Synthesis and antiplatelet evaluation of novel aryl-sulfonamide derivatives, from natural safrole

Article abstract of DOI:10.1016/S0031-6865(99)00004-7

In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antiplatelet prototype compounds, exploring bioisosterism principles for molecular design, we describe in this paper the synthesis of new aryl-sulf

Full text of DOI:10.1016/S0031-6865(99)00004-7

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Pharmaceutica Acta Helvetiae 73 1999 281–292
Synthesis and antiplatelet evaluation of novel aryl-sulfonamide
1
derivatives, from natural safrole
a,b
a
a
a
Lıdia M. Lima , Claudia B. Ormelli , Fernanda F. Brito , Ana L.P. Miranda ,
´
´
a
a,b,)
Carlos A.M. Fraga , Eliezer J. Barreiro
a
(
)
Laboratorio de AÕaliac¸ao e Sıntese de Substancias BioatiÕas LASSBio, http:rrwww.ufrj.brr; pharmarlassbio , Faculdade de Farmacia,
´
˜
´
ˆ
´
UniÕersidade Federal do Rio de Janeiro, CP 68.006, ZIP 21944-970, Rio de Janeiro, RJ, Brazil
b Instituto de Quımica, UniÕersidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
´
Received 10 August 1998; received in revised form 2 December 1998; accepted 13 January 1999
Abstract
In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antiplatelet prototype
compounds, exploring bioisosterism principles for molecular design, we describe in this paper the synthesis of new aryl-sulfonamides
derivatives, structurally similar to known thromboxane A2 receptor antagonists. The synthetic route used to access the new compounds
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described herein starts from safrole, an abundant Brazilian natural product, which occurs in Sassafras oil Ocotea pretiosa . The results
from preliminary evaluation of these novel aryl-sulfonamide compounds by the platelet aggregation inhibitory test, using rabbit PRP,
w
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.
x
induced by ADP, collagen, arachidonic acid, and U46619, identified the N- 2- 4-carboxymethoxyphenyl ethyl -6-methyl-3,4-methylen-
edioxyphenyl-sulfonamido derivative as the most active among them, presenting an IC50 value for the U-46619-induced platelet
aggregation in rabbit platelet-rich plasma: 329 mM. q 1999 Elsevier Science B.V. All rights reserved.
Keywords: Aryl-sulfonamide derivatives; TXA2 antagonists; Antiplatelet activity; Safrole in synthesis
1. Introduction
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Thromboxane A2 TXA2 , 1 is an important prostanoidal autacoid, generated mainly by platelets through sequential
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actions of prostaglandin-H2 synthase PGHS and thromboxane synthase TXS . TXA2 is a potent vasoconstrictor and
platelet-aggregating agent, responsible for stimulation of blood platelet aggregation and constriction of the vascular and
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respiratory smooth muscles Hamberg et al., 1975; Moncada and Vane, 1979; Bunting et al., 1983; Bhagwat et al., 1985 .
Ž .
TXA2 1 has been implicated in a wide variety of cardiovascular diseases including unstable angina, thrombosis and
hypertension Haluska and Lefer, 1987; Ogletree, 1987 . Therefore, considerable efforts has been devoted to modulate the
actions of TXA2 by design of TXS-inhibitors TXSi Kato et al., 1985 or TXA2 antagonists TPant. Collington and
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Corresponding author. Tel.: q55-21-280-1784, ext. 238; fax: q55-21-260-2299 or 273-3890; e-mail: eliezer@pharma.ufrj.br
1
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Taken, in part, from the M.Sc. Thesis of LML; This paper represents the contribution a30 from LASSBio, UFRJ Br. .
0031-6865r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
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PII: S0031-6865 99 00004-7

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.
Finch, 1990; Hall, 1991 . The former approach has received enormous attention due, in part, to the hypothesis that PGH2
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2 produced in the platelet could be secreted and taken up by endothelial cells for conversion to the anti-aggregatoric and
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anti-spasmodic prostacyclin PGI2 , 3 by action of prostacyclin synthase Chart 1 Bertele and De Gaetano, 1982 . In fact,
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this approach has found to be valid in therapeutics, as illustrated by discovery of ozagrel 4 an important antithrombotic,
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anti-asthmatic agent which acts as TXSi Ong and Allen, 1988 . Meanwhile, a potential possible disadvantage of this
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approach is that PGH2 2 possesses a similar pharmacological profile to that of TXA2 Fitzgerald et al., 1985; Fiddler
1
.
and Lumley, 1990 . Therefore, an alternative approach to preventing the action of TXA2 is the use of compounds which
would act as antagonist of TP receptor level. This approach has the advantage, because it blocks not only the actions of
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TXA2 1 , but also its biosynthetic precursor, PGH2
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.
2
Patscheke, 1990; Watts et al., 1991 . Combination of TPant and
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TXSi Jakubowski et al., 1992; Soyka et al., 1994; Ackerley et al., 1995 activity should have the beneficial effect on
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blocking the action of PGH2 at TP level even allowing conversion to PGI2 Chart 1 Collington and Finch, 1990;
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Jakubowski et al., 1992 . Preclinical studies employing stable TXA2 receptor antagonists have suggested that these
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compounds may be useful in the treatment of the diseases mentioned above Hall, 1991 . Thus, TP antagonists possessing
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suitable pharmacokinetic and pharmacodynamic properties may serve as useful therapeutic agent Hall, 1991 . For instance,
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administration of TPant sulotroban 5 to human volunteers gave a stronger inhibition of platelet aggregation ex vivo when
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combined with TXSi agent Gresele et al., 1987 . Structure-activity relationships SAR concerning TPant is relatively
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well-known, being substantially based on the presence of an hydrogen bond acceptor group e.g., sulfonamide group , a
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hydrophobic moiety e.g., phenyl ring para-substituted and an ionic interaction site e.g., carboxylic acid Chart 2 Jin
.
and Hopfinger, 1994; Albuquerque et al., 1995 .
In an attempt to identify new lead-candidate compounds with TPant activity, exploring the previous knowledge on the
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structure–activity relationships of the aryl-sulfonamide derivatives 5–9 Patscheke and Stegmeyer, 1984; Stegmeier et al.,
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.
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1986; Prous, 1994; Sato et al., 1995 Chart 2 , we wish to report in this paper the design, synthesis and antiplatelet
properties of novel aryl-sulfonamide derivatives 10–11 , structurally planned as hybrid of sulotroban 5 and daltroban 6 ,
.
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and also the acyl-sulfonyl hydrazide derivative 12 . These compounds were synthesized from natural safrole 13 , an
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abundant Brazilian natural product, occurring in the Sassafras oil Ocotea pretiosa Barreiro et al., 1982, 1985; Perreira et
.
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al., 1989; Cabral and Barreiro, 1995; Lages et al., 1998 . These new derivatives 10–12 were planned exploring the
classical aromatic ring bioisosteric replacement Patani and LaVoie, 1996 between the 1,3-benzodioxole ring, present in the
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natural product used as synthetic starting material, and the phenyl ring of bioactive benzenesulfonamides 5–9 . Considering
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that sulfonamide derivatives bearing a phenoxyacetic framework as sulotroban 5, BM 13.177 Patscheke and Stegmeyer,
.
1984; Stegmeier et al., 1986 , could possess a poor bioavailability profile due the enzymatic oxidative O-dealkylation step,
promoted by hepatic metabolism Testa, 1995 we decided to introduce the phenoxypropionyl chain in the new derivative
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11 , as a possible more metobolically stable analogue of 10 . Otherwise, compound 12 was designed as a new

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L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
283
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non-interphenylenic sulfonamide analogue of previous derivatives 10, 11 , that contain supplementary hydrogen donor–
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acceptor moiety in the sulfonamide chain, which may promote additional favorable interactions with the TP Chart 3 .
2. Experimental section
2.1. Chemistry
Melting points were determined with a Thomas–Hoover apparatus and are uncorrected. Proton magnetic resonance
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H NMR spectra are determined in deuterated solvents, using tetramethylsilane as an internal standard with a Brucker AC

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284
L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
200 spectrometer. Splitting patterns were as follows: s, singlet; d, doublet; dd, double doublet; t, triplet; q, quartet; qt,
Ž13
.
quintet; m, multiplet; br, broad. The carbon magnetic resonance spectra C NMR are determined in the same spectrometer
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described above at 50 MHz, using deuterated chloroform as internal standard. Infrared spectra IR were obtained with a
Perkin-Elmer 1600 spectrometer as neat film and KBr pelets. The mass spectra MS were obtained on a GCrVG
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Micromass 12 at 70 eV.
The progress of all reactions was monitored by TLC which was performed on 2.0 cm=5.0 cm aluminum sheets
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precoated with silica gel 60 HF-254, Merck to a thickness of 0.25 mm. The developed chromatograms were visualized
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under ultraviolet light 254–265 nm . For column chromatography Merck silica gel 70–230 mesh was used. Solvents used
in reactions were dried, redistillated prior to use and stored over 3–4 A molecular sieves.
(
) [
]
2.1.1. Potassium 6-methyl-3,4-methylenedioxyphenylsulfonate 15 Fraga, 1991 .
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To a solution of 3,4-methylenedioxytoluene 14 0.1 g; 0.74 mmol in 2.2 ml of ethyl acetate containing acetic
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anhydride 0.21 ml; 2.2 mmol cooled at 08C, was added, dropwise, a solution of sulfuric acid 0.06 ml; 0.93 mmol;
ds1.84 in ethyl acetate. To this reaction mixture, after stirring for 2 h at room temperature, was next added a solution of
potassium acetate 0.11 g; 1.1 mmol in ethanol 0.6 ml and the suspension formed was maintained under stirring at room
.
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temperature for additional 30 min. The resulting precipitates were removed by filtration, washed with ethyl acetate to give
1
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0.16 g 91% of 15 as colorless powder, mp 1898C. H NMR D2O d: 2.45 s, 3H, ArC H3 , 5.89 s, 2H, OC H2O , 6.70
y1
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s, 1H, Ar-H5 , 7.27 s, 1H, Ar-H2 ppm; IR KBr : 1346 n S–O , 1242 n C–O , 1055 n S–O–C cm
.
(
) [
]
2.1.2. 6-Methyl-3,4-methylenedioxyphenylsulfonyl chloride 16 Fraga, 1991 .
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A mixture of thionyl chloride 3.9 ml; 53.4 mmol and dry dimethylformamide 0.1 ml was added to the dry potassium
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salt derivative 15 2.5 g; 10.2 mmol . The reaction mixture, maintained under a nitrogen atmosphere, was stirred at reflux
for 4 h and then poured into crushed ice and extracted with methylene chloride 5=30 ml . The organic extracts were dried
over anhydrous Na2 SO4 and evaporated at reduced pressure to give 1.8 g 74% of the sulfonyl chloride derivative 16 as
yellow prisms: mp 798C. H NMR CDCl3 d: 2.70 s, 3H, ArC H3 , 6.10 s, 2H, OC H2O , 6.81 s, 1H, Ar-H5 , 7.49 s,
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1
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1H, Ar-H2 ppm; IR KBr : 1359 n S–O , 1251 n C–O , 1160 n S–O–C cm
.
[ (
)
]
(
) [
]
2.1.3. N- 2- 4-Hydroxyphenyl ethyl -6-methyl-3,4-methylenedioxyphenylsulfonamido 18 adapted from Fraga, 1991 .
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To a solution of the sulfonyl chloride derivative 16 0.3 g; 1.28 mmol in ethyl acetate 10 ml was added a solution of
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4-hydroxy-phenylethylamine hydrochloride 17 0.22 g; 1.28 mmol in 10% aqueous NaHCO3 2 ml . The reaction mixture
was stirred at room temperature for 2 h, then poured into water 5 ml and extracted with ethyl acetate 3=10 ml . The
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resulting organic layer was next washed with brine and dried over anhydrous Na2 SO4, and evaporated at reduced pressure
1
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to give 0.39 g 91% of the sulfonamide derivative 18 as colorless powder: mp 128–1308C. H NMR DMSO-d6 d: 2.42
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s, 3H, ArC H3 , 2.50 t, 2H, Js7.5 Hz, ArC H2CH2 NH , 2.90 q, 2H, Js7.0 Hz, ArCH2C H2 NH , 6.10 s, 2H,
.
.
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.
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.
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.
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X
X
OC H2O , 6.60 d, 2H, Js8.5 Hz, Ar-H2 , 6.90 d, 2H, Js8.5 Hz, Ar-H3 , 6.95 s, 1H, Ar-H5 , 7.25 s, 1H, Ar-H2 ,
13
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7.60 t, 1H, Js5.8 Hz, ArCH2CH2 NH ppm; C NMR DMSO-d6 d: see Table 1; IR KBr : 3420 n O–H , 3260 n
y1
H
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.
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.
.
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.
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.
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.
N–H , 1370 and 1176 n S–N , 1250 n C–O cm ; MS mrz : 335 M , 24% , 135 62% , 199 100% , 77 37% , 228
Ž
.
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.
29% , 107 56% .
[ (
)
]
( ) [
2.1.4. N- 2- 4-Methoxycarbonylmethoxy ethyl -6-methyl-3,4-methylenedioxyphenyl sulfonamido 19 adapted from Fraga
]
and Barreiro, 1992 .
To a suspension of the sulfonamide derivative 18 0.4 g; 1.19 mmol and potassium carbonate 0.16 g; 1.19 mmol in
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.
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.
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.
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dry acetone 30 ml was added ethyl 2-bromoacetate 0.13 ml; 1.19 mmol . The reaction mixture was stirred at room
temperature, under argon atmosphere, for 24 h and then the solvent was evaporated at reduced pressure. The residue was
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diluted with 5% aqueous NaOH solution ca. 15 ml and next extracted with ethyl acetate 4=10 ml . The organic layer
was washed with brine, dried over anhydrous Na2 SO4 and evaporated at reduced pressure. The residue was purified by
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silica gel column chromatography using CH2Cl2rMeOH 10:0, 10:1 then 10:2 to give 0.1 g 37% of the mono-alkylated
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.
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derivative 19 and 0.05 g 18% of O,N-bis-alkylated derivative 20 as yellow oils.
1
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Compound 19 : H NMR CDCl3 d: 1.30 t, 3H, Js7.1 Hz, COOCH2C H3 , 1H , 2.37 s, 3H, ArC H3 , 2.70 t, 2H,
.
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.
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Js6.6 Hz, ArC H2CH2 NH , 3.15 q, 2H, Js6.7 Hz, ArCH2C H2 NH , 4.25 q, 2H, Js7.1 Hz, COOC H2CH3 , 4.45 t,
.
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1H, Js6.0 Hz, ArCH2CH2 NH , 4.60 s, 2H, ArOC H2CsO , 6.05 s, 2H, OC H2O , 6.70 s, 1H, Ar-H5 , 6.82 d, 2H,
13
.
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.
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.
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.
X
X
Js8.8 Hz, Ar-H3 , 7.02 d, 2H, Js8.5 Hz, Ar-H2 , 7.41 s, 1H, Ar-H2 ppm; C NMR CDCl3 d: 14.0 COOCH2CH3 ,

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L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
285
Table 1
13
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C nuclear magnetic resonance 50 MHz of the compounds 18, 10, 11, 12
Compounds d: ppm
a
a
b
a
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18
.
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10
.
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11
.
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12
C-1
C-2
C-3
C-4
C-5
C-6
132.2
110.6
145.2
150.2
112.0
128.7
155.7
115.1
129.4
131.3
129.5
115.2
102.1
19.7
132.7
110.2
145.5
150.8
112.3
129.8
156.6
114.6
129.6
130.7
129.4
114.8
101.9
19.9
132.8
109.8
145.5
150.8
112.0
130.2
156.1
115.2
129.8
130.7
129.7
115.3
102.0
19.9
134.6
109.7
144.9
150.8
119.9
129.1
X
C-1
C-2X
C-3X
C-4X
C-5X
C-6X
OCH2O
ArCH3
HNCH2
CH2 Ph
OCH2CO2 R
OCH2CO2 R
102.2
20.3
44.2
34.5
43.9
34.6
65.3
169.8
43.8
34.6
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OCH CH3 CO2 R
170.2
72.4
18.3
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OCH CH3 CO2 R
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OCH CH3 CO2 R
HNCO
170.7
32.5
20.2
32.0
174.0
HNCOCH2
CH2CH2CH2
CH2CO2 R
COOH
aca. 50 mg of compound in 0.7 ml of DMSO-d6.
bca. 50 mg of compound in 0.7 ml of CDCl3.
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19.9 ArCH3 , 34.6 ArCH2CH2 NH , 43.9 ArCH2CH2 NH , 61.3 COOCH2CH3 , 65.3 ArOCH2C5O , 101.9
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.
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.
.
X
X
X
OCH2O , 110.2 C2 , 112.2 C5 , 114.6 and 114.8 C2 , 129.6 C3 , 129.8 C6 , 130.7 C4 , 132.7 C1 , 145.5 C3 ,
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X
150.8 C4 , 156.6 C1 , 168.7 ArOCH2C5O ppm; IR neat : 3306 n N–H , 1750 n C5O 1325 and 1100 n S–N ,
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1210 and 1248 n C–O cm
.
Anal. Calcd. for C20 H23O7 NS: C, 57.00; H, 5.50; N, 3.32. Found: C, 56.90; H, 5.51; N, 3.29.
Compound 20 : 1H NMR CDCl3 d: 1.25 t, 3H, Js7.0 Hz, NCH2COOCH2C H3 1.32 t, 3H, Js7.1 Hz,
Ž
.
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.
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.
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.
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.
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.
OCH2COOCH2C H3 , 2.42 s, 3H, ArC H3 , 2.77 t, 2H, Js6.4 Hz, ArC H2CH2 NH , 3.50 t, 2H, Js6.6 Hz,
.
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.
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.
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ArCH2C H2 NH , 4.00 s, 2H, NC H2COOCH2CH3 , 4.15 q, 2H, Js7.1 Hz, NCH2COOC H2CH3 , 4.28 q, 2H, Js7.1
.
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.
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.
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Hz, OCH2COOC H2CH3 , 4.60 s, 2H, OC H2COOCH2CH3 , 6.02 s, 2H, OC H2O , 6.67 s, 1H, Ar-H5 , 6.79 d, 2H,
.
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.
X
X
Js8.8 Hz, Ar-H3 , 7.03 d, 2H, Js8.5 Hz, Ar-H2 , 7.44 s, 1H, Ar-H2 ppm.
[ (
)
]
(
) [
2.1.5. N- 2- 4-Carboxymethoxyphenyl ethyl -6-methyl-3,4-methylenedioxyphenyl sulfonamido 10 adapted from Barrow et
]
al., 1995 .
To a solution of the methyl ester derivative 19 0.2 g; 0.4 mmol in THF 10 ml was added 1 N aqueous LiOH
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.
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.
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solution 0.173 g; 7.2 mmol and the resulting emulsion was stirred at room temperature for 45 min. The reaction mixture

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L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
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.
was diluted with ethyl ether 20 ml and carefully acidified with 1 N aqueous HCl. The organic layer was washed with
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.
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brine, dried over anhydrous Na2 SO4 and evaporated at reduced pressure to give 0.15 g 85% of the acid derivative 10 as
1
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.
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.
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.
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colorless oil. H NMR CD3OD d: 2.40 s, 3H, ArC H3 , 2.63 t, 2H, Js6.9 Hz, ArC H2CH2 NH , 3.08 q, 2H, Js7.0
.
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.
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.
Ž
.
Ž
Hz, ArCH2C H2 NH , 4.60 s, 2H, OC H2C5O , 6.03 s, 2H, OC H2O , 6.78 s, 1H, Ar-H5 , 6.98 d, 2H, Js8.5 Hz,
13
.
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.
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.
.
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.
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.
X
X
Ar-H3 , 7.0 d, 2H, Js8.7 Hz, Ar-H2 , 7.30 s, 1H, Ar-H2 ppm; C NMR DMSO d6 d: see Table 1; MS mrz : 383
H
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.
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.
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.
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.
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.
M , 16% , 135 77% , 199 100% , 77 76% , 228 34% , 107 86% , 165 31% .
{ [ (
)
]
}
( ) [
2.1.6. N- 2- 4- 1-Methoxycarbonylethoxy phenyl ethyl -6-methyl-3,4-methylenedioxy phenyl sulfonamido 21 adapted
]
from Hawker and Frechet, 1990 .
´
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.
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.
To a suspension of the sulfonamide derivative 18 0.25 g; 0.75 mmol and potassium carbonate 0.1 g; 0.75 mmol in
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.
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.
dry acetone 20 ml was added methyl 2-bromopropionate 0.1 ml; 0.75 mmol . The reaction mixture was stirred at room
temperature, under argon atmosphere, for 36 h and then poured into water 5 ml and next extracted with ethyl acetate
3=10 ml . The organic layer was washed with brine, dried over anhydrous Na2 SO4 and evaporated at reduced pressure.
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.
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The residue was purified by flash chromatography using n-hexanerethyl acetate 10:1 to give 0.23 g 74% of the
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O-alkylated derivative 21 as yellow oil. H NMR CDCl3 d: 1.60 d, 3H, Js6.9 Hz, OCH C H3 COOCH3 , 2.38 s,
.
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.
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.
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3H, ArC H3 , 2.70 t, 2H, Js6.8 Hz, ArC H2CH2 NH , 3.10 q, 2H, Js6.6 Hz, ArCH2C H2 NH , 3.75 s, 3H,
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.
.
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.
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.
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.
.
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OCH CH3 COOC H3 , 4.57 t, 1H, Js6.0 Hz, ArCH2CH2 NH , 4.73 q, 1H, Js6.9 Hz, OC H CH3 COOCH3 , 6.02 s,
.
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.
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.
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X
X
2H, OC H2O , 6.73 s, 1H, Ar-H5 , 6.80 d, 2H, Js8.8 Hz, Ar-H3 , 6.98 d, 2H, Js8.5 Hz, Ar-H2 , 7.42 s, 1H,
.
Ar-H2 ppm.
Anal. Calcd. for C20 H23O7 NS: C, 57.00%; H, 5.50%; N, 3.32%. Found: C, 56.97%; H, 5.55%; N, 3.30%.
{ [ (
)
]
}
( ) [
2.1.7. N- 2- 4- 1-Carboxyethoxy phenyl ethyl -6-methyl-3,4-methylenedioxyphenyl sulfonamido 11 adapted from Barrow
]
et al., 1995 .
To a solution of methyl ester 21 0.3 g; 0.72 mmol in tetrahydrofuran 20 ml was added 1 N aqueous LiOH solution
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.
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.
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0.07 g; 3.1 mmol and the resulting emulsion was stirred at room temperature for 1 h. The reaction mixture was diluted
with ethyl ether 20 ml and acidified with 1 N aqueous HCl. The organic layer was washed with brine, dried over
anhydrous Na2 SO4 and evaporated at reduced pressure to give 0.3 g 62% of the corresponding acid derivative 11 as
Ž
.
Ž
.
Ž .
1
Ž
.
Ž
Ž
.
.
Ž
.
Ž
.
Ž
colorless oil. H NMR CDCl3 d: 1.62 d, 3H, Js6.8 Hz, OCH C H3 COOCH3 , 2.38 s, 3H, ArC H3 , 2.70 t, 2H,
.
Ž
Ž
Ž
.
Ž
Js6.7 Hz, ArC H2CH2 NH , 3.10 q, 2H, Js6.3 Hz, ArCH2C H2 NH , 4.63 br, 1H, ArCH2CH2 NH , 4.76 m, 1H,
Ž
.
.
.
.
Ž
.
.
Ž
Ž
X
OC H CH3 COOCH3 , 6.02 s, 2H, OCH2O , 6.68 s, 1H, Ar-H5 , 6.78 d, 2H, Js8.4 Hz, Ar-H3 , 6.98 d, 2H, Js8.3
13
Ž
.
Ž
.
.
Ž
.
Ž
.
.
Ž
X
Hz, Ar-H2 , 7.40 s, 1H, Ar-H2 ppm; C NMR CDCl3 d: see Table 1; IR neat : 3315 n O–H , 3250 n N–H , 1726
y1
H
Ž
.
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
.
.
n C5O , 1302 and 1116 n S–N , 1245 n C–O cm ; MS mrz : 407 M , 6% , 135 56% , 199 100% , 77 66% ,
Ž
.
Ž
.
228 25% , 107 78% .
Anal. Calcd. for C25 H31O9 NS: C, 57.57%; H, 5.99%; N, 2.69%. Found: C, 57.18%; H, 5.05%; N, 2.80%.
(
) [
]
2.1.8. 6-Methyl-3,4-methylenedioxyphenylsulfonylhydrazine 22 adapted from Fraga, 1991 .
Ž
. Ž
.
Ž
.
To a solution of sulfonyl chloride derivative 16 0.4 g; 1.7 mmol in chloroform 20 ml , cooled at 08C, was added
Ž
.
dropwise 40% aqueous hydrazine hydrate 5 ml . The reaction mixture was stirred for 2 h at 08C, then poured into water
Ž
.
Ž
.
10 ml and extracted with chloroform 4=10 ml . The organic layer was washed with brine, dried over anhydrous Na2 SO4
Ž
.
.
Ž .
and evaporated at reduced pressure to give 0.36 g 92% of the sulfonylhydrazine derivative 22 as colorless prisms: mp
120–1218C. H NMR DMSO-d6 d: 2.50 s, 3H, ArC H3 , 6.10 s, 2H, OC H2O , 6.97 s, 1H, Ar-H5 , 7.28 s, 1H, Ar-H2
ppm; C NMR DMSO-d6 d: 19.8 ArCH3 , 102.1 OCH2O , 109.9 C2 , 111,8 C5 , 128.7 C6 , 133.2 C1 , 145.1
1
Ž
.
Ž
Ž
.
.
Ž
.
Ž
.
13
Ž
.
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
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.
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.
C3 , 150.4 C4 ppm; IR KBr : 3398 and 3370 n H–N–H , 3327 n N–H , 1348 and 1117 n S–N , 1213 n C–O
H
cmy1; MS mrz : 230 M , 28% , 200 100% , 135 79% , 151 67% , 77 66% .
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
(
)
(
)
(
) [
2.1.9. 1- 5-Carboxybutyryl -2- 6-methyl-3,4-methylenedioxyphenylsulfonyl hydrazine 12 adapted from Grahan et al.,
]
1990 .
To a solution of the sulfonylhydrazine derivative 22 0.2 g; 0.87 mmol in chloroform 15 ml was added glutaric
Ž
. Ž
.
Ž
.
Ž
.
anhydride 0.09 g; 0.87 mmol and the mixture was stirred at room temperature for 8 h, and evaporated. The residue was
poured into crushed ice and the resulting precipitates were collected by filtration to give 0.25 g 84% of the
sulfonylhydrazine derivative 12 as colorless powder: mp 201–2048C. H NMR DMSO-d6 d: 1.55 qt, 2H, Js7.0 Hz,
O5CCH2C H2CH2COOH , 2.05 m, 4H, O5CC H2CH2C H2COOH , 2.55 s, 3H, ArC H3 , 6.05 s, 2H, OC H2O , 6.96 s,
Ž
.
1
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž

(
)
L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
287
Scheme 1.
.
Ž
.
Ž
.
Ž
.
1H, Ar-H5 , 7.22 s, 1H, Ar-H2 , 9.60 d, 1H, Js2.8 Hz, O5CNH NHSO2 , 9.95 d, 1H, Js2.8 Hz, O5CNHNHSO2 ,
13
Ž
.
Ž
.
Ž
.
Ž
.
Ž
12.00 br, 1H, O5CCH2CH2CH2COOH ppm; C NMR DMSO-d6 d: see Table 1; IR KBr : 3500 n O–H , 3250 n
N–H , 1750 n C5O , 1670 n C5O , 1302 and 1116 n S–N , 1250 n C–O cm ; MS mrz : 344 M , 6% , 135
78% , 200 72% , 151 44% , 77 100% , 87 53% .
y1
H
.
.
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
Ž
.
Ž
Ž
.
Ž
.
Anal. Calcd. for C13 H16O7 N2 S: C, 45.35; H, 4.68; N, 8.14. Found: C, 45.37; H, 4.67; N, 8.16.
2.2. Pharmacology
Ž
Blood was collected from rabbits weighing 2.5–3.0 kg by puncture of the ear central artery into 3.8% sodium citrate 9:1
.
Ž
.
vrv . Platelet-rich plasma PRP was prepared by centrifugation, 500=g for 10 min, at room temperature, and platelet
count was adjusted to 5=108 plateletsrml.

(
)
288
L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
Ž
.
Platelet aggregation was monitored by the turbidimetric method Born and Cross, 1963 in a Chrono-Log aggregometer.
Ž
.
Ž
PRP 400 ml was incubated at 378C for 1 min. with continuous stirring at 900 rpm and then stimulated with ADP 5 mM in
.
Ž
.
Ž
Ž
.
Ž
.
distilled water , collagen 5 mgrml in saline , arachidonic acid AA-200 mM in ethanol or U-46619 3 mM in ethanol .
.
The aryl-sulfonamide derivatives and the dissolution solvent 0.5% DMSO, 2 ml in volume were added to the PRP
Ž
.
samples 5 min before addition of the aggregating agent. Indomethacin 10 mM , a classical cyclooxygenase inhibitor
Ž
.
Scherrer, 1974 , was used as standard.
The platelet aggregation rates were expressed as percentage of aggregation for ADP, AA and U-46619 and as the
Ž
.
maximum rate of aggregation slope for collagen.
Data were analyzed statistically by Student’s t-test for a p value of -0.05 and are expressed as mean"SD. for n
experiments in triplicate.
3. Results and discussion
Ž
.
The target new acid derivatives 10–12 were synthesized by using the methodology illustrated in Scheme 1. Our
Ž
.
synthetic approach to these new compounds identify the 6-methyl-3,4-methylenedioxy-phenylsulfonyl chloride 16 as the
key intermediate. This compound can be obtained by exploring regioselective aromatic electrophilic substitution at the C-6
Ž
.
position of phenyl ring of the precursor 3,4-methylenedioxytoluene 14 , which was obtained in ca. 49% overall yield from
Ž
.
natural safrole 13 , using base catalyzed isomerization of double bond, oxidative cleavage followed by Wolff–Kishner
reaction Fraga, 1991; Barreiro and Lima, 1992; Fraga and Barreiro, 1992; Silva and Barreiro, 1993 . The sulfonyl chloride
derivative 16 was prepared, in two steps from 14 , applying methodology previously used in this laboratory Fraga and
Barreiro, 1992 to promote a mild sulfonation of sensitive 1,3-benzodioxole derivatives. Thus, treatment of 14 with a
Ž
.
Ž
.
.
Ž
.
Ž
Ž .
mixture of acetic anhydride in ethyl acetate, containing 1.1 eq of sulfuric acid, at 08C, followed by slow addition of a
solution of potassium acetate in ethanol, furnished, as the only product, the potassium salt of 6-methyl-3,4-methylenedioxy-
1
Ž
.
Ž .
phenylsulfonic acid 15 in 91% yield. The analysis of H NMR spectra of compound 15 confirmed the anticipated
regioselectivity of this process as evidenced by the presence of two singlets signals at d 6.70 and d 7.27 ppm, performing a
Ž
.
typical AB-pattern due to tetrasubstituted phenyl system present in 15 .
Ž
.
Next, treatment of the potassium salt 15 with thionyl chloride, catalyzed by Vilsmeyer–Haack complex, afforded the
corresponding sulfonyl chloride derivative 16 in 74% yield. Using this experimental conditions we are able to obtain the
desired compound 16 , preventing the possible 1,3-benzodioxole ring cleavage that occurs by the use of classical Lewis
Ž
.
Ž
.
Ž
.
acids as POCl3 and PCl5 Fraga, 1991 .
With an attractive method to access to the key intermediate 16 in hands, we next performed the condensation step of
. Ž
Ž
.
Ž
.
Ž
this compound with tyramine 17 , in order to obtain the corresponding phenol-sulfonamide derivative 18 Fraga and
.
Ž
.
Ž
Barreiro, 1992 . In this process we were able to obtain the crystalline solid 18 , as the only product, in 91% yield Scheme
1 . Then, the synthesis of the target compounds 10 and 11 could be concluded by chemoselective O-alkylation of phenol
moiety of sulfonamide 18 , using ethyl 2-bromoacetate or methyl 2-bromopropionate as alkylating agents. In the case of
applying the classical method of phenol alkylation Hawker and Frechet, 1990 with potassium carbonate in acetone at room
.
Ž
.
Ž .
Ž
.
Ž
.
´
temperature, using ethyl 2-bromoacetate as alkylating agent, an unexpected mixture of products, characterized as the
Ž
.
Ž
. Ž
.
Ž
.
mono-alkylating 19 , and bis-alkylated 20 Chart 4 , were obtained in 37 and 18% yield, respectively Scheme 1 . This
Ž
. Ž
result can be rationalized by the similar pKa values of phenol and sulfonamide moieties present in 18 Larsen and Lish,
1964 . Unfortunately, in spite of several attempts to outline this limitation by varying the experimental conditions e.g.,
.
Ž
.
temperature, solvent, equivalent of base we have not able to introduce an adequate chemo-control favoring the O-alkylated
. Ž
Ž
.
Ž
.
Ž .
derivative 19 Magnera et al., 1984 . Thus, compounds 19 and 20 were obtained, in high degree of purity, after careful
1
Ž
.
column chromatographic separation over silica gel. The H NMR spectrum of the desired mono-alkylated compound 19
presents a singlet signal at d 4.60 ppm, attributed to a-carbonyl hydrogen’s, while the corresponding spectrum of
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.
bis-N,O-alkylated compound 20 presents an additional singlet signal at d 4.15 ppm, referred to a-nitrogen hydrogen’s.
Ž
.
Fortunately, alkylation of sulfonamide 18 with methyl 2-bromopropionate by using the same experimental conditions
described above, furnished, as the only product, the desired O-alkylated derivative 21 in 74% yield Scheme 1 . The
analysis of the H NMR spectrum of 21 indicated the presence of propionyl moiety, as shown by the quartet signal
centered at d 4.73 ppm due to a-carbonyl hydrogen’s. These results indicate that the chemoselective alkylation of 18 ,
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.
Ž
.
1
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.
Ž
.
using a secondary alkyl halide, occurs in good yield and could be rationalized as due to steric hindrance, which prevents the

Table 2
Effect of aryl-sulfonamide derivatives 10–12 and indomethacin on in vitro platelet aggregation of citrated rabbit platelet-rich plasma induced by U-46619, ADP, collagen and arachidonic acid
Ž
.
Ž
.
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.
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.
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.
Ž
.
Compound
C mM
n
U-46619 3 mM
n
ADP 5 mM
n
Collagen 5 mgrml
n
Arachidonic Acid 200 mM
Aggregation
Inhibition
Aggregation
Inhibition
Aggregation Inhibition
Aggregation Inhibition
Ž .
%
Ž .
%
Ž .
%
Ž .
%
Ž
.
Ž .
%
Ž
.
%
Ž .
%
slope
Control
Indomethacin
10
–
10
4
5
3
3
3
3
3
3
3
3
3
3
3
3
3
52.2"5.9
–
–
1.0
7.8
5
3
3
45.4"3.9
–
38.0"6.4
–
1.2
16.3
5
4
3
7.3"0.4
–
5.2"0.2
–
6
4
3
66.1"3.3
–
60.4"5.1
–
94.8U
28.8U
100.0U
8.6
100
200
400
600
800
100
200
400
600
100
200
400
600
48.1"2.5
27.6"6.1
16.2"5.1
7.3"3.0
5.9"1.5
54.3"5.6
47.5"7.4
39.4"7.3
36.9"7.1
56.5"4.6
49.4"7.0
45.4"8.5
48.1"9.1
47.1 U
68.9 U
86.0 U
88.7 U
11
12
y4.0
9.0
24.5
29.3
y8.2
5.4
3
3
33.2"6.4
43.0"3.8
26.8
5.3
3
3
5.7"0.2
5.7"0.7
21.9
21.9
3
3
64.6"1.7
59.2"5.4
2.3
10.4
13.0
7.8
a The values of the aggregation represents the means"SD; Csfinal concentration; nsnumber of independent experiments carried out in triplicate.
U
Ž
.
p-0.05 Compared to appropriate control Student’s t-test .

(
)
290
L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
access of the primary sulfonamide anion to the more bulky alkylating agent used, preventing the formation of undesirable
. Ž
Ž
.
N-alkylated product 23 Chart 4 and introducing an attractive chemocontrol in this process.
Ž
.
Ž .
Finally, the corresponding new phenoxyalkanoic acid derivatives 10 and 11 were next prepared by chemoselective
Ž
.
Ž
.
w
x
Ž
.
ester hydrolysis of 19 and 21 by using LiOH in acetone 41 , in 85 and 62% yield, respectively Scheme 1 .
The synthesis of the non-interphenylenic sulfonamide analogue 12 , was performed as illustrated in Scheme 1. The
common sulfonyl chloride intermediate 16 was first condensed with 40% aqueous hydrazine hydrate in chloroform at 08C,
Ž
.
Ž
.
Ž
.
Ž
.
Ž .
furnishing the respective sulfonylhydrazine 22 in 92% yield Fraga, 1991 . The infrared spectrum of 22 indicated the
sulfonylhydrazine moiety by presence of absorptions at 3398, 3370 and 3327 cmy1, typical to group –NH-NH2 of
Ž
.
hydrazines. With sulfonylhydrazine 22 in hands, we performed the chemoselective acylation step using glutaric anhydride
Ž
.
Ž
in chloroform, at room temperature, in order to obtain the desired 1- 4-carboxybutyryl -2- 6-methyl-3,4-methylenedio-
xyphenylsulfonyl -hydrazine 12 in 84% yield Scheme 1 . The C NMR spectrum of compound 12 presents two C5O
signals at d 170.7 and 174.0 ppm, corresponding to amide and terminal carboxylic acid moieties, respectively Table 1 .
13
.
Ž
.
Ž
.
Ž .
Ž
.
Ž
.
The observed antiplatelet activity of these new aryl-sulfonamide derivatives 10–12 is shown in Tables 2 and 3.
Indomethacin used as standard at 10 mM concentration does not interfere with the aggregation induced by ADP, but inhibits
Ž
.
100% and 94.8% the aggregation induced by arachidonic acid and collagen respectively Table 2 .
Ž
.
Curiously, at the first screening concentration of 100 mM, none of these new compounds 10–12 showed an attractive
antiaggregation inhibitory activity on ADP-, AA- or U-46619-induced rabbit platelet aggregation, when compared to the
Ž
.
Ž
.
control. In contrast, compound 10 presented a moderated platelet aggregation inhibitory activity 28.8% on the
Ž
.
collagen-induced aggregation, at screening concentration i.e., 100 mM , as illustrated in Table 2. On the other hand, this
compound strongly inhibited 83.6% the second wave of the aggregation induced by ADP on human PRP Table 3 .
Therefore these results lead us to study the concentration–response for these new compounds. Compound 10 inhibited, in
a concentration-dependent manner, the platelet aggregation induced by 3 mM of U-46619, with an IC50 of 329 mM Table
2 . In contrast, compounds 11 and 12 , even at 600 mM, were not able to inhibited at 30% level the U-46619-induced
aggregation on rabbit PRP Table 2 .
Ž
.
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.
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.
Ž
.
Ž
Ž
.
Ž .
.
These results indicate that the presence of the methylenedioxy bridge, in the phenyl ring of the sulfonamide framework,
is deleterius to the anti-platelet activity. The presence of hydrogen-bond acceptors in the aryl-sulfonamide moiety, as the
methylenedioxy bridge, seems to give no contribution to improve the TP recognition for this class of derivatives.
Ž
.
Substitution of a-carboxylic acid hydrogen atom by a methyl group 11 , almost abolish the anti-platelet activity, probably
due to steric constrain. The importance of phenyl ring in the spacer between the carboxylic acid terminus and the
Table 3
Ž
.
Effect of aryl-sulfonamides derivatives 10–12 and indomethacin on in vitro platelet aggregation of citrated human platelet-rich plasma induced by ADP
Ž
.
Ž .
ADP 5 mM Human PRP
Compounds
C mM
n
Ž
.
Aggregation %
Inhibition
Inhibition
Ž .
%
Ž .
%
Ž
.
% of 2nd wave
Control
Indomethacin
10
11
12
–
10
100
100
100
4
5
4
3
4
75.0"3.6
39.4"3.5
45.2"2.5
58.3"4.4
59.9"7.0
–
–
47.5U
39.7U
22.3
100.0U
83.6U
46.9
20.1
42.3
a The values of the aggregation represents the means"SD; Csfinal concentration; nsnumber of independent experiments carried out in triplicate.
U
Ž
.
p-0.05 Compared to appropriate control Student’s t-test .

(
)
L.M. Lima et al.rPharmaceutica Acta HelÕetiae 73 1999 281–292
291
Ž
.
aryl-sulfonamide moiety is demonstrated by the lack of anti-platelet activity in compound 12 , possessing an acyl-
Ž
.
sulfonylhydrazine chain instead the N-phenoxyethyl unit 10 and 11 .
4. Conclusion
Ž
.
Ž .
The effect on platelets of the new derivative 10 , synthesized in good overall yield from natural safrole 13 , obtained
from sassafraz oil, represents a moderate anti-platelet profile, probably through thromboxane receptor. The synthetic route
Ž
.
described herein exploring the 6-methyl-3,4-methylenedioxybenzenosulfonyl chloride 16 as a key intermediate represents
Ž
.
a useful, efficient and high yield method to access to new aryl-sulfonamide derivatives 10–12 , structurally related to
known phenylsulfonamides TPant class.
Acknowledgements
Ž
.
Ž
.
Ž
We are grateful to the CNPq BR., grant a52.0033r96-5 , FAPERJ BR., grant a171.299r95 , Pronex BR, FINEP
.
Ž
.
0888 and FUJB BR., grant a6276-6; a6487-4 and a7675-9 for the financial support of this work and to CNPq and
CAPES BR. for fellowships LML, CBO, ALPM, CAMF and EJB . The technical assistance of Mr. Renato Moreira
LASSBio is also gratefully acknowledged.
Ž
.
Ž
.
Ž
.
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