.
Chemother. 1968, 32, 295.
27. Hunskaar, S.; Hole, K. Pain 1987, 30, 103.
The authors thank Central Analítica (IQ-UFRJ) for technical facil-
ities and CAPES (BR), CNPq (BR), FAPERJ (BR), PRONEX (BR) and
INCT-INOFAR (BR, #573.564/2008-6) for financial support and
fellowships.
28. Kuraishi, Y.; Harada, Y.; Aratani, S.; Satoh, M.; Takagi, H. Brain Res. 1983, 273, 245.
29. Lavich, T. R.; Cordeiro, R. S. B.; Silva, P. M. R.; Martins, M. A. Braz. J. Med. Biol.
Res. 2005, 38, 445.
Supplementary data
30. Ferreira, S. H. J. Pharm. Pharmacol. 1979, 31, 648.
31. Born, G. V. R.; Cross, M. J. J. Physiol. 1963, 168, 178.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.009.
32. Enraf-Nonius COLLECT. Nonius BV, Delft, The Netherlands, 1997–2000.
33. Otwinowski, Z.; Minor, W.. In Methods in Enzymology; Carter, C. W., Jr., Sweet, R.
M., Eds.; Academic Press: New York, 1997; Vol. 276, pp 307–326.
34. Sheldrick, G. M. Acta Crystallogr., Sect. A 2007, 64, 112–122.
35. Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837.
References and notes
36. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
37. Bruno, I. J.; Cole, J. C.; Edgington, P. R.; Kessler, M. K.; Macrae, C. F.; McCabe, P.;
Pearson, J.; Taylor, R. Acta Crystallogr., Sect. B 2002, 58, 389.
1. Fraga, C. A. M.; Barreiro, E. J. Curr. Med. Chem. 2006, 13, 167.
2. Rollas, S.; Küçükgüzel, S. G. Molecules 2007, 12, 1910.