Studies towards the identification of putative bioactive conformation of potent vasodilator arylidene N-acylhydrazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.04.044

In this report we disclose the synthesis, vasodilatory activity, and identification of bioactive conformation of new N-acylhydrazone and N-methyl-N-acylhydrazone derivatives, structurally designed by bioisosteric replacements of previously described cardioactive compounds LASSBio-294 and its N-methyl derivative LASSBio-785. Some of these novel derivatives presented improved vasorelaxant properties, being new cardiovascular drug candidates.

Full text of DOI:10.1016/j.ejmech.2009.04.044

European Journal of Medicinal Chemistry 44 (2009) 4004–4009
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Studies towards the identification of putative bioactive conformation of potent
vasodilator arylidene N-acylhydrazone derivatives
,
Arthur E. Ku¨mmerle a b, Juliana M. Raimundo c, Carla M. Leal c, Givanildo S. da Silva d, Tatiane L. Balliano e,
,
b
Mariano A. Pereira d, Carlos A. de Simone d, Roberto T. Sudo c, Gisele Zapata-Sudo c, Carlos A.M. Fraga a
,
,b,c,
Eliezer J. Barreiro a
*
a
´
˜
´
ˆ
´
Laboratorio de Avaliaçao e Sıntese de Substancias Bioativas (LASSBio), Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, RJ 21941-902, Brazil
b
c
´
˜
´
´
Programa de Pos-Graduaçao em Quımica, Instituto de Quımica, Universidade Federal do Rio de Janeiro, RJ 21949-900, Brazil
Programa de Pos-Graduaçao em Farmacologia e Quımica Medicinal, ICB, Universidade Federal do Rio de Janeiro, RJ 21941-590, Brazil
´
˜
´
d Departamento de Qu´ımica, Universidade Federal de Alagoas, Al 57072-970, Brazil
e Centro de Biotecnologia Molecular Estrutural, Instituto de F´ısica de S˜ao Carlos, Universidade de S˜ao Paulo, SP 13.566-970, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this report we disclose the synthesis, vasodilatory activity, and identification of bioactive conformation
of new N-acylhydrazone and N-methyl-N-acylhydrazone derivatives, structurally designed by bio-
isosteric replacements of previously described cardioactive compounds LASSBio-294 and its N-methyl
derivative LASSBio-785. Some of these novel derivatives presented improved vasorelaxant properties,
being new cardiovascular drug candidates.
Received 5 February 2009
Received in revised form
16 April 2009
Accepted 20 April 2009
Available online 8 May 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
N-Acylhydrazone
Vasodilation
Bioactive conformation
1. Introduction
enabled us to recognize the structural similarity between N-acyl-
hydrazone (NAH) derivatives (2) and these cardioactive compounds
Hypertension is the most common cardiovascular disease, and
the one which represents the major risk factor for coronary artery
disease, heart failure, stroke and renal failure [1]. The reduction in
blood pressure achieved with most of the currently used antihy-
pertensive agents, such as angiotensin-converting enzyme inhibi-
[5]. The inclusion of the 1,3-benzodioxolyl ring, coming from Bra-
zilian natural safrole (3) [6] attached to the acyl group of NAH
moiety [7,8] and replacing the phenyl ring attached to the imine
moiety by the bioisosteric 2-thienyl ring [9] resulted in the design
of 3,4-methylenedioxybenzoyl-2-thienyl-hydrazone, named LASS-
Bio-294 (4) (Chart 1) [5].
tors, calcium antagonists,
b-blockers and diuretics, is direct or
indirectly related to the relaxation of the vascular smooth muscle
[2]. Several direct vasodilators have been synthesized but none of
them has presented a specific action while being free of side effects,
reinforcing the importance of identifying new clinically safe useful
vasodilator agents [3].
LASSBio-294 (4) was described as a potent positive cardio-
inotropic agent due to an increase in the Ca2þ accumulation in the
sarcoplasmic reticulum (SR) [10]. In addition, LASSBio-294 (4) also
promoted vasodilation in aortic rings, mediated by the guanylate
cyclase/cyclic guanylate monophosphate pathway [11].
The strategy of molecular simplification on pyridazinone phos-
phodiesterase (PDE) inhibitors (1) [4], represented by a simple
bond rupture in a and the elimination of the stereogenic center in b,
Considering that the N-acylhydrazone subunit present in
LASSBio-294 (4) has an isosteric relationship with the pyridazinone
ring shared by phosphodiesterase (PDE) inhibitors [12], the bio-
profiles of several synthetic analogues planned by structural opti-
mization of the lead compound LASSBio-294 (4) were investigated
in the vascular smooth muscle [13]. Among the derivatives tested
on the contractile response of the vascular smooth muscle of Wistar
rats, we observed that changes in electronic density of the thienyl
subunit did not increase the vasodilatory effects [13]. However, the
* Corresponding author. Laborato´rio de Avaliaça˜o
e Sı´ntese de Substaˆncias
´
Bioativas (LASSBio), Faculdade de Farmacia, Universidade Federal do Rio de Janeiro,
RJ 21941-902, Brazil. Tel./fax: þ55 21 25626644.
E-mail address: ejbarreiro@ccsdecania.ufrj.br (E.J. Barreiro).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.044

A.E. Ku¨mmerle et al. / European Journal of Medicinal Chemistry 44 (2009) 4004–4009
4005
derivatives LASSBio-785 (5), LASSBio-1003 (9a), LASSBio-1004 (9b)
and LASSBio-1456 (9c) in yields ranging from 88% to 95% after
recrystallization.
The careful analysis of 1H NMR spectra of the new acylhy-
drazone derivatives demonstrated the selective N-alkylation by the
presence of a single N-methyl hydrogen signal ranging from
3.6 ppm and the presence of only one imino hydrogen signal
ranging from 7.7 to 7.9 ppm (N-methyl-NAH derivatives) and 8.3
d 3.4 to
d
d
to 8.6 ppm (NAH derivatives), attributed to the (E)-diastereomer on
the basis of several previous reports from our group describing the
configuration of bioactive NAH derivatives [7,8,13–17], as well as an
only amide hydrogen signal for the NAH derivatives ranging from
d
11.6 to 11.7 ppm.
Vasodilatory activity of these new derivatives was investigated
in aortic rings pre-contracted with 10 mM phenylephrine [13]. Fig. 1
shows concentration–response curves for NAH derivatives in aorta
with intact endothelium. As it has been shown in LASSBio-294 (4)
and LASSBio-785 (5) [13], the N-alkylation of N-acylhydrazone
compounds, generating the corresponding N-methyl-NAH deriva-
tives, led to intense and concentration-dependent aorta relaxation
Chart 1. Structural planning of LASSBio-294.
introduction of small alkyl groups linked to the amide nitrogen of
the NAH moiety of LASSBio-294 (4), especially the methyl group,
led to the discovery of derivative LASSBio-785 (5), which presented
a significant improvement in vasodilator properties (IC50 was
(Fig. 1B). At 20 mM, LASSBio-1004 (9b) completely relaxed the
phenylephrine-induced contraction, while its non-alkylated
analogue (LASSBio-123) (6b) produced a relaxation of 70.7 ꢀ 2.7%
only at 500 mM. Among the N-methyl-NAH derivatives tested,
10.2 ꢀ 0.5
mM), being seven times more potent than LASSBio-294
(4) in the same bioassay [13].
LASSBio-1004 (9b) and LASSBio-1456 (9c) were more potent than
LASSBio-1003 (9a) in reducing the phenylephrine-induced
Herein we explore the synthesis of new arylidene N-acylhydra-
zones (6a–c) and their corresponding N-methyl derivatives (9a–c)
planned by classical bioisosteric replacement of the thienyl ring
from LASSBio-294 (4) and LASSBio-785 (5) for the phenyl, furanyl
and pyrrolyl rings, aiming at determining the structure-associated
events of the methyl group to the vasodilator profile of these NAH
compounds.
contracture with IC50 of 3.5 ꢀ1.1, 3.1 ꢀ1.1 and 40.8 ꢀ 16.2
mM,
respectively (Table 1). Our results indicated that bioisosteric
replacements of the aromatic rings bridged to the imine group (i.e.,
thienyl, pyrrolyl and phenyl) did not drastically change the vaso-
dilator activity, indicating that the interaction of this bioisosteres
with a putative receptor would occur mainly through hydrophobic
interactions. On the other hand, the furanyl bioisoster (9a) was the
only N-methyl-NAH derivative to present some reduction in
activity, may be due to the remarkable electronegative properties of
its oxygen atom.
2. Results and discussion
The compounds were also evaluated in aortic rings without
a functional endothelium. Removal of the endothelium did not
reduce the maximum relaxation and only slightly reduced the
vasodilatory potency promoted by N-methyl-NAH derivatives (9a–
c), but abolished the vasodilatory effect of NAH derivative LASSBio-
The synthesis of the new N-acylhydrazone derivatives (6a–c)
was carried out as outlined in Scheme 1. The starting material was
3,4-methylenedioxybenzoylhydrazine (8) that can be obtained
from natural safrole (3) in 57% overall yield as described previously
[7]. The acid-catalyzed condensation of the hydrazide (8) with the
corresponding aromatic or heteroaromatic aldehydes resulted in
LASSBio-294 (4) and its desired isosteric N-acylhydrazone deriva-
tives LASSBio-129 (6a), LASSBio-123 (6b) and LASSBio-1028 (6c) in
very good yields (Scheme 1). Next, N-methyl-NAH series was
synthesized using the corresponding N-acylhydrazones as precur-
sors after treatment with potassium carbonate in acetone followed
by the addition of methyl iodide at 50 ꢁC. Through this procedure
we were able to obtain, regioselectively, the N-methylated
123 (6b) until the concentration of 500 mM was reached, as has
been seen for LASSBio-294 (4) (Table 1), indicating a possible
distinct mechanism of action between these two NAH series or an
additional vasodilator mechanism for the N-methyl-NAH related to
the NAH series.
Despite the original structural planning based on the molecular
simplification of PDE4 inhibitors, the compounds did not present
any significant in vitro activity in this enzyme, as well as in the PDE5
Scheme 1. Synthetic route exploited in the preparation of the target N-acylhydrazones (8) and their corresponding N-alkylated analogues (9).

4006
A.E. Ku¨mmerle et al. / European Journal of Medicinal Chemistry 44 (2009) 4004–4009
Fig. 1. Effects of NAH derivatives (6a–c and 9a–c) on phenylephrine (Phe)-induced contracture in rat aortic rings. (A) Concentration–response curves for N-methyl-NAH derivatives
(6a–c and 9a–c) in aorta with intact endothelium. (B) Concentration–response curves for NAH derivatives (6a–c and 9a–c) in aorta with intact endothelium. Data represent
mean ꢀ SEM of 8 experiments.
and PDE2A subtypes, showing that this series has a different
mechanism of vasodilatation [18].
To best understand the influence of the methyl group for the
bioprofile of these NAH derivatives, we performed a structural
study by using X-ray diffraction and molecular modeling as tools to
investigate possible conformation-associated events.
The X-ray analysis of LASSBio-294 (4), LASSBio-785 (5), LASSBio-
123 (6b) and LASSBio-1004 (9b) monocrystals obtained from an
ethanol saturated solution showed the exclusive formation of (E)-
diastereomer and the regioselective N-methylation of the NAH
derivatives as previously attributed in the 1H NMR spectra (Fig. 2).
The most stable structural conformers of the NAH and N-
methyl-NAH series in the solid state differ between themselves
[19]. The first one presented a preferential flat conformation (I)
(amide hydrogen antiperiplanar to the carbonyl oxygen) while the
second one presented a tendency to turn the amide bond by 180ꢁ
(II) (amide hydrogen synperiplanar to the carbonyl oxygen) form-
ing a folded structure (Fig. 2) in a similar manner as described in
some aromatic methyl amides [20,21].
in the vasodilatory activity of these compounds by inducing
conformational changes supported by crystallography, molecular
modeling and UV studies. The structural modifications introduced
by the methyl group are certainly required to the vasodilator
effectiveness and potency increase observed for the N-methyl-NAH
series, as well as to promote changes in its molecular mechanism of
action. This investigation enabled the discovery of two new bio-
isosteric N-methyl-NAH prototypes with potent vasodilator prop-
erties, i.e., LASSBio-1004 (9b) and LASSBio-1456 (9c), which were
elected for further pharmacological evaluation on in vivo hyper-
tension models in order to confirm their potential as antihyper-
tensive drug candidates, as will be disclosed in a forthcoming paper.
4. Experimental
4.1. Synthesis
4.1.1. General information
Melting points were determined with a Quimis 340 apparatus
and are uncorrected. 1H NMR spectra were determined in deuter-
ated chloroform or dimethyl sulfoxide containing ca. 1% tetrame-
thylsilane as an internal standard, using a Bruker DPX-200 at
200 MHz. 13C NMR spectra were determined in the same spec-
trometer at 50 MHz, employing the same solvents. Microanalyses
were carried out using a Perkin Elmer 240 analyzer and Perkin
Elmer AD-4 balance. The progress of all reactions was monitored by
The synperiplanar conformation (II) adopted after the amide
bond 180ꢁ rotation is probably due to a steric hindrance introduced
by the methyl group in relation to the hydrogen present in position
2 of the 1,3-benzodioxolyl ring (Fig. 2). The preferential folded
conformation (II) could be explained by a high energy barrier
(DHf ¼ 17 kcal/mol) [20] calculated for LASSBio-1004 (9b), required
for the interconversion of this form (II) into the flat antiperiplanar
conformer (I) (Fig. 2).
In addition, the assumption of conformational differences when
in solution was also supported by changes in UV spectra for
acetonitrile, between the NAH and N-methyl-NAH series. The most
remarkable difference between the UV spectra of derivatives
belonging to these two series was the antiperiplanar conformation
(I, Fig. 2) of non-alkylated derivatives, e.g. LASSBio-123 (6b), which
Table 1
Effects of NAH derivatives (4), (5), (6a–c) and (9a–c) in phenylephrine-induced
contracture of aorta.
Compounds
IC50 (mM)
With endothelium
Without endothelium
presented absorption at 306 nm (log
3
¼ 4.00), whereas the anti-
N-Acylhydrazones
LASSBio-123 (6b)
LASSBio-129 (6a)
LASSBio-294 (4)
LASSBio-1028 (6c)
periplanar conformation (I, Fig. 2) adopted by the corresponding N-
methyl-NAH derivative LASSBio-1004 (9b) shifted it to a shorter
wavelength (291 nm) and its absorbance was diminished
81.0 ꢀ 27.2
ND (500
ND (500
ND (100
ND (200
m
m
M)
M)
ND (500
74.0 [11]
ND (200
m
M)
m
m
M)
M)
m
M)
(log
3
¼ 3.78) due to a difference in the electronic transition in
aromatic moieties between the series caused by conformational
variations. These results are in agreement with previous confor-
mational studies of secondary and tertiary aromatic amides [20,22].
N-Methyl-N-acylhydrazones
LASSBio-785 (5)
LASSBio-1003 (9a)
LASSBio-1004 (9b)
LASSBio-1456 (9c)
10.2 ꢀ 0.5 [13]
40.8 ꢀ 16.2
3.5 ꢀ 1.1
18.5 ꢀ 3.6
15.7 ꢀ 4.4
11.1 ꢀ 0.9*
17.8 ꢀ 7.2
3.1 ꢀ 1.1
3. Conclusions
ND ¼ not determined, because the maximal concentration tested did not yield 50%
of vasorelaxant activity. Numbers in brackets indicate the maximal concentration
tested.
In this report, we have showed for the first time that the methyl
group, bridged to the amide bond of NAH, plays an important role
*P < 0.05 compared to with endothelium.

A.E. Ku¨mmerle et al. / European Journal of Medicinal Chemistry 44 (2009) 4004–4009
4007
Fig. 2. ORTEP view of representative NAH and N-methyl-NAH derivatives LASSBio-294 (4), LASSBio-785 (5), LASSBio-123 (6b) and LASSBio-1004 (9b) showing the alkylation-
associated conformational changes.
thin layer chromatography, which was performed on 2.0 $ 6.0 cm
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 (254–265 nm) and treated with
iodine vapor. Reagents and solvents were purchased from
commercial suppliers and used as received.
H6, J1–2 ¼ 8.2 Hz and J1–3 ¼ 1.4 Hz), 7.43 (d, 1H, H2, J ¼ 1.4 Hz), 7.04
0
0
(d, 1H, H5, J ¼ 8.2 Hz), 6.91 (d, 1H, H3 , J ¼ 3.2 Hz), 6.63 (dd, 1H, H4 ,
J1–2 ¼ 3.2 Hz and J1–3 ¼1.3 Hz), 6.12 (s, 2H, O–CH2–O). MS m/z: 258
(Mþ, 12%), 165 (20%), 149 (100%), 121 (22%), 93 (5%). Anal. Calcd for
C13H10N2O4: C 60.47, H 3.90, N 10.85; found: C 60.65, H 3.88, N
10.65.
4.1.2. General procedure for the preparation of 3,4-
methylenedioxybenzoyl-acylhydrazones LASSBio-123 (6b), LASSBio-
129 (6a), LASSBio-294 (4), and LASSBio-1028 (6c)
4.1.2.3. (2-Thienylidene)3,4-methylenedioxybenzoylhydrazine (LASS-
Bio-294) (4). Derivative LASSBio-294 (4) was obtained as a white
solid by condensation of 8 with 2-thiophenecarboxyaldehyde in
90% yield, mp 204–205 ꢁC. 1H NMR data are in agreement with
those previously published [13]. MS m/z: 274 (Mþ, 9%), 165 (32%),
149 (100%), 121 (12%), 109 (6%). Anal. Calcd for C13H10N2O3S$H2O: C
53.42, H 4.14, N 9.58; found: C 53.34, H 4.11, N 9.25.
To
a solution of 3,4-methylenedioxybenzoylhydrazine (8)
(0.150 g, 0.83 mmol), prepared as previously described [7], in
absolute EtOH (7 mL) containing two drops of 37% hydrochloric
acid, 0.87 mmol of the corresponding aromatic or heteroaromatic
aldehyde derivative was added. The mixture was stirred at room
temperature for 30 min until extensive precipitation was visual-
ized. Afterwards, the solvent was partially concentrated at reduced
pressure and the resulting mixture was poured into cold water.
After neutralization with 10% aqueous sodium bicarbonate solu-
tion, the precipitate formed was filtered out and dried under
vacuum producing the desired acylhydrazone derivatives LASSBio-
123 (6b), LASSBio-129 (6a), LASSBio-294 (4), and LASSBio-1028
(6c), as described below.
4.1.2.4. (2-Pyrrolidene)3,4-methylenedioxybenzoylhydrazine (LASS-
Bio-1028) (6c). Derivative LASSBio-1028 (6c) was obtained as
a cream solid by condensation of 8 with 2-pyrrolecarboxyaldehyde
in 88% yield, mp 181–182 ꢁC. 1H NMR (200 MHz) DMSO-d6:
d 11.57
(s, 1H, CONH–), 11.42 (s, 1H, NH-pyrrol), 8.20 (s, 1H, ]CH), 7.52 (d,
1H, H6, J ¼ 8.2 Hz), 7.40 (s, 1H, H2), 7.02 (d, 1H, H5, J ¼ 8.2 Hz), 6.92
0
0
0
(d, 1H, H5 , J ¼ 1.3 Hz), 6.48 (d, 1H, H3 , J ¼ 3.2 Hz), 6.18 (dd, 1H, H4 ,
J1–2 ¼ 3.2 Hz and J1–3 ¼1.3 Hz), 6.12 (s, 2H, O–CH2–O). Anal. Calcd
for C13H11N3O3$H2O: C 56.76, H 4.76, N 15.27; found: C 56.86, H
4.75, N 14.47.
4.1.2.1. (Benzylidene)3,4-methylenedioxybenzoylhydrazine (LASSBio-
123) (6b). Derivative LASSBio-123 (6b) was obtained as a white
solid by condensation of 8 with benzaldehyde in 92% yield, mp
180–181 ꢁC. 1H NMR data are in agreement with those previously
published [7]. MS m/z: 268 (Mþ, 5%), 165 (38%), 149 (100%), 121
(17%), 103 (5%). Anal. Calcd for C15H12N2O3$H2O: C 62.93, H 4.93, N
9.79; found: C 62.88, H 4.91, N 9.48.
4.1.3. General procedure for selective N-methylation of compounds
LASSBio-123 (6b), LASSBio-129 (6a), LASSBio-294 (4) and LASSBio-
1028 (6c)
The correspondent N-acylhydrazone (1.0 mmol) and potassium
carbonate (3.0 mmol) were suspended in 5 mL of acetone. The
suspension was thoroughly mixed under vigorous stirring for 5 min
and then methyl iodide (3.0 mmol) was added. The reaction was
heated at 50 ꢁC and maintained under stirring for 24 h. Afterwards,
the reaction was evaporated under reduced pressure, the residual
solid suspended in 2 mL of ethanol and then poured into cold water.
The precipitate formed was filtered out, dried under vacuum and
4.1.2.2. (2-Furanylidene)3,4-methylenedioxybenzoylhydrazine(LASSBio-
129) (6a). Derivative LASSBio-129 (6a) was obtained as a cream
solid by condensation of 8 with 2-furanecarboxyaldehyde in 88%
yield, mp 215–216 ꢁC. 1H NMR (200 MHz)0 DMSO-d6:
d 11.66 (s, 1H,
CONH–), 8.32 (s, 1H, ]CH), 7.84 (d, 1H, H5 , J ¼ 1.3 Hz), 7.50 (dd, 1H,

4008
A.E. Ku¨mmerle et al. / European Journal of Medicinal Chemistry 44 (2009) 4004–4009
washed with petroleum ether producing the desired N-methyl-N-
acylhydrazone derivatives LASSBio-785 (5), LASSBio-1003 (9a),
LASSBio-1004 (9b), and LASSBio-1456 (9c), as described below.
contracted aorta was greater than 80%. Removal of functional
endothelium was confirmed by the lack of relaxation (<10%) in the
presence of acetylcholine. The derivatives were dissolved in
dimethyl sulfoxide (DMSO) as stock solutions of 50 mg/mL. Control
experiments performed in the presence of DMSO alone, at the same
concentrations as those used with the derivatives tested, demon-
strated that the solvent did not affect the contractile response of
isolated aorta. Phenylephrine and acetylcholine were obtained
from Sigma Chemical Co. (St. Louis, MO). All data were expressed as
mean of percentage of maximal tension ꢀ SEM. Differences
between different concentrations were considered statistically
significant when P < 0.05 using paired Student’s test. For compar-
ison between two groups with a non-normal distribution, the
Mann–Whitney test was used.
4.1.3.1. N-Methyl (2-thienylidene)3,4-methylenedioxybenzoylhydrazine
(LASSBio-785) (5). Derivative LASSBio-785 (5) was obtained as
a white solid by N-alkylation of LASSBio-294 (4) with methyl iodide,
in 92% yield, mp 142–143 ꢁC. 1H NMR data are in agreement with
those previously published [13]. MS m/z: 288 (Mþ, 5%),179 (15%),149
(100%), 121 (13%), 109 (4%). Anal. Calcd for C14H12N2O3S: C 58.32, H
4.20, N 9.72; found: C 58.39, H 4.19, N 9.34.
4.1.3.2. N-Methyl (2-furanylidene)3,4-methylenedioxybenzoylhydrazine
(LASSBio-1003) (9a). Derivative LASSBio-1003 (9a) was obtained as
a white cream solid by N-alkylation of LASSBio-129 (6a) with methyl
iodide, in 89% yield, mp 104–105 ꢁC. 1H NMR (200 MHz) CDCl3:
d
7.66
4.3. X-ray crystallography
0
(s, 1H, ]CH), 7.47 (d, 1H, H5 , J ¼ 1.8 Hz), 7.42 (dd, 1H, H6, J1–2 ¼ 8.2 Hz
and J1–3 ¼ 1.4 Hz), 7.34 (d, 1H, H2, J ¼ 1.4 Hz), 6.85 (d, 1H, H5,
Data pertaining to the X-ray crystallographic determination of 4,
5, 6b and 9b have been deposited with the Cambridge Crystallo-
graphic Data Centre 60 H.N. as supplementary publication no.:
LASSBio-123 (6a) CCDC 707595; LASSBio-294 (4) CCDC 707596;
LASSBio-785 (5) CCDC 707597; and LASSBio-1004 (9b) CCDC
707598. This information may be freely obtained by applying to
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44 (0) 1223
336033 or e-mailing to deposit@ccdc.cam.ac.uk).
0
0
J ¼ 8.2 Hz), 6.62 (d, 1H, H3 , J ¼ 3.3 Hz), 6.45 (dd, 1H, H4 , J1–2 ¼ 3.3 Hz
and J1–3 ¼1.8 Hz), 6.02 (s, 2H, O–CH2–O), 3.50 (s, 1H, N–CH3). MS m/z:
272 (Mþ, 9%), 179 (14%), 149 (100%), 121 (15%), 93 (5%).
4.1.3.3. N-Methyl (benzylidene)3,4-methylenedioxybenzoylhydrazine
(LASSBio-1004) (9b). Derivative LASSBio-1004 (9b) was obtained as
a white solid by N-alkylation of LASSBio-123 (6b) with methyl
iodide, in 95% yield, mp 109–110 ꢁC. 1H NMR (200 MHz) CDCl3:
0
d
7.75 (s, 1H, ]CH), 7.55 (d, H2 , J ¼ 4.0 Hz), 7.35–7.27 (m, 5H, H2, H6,
4.4. Molecular modeling
0
0
H3 , H4 ), 6.86 (d, H5, J ¼ 8.2 Hz), 6.03 (s, 2H, O–CH2–O), 3.54 (N–
CH3). MS m/z: 282 (Mþ, 6%), 179 (17%), 149 (100%), 121 (15%), 103
(5%). Anal. Calcd for C16H14N2O3: C 68.07, H 5.00, N 9.92; found: C
67.92, H 4.99, N 9.54.
The sketching, geometry optimization and conformational
search of compounds were performed in BioMedCAche 6.0 soft-
ware [20]. The local energy minimization was obtained with
semiempirical AM1 method followed by application of the same
method for a global minimization using dihedral angle search. The
energy barrier introduced by the methyl group was calculated
using AM1 method with a dihedral angle search in 24 steps of 15ꢁ
on the amide bond.
4.1.3.4. N-Methyl (2-pyrrolidene) 3,4-methylenedioxybenzoylhy-
drazine (LASSBio-1456) (9c). Derivative LASSBio-1456 (9c) was
obtained as a cream solid by N-alkylation of LASSBio-1028 (6c) with
methyl iodide, in 86% yield, mp 106–107 ꢁC. 1H NMR (200 MHz)
CDCl3: d 10.82 (s, 1H, NH-pyrrol), 7.87 (s, 1H, ]CH), 7.23 (dd, 1H, H6,
J1–2 ¼ 8.2 Hz and J1–3 ¼ 1.4 Hz), 7.17 0(d, 1H, H2, J ¼ 1.4 Hz), 6.94 (d,
Acknowledgments
0
1H, H5, J ¼ 8.2 Hz), 6.85 (d, 1H, H5 , J ¼ 1.5 Hz), 6.40 (d, 1H, H3 ,
0
J ¼ 3.2 Hz), 6.11 (dd, 1H, H4 , J1–2 ¼ 3.2 Hz and J1–3 ¼1.5 Hz), 6.07 (s,
Special thanks are due to CAPES (BR.), CNPq (BR.), PRONEX (BR.)
and FAPERJ (BR.) for financial support and fellowships.
2H, O–CH2–O), 3.41 (s,1H, N–CH3). MS m/z: 271 (Mþ, 8%),179 (15%),
149 (100%), 121 (14%), 92 (9%). Anal. Calcd for C14H13N3O2: C 61.99,
H 4.83, N 15.49; found: C 61.51, H 4.82, N 14.97.
Appendix. Supporting information
4.2. Pharmacology
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2009.04.044.
The Animal Care and Use Committee at the Universidade Federal
do Rio de Janeiro approved the protocol described as follows.
Thoracic aorta was dissected from male Wistar rats (240–280 g)
and prepared for isometric tension recording. Aorta was cut in rings
of 2–3 mm and placed in a vertical chamber filled with Tyrode’s
solution composed of (in mM): NaCl, 120; KCl, 5.9; MgCl2, 1.2;
NaH2PO4, 0.9; NaHCO3, 24; CaCl2, 2.5; glucose, 11 (pH 7.4) and
oxygenated with carbogen gas (95% O2/5% CO2) at 37 ꢀ 0.5 ꢁC. Each
aorta ring was mounted between two hooks and attached to a force
transducer (Grass, mod. FT-03). The transducer signal was condi-
tioned by a Cyberamp (Axon Instruments, Inc.) and then displayed
and stored in a computer for future analysis using Axoscope soft-
ware (Axon Instruments, Inc.). Preparations were stabilized under
1 g resting tension for 2 h and then the contractile response to
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