Novel furfurylidene N-acylhydrazones derived from natural safrole: Discovery of LASSBio-1215, a new potent antiplatelet prototype

Article abstract of DOI:10.3109/14756366.2011.578575

We describe herein the discovery of (E)-N-methyl-N'-((5-nitrofuran-2-yl) methylene)benzo[d]^1,3 dioxole-5-carbohydrazide (9e), named LASSBio-1215, as a novel antiplatelet agent belonging to the N-methyl-N-acylhydrazone class, which exert their antiaggregating actions on human and rabbit platelets induced by different agonists, through cyclooxygenase-1 (COX-1) or thromboxane synthase inhibition. This compound was elected after screening of a series of functionalized furyl N-acylhydrazone derivatives, synthesized from natural safrole 10. In vitro assays showed that compound 9e presents platelet-aggregating activity in rabbit platelet-rich plasma (PRP) induced by arachidonic acid (IC₅₀=0.7 M) and collagen (IC₅₀=4.5 M). Moreover, LASSBio-1215 also inhibited almost completely the second wave of adenosine diphosphate-induced platelet aggregation in human PRP, and this effect was correlated with their ability to block the production of pro-aggregating autacoid thromboxane A2.

Full text of DOI:10.3109/14756366.2011.578575

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(1): 101–109
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.578575
ReseaRch aRtIcle
Novel furfurylidene N-acylhydrazones derived from natural
safrole: discovery of LASSBio-1215, a new potent antiplatelet
prototype
Ana Paula C. Rodrigues1,2, Luciana M.M. Costa1,3, Bruna L.R. Santos1,4, Rodolfo C. Maia1,2,
Ana L.P. Miranda1,3,4, Eliezer J. Barreiro1,2,4, and Carlos A.M. Fraga1,2,4
1Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculdade de Farmácia, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, 2Programa de Pós-Graduação em Química, Instituto de Química,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, 3Programa de Pós-Graduação em Ciências
Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, and
4Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
abstract
We describe herein the discovery of (E)-N-methyl-N’-((5-nitrofuran-2-yl)methylene)benzo[d][1,3]dioxole-5-
carbohydrazide (9e), named LASSBio-1215, as a novel antiplatelet agent belonging to the N-methyl-N-acylhydrazone
class, which exert their antiaggregating actions on human and rabbit platelets induced by different agonists, through
cyclooxygenase-1 (COX-1) or thromboxane synthase inhibition. This compound was elected after screening of a
series of functionalized furyl N-acylhydrazone derivatives, synthesized from natural safrole 10. In vitro assays showed
that compound 9e presents platelet-aggregating activity in rabbit platelet-rich plasma (PRP) induced by arachidonic
acid (IC50 =0.7 µM) and collagen (IC50 =4.5 µM). Moreover, LASSBio-1215 also inhibited almost completely the second
wave of adenosine diphosphate-induced platelet aggregation in human PRP, and this effect was correlated with their
ability to block the production of pro-aggregating autacoid thromboxane A2.
Keywords: N-acylhydrazone, 1;3-benzodioxole, N-methyl-N-acylhydrazone, antiplatelet drugs, COX inhibitors
Introduction
clopidogrel 2; (iii) glycoprotein IIB/IIIA receptor antago-
Platelet activation and aggregation are crucial events to
the pathophysiology of several acute and chronic arte-
rial vascular diseases that are responsible for important
impact on morbidity, mortality, and health care costs
worldwide1,2. Despite the worrying status of these patho-
logical states associated with thrombus formation, the
currently available therapies are limited to few antiplate-
let drugs3 belonging to one of these four main classes:
(i) cyclooxygenase-1 (COX-1) inhibitors, for example,
aspirin 1; (ii) adenosine diphosphate (ADP) receptor
antagonists, represented by thienopyridine derivatives as
nists, for example, tirofiban 3; (iv) selective phospho-
diesterase 3 inhibitors, for example cilostazol 4 (Figure
1). All of them presented therapeutic limitations, for
example reduced oral effectiveness4, characteristic side
effects such as bleeding or are able to induce resistance
after long-term treatment5.
In this context, we have described previously the
platelet antiaggregating profile of a series of 2-pyridynyl-
hydrazones 56 and N-acylhydrazone (NAH) derivatives
from phenothiazine 67 and 1,3-benzodioxolyl series,
for example LASSBio-294 7 and LASSBio-785 88, which
Address for Correspondence: Prof. Carlos Alberto Manssour Fraga, Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio),
Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, P.O. Box 68.023, 21941-902, Rio de Janeiro, RJ, Brazil. Tel: +55-21-25626503.
Fax: +55-21-2562–6503. E-mail: cmfraga@ccsdecania.ufrj.br
(Received 29 October 2010; revised 28 March 2011; accepted 04 April 2011)
101

102 A.P.C. Rodrigues et al.
have also potent inotropic9 and vasodilator properties10,11
(Figure 2).
e NAH subunit has been described as the phar-
macophoric framework of several bioactive substances
from different therapeutic classes12,13, indicating the
privileged status of this moiety, as has been recently
reviewed14.
of new platelet antiaggregating lead-compound can-
didates, we developed new functionalized furyl 1,3-
benzodioxolyl-N-acylhydrazone derivatives 9 (Figure 2),
designed as isosteres of the antiplatelet thienyl-NAH
derivatives 7 and 8.
ese target compounds 9a–j were planned by inser-
tion of the furane ring present in antiplatelet derivatives
5 and 6 at the imine subunit of 1,3-benzodioxolyl-NAH
derived from Brazilian natural product safrole 1015. In
So, in the scope of a research program aiming to design,
synthesize, and perform the pharmacological evaluation
Figure 1. Currently available platelet antiaggregating drugs.
Figure 2. Design concept of new functionalized furylydene 1,3-benzodioxolyl-N-acylhydrazine derivatives 9a–f and the vinylogous
analogues 9g–j.
Journal of Enzyme Inhibition and Medicinal Chemistry

Discovery of LASSBio-1215 103
order to define the structure–activity relationships based
on steric, electronic, and lipophilic effects of substituents
at C5-position of the furane subunit, we proposed the
substitution of the hydrogen by bromine and nitro groups
(Figure 2). In addition, considering the well-known
conformational effects promoted by methylation of the
amide group at NAH moiety11, we decided to prepare the
corresponding N-methyl-NAH derivatives 9d–fto explore
the effect of structural change on the platelet antiaggre-
gating profile. Finally, the influence of the length of the
spacer between the heteroaromatic 1,3-benzodioxole
and furane rings on the molecular recognition by target
bioreceptor was investigated through the planning of
the vinylogous analogues 9g–h and their correspond-
ing N-methyl derivatives 9i–j. Previous results showed
that NAH derivatives displayed their platelet aggregating
actions mainly through the blockage of thromboxane A2
(TXA2) production8. ese actions at arachidonic acid
(AA) metabolism level seem to be dependent on the rela-
tionship between NAH framework and the bis-allyl unit
of this unsaturated fatty acid7.
and the precipitate formed was filtered out affording the
title compound in 80% yield, as a white solid, m.p. 53°C
[Lit. (16) 53°C]. See analytical data in supplementary
material.
3,4-Methylenedioxybenzoylhydrazine 13
To a solution of the ester 12 (6.42 g, 35.6 mmol) in
25 ml of ethanol, was added 32.5 ml of 80% hydrazine
monohydrate. e reaction mixture was maintained
under reflux for 3.5 h, when TLC indicated the end of
the reaction. en the media was poured on ice and
the resulting precipitate was filtered out affording the
title compound in 73% yield, as a white solid, m.p.
170–171°C [Lit. (16) 170–171°C]. See analytical data in
supplementary material.
General procedure for preparation of 3,4-
methylenedioxybenzoylhydrazones 9a–c and 9g–h
To a solution of 0.150g (0.83 mmol) of the hydrazine 13
in absolute ethanol (20 ml) containing three drops of 37%
hydrochloric acid, was added 0.87 mmol of the corre-
sponding functionalized furfural. e mixture was stirred
at room temperature for 30 min, until extensive precipi-
tation was visualized. Next, the solvent was partially con-
centrated at reduced pressure and the resulting mixture
was poured into cold water. e precipitate formed was
filtered out and dried under reduced pressure to give the
desired NAH derivatives, as described next.
Methods
Chemistry
Melting points were determined using a Quimis instru-
ment and are uncorrected. 1H-NMR spectra were
determined otherwise in dimethylsulfoxide (DMSO)-d6
containing ~1% tetramethylsilane as an internal stan-
dard, with Bruker DPX-200 or Bruker DRX-300 spectrom-
eters at 200 or 300 MHz, respectively. 13C-NMR spectra
were determined in the same spectrometers described
above at 50 or 75 MHz, employing the same solvents.
Infrared spectra were obtained using a Nicolet Magna IR
760 spectrometer. Samples were examined as potassium
bromide (KBr) disks. Microanalysis was obtained with
ermofinnigan EA1112 analyzer, using a Metler MX5
electronic balance. Reactions were routinely monitored
by thin-layer chromatography (TLC) in silica gel (F245
Merck plates), and the products were visualized using
iodine or ultraviolet lamp (254 and 365 nm). Column
chromatography purifications were performed using
silica gel Merck 230–400 mesh. Unless stated otherwise,
starting materials used were high-grade commercial
products. All organic solutions were dried over anhy-
drous sodium sulphate, filtered, and concentrated, under
reduced pressure, in rotary evaporators.
2-Furfurylidene-3,4-methylenedioxybenzoylhydrazine 9a
is compound was obtained as a beige solid by conden-
sation of 13 with furfural in 94% yield, m.p. 215–216°C
[Lit. (11) 215–216°C]. See analytical data in supplemen-
tary material.
5-Nitro-2-furfurylidene-3,4-
methylenedioxybenzoylhydrazine 9b
is compound was obtained as a yellow solid by con-
densation of 13 with 5-nitro-furfural in 90% yield, m.p.
233–234°C. 1H-NMR (DMSO-d6; 300 MHz) δ 12.07 (s,
CO-NH), 8.37 (s, N=CH), 7.77(d, Furan-H3’, J= 3.5 Hz),
7.54 (d, Ar-H6, J= 8.0 Hz), 7.44 (s, Ar-H2), 7.23 (d, Ar-H4,
J= 3.5 Hz), 7.05 (d, Ar-H5, J= 8.0 Hz), 6.13 (s, O-CH2-O);
13C-NMR (DMSO-d6; 75 MHz) δ 162.9 (C=O), 152.3
(Furan-C5’), 151.0 (Ar-C4), 147.9 (Ar-C3), 135.5 (N=CH),
126.9 (Ar-C1), 123.7 (Ar-C6), 115.6 (Furan-C4’), 115.1
(Furan-C3’), 108.6 (Ar-C5), 108.2 (Ar-C2), 102.4 (O-CH2O);
IR (KBr, cm−1) 3291 (N-H), 1661 (C=O), 1607 (C=N),
1513.2–1472.0 (NO2), 1256 (C-O). Elemental Analysis
(CHN) Calculated: C, 51.49; H, 2.99; N, 13.86. Found: C,
51.42; H, 2.93; N, 13.75.
Methyl 3,4-methylenedioxybenzoate 12
To a solution of piperonal 11 (5.0 g, 33.3 mmol) in abso-
lute methanol (190 ml) cooled at 0°C, were successively
added solutions of KOH (5.6 g, 99.9 mmol) in absolute
methanol (190 ml) and iodine (10.98 g, 43.2 mmol) in
absolute methanol (380 ml) 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 colour.
Next, the methanol was almost totally evaporated under
reduced pressure. To the residue was added cold water,
5-Bromo-2-furfurylidene-3,4-
methylenedioxybenzoylhydrazine 9c
is compound was obtained as a beige solid by con-
densation of 13 with 5-bromo-furfural in 92% yield,
m.p. 155–156°C. See analytical data in supplementary
material.
© 2012 Informa UK, Ltd.

104 A.P.C. Rodrigues et al.
2-Furylpropenylidene-3,4-methylenedioxybenzoylhydrazine
9g
(2-Furylpropenylidene)-N-methyl-3,4-
methylenedioxybenzoylhydrazine 9i
is compound was obtained as a beige solid by con-
densation of 13 with 3-(2-furyl)acrolein in 51% yield,
m.p. 222–223°C. See analytical data in supplementary
material.
is compound was obtained as a beige solid by the
N-methylation of 9g in 92% yield, m.p. 109–110°C. See
analytical data in supplementary material.
N-Methyl-(5-nitro-2-furylpropenylidene)-3,4-
5-Nitro-2-furylpropenylidene-3,4-methylenedioxybenzoyl-
hydrazine 9h
is compound was obtained as a brown solid by conden-
sation of 13 with 5-nitro-3-(2-furyl)acrolein in 50% yield,
m.p. 241–242°C. See analytical data in supplementary
material.
methylenedioxybenzoylhydrazine 9j
is compound was obtained as a beige solid by the
N-methylation of 9h in 79% yield, m.p. 241–242°C. See
analytical data in supplementary material.
Pharmacology
Collagen, sodium arachidonate, 9,11-dideoxy-11α,9α-
epoxy methanoprostaglandin F2α (U-46619), platelet-
activating factor (PAF), ADP, DMSO, acetylsalicylic acid
(ASA) were purchased from Sigma Chem. Co. (USA).
Indomethacin was purchased from Merck (USA).
romboxane B2 enzyme immuno-assay kit was pur-
chased from Amersham Bioscience.
General procedure for selective N-methylation of 3,4-
methylenedioxybenzoylhydrazones 9a–c and 9g–h
To a solution of 0.75 mmol of corresponding acylhydra-
zone derivative and 0.31 g (2.25 mmol) of K2CO3 in 4 ml
acetone, was added 1.5 mmol of CH3I. e reaction mix-
ture was maintained under reflux for 3 h, when TLC indi-
cated the end of the reaction. To the reaction mixture was
added 5 ml of acetone and the mixture was filtered out.
e residual solid was washed with 20 ml of acetone and
the suspension was filtered. en, the organic layer was
concentrated under reduced pressure to give the desired
N-methyl-N-acylhydrazone derivatives 9d–f and 9i–h, as
described next.
Animals
New Zealand white rabbits weighing 2.5–3.0 kg of both
sexes were obtained from the LASSBio breeding unit
(Faculty of Pharmacy, UFRJ, Brazil). All animals were
kept under standardized conditions, maintained in a
12-h light/dark cycle with water and food ad libitum
until use. Animal experiments were performed accord-
ing to the ‘Principles of Laboratory Animal Care and
Use in Research’ (Colégio Brasileiro de Experimentação
Animal-COBEA/Instituto Brasileiro Carlos Chagas Filho-
IBCCF, Brazil), based on international guidelines for the
care and use of laboratory animals.
(2-Furfurylidene)-N-methyl-3,4-
methylenedioxybenzoylhydrazine 9d
is compound was obtained as a beige solid by the
N-methylation of 9a in 91% yield, m.p. 115–116°C [Lit.
(11) 104–105°C]. See analytical data in supplementary
material.
Human volunteers
N-Methyl-(5-nitro-2-furfurylidene)-3,4-
methylenedioxybenzoylhydrazine (LASSBio-1215) 9e
is compound was obtained as a yellow solid by the
Blood samples were obtained from healthy volunteers
who had no apparent thromboembolic or hemorrhagic
diseases, renal, hepatic, or malignant conditions, and
had not taken any medication for at least 2 weeks prior
to blood collection, following their informed consent and
approval by the local ethics committee.
1
N-methylation of 9b in 67% yield, m.p. 175–176°C. H-
NMR (DMSO-d6; 300 MHz) δ 7.98 (s, N=CH), 7.73 (s,
Furan-H4’), 7.31–7.24 (m, Ar-H2 and Ar-H6), 6.99–6.92
(m, Furan-H3’ and Ar-H5), 6.10 (s, O-CH2-O), 3.32
(N-CH3); 13C-NMR (DMSO-d6; 75 MHz) δ 168.6 (C=O),
152.6 (Ar-C4), 149.2 (Furan-C5’), 147.8 (Ar-C3), 146.4
(Furan-C2’), 128.5 (N=CH), 127.5 (Furan-C3’), 125.4
(Furan-C4’), 114.7 (Ar-C1), 113.6 (Ar-C6), 110.3 (Ar-C2),
107.2 (Ar-C5), 101.5 (O-CH2O); 29.5 (N-CH3); IR (KBr,
cm−1) 3451 (N-H), 1670 (C=O), 1604 (C=N), 1521.7–
1457.2 (NO2), 1259 (C-O). Elemental Analysis (CHN)
Calculated: C, 53.00; H, 3.49; N, 13.24. Found: C, 53.07;
H, 3.55; N, 13.12.
Rabbit and human platelet-rich plasma preparation
e rabbit and human blood were obtained by punc-
ture of the central ear artery and by vein puncture of the
median cubital vein, respectively, into 3.8% trisodium
citrate (9:1 v/v) solution. Platelet-rich plasma (PRP) was
prepared by centrifugation at 500×g for 10 min at room
temperature. e platelet pour plasma (PPP) was pre-
pared by centrifugation of the pellet at 1800×g for 10 min
at room temperature. Platelet count was adjusted to
6 × 108 platelets/ml and 3 × 108 platelets/ml for rabbit and
human PRP, respectively.
(5-Bromo-2-furfurylidene)-N-methyl-3,4-
methylenedioxybenzoylhydrazine 9f
is compound was obtained as a beige solid by the
N-methylation of 9c in 83% yield, m.p. 135–137°C. See
analytical data in supplementary material.
Platelet aggregation studies
Platelet aggregation was conducted by the turbidimet-
ric method of Born and Cross17 and monitored using a
Journal of Enzyme Inhibition and Medicinal Chemistry

Discovery of LASSBio-1215 105
platelet lumi-aggregometer (Model 560CA, Chrono-log
Corporation, Harvertown, PA, USA). PRP (400 μl) was
incubated at 37°C for 1 min with continuous stirring at
1200 rpm. Aggregation of PRP was induced by ADP (5
μM), collagen (5 μg/ml), AA (200 μM), U-46619 (3 μM),
and PAF (1 μM).
Test compounds and the vehicle (0.5% DMSO) were
added to the PRP samples 5 min before the addition of
the aggregating agent. DMSO used as vehicle did not
have either pro- or antiplatelet aggregation activity. On
the one hand, the platelet antiaggregating activity was
expressed as % of inhibition of aggregation induced by
ADP, AA, U-46619, and PAF. On the other hand, for col-
lagen-induced aggregation, it was expressed as % inhibi-
tion of maximum rate of aggregation (slope).
to evidence that they were obtained as a single geometri-
cal isomer at imine double bound level, which were char-
acterized as presenting relative (E)-configuration taking
on basis the previous crystallographic studies performed
with thienyl-NAH derivatives 7 and 811. e diastereose-
lective profile of the imine bond formation was also rep-
resentatively confirmed in acylhydrazone derivative 9a
by using mass spectrometry as auxiliary tool20, through
the appearance of the fragment (m/z= 165, relative
abundance 21%) of McLafferty rearrangement, which is
formed due to the favourable orientation between the
carbonyl oxygen atom and the γ-hydrogen in the (E)-
diastereomer (Figure 3).
Moreover, the vinylogous derivatives 9g–j presented
the same relative configuration (E) derived from α,β-
unsaturated 2-furaldehydes 14d and 14e used at the
condensation step with acylhydrazine 13. e 1H-
NMR spectra of these 2-furylpropenylidene NAHs 9g–j
showed a typical AB pattern (J= 15.9 Hz) resulting from
vicinal coupling between the vinylic hydrogens with the
expected (E)-configuration.
Finally, the N-methyl-NAH derivatives 9d–f and 9i–j
were obtained from selective alkylation of the corre-
sponding N-acylhydrazones 9a–c and 9g–h after initial
treatment with potassium carbonate in acetone followed
by addition of methyl iodide (Scheme 1).
e structure of all furyl N-acylhydrazone or
N-methyl-NAH derivatives described herein was com-
pletely characterized by usual spectroscopic methods
(see experimental section) before the investigation of
their antiplatelet properties.
TXB2 measurements
TXB2 levels were measured in human PRP samples
submitted to aggregation induced by AA (500 µM) as
described above. e TXB2 formation reaction was
stopped by indomethacin 10 µM and ethylenediami-
notetraacetic acid (EDTA) 2 mM 5 min after the AA addi-
tion. Plasma samples were then centrifuged at 3600×g for
5 min. Supernatant samples were collected and stored in
Eppendorf tubes at −20°C. TXB2 production was deter-
mined using an enzyme immuno-assay kit according to
the manufacturer’s instructions18,19
.
Statistical analysis
Results were expressed as mean SEM of independent
experiments carried out in duplicate and compared with
vehicle control groups. Data were statistically analyzed by
the Student’s t-test or analysis of variance (ANOVA) one-
way (Dunnet posttest) for a significance level of P< 0.05.
When appropriate, the IC50 values (i.e., the concentration
able to inhibit 50% of the maximum effect observed) were
determined by nonlinear regression using GraphPad
Prism software v. 4.0.
Pharmacological assays
e in vitro activity of the functionalized furfurylidene
NAH and N-methyl-NAH derivatives (9) was evalu-
ated on rabbit platelet aggregation model with AA (200
µM), collagen (5 µg/ml), and adenosine 5-diphosphate
(ADP, 5 µM) as the inducers (Table 1)17. As a general
trend, NAH derivatives 9a–j showed to be more active
as inhibitors of AA-induced platelet aggregation, when
compared with the two other exploited agonists. e
best antiplatelet profile was evidenced for N-methyl
nitrofuryl NAH derivative 9e, which presented an IC50
of 0.7 and 4.5 µM in AA- and collagen-induced platelet
aggregation, respectively.
Moreover, all unsubstituted NAH derivatives 9a–c,
9g–h were less active as platelet antiaggregating agents
than the corresponding N-methyl NAH derivatives 9d–f,
9i–j, possibly indicating that alkylation could change the
conformational behaviour of these compounds, as pre-
viously described for LASSBio-294 7 and LASSBio-785
811. On the contrary, homologation of NAH framework
with a vinyl group does not improve or reduce the anti-
platelet activity displayed by the corresponding NAH or
N-methyl-NAH derivative, as could be evidenced through
the comparison of the IC50 value of compounds 9e vs. 9j,
that is 0.7 and 23.2 µM in AA-induced assay, respectively
(Table 1).
Results and discussion
Chemistry
e novel series of 1,3-benzodioxole N-acylhydrazone
derivatives 9 were prepared by applying classical syn-
thetic methodologies exploiting piperonal 11, obtained
from isomerization and oxidative cleavage of safrole
1015, as starting material (Scheme 1). e desired key-
intermediate (acylhydrazine, 13) was prepared from
aromatic aldehyde 11 in 75% yield (two steps), exploit-
ing the initial oxidation to the corresponding methyl
ester 12 followed by acylation of hydrazine, as described
previously11,16. e target NAH derivatives 9a–c and 9g–h
were prepared through the acid catalyzed condensation
of13withthecorrespondingfunctionalizedfuraldehydes
14a–e11,16, as depicted in Scheme 1 (see Supplementary
Table S1).
e careful analysis of the imine hydrogen shifts on
1H-NMR spectra of all these NAH derivatives allowed us
© 2012 Informa UK, Ltd.

106 A.P.C. Rodrigues et al.
Scheme 1. Reaction conditions: a) I2, KOH, MeOH, rt, 6h, 90%; b) NH2NH2.H2O 80%, EtOH, reflux, 3.5h, 84%; c) Functionalized 2-furyl-
(CH=CH)n-CHO (14a–e), EtOH, rt, 30min, see Table 1; d) K2CO3, acetone, MeI, 40°C, 24h, see supplementary Table S1.
Figure 3. McLafferty rearrangement peak in the mass spectra of (E)-(2-furfurylidene) 3,4-methylenedioxybenzoylhydrazine derivative 9a.
Journal of Enzyme Inhibition and Medicinal Chemistry

Discovery of LASSBio-1215 107
Since the distinct biological profile of derivatives 9b,
9e, and their corresponding vinylogous analogues 9h
and 9j could be associated with their different confor-
mations, we decided to investigate their comparative
conformational behaviour through molecular model-
ling studies.
In this context, their structures were built using
SPARTAN 8.0 software21 and full geometrically opti-
mized using the standard molecular mechanics force
field followed by systematic conformational analysis
performed using Hartree-Fock 3-21G(*) method22.
Supplementary Figure S4 shows that minimum energy
conformer of NAH derivatives 9b and 9h presented
antiperiplanar arrangement between carbonyl and
N-H bond of amide moiety, whereas in N-methyl-NAH
derivatives 9e and 9j the geometry adopted is mainly
synperiplanar between carbonyl and N-CH3 groups
(11), changing the general conformation from a linear
to a folded conformation, which contributes to improve
the antiplatelet potency.
e comparison of the lowest energy conformations
of 5-nitrofuryl compounds 9e and 9j with respect to the
influence of the spacer unit between the NAH moiety
and the heteroaromatic unit corroborates the impor-
tance of maintaining an adequate distance and orienta-
tion between the groups attached to NAH moiety, the
pharmacophore, once the introduction of vinyl group
does not present a favourable influence on the platelet
antiaggregating activity (Figure S4).
It is interesting to observe that the introduction of
hydrophobic bromo substituent at C-5 position of the
furan ring of 9c improves the platelet antiaggregating
profile in unsubstituted NAH series, whereas its effect on
N-methyl NAH series was less pronounced than hydro-
philic and H-bond acceptor nitro group, as could be evi-
denced by the respective antiplatelet potency order, that
is 9c>9a>9b vs. 9e>9d>9f.
Similar structure–activity relationships were found
in platelet aggregation induced by collagen, allowing
us to distinguish two groups of compounds taking into
account the observed antiplatelet potency, that is a group
formed by the N-methyl-NAH derivatives 9d, 9e, 9f, and
9i with IC50 ranging from 4 to 8 µM and a second one
constituted by compounds 9c and 9j, which presented
IC50 of 47.7 and 41.2 µM, respectively. On the contrary,
only 5-nitro-furyl-NAH derivatives 9e and 9j were able
to inhibit moderately the platelet aggregation induced by
ADP in rabbit PRP (25.7% and 22.6%, respectively) at 100
µM concentration (Table 1).
Aiming to better understanding the molecular mecha-
nisms associated with the antiplatelet profile of NAH and
N-methyl-NAH derivatives, the most active compounds
9a, 9c–9f, 9i, and 9j were evaluated on in vitro human
platelet aggregation induced by ADP (3 µM) (Table 2).
In this assay is observed a characteristic second wave of
aggregation, dependent on ADP concentration, which
is associated with TXA2 production, that is almost com-
pletely inhibited by ASA or other COX-1 inhibitors, for
example indomethacin18,23
.
Despite only compounds 9c and 9e have inhibited
significantly in 30% and 38%, respectively, the platelet
aggregation induced by ADP in human PRP, most of the
furyl NAH derivatives were able to block completely the
second wave of this process (Table 2). e exceptions were
derivatives 9a and 9j, which also were not significantly
active against ADP. is profile indicates that platelet
antiaggregating activity exhibited by these compounds is
related to their ability to modulate AA cascade enzymes
responsible for TXA2 biosynthesis, for example COX-1,
isoform predominantly on platelets, or thromboxane
synthase (TXS).
None of the NAH or N-methyl-NAH derivatives 9
was able to inhibit significantly the first wave of platelet
aggregation, suggesting that their antiplatelet actions are
Table 1. Effect of functionalized 2-furyl N-acylhydrazone derivatives 9a–j on platelet aggregation induced by arachidonic acid (AA),
collagen, and ADP in rabbit platelet-rich plasma.
IC50 valuesa (µM) or inhibition (%) at 100 µM
Inhibition (%) at 100 µM
Compoundb
Arachidonic acid (200 µM)
Collagen (5 µg/ml)
3.1c
ADP (5 µM)
—
Indomethacin
0.6c
9a
30.1 (29.8–30.4)
0.0%
156.1 (114.4–212.9)
3.2%
14.1
9b
15.2
9c
7.9 (6.2–10.2)
1.2 (0.4–3.4)
0.7 (0.6–1.1)
2.0 (1.9–2.2)
47.4 (43.8–51.3)
11.4%
47.7 (39.7–57.3)
8.2 (6.0–11.1)
4.5 (3.9–5.1)
6.0 (4.9–7.4)
17.7%
15.0
9d
2.2
9e
25.7*
18.7
9f
9g
15.6
9h
2.1%
18.3
9i
1.7 (0.9–3.1)
23.2 (23.1–23.4)
15.3c
6.1 (4.3–8.5)
41.2 (32.7–51.7)
18.3c
1.4
9j
22.6*
14.7c
LASSBio-294
an=ree independent experiments in duplicate; 95% confidence interval for IC50 values is shown in parentheses. bN-acylhydrazone
derivatives 9a–j were incubated with platelet-rich plasma 5min before the addition of the agonist (AA, collagen, or ADP) . cData obtained
from reference (8). *P<0.05 (ANOVA one-way; Dunnet posttest).
© 2012 Informa UK, Ltd.

108 A.P.C. Rodrigues et al.
Table 2. Effect of ASA and functionalized 2-furyl N-acylhydrazone derivatives 9a, 9c–e, and 9i–j on platelet aggregation induced by ADP
(3 µM) in human platelet-rich plasma.
Inhibition (%)
Compounda
nb
4
Aggregationc (%)
64.0 4.4
69.3 7.8
53.0 5.5
58.0 6.0
48.5 3.0
52.5 6.0
42.5 3.8
55.8 6.6
55.5 3.8
69.0 7.6
—
Total inhibition (%)
First wave
—
Second wave
—
ADP
—
DMSO
4
—
—
—
ASA
3
23.5
16.2
30.0*
24.5
38.6*
19.5
19.9
0.4
−4.0 n.s.
−0.5 n.s.
0.5 n.s.
−2.9 n.s.
9.7 n.s.
−8.8 n.s.
−4.4 n.s.
−7.9 n.s.
—
96.5*
41.7*
88.3*
96.1*
97.4*
94.7*
97.0*
22.4
9a
4
9c
4
9d
4
9e
4
9f
4
9i
4
9j
4
LASSBio-294
—
40.9d
—
aCompounds were assayed at 100 µM concentration. bn=number of independent experiments in duplicate. cResults are expressed as
mean SEM. dData obtained from reference (8). N-acylhydrazone derivatives 9 were incubated with plasma-rich platelet 5min before the
addition of the ADP. *P<0.05 (ANOVA one-way; Dunnet posttest).
the production of TXB2 in human platelets activated
by AA (500 µM). Similarly, nonselective COX inhibi-
tor indomethacin (10 µM), used as standard, inhib-
ited almost completely the TXB2 biosynthesis in this
experiment.
On the contrary, vinylogous N-methyl-NAH derivative
9j does not inhibit significantly the production of TXB2
(Figure 4), indicating that its antiplatelet profile should
be dependent on a different mechanism of action those
of COX-1 or TXS blockage.
Moreover, none of the NAH derivatives 9a–j (100 µM)
was able to interfere with the rabbit PRP aggregation
induced by U-46619 (3 µM) (e.g., vehicle 60.8 1.8 × 9e
52.1 2.8) or PAF (1 µM) (e.g., vehicle 71.2 1.4 × 9e
70.3 1.8), indicating that their antiplatelet effects are not
dependent of TP or PAF receptor antagonism.
Figure 4. Effect of NAH derivatives 9a, 9e, and 9j on the TXB2
production in arachidonic acid (500 µM)-induced platelet
aggregation in human PRP. Results are expressed in terms of
mean SEM (n=3–5 independent experiments). *P<0.05 (ANOVA
one-way; Dunnet posttest); N-acylhydrazone derivatives 9 were
incubated with plasma-rich platelet 5min before the addition
of AA; e vehicle concentration in the samples does not exceed
0.3%; INDO=indomethacin.
conclusions
We described herein the discovery of (E)-N-methyl-
N’-((5-nitrofuran-2-yl)methylene)benzo[d][1,3]diox-
ole-5-carbohydrazide 9e, named LASSBio-1215, as a
novel antiplatelet agent belonging to the N-methyl-N-
acylhydrazone class, which exert their antiaggregating
actions on human and rabbit platelets induced by dif-
ferent agonists, through COX-1 or TXS inhibition. is
compound was elected for further pharmacological
evaluation on in vivo thromboembolic disease models to
confirm its potential as a drug candidate for treatment of
different cardiovascular disorders and these results will
be described in a forthcoming paper.
not related to the blockage of purinergic P2Y1 or P2Y12
receptors (24).
Furthermore, in order to confirm the action of some of
these NAH derivatives on AA metabolism, we performed
studies on the biosynthesis of TXA2 in the activated
human PRP by measuring the production of their stable
metabolite TXB2 (19).
Compounds (9d) and (9e) were selected to this study
duetotheirantiplateletpotencyinbothrabbitandhuman
platelets. Compound (9j) was also elected because of
its ability to completely inhibit the platelet aggregation
induced by AA in rabbit PRP, although it has not inhib-
ited the second wave of platelet aggregation induced by
ADP in human PRP.
Declaration of interest
e authors thank CAPES (BR), CNPq (BR), FAPERJ (BR),
PRONEX (BR), and INCT-INOFAR (BR, #573.564/2008-6)
for financial support and fellowships. e authors report
no declarations of interest.
e results depicted in Figure 4 showed that com-
pounds LASSBio-1003 9d and LASSBio-1215 9e at 100
µM were able to inhibit by 92% and 90%, respectively,
Journal of Enzyme Inhibition and Medicinal Chemistry

Discovery of LASSBio-1215 109
arylidene N-acylhydrazone derivatives. Eur
2009;44:4004–4009.
J Med Chem
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