Synthesis and anti-platelet activity of novel arylsulfonate-acylhydrazone derivatives, designed as antithrombotic candidates

Article abstract of DOI:10.1016/j.ejmech.2007.03.032

In this work, we describe a new class of promising anti-platelet drug candidates with significant antithrombotic activity in vivo. This new series of compounds was structurally planned by modification of known thrombin inhibitors based on the use of acylh

Full text of DOI:10.1016/j.ejmech.2007.03.032

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European Journal of Medicinal Chemistry 43 (2008) 348e356
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-platelet activity of novel arylsulfonateeacylhydrazone
derivatives, designed as antithrombotic candidates
a,
b
a
Lıdia M. Lima , Flavia S. Frattani , Jean L. dos Santos , Helena C. Castro
b,1
*
´
´
,
Carlos Alberto M. Fraga a, Russolina B. Zingali b, Eliezer J. Barreiro
a,**
a Laborato´rio de Avaliac¸~ao e S´ıntese de Substaˆncias Bioativas (LASSBio), Faculdade de Farma´cia,
Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, PO Box 68006, 21944-910, Brazil
b Laborato´rio de Hemostase e Venenos (LabHemoVen), Programa de Biologia Estrutural,
Instituto de Bioqu´ımica Me´dica UFRJ, Rio de Janeiro, RJ, Brazil
Received 25 January 2007; received in revised form 26 March 2007; accepted 28 March 2007
Available online 14 April 2007
Abstract
In this work, we describe a new class of promising anti-platelet drug candidates with significant antithrombotic activity in vivo. This new
series of compounds was structurally planned by modification of known thrombin inhibitors based on the use of acylhydrazone subunit, as a non-
peptide scaffold, and variations at P1 moiety. Three different families of arylsulfonateeacylhydrazone derivatives were designed. The bioassays
indicated the first class of derivatives represented by 4f (LASSBio-693) and 4j (LASSBio-743), which were active in inhibiting the platelet ag-
gregation induced by thrombin. The second class represented by compounds 4e (LASSBio-774) and 4h (LASSBio-480) that selectively inhibit
the platelet aggregation involving TXA2 formation. Finally, the third class of derivatives was identified acting as a novel symbiotic agent able to
inhibit the platelet aggregation induced by collagen or AA and by thrombin, represented by compounds 4b (LASSBio-694) and 4g (LASSBio-
770).
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Anti-platelet activity; N-Acylhydrazone (NAH); Thrombin
1. Introduction
enzymatic activation of coagulation factors, but also by direct
stimulation of several cell types such as platelets [4,5].
Platelets are also involved in the thrombotic pathologies.
They play a major role in vascular occlusive diseases such
as angina, myocardial infarction, and stroke, as a consequence
of their inappropriate and sustained activation, being thus con-
sidered also important targets for antithrombotic drugs [6]. In-
hibitors of thrombin have long been recognized as potential
therapeutic agents for the treatment of a variety of thrombotic
disorders [7]. In fact, intravenous (e.g. argatroban) and oral
(e.g. ximelagatran) chiral thrombin inhibitors have shown
promising results in human clinical trials [7,8]. It was often
observed that thrombin inhibitors with strongly basic P1 moi-
eties have low selectivity against other serine proteases and
poor oral bioavailability. Therefore, derivatives bearing P1 el-
ements with modulated basicity (between pKa 6.5 and 9.0)
may be considered more promising lead-compounds [9].
Thromboembolic disorders are the major cause of morbid-
ity and mortality in Western societies. Clinical regulation of
thrombosis involves the anti-platelet therapy (e.g. acetylsali-
cylic acid), thrombolytic agents (e.g. streptokinase), and drugs
that affect the activity and generation of thrombin (e.g. hepa-
rin, argatroban) [1e3].
Thrombin is a multifunctional serine protease that plays
a central role in thrombosis and hemostasis, not only by
* Corresponding author.
** Corresponding author. Tel./fax: þ55 21 25626644.
E-mail addresses: lidia@pharma.ufrj.br (L.M. Lima), ejbarreiro@
ccsdecania.ufrj.br (E.J. Barreiro).
1
´
´
´
Present address. Laboratorio de Antibioticos, Bioquımica e Modelagem
Molecular (LABioMol), Instituto de Biologia, UFF, R.J., Brazil.
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.032

L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
349
In this paper, we report the design, synthesis and anti-plate-
let activity of novel arylsulfonateeacylhydrazone derivatives
(4aej). These compounds were planned by applying molecu-
lar hybridization strategy on classical thrombin inhibitors (1)
[11] and on anti-platelet N-acylhydrazone (NAH) compound
(2) [12], elected as prototypes (Scheme 1). The main structural
features of the new arylsulfonateeacylhydrazone derivatives
(4aej) were based on classic isosteric replacement [20] of
chlorine atom, of the structure of anti-thrombin prototype
(1), by methyl group and the introduction of the NAH moiety
instead of the alkylealkyloxy subunit present in 1. Otherwise,
considering the azaevinylogue relationship of NAH group
with the amide present in the structures of ximelagatran (3),
it seems reasonable to introduce the NAH moiety as a spacer
between the subunits arylsulfonate (A) and N-hydroxy benza-
midine (B) (Scheme 1). Finally, variations at terminal basic
N-hydroxy-benzamidine subunit B by groups like aniline
and pyridine were done in order to obtain compounds bearing
P1 moiety with modulated basicity. Otherwise, the chemical
nature of the subunit B can also be modified aiming to
investigate the contribution of neutral and acid groups in the
activity of this new series of compounds, designed as antith-
rombotic candidates.
regioselective aromatic electrophilic substitution at the C-6
position of 3,4-methylenedioxytoluene (6), which was ob-
tained in ca. 50% overall yield from natural safrole (5), an
abundant natural product [12,13]. As previously described
[10], simple distillation of sassafras oil generates 5 in 85%
yield. Basic catalyzed isomerization of the double bond fol-
lowed by oxidative cleavage and WolffeKishner reduction
gave 6 in appropriated yield. The arylsulfonyl chloride deriv-
ative (8) was next prepared in two steps from 6, applying an
efficient and mild sulfonation of 1,3-benzodioxole derivative
6 followed by the treatment of the potassium salt (7) with thi-
onyl chloride, catalyzed by the VilsmeyereHaack complex, to
afford derivative 8 in 74% yield [13].
With an attractive method for access to the key intermediate
(8), we next performed the condensation of this compound
with phenols (e.g. 3-hydroxybenzaldehyde, 4-hydroxybenzal-
dehyde and 2-hydroxybenzaldehyde) in chloroform at room
temperature, in the presence of triethylamine, to afford the cor-
responding arylsulfonateealdehyde derivatives 9aec in 81e
98% yield. The synthesis of NAH-derivatives (4bef, 4i, 4j)
was carried out by exploring successive classical functional
groups interconversions on compounds 9aec. Thus, the treat-
ment of 9aec with sodium cyanide and manganese oxide in
methanol at room temperature [14] furnished the ester interme-
diates (10aec) in 60e97% yield. The hydrazinolysis of the
methyl esters (10aec) furnished the corresponding hydrazide
derivatives (11aec) in 75e81% yield. Condensation of 11ae
c with the appropriate aromatic aldehydes yielded the new se-
ries of sulfonateeacylhydrazone derivatives (4bef, 4i, 4j) in
2. Chemistry and biological activity testing
The target derivatives (4aej) were synthesized as depicted
in Scheme 2. In the synthetic approach compound 2-methyl-
4,5-methylenedioxy-phenyl-sulfonyl chloride (8) was identi-
fied as the key precursor. This compound can be obtained by
1
excellent yield. Analysis of the H NMR spectra of the new
A
basic P1 moiety
Cl
NAH-moiety
alkyl-alkyloxy
O spacer
H
N
N
H
O
NH2
NH
N
O
S
H
C
O
O
H
N
CH3
A+C+B
Molecular
S
anti-platelet prototype (2)
anti- thrombin prototype (1)
Isosteric
Hybridization Replacement
basic P1 moietyOH
N
meta
A
NAH-moiety
CH3
O
orto
para
O
spacer
O
NH2
C
N
N
O
S
B
H
O
O
H
4g
Congenere series
CH3
NH2
NO2
N
O
O
O
replaced by
H
basic P1 moiety
N
B
N
H
N
(a, meta)
(b, meta) (c, meta)
(d, meta)
O
NH2
CN
CO2H
CO2H
CO2H
N
OH
B
ximelagatran (3)
(e, meta)
(f, meta)
(i, para)
(j, orto)
Scheme 1.

350
L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
CH3
CH3
OK
O
O
O
O
O
O
b)
a)
S
ref. 10,11
O
O
5
7
6
CH3
O
O
CH3
CH3
O
O
O
O
O
c)
O
d)
O
Cl
O
S
H
S
OCH3
S
O
O
orto (a),
meta (b),
para (c)
O
O
O
O
8
orto (a),
meta (b),
para (c)
e)
9a-c
10a-c
i)
CH3
O
CH3
O
O
N
H
O
H
N
O
O
O
S
NH2
S
N
N
H
O
O
O
O
O
orto (a),
meta (b),
para (c)
4h
f)
11a-c
CH3
O
O
O
O
N
W
N
S
H
O
O
H
W = Ph- 4 - NH2 (a, meta)
W = 4 - Py ( b, meta )
W = Ph ( c, meta)
g)
W = Ph - 4 - NO2 ( d, meta )
W = Ph - 4 - CN ( e, meta )
4a - j
h)
W = Ph - 4 - CO2H ( f, meta )
W = Ph - 4 - C=N( -OH ) NH2 (g, meta)
W = Ph - 4 - CO2H ( i, para )
W = Ph - 4 - CO2H ( j, orto )
Scheme 2. Conditions: (a) (1) H2SO4, Ac2O, AcOEt, 0 ꢁC, r.t., 2 h, (2) AcOK, EtOH, r.t., 30 min, 91%; (b) SO2Cl, DMF cat, reflux, 4 h, 74%; (c) 3-hydroxyben-
zaldehyde (or 2-hydroxybenzaldehyde or 4-hydroxybenzaldehyde), Et3N, CH2Cl2, r.t., 2 h, 81e98%; (d) MnO2, NaCN, MeOH, r.t., 24 h, 60e97%; (e) N2H4$H2O,
EtOH, r.t., 2 h, 75e81%; (f) ArCHO, EtOH, HCl cat, reflux, 1 h, 84e98%; (g) Fe, NH4Cl, EtOH/H2O (2:1), reflux, 30 min, 75%; (h) NH2OH$HCl, Na2CO3, EtOH,
H2O, reflux, 8 h, 69%; (i) isoniazide, EtOH, HCl cat, r.t., 30 min,78%.
derivatives 4bef and 4i, 4j showed the presence of a single sig-
nal referring to ylidenic hydrogen, attributed to the E-diaste-
reomer in agreement with the previous report [15]. Finally,
compounds 4a and 4g were prepared by interconvertion of ni-
tro and nitrile groups, respectively, as follow. Compound 4a
was prepared by reduction of derivative 4d in the presence of
iron powder and ammonium chloride in a mixture of ethanol
and water [16], while compound 4g was synthesized by the
treatment with hydroxylamine in ethanol at reflux in 69% yield
[17]. Finally, compound 4h was prepared in good yield by con-
densation of hydrazide 9a with isoniazide, using hydrochloric
rich plasma (PRP) [18] induced by thrombin (2 nM), ADP
(5 mM), collagen suspension (5 mg.mLꢀ1) and arachidonic
acid (AA) (100 mM), using acetylsalicylic acid (AAS,
150 mM) as a positive control.
3. Results and discussion
The primary screening data for all compounds (150 mM)
are shown in Table 1. The results, displayed in Table 1,
demonstrate that compounds 4b (LASSBio-694) and 4g
(LASSBio-770) having a basic P1 subunit (pyridine and N-
hydroxybenzamidine, respectively) were able to inhibit the
platelet aggregation induced by thrombin in 72.5% and 81%,
respectively, while compound 4a (LASSBio-751), having ami-
nophenyl P1 subunit, was inactive. Nevertheless, the anti-
platelet activity found for these compounds, when the platelets
were induced either by AA or collagen, indicates that 4b and
4g could have dual mechanism of action. In fact, its known
1
acid as catalyst. The analysis of the H NMR spectra of the
hydrazone derivative 4h showed the presence of two ylidenic
hydrogen signals at d 7.9 and 8.5 attributed to the Z- and
E-diastereomers (1:4), respectively.
The in vitro anti-platelet activity of these novel arylsulfon-
ateeacylhydrazone derivatives (4aej, 150 mM) was evaluated
by their ability to inhibit platelet aggregation of rabbit platelet-

L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
351
that in rabbit PRP, the collagen and AA induced aggregation is
mediated by thromboxane (TXA2) formation [19]. Intriguing,
the evaluation of the retroisoster [20] of compound 4b,
derivative 4h (LASSBio-480), revealed loss of activity upon
thrombin induced platelet aggregation and potentiated the
anti-platelet activity mediated by AA or collagen. As antici-
pated, none of the derivatives having a neutral P1 subunit
was active in inhibiting the aggregation induced by thrombin.
However, compounds 4c (LASSBio-744), 4d (LASSBio-750)
and 4e (LASSBio-774) effectively inhibited the platelet aggre-
gation induced by AA and collagen, indicating a distinct profile
when compared to 4b and 4g (Table 1). Curiously, derivative 4f
(LASSBio-693), having an acidic P1 subunit, showed a great
selectivity and activity on inhibition of platelet aggregation in-
duced by thrombin (95% inhibition). With these results in
hands, the regioisomers of compound 4f (meta-isomer), i.e., de-
rivatives 4i ( para-isomer) and 4j (ortho-isomer), were synthe-
sized and evaluated with the same bioassay (Table 1). As
showed in Table 1, the replacement of N-acylhydrazone moiety
at the position 3 (meta, 4f) either to the position 4 ( para, 4i) or
the position 2 (ortho, 4j) results in activity on thrombin level,
although the compound 4j loses the selectivity. These results
indicated that geometric modifications, exploring the space
relationship between the NAH and sulfonate subunits, can
address the activity on different physiological agonists of plate-
let aggregation process. Otherwise, electronical parameters
also seem to be important and only compounds bearing basic
or acid groups at the P1 subunit were able to prevent platelet
aggregation induced by thrombin.
Molecular modeling studies were carried out using SYBYL
molecular mechanics in order to establish the conformer distri-
bution in compounds 4f (LASSBio-693), 4i (LASSBio-752) and
4j (LASSBio-743). Then, the global minimum conformer of 4f,
4i and 4j were selected to perform the equilibrium geometry us-
ing the semiempirical AM1 Hamiltonian (Table 2) [21].
The results from conformational analysis of derivatives 4f, 4i
and 4j provided a rational for the differences found in the phar-
macological profile of ortho-isomer 4j, when compared to
meta- 4f and para-isomers 4i (Table 2). As shown in Table 2,
these regioisomers also differ in their molecular shape, area
and volume, having distinct polarity. Compounds 4f and 4i
are more similar in this series.
More attractive compounds 4b, 4f, 4i and 4j were selected
to evaluate their antithrombotic effects in a pulmonary throm-
boembolism model induced by thrombin in mouse [23]. As
shown in Table 3, all compounds were able to increase the sur-
vival rate when given orally at a dose of 100 mmol/kg.
4. Conclusions
In summary, we demonstrated that the novel series of arylsul-
fonateeacylhydrazone derivatives(4aej) represent a promising
anti-platelet lead-candidate with significant antithrombotic
activity in vivo. Structural modifications at P1 subunit exploring
neutral, acid or basic groups can address the activity onthe plate-
let, resulting in compounds that are more or less selective on
thrombin levels. Otherwise, the results from the bioassays

352
L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
Table 2
Minimum energy conformer of arylsulfonateeacylhydrazone derivatives (4f, 4i and 4j). (A) Space-filling (CPK) model for compounds 4f, 4i and 4j; B) tube model
for compounds 4f, 4i and 4j
Compound
DHf (kcal/mol)
Diastereomer
Conformer
Area (A2)
Volume (A3)
Dipole (debye)
4f
4i
4j
ꢀ169.23
ꢀ169.55
ꢀ163.71
E
E
E
S-cis
S-trans
S-cis
494.02
498.20
490.28
488.74
489.81
488.01
7.012
9.121
4.648
identify a new class of derivatives, the first class, represented by
compounds 4f and 4j, which were activein inhibiting the platelet
aggregation induced by thrombin, the second class of deriva-
tives, represented by compounds 4e and 4h, that inhibit the
platelet aggregation involving TXA2 formation and finally the
third class of derivatives acting as a novel symbiotic candidates,
able to inhibit simultaneously the platelet aggregation induced
by collagen or AA and by thrombin, represented by compounds
4b and 4g.
performed on 2.0 ꢃ 6.0 cm aluminium sheets precoated with
silica gel 60 (HF-254, E. Merck) to a thickness of 0.25 mm.
The developed chromatograms were viewed under ultraviolet
light at 254 nm. E. Merck silica gel (60e200 mesh) was
used for column chromatography.
5.1. Anti-platelet activity
Blood was withdrawn from rabbit ear central artery and
mixed with 3.8% trisodium citrate (9:1 v/v). Platelet-rich
plasma (PRP) was prepared by centrifugation at 375 ꢃ g for
10 min at room temperature. The platelet-poor plasma (PPP)
was prepared by centrifugation of the pellet at 1800 ꢃ g for
10 min at room temperature. Platelet aggregation was moni-
tored by the turbidimetric method of Born and Cross [18] us-
ing a Chrono-Log aggregometer. PRP (300 mL) was incubated
5. Experimental
Melting points were determined with a Quimis 340 appara-
1
tus and are uncorrected. H NMR spectra, unless otherwise
stated, were obtained in deuterated dimethylsulfoxide or deu-
terated chloroform containing ca. 1% tetramethylsilane as an
internal standard using a Bruker AC 200 spectrometer at
200 MHz. Splitting patterns are as follows: s, singlet; d, dou-
blet; dd, double doublet; dt, double triplet; br, broad; m, mul-
tiplet. 13C NMR spectra were obtained using the same
spectrometer described above at 50 MHz, using deuterated di-
methylsulfoxide as internal standard. IR spectra were obtained
using Bruker IFS66 spectrophotometer employing potassium
bromide plates. Microanalysis data were obtained using
a Perkin-Elmer 240 analyser, using a Perkin-Elmer AD-4 bal-
ance. The progress of all reactions was monitored by TLC
Table 3
Antithrombotic effect of compounds (4b, 4f, 4j and 4i) in a pulmonary throm-
boembolism model induced by thrombin in mouse
Compound
Dose (p.o.) mmol/kg
(0.1 mL/20 g)
Survive (%)
Control
e
16.67 ꢂ 3.33
33.33 ꢂ 6.67
53.33 ꢂ 6.67
80.00 ꢂ 11.5
80.00 ꢂ 1.00
4b (LASSBio-694)
4f (LASSBio-693)
4j (LASSBio-743)
4i (LASSBio-752)
100
100
100
100

L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
353
at 37 ꢁC for 1 min with continuous stirring at 900 rpm. Platelet
aggregation was induced by ADP (5 mM), collagen suspension
(5 mg.mLꢀ1), thrombin (2 nM) or arachidonic acid (AA,
100 mM). Compound (150 mM) or vehicle DMSO (0.5% v/v)
was added to the PRP samples 1 min before addition of the ag-
gregating agent. Acetylsalicylic acid (AAS, 150 mM), a classi-
cal PGHS inhibitor, was used as positive control.
122.93 (C20 and C60); 126.27 (C40); 131.50 (C30 and C50);
134.97 (C1); 135.53 (C6); 146.11 (C4); 152.77 (C3); 154.01
(C10); 190.78 (ArCOH) ppm.
5.3.3. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid
2-formyl-phenyl ester (9c)
Derivative 9c was obtained as brown solid in 81% yield, mp
1
98e100 ꢁC. H NMR (300 MHz, CDCl3) d: 2.66 (s, ArCH3);
5.2. Pulmonary thromboembolism model induced by
thrombin in mouse [23]
6.09 (s, OCH2O); 6.85 (s, H2); 7.09 (d, J ¼ 8.1 Hz, H60); 7.29
(s, H5); 7.30 (dt, J ¼ 7.1 Hz and 8.1 Hz, H50); 7.58 (dt,
J ¼ 7.1 Hz and 7.7 Hz, H40); 7.95 (d, J ¼ 7.7 Hz, H30); 10.20
(s, ArCOH ) ppm. 13C NMR (75 MHz, CDCl3) d: 20.80
(ArCH3); 102.70 (OCH2O); 110.40 (C5); 112.60 (C2);
123.48 (C60); 125.78 (C20); 127.67 (C1); 128.89 (C40);
129.74 (C30); 135.46 (C50); 135.78 (C6); 146.24 (C4);
151.38 (C10); 152.98 (C3); 187.85 (ArCOH) ppm.
Arylsulfonateeacylhydrazones derivatives (4b, 4f, 4j and
4i) were given orally (100 mmol/kg; 0.1 mL/20 g) as a suspen-
sion in 5% arabic gum in saline (vehicle), 60 min before i.v.
injection of the thrombogenic stimulus. Thrombotic event
was induced by human thrombin (2000 UI/kg animal) and
the survival rate was evaluated 15 min after thrombotic event.
Results shown represent the mean ꢂ SD of three groups of five
animals each.
5.4. General procedure for the preparation of
(6-methyl-benzo[1,3]dioxole-5-sulfonyloxy)-benzoic acid
methyl ester derivatives (10aec)
5.3. General procedure for the preparation of 6-methyl-
benzo[1,3]dioxole-5-sulfonic acid-formyl-phenyl ester
derivatives (9aec)
To a mixture of 0.81 mmol of aldehyde derivatives (9aec)
in methanol (30 mL) was added 4.1 mmol of sodium cyanide
and 12.2 mmol of activated manganese dioxide. The reactants
were stirred at room temperature for 24 h and the suspension
was filtered through CeliteÒ and treated with dichloromethane
(3 ꢃ 30 mL) and water (100 mL). The organic layers were
joined, dried over anhydrous sodium sulfate, filtered and evap-
orated under reduced pressure to furnish methyl esters 10aec
in good yields.
To a solution of 1.45 mmol of sulfonyl chloride derivative
(8) in 10 mL of methylene chloride, were added 1.45 mmol
of the appropriate hydroxybenzaldehydes and 1.54 mmol of
triethylamine. The reaction mixture was stirred for 2 h at
room temperature, when the end of reaction was observed
by TLC. The products (9aec) were isolated by addition of
40 mL of methylene chloride and extraction with 10% aq.
HCl (4 ꢃ 20 mL) and brine (3 ꢃ 20 mL). The organic layer
was dried over anhydrous sodium sulfate, filtered and evapo-
rated under reduced pressure to furnish the aldehyde deriva-
tives (9aec) in excellent yields.
5.4.1. 3-(6-Methyl-benzo[1,3]dioxole-5-sulfonyloxy)-
benzoic acid methyl ester (10a)
Derivative 10a was obtained as yellow solid in 97% yield,
mp 178e180 ꢁC. 1H NMR (200 MHz, CDCl3) d: 2.58 (s,
ArCH3); 3.80 (s, ArCO2CH3); 5.94 (s, OCH2O); 6.73 (s,
H2); 7.14 (s, H5); 7.15 (d, J ¼ 8.1 Hz, H60); 7.29 (dt,
J ¼ 8.1 Hz and 7.8 Hz, H50); 7.58 (s, H20); 7.84 (d,
J ¼ 7.8 Hz, H40) ppm. 13C NMR (50 MHz, CDCl3) d: 20.74
(ArCH3); 52.56 (s, ArCO2CH3); 102.60 (OCH2O); 110.63
(C5); 112.35 (C2); 123.46 (C20); 126.38 (C60); 126.79 (C50);
128.32 (C40); 129.89 (C30); 132.18 (C1); 135.48 (C6);
146.04 (C4); 149.64 (C10); 152.61 (C3); 165.83
(ArCO2CH3) ppm.
5.3.1. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid
3-formyl-phenyl ester (9a)
Derivative 9a was obtained as brown solid in 98% yield,
mp 65e66 ꢁC. 1H NMR (200 MHz, CDCl3) d: 2.68 (s,
ArCH3); 6.04 (s, OCH2O); 6.83 (s, H2); 7.22 (s, H5); 7.33e
7.35 (m, H50); 7.47e7.51 (d, J ¼ 8.1 Hz, H60); 7.50 (s, H20);
7.78 (d, J ¼ 7.8 Hz, H40), 9.94 (s, ArCOH ) ppm. 13C NMR
(50 MHz, CDCl3) d: 20.69 (ArCH3); 102.64 (OCH2O);
110.49 (C5); 112.38 (C2); 122.77 (C20); 126.08 (C30);
128.29 (C60); 128.50 (C50); 130.64 (C40); 135.46 (C1);
138.03 (C6); 146.04 (C4); 150.21 (C10); 152.68 (C3); 190.75
(ArCOH) ppm.
5.4.2. 4-(6-Methyl-benzo[1,3]dioxole-5-sulfonyloxy)benzoic
acid methyl ester (10b)
Derivative 10b was obtained as yellow solid in 80% yield,
mp 99e101 ꢁC. 1H NMR (200 MHz, CDCl3) d: 2.66 (s,
ArCH3); 3.88 (s, ArCO2CH3); 6.04 (s, OCH2O); 6.80 (s,
H2); 7.09 (d, J ¼ 8.6 Hz, H20 and H60); 7.22 (s, H5); 7.99
(d, J ¼ 8.6 Hz, H30 and H50) ppm. 13C NMR (50 MHz,
CDCl3) d: 20.70 (ArCH3); 52.48 (ArCO2CH3); 102.64
(OCH2O); 110.60 (C5); 112.32 (C2); 122.18 (C20 and C60);
126.29 (C40); 129.04 (C1); 131.52 (C30 and C50); 135.45
(C6); 146.04 (C4); 152.65 (C3); 153.06 (C10); 166.11
(ArCO2CH3) ppm.
5.3.2. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid
4-formyl-phenyl ester (9b)
Derivative 9b was obtained as yellow solid in 89% yield,
mp 104e106 ꢁC. 1H NMR (200 MHz, CDCl3) d: 2.58 (s,
ArCH3); 5.95 (s, OCH2O); 6.73 (s, H2); 7.12 (d, J ¼ 8.6 Hz,
H20 and H60); 7.15 (s, H5); 7.75 (d, J ¼ 8.6 Hz, H30 and
H50); 9.87 (s, ArCOH ) ppm. 13C NMR (50 MHz, CDCl3) d:
20.71 (ArCH3); 102.70 (OCH2O); 110.55 (C5); 112.40 (C2);

354
L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
5.4.3. 2-(6-Methyl-benzo[1,3]dioxole-5-sulfonyloxy)benzoic
acid methyl ester (10c)
desired N-acylhydrazone derivatives (4bej), that were purified
by recrystallization in ethanol, yielding compounds 4bej in
excellent yields.
Derivative 10c was obtained as yellow solid in 60% yield,
mp 106e108 ꢁC. 1H NMR (300 MHz, CDCl3) d: 2.64 (s,
ArCH3); 3.87 (s, ArCO2CH3); 6.04 (s, OCH2O); 6.81 (s,
H2); 6.92 (d, J ¼ 8.1 Hz, H60); 7.21 (s, H5); 7.32 (dt,
J ¼ 6.3 Hz and 7.6 Hz, H40); 7.34 (dt, J ¼ 8.1 Hz and 6.3 Hz,
H50); 7.89 (d, J ¼ 7.6 Hz, H30) ppm. 13C NMR (75 MHz,
CDCl3) d: 20.84 (ArCH3); 52.59 (s, ArCO2CH3); 102.56
(OCH2O); 110.40 (C5); 112.31 (C2); 123.57 (C60); 126.07
(C20); 127.17 (C1); 127.17 (C40); 132.12 (C30); 133.34
(C50); 135.60 (C6); 145.91 (C4); 147.97 (C10); 152.43 (C3);
165.46 (ArCO2CH3) ppm.
5.6.1. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid 3-
(pyridin-4-ylmethylene-hydrazinocarbonyl)-
phenyl ester (4b)
Derivative 4b was obtained as a white solid by condensa-
tion of 11a with pyridine-4-carboxaldehyde in 89% yield.
1H NMR (200 MHz, DMSO-d6) d: 2.55 (s, ArCH3); 6.17 (s,
OCH2O); 7.20 (d, J ¼ 7.9 Hz, H60); 7.22 (s, H2); 7.29 (s,
H5); 7.58 (dt, J ¼ 7.7 Hz and 7.9 Hz, H50); 7.69 (s, H20);
7.94 (m, H40, H200 and H600); 8.54 (s, CH]N); 8.77 (m,
J ¼ 7.9 Hz, H300 and H500); 12.58 (br, CONH ) ppm. 13C
NMR (50 MHz, DMSO-d6) d: 19.87 (ArCH3); 102.90
(OCH2O); 109.39 (C5); 112.44 (C2); 121.52 (C20); 121,99
(C60); 124.92 (C40); 125.51 (C600); 126.76 (C200); 130.45
(C50); 134.67 (C30); 135.09 (C1); 135.61 (C6); 144.26 (C100);
145.08 (CH]N); 147.64 (C300 and C500); 145.76 (C4);
148.95 (C10); 152.52 (C3); 162.56 (ArCONH) ppm.
5.5. General procedure for the preparation of 6-methyl-
benzo[1,3]dioxole-5-sulfonic acid-hydrazinocarbonyl-
phenyl ester derivatives (11aec)
A solution of 1.14 mmol of the appropriate methyl ester de-
rivatives (10aec) and 6 mL of 80% hydrazine hydrate in
15 mL of ethanol, was stirred at room temperature for 2 h,
when the end of reaction was observed by TLC. Then, the
mixture was cooled and neutralized with concentrated HCl, re-
sulting in the formation of a white precipitate, which was fil-
tered, washed with water and dried under reduced pressure to
give hydrazide derivatives (11aec) in good yields.
5.6.2. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid
3-(benzylidene-hydrazinocarbonyl)-phenyl ester (4c)
Derivative 4c was obtained as a white solid by condensa-
tion of 11a with benzaldehyde in 84% of yield. 1H NMR
(200 MHz, DMSO-d6) d: 2.61 (s, ArCH3); 6.15 (s, OCH2O);
7.08e7.16 (m, H2, H5 and H60); 7.45e7.49 (m, H20, H40
and H50); 7.82e7.89 (m, H200, H300, H400, H500 and H600);
8.45 (s, CH]N); 11.90 (br, CONH ) ppm. 13C NMR
(50 MHz, DMSO-d6) d: 20.34 (ArCH3); 103.37 (OCH2O);
109.89 (C5); 112.91 (C2); 121.76 (C20); 125.45 (C60);
126.96 (C40); 127.67 (C300 and C500); 129.34 (C200 and 600);
130.73 (C50); 130.85 (C400); 134.62 (C30); 135.55 (C100);
135.72 (C1); 136.25 (C6); 148.99 (C4); 149.00 (CH]N);
149.45 (C10); 152.99 (C3); 162.03 (ArCONH) ppm.
5.5.1. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid-3-
hydrazinocarbonyl-phenyl ester derivatives (11a)
Derivative 11a was obtained as white solid in 78% yield,
mp 166e168 ꢁC. IR (KBr): 3343 and 3279 (NeH), 1673
(C]O), 1350 (S]O) cmꢀ1
.
5.5.2. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid-4-
hydrazinocarbonyl-phenyl ester derivatives (11b)
Derivative 11b was obtained as white solid in 81% yield,
mp 130e131 ꢁC. IR (KBr): 3348 and 3279 (NeH), 1668
5.6.3. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid 3-
(4-nitro-benzylidene-hydrazinocarbonyl)-phenyl ester (4d)
Derivative 4d was obtained as a yellow solid by condensa-
tion of 11a with 4-nitrobenzaldehyde in 98% of yield. 1H
NMR (300 MHz, DMSO-d6) d: 2.67 (s, ArCH3); 6.04 (s,
OCH2O); 6.82 (s, H2); 7.13 (d, J ¼ 7.4 Hz, H60); 7.22 (s,
H20); 7.26 (s, H5); 7.40 (m, H50); 7.65 (m, H40); 7,85 (d,
J ¼ 7.6 Hz, H200 and H600); 8.22 (d, J ¼ 7.6 Hz, H300 and
H500); 8.31 (s, CH]N); 11.65 (br, CONH ) ppm. NMR 13C
(75 MHz, DMSO-d6) d: 20.39 (ArCH3); 103.25 (OCH2O);
109.89 (C5); 112.90 (C2); 121.60 (C20); 121.83 (C100);
123.75 (C300 and C500); 125.21 (C60); 125.61 (C50); 126.84
(C40); 129.49 (C200 and C600); 130.68 (C30), 135.59 (C1);
136.20 (C6); 146.29 (C4); 149.38 (CH]N); 150.21 (C10);
150.85 (C400); 153.10 (C3); 161.53 (ArCONH) ppm.
(C]O), 1357 (S]O) cmꢀ1
.
5.5.3. 6-Methyl-benzo[1,3]dioxole-5-sulfonic
acid-2-hydrazinocarbonyl-phenyl ester derivatives (11c)
Derivative 11c was obtained as white solid in 75% yield,
mp 128e130 ꢁC. IR (KBr): 3340 and 3275 (NeH), 1669
(C]O), 1355 (S]O) cmꢀ1
.
5.6. General procedure for the preparation of 6-methyl-
benzo[1,3]dioxole-5-sulfonic acid-benzylidene-
hydrazinocarbonyl-phenyl ester derivatives (4bej)
An equimolar amount of appropriate aromatic aldehyde
was added to a solution of hydrazide derivatives 11aec
(1.05 mmol) in 20 mL of ethanol, in the presence of catalytic
amount of hydrochloric acid. The reaction was stirred for 0.5e
1.0 h, at reflux, and the solvent was evaporated under reduced
pressure. The colored precipitate was collected by filtration,
washed with cold water and dried under vacuum to give the
5.6.4. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid 3-
(4-cyano-benzylidene-hydrazinocarbonyl)-phenyl ester (4e)
Derivative 4e was obtained as a white solid by condensation
of 11a with 4-cyanobenzaldehyde in 94% of yield. 1H
NMR (200 MHz, DMSO-d6) d: 2.60 (s, ArCH3); 6.14 (s,

L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
355
OCH2O); 7.18 (d, J ¼ 8.8 Hz, H60); 7.20 (s, H2); 7.26 (s, H5);
7.56 (dt, J ¼ 8.1 Hz and 7.8 Hz, H50); 7.64 (s, H20); 7.86e7.94
(m, H40, H200, H600, H300 and H500); 8.48 (s, CH]N); 12.17 (br,
CONH ) ppm. 13C NMR (50 MHz, DMSO-d6) d: 20.38
(ArCH3); 103.42 (OCH2O); 109.93 (C5); 112.60 (C400);
112.96 (C2); 119.15 (ArCN); 121.90 (C20); 125.49 (C60);
125.79 (C50); 127.14 (C40); 128.25 (C200 and C600); 130.68
(C30), 133.28 (C300 and C500); 135.45 (C100); 135.61 (C1);
136.12 (C6); 146.29 (C4); 147.06 (CH]N); 149.50 (C10);
153.05 (C3); 162.35 (ArCONH) ppm.
132.36 (C1); 132.85 (C400); 135.68 (C6); 138.80 (C100);
147.45 (CH]N); 147.46 (C4); 151.85 (C3); 153.11 (C10);
162.78 (ArCONH); 167.46 (ArCO2H) ppm.
5.6.8. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid 2-
(4-carboxy-benzylidene-hydrazinocarbonyl)-phenyl ester (4j)
Derivative 4j was obtained as a white solid by condensation
1
of 11c with 4-carboxybenzaldehyde in 89% yield. H NMR
(200 MHz, DMSO-d6) d: 2.61 (s, ArCH3); 6.15 (s, OCH2O);
7.14 (s, H2); 7.21 (s, H5); 7.23 (d, J ¼ 8.5 Hz, H60); 7.85e
8.03 (m, H30, H40, H50, H200, H300, H500 and H600); 8.47 (s,
CH]N); 12.06 (s, RCO2H ) ppm. 13C NMR (50 MHz,
DMSO-d6) d: 20.37 (ArCH3); 103.45 (OCH2O); 109.99
(C5); 112.98 (C2); 121.84 (C60 and C40); 125.48 (C20);
127.68 (C1 and C30); 130.32 (C200, C600, C300 and C500);
132.31 (C400); 132.79 (C100); 135.64 (C50); 138.76 (C6);
146.28 (C4); 147.43 (CH]N); 151.82 (C3); 153.06
(C10);162.77 (ArCONH); 167.43 (ArCO2H) ppm.
5.6.5. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid 3-
(4-carboxy-benzylidene-hydrazinocarbonyl)-phenyl ester (4f)
Derivative 4f was obtained as a white solid by condensation
1
of 11a with 4-carboxybenzaldehyde in 91% yield. H NMR
(200 MHz, DMSO-d6) d: 2.50 (s, ArCH3); 6.04 (s, OCH2O);
7.18 (s, H2); 7.20 (s, H5); 7.22 (d, J ¼ 7.9 Hz, H60); 7.56
(dt, J ¼ 8.1 Hz and 7.9 Hz, H50); 7.62 (s, H20); 7.84 (d,
J ¼ 8.3 Hz, H200 and H600); 7,86 (d, J ¼ 8.1 Hz, H40); 8.01 (d,
J ¼ 8.3 Hz, H300 and H500); 8.49 (s, CH]N); 11.95 (br,
CONH ); 12.05 (br, COOH ) ppm. 13C NMR (50 MHz,
DMSO-d6) d: 20.35 (ArCH3); 103.38 (OCH2O); 109.89
(C5); 112.93 (C2); 121.83 (C20); 125.48 (C60); 125.81 (C50);
127.04 (C40); 127.68 (C200 and C600); 129.01 (C30), 130.29
(C300 and C500); 132,36 (C400); 135.58 (C1); 135.61 (C100);
138.66 (C6); 146.27 (C4); 147.82 (CH]N); 149.46 (C10);
153.01 (C3); 162.23 (ArCONH); 167.38 (ArCOOH) ppm.
5.7. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid
3-(4-amino-benzylidene-hydrazino
carbonyl)-phenyl ester (4a)
A mixture of nitro-acylhydrazone 4d (0.41 mmol), iron
powder (0.123 g) and ammonium chloride (0.034 g;
0.24 mmol) in 15 mL of a mixture of ethanol and water
(2:1) was refluxed for 30 min. Then, the reaction mixture
was filtered hot, and the filtrate was evaporated to half of its
original volume, followed by addition of 20 mL of water
and extraction with AcOEt (3 ꢃ 20 mL). The organic layer
was dried (Na2SO4), filtered and evaporated under reduced
pressure to give the desired amine-derivative in 75% yield.
1H NMR (300 MHz, DMSO-d6) d: 2.60 (s, ArCH3); 5.63
(br, ArNH2); 6.15 (s, OCH2O); 6.60 (d, J ¼ 8.4 Hz, H300 and
H500); 7.2 (m, H2 and H5 and H60); 7.40 (d, J ¼ 8.4 Hz, H200
and H600); 7,53 (dt, J ¼ 8.1 Hz and 8.0 Hz, H50); 7.60 (s,
H20); 7.85 (d, J ¼ 8.1 Hz, H40); 8.31 (s, CH]N); 11.58 (br,
CONH ) ppm. 13C NMR (75 MHz, DMSO-d6) d: 20.38
(ArCH3); 103.40 (OCH2O); 109.93 (C5); 112.95 (C2);
114.12 (C300 and C500); 121.62 (C20); 121.80 (C100); 125.21
(C60); 125.49 (C50); 126.83 (C40); 129.33 (C200 and C600);
130.78 (C30), 135.58 (C1); 136.17 (C6); 146.28 (C4); 149.46
(CH]N); 150.16 (C10); 151.68 (C400); 153.02 (C3); 161.49
(ArCONH) ppm.
5.6.6. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid 3-
[(pyridine-4-carbonyl)-hydrazonomethyl]-phenyl ester (4h)
Derivative 4h was obtained as a white solid by condensa-
tion of aldehyde derivative 9a with isonicotinic acid hydrazide
in 78% yield. 1H NMR (200 MHz, DMSO-d6) d: 2.50 (s,
ArCH3); 2.56 (ArCH3); 6.12 (s, OCH2O); 7.04 (d,
J ¼ 7.2 Hz, H60); 7.11 (s, H2); 7.16 (s, H5); 7.42 (d,
J ¼ 8.9 Hz, H200 and H600); 7.64 (m, H40); 7.94 (s, CH]N);
8.01 (m, H20 and H50); 8.51 (s, CH]N); 8.9 (m, H300 and
H500); 12.37 (br, CONH ); 12.53 (br, CONH ) ppm. 13C NMR
(50 MHz, DMSO-d6) d: 20.35 (ArCH3); 103.38 (OCH2O);
109.86 (C5); 112.90 (C2); 120.42 (C20); 123,48 (C60);
124.00 (C200 and C600); 125.54 (C40); 127.01 (C50); 131.21
(C30); 135.52 (C6); 136.59 (C1); 143.45 (C100); 146.11
(CH]N); 146.27 (C4); 148.18 (CH]N); 148.40 (C300 and
C500); 149.83 (C10); 152.99 (C3); 161.38 (ArCONH) ppm.
5.6.7. 6-Methyl-benzo[1,3]dioxole-5-sulfonic acid 4-
(4-carboxy-benzylidene-hydrazinocarbonyl)-phenyl ester (4i)
Derivative 4i was obtained as a white solid by condensation
5.8. 6-Methyl-benzo[1,3]dioxol-5-sulfonic acid 3-
[4-(N-hydroxycarbamimidoyl)-benzylidene-hydrazino
carbonyl]-phenyl ester (4g)
1
of 11b with 4-carboxybenzaldehyde in 89% yield. H NMR
(200 MHz, DMSO-d6) d: 2.61 (s, ArCH3); 6.16 (s, OCH2O);
7.16 (s, H2); 7.21 (s, H5); 7.23 (d, J ¼ 7.8 Hz, H20 and H60);
7.83 (d, J ¼ 8.1 Hz, H200 and H600); 7.92 (d, J ¼ 7.8 Hz, H30
and H50); 8.0 (d, J ¼ 8.1 Hz, H300 and H500); 8.46 (s,
CH]N); 12.05 (s, ArCONH ) ppm. 13C NMR (50 MHz,
DMSO-d6) d: 20.41 (ArCH3); 103.49 (OCH2O); 110.02
(C5); 113.03 (C2); 122.47 (C20 and C60); 125.54 (C40);
127.72 (C30 and C50); 130.36 (C200, C300, C500 and C600);
Hydroxylamine hydrochloride (0.97 mmol) was added to
a solution of cyano-acylhydrazone 4e (0.32 mmol) and
Na2CO3 (1.3 mmol) in ethanol (10 mL) and H2O (5 mL).
The mixture was heated at reflux temperature for 8 h, then,
it was cooled and neutralized with concentrated HCl, followed
by extraction with AcOEt (3 ꢃ 20 mL). The organic layer was
dried (Na2SO4), filtered and evaporated under reduced
pressure. The crude material was purified by column

356
L.M. Lima et al. / European Journal of Medicinal Chemistry 43 (2008) 348e356
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(yield: 69%). 1H NMR (200 MHz, DMSO-d6) d: 2.49 (s,
ArCH3); 3.49 (br, NH2); 5.95 (s, OCH2O); 7.1 (s, H2); 7.18
(s, H5); 7.25 (d, J ¼ 7.9 Hz, H60); 7.56 (dt, J ¼ 8.1 Hz and
7.9 Hz, H50); 7.65 (s, H20); 7,85 (d, J ¼ 8.1 Hz, H40); 7.87
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(CH]N); 153.05 (C10); 157.96 (C3); 168.07 (ArCONH);
168.67 (ArC](NeOH)NH2).
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5.9. Molecular modeling
The conformer distribution of 6-methyl-benzo[1,3]dioxole-
5-sulfonic acid (4-carboxy-benzylidene-hydrazinocarbonyl)-
phenylester derivatives(4f, 4i, 4j) was carried out usingSYBYL
molecular mechanics. The geometry optimization was per-
formed using the semiempirical AM1 Hamiltonian [21] with
SPARTAN 1.0.5 program [22] on Pentium IV 1.5 GHz.
ˇ ˇ
(h) A. Kranjc, L.P. Masie, S. Reven, K. Mikic, A. Preꢀzelj, M. Stegnar,
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S. Yu, B.K. Wong, C.J. Kochansky, M.R. Anari, Y. Yan, J.P. Vacca, Bio-
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Acknowledgements
The authors wish to thank financial support from IM-INO-
FAR (CNPq-BR # 420.015/05-1), PRONEX (BR), FAPERJ
(BR), and fellowships (CNPq (BR) LML, CAMF, EJB). We
also thank Mrs. Dione M. Silva for technical assistance.
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