New Antithrombotic Aryl-sulfonylthiosemicarbazide Derivatives Synthesized from Natural Safrole

No âmbito de uma linha de pesquisas que trata da síntese e avaliação farmacológica de novos candidatos a protótipos de agentes antitrombóticos, explorando a hibridação molecular como estratégia de planejamento estrutural, descrevemos neste trabalho a síntese e avaliação farmacológica de novas sulfonilsemicarbazidas (10a-d), planejadas por analogia estrutural a antagonistas de receptores de TXA2, utilizando o safrol (9), produto natural brasileiro abundante, isolado do óleo de Sassafrás, como matéria-prima sintética. A avaliação das propriedades antiagregantes plaquetárias das sulfonilsemicarbazidas (10a-d) no modelo induzido por ADP, colágeno, ácido araquidônico e U46619, permitiu evidenciar um importante perfil antitrombótico ao nível da cascata do ácido araquidônico para o derivado 6-metil-3,4-metilenodioxifenil-sulfonil-N-feniltiosemicarbazida (10d), que representa um novo protótipo de agentes anti-trombóticos.

As part of an ongoing research program devoted to identifying new antithrombotic candidates on the basis of the structure-activity relationships of TP antagonists, we described previously a new series of sulotroban ana-logues 16 , e.g.derivative (8), exploring natural safrole (9), an abundant Brazilian natural product occurring in the Sassafras oil 17,18 , as starting material.Thus, we describe herein the planning, the synthesis and the anti-platelet properties of new aryl-sulfonylsemicarbazide derivatives with the general structure (10), which were structurally planned as a hybrid of both SQ 29.548 (4) and sulotrobanrelated derivative (8) (Fig. 1).
All these new compounds (10a-d) were structurally designed in order to explore a new possible bioisosteric relationship between the 1,3-benzodioxole ring, present in the natural product used as starting material, and the oxabicyclo[2.2.1]heptane system of (4-6), considering the hydro- gen-bonding acceptor character of both subunits as represented by the oxygen atom indicated in Figure 1.The introduction of the sulfonyl group on the semicarbazide side chain, was performed with the aim to study the eventual contribution of this multiple hydrogen-bonding acceptor/donor functionality in the antiplatelet activity of these new compounds (10a-d).

Results and Discussion
The new target derivatives, aryl-sulfonylsemicarbazides, (10a-d) were synthesized as depicted in Scheme 1.Our synthetic approach to these new compounds identified 6methyl-3,4-methylenedioxy-phenylsulfonyl chloride (14)  as the key intermediate (Scheme 1).This compound can be obtained by regioselective aromatic electrophilic substitution at the C-6 position of the precursor 3,4-methylenedioxytoluene (12), which was obtained in ca.50% overall yield from natural safrole (9).Simple distillation of Sassafras oil yielded 9 in 85% yield.Base catalysed isomerization of the double bond followed by oxidative cleavage and Wolff-Kishner reduction yielded 12 [19][20][21][22] .The arylsulfonyl chloride derivative (14) was next prepared in two steps from (12), applying methodology previously used in this laboratory to promote an efficient and mild sulfonation of the sensitive 1,3-benzodioxole derivatives 16,20 .Thus, treatment of (12) with a mixture of acetic anhydride in ethyl acetate, containing 1.1 eq. of sulfuric acid, at 0 °C, followed by careful addition of a solution of potassium acetate in ethanol, furnished as the only product, the potassium salt of 6-methyl-3,4-methylenedioxy-phenylsulfonic acid (13)  in 91% yield.The analysis of 1 H-NMR spectra of compound (13), confirmed the anticipated regioselectivity of this process as evidenced by the presence of two singlet signals at δ 6.70 and 7.27.The next step in the planned synthetic route was the treatment of the potassium salt (13)  with thionyl chloride, catalyzed by the Vilsmeyer-Haack complex, to afford the desired arylsulfonyl chloride derivative (14) in 74% yield 16,19,20 .
With an attractive method for access to the key intermediate (14) in hand, we next performed the condensation step of this compound with 40% aqueous hydrazine hydrate in chloroform at 0 °C to obtain the corresponding arylsulfonylhydrazine (15) in 92% yield 16,18 .The infrared spectrum of (15) indicated the sulfonylhydrazine moiety by the presence of absorptions at 3398, 3370 and 3327 cm -1 , typical for the group -NH-NH 2 .Finally, the synthesis of the novel arylsulfonylsemicarbazide (10a-d) was concluded in good yield (Table 1), by treatment of the arylsulfonylhydrazine derivative (15) with phenylisocyanate, 4-chlorophenylisocyanate, 4-bromophenylisocyanate and phenylisothiocyanate, respectively, in tetrahydrofuran at room temperature 23 (Scheme 1).The 13 C-NMR spectrum of compounds (10a-d) (Table 2) shows the C=O and C=S signals at δ 154.7 and 180.8, corresponding to semicarbazide and thiosemicarbazide moieties, respectively.
The antithrombotic activity of these novel arylsulfonylsemicarbazide derivatives (10a-d) was evaluated by their ability to inhibit platelet aggregation of rabbit platelet-rich plasma (PRP) induced by adenosine diphosphate (ADP, 5 µM), or collagen (90 µM), or arachidonic acid (AA, 200 µM) or U-46619 (3 µM), a stable analogue of TXA 2 , used as a pharmacological probe to TP.The results of the antiplatelet profile of (10a-d) are displayed in Table 3.
Consistent with these compounds exerting their effects at the arachidonic acid cascade level was that none of them were effective in preventing platelet aggregation induced by ADP.Compound (10d), belonging to the phenylthiosemicarbazide class, was able to significantly inhibit the platelet aggregation induced by collagen (25.9%) at 90 µM concentration.In contrast, this compound was not able to promote any platelet inhibition when induced by arachidonic acid.It is known that in rabbit PRP, the collagen induced aggregation is partially mediated by TXA 2 formation 24 .Thus, in order to evaluate the activity profile of these compounds at different concentrations, we decided to investigate the biological response at 200 µM concentration.At this concentration, compound (10d) presented a very important anti-platelet profile, inhibiting by 99.8% and 49.1% the aggregation induced by arachidonic acid and collagen respectively.These results indicate an antithrombotic activity at the arachidonic acid cascade level, and consequently it was decided to evaluate the effect of these compounds on the platelet aggregation induced by U-46619 (3 µM), in order to verify any eventual TPant properties.In this bioassay (Table 3) compound (10d) effectively inhibited the platelet aggregation by 24.4% at a O O H O O O 6 (10a) X=O, W=H (10b) X=O, W=Cl (10c) X=O, W=Br (10d) X=S, W=H Scheme 1. a) 3 N KOH/nBuOH, reflux, 6 h, 96%; b) 1) O2/O3, AcOH, 0 °C; 2) Zn, AcOH, 0 °C, 4 h, 70%; c) KOH, N2H4.H2O, ethyleneglicol, reflux, 6 h, 73%; d) 1) H2SO4, Ac2O, AcOEt, 0 °C; 2) AcOK, EtOH.r.t., 30 min., 91%; e) SOCl2, DMF(cat.), reflux, 4 h, 74%; f) 40% aq.N2H4.H2O, CHCl3, 0 °C, 2 h, 92%; g) 4-W-C6H4NCX, THF, r.t., 12 h.200 µM concentration in this assay.These results seem to indicate that (10d) does not act at the TP level but does prevent platelet aggregation by acting upon the arachidonic acid cascade, probably at the TXS level.It is important to mention that compound (10c), belonging to the paraphenylsemicarbazide substituted class, at 200 µM concentration, was the most active one (26%) in the assay induced by U-46619.The activity presented by both para-halogenated phenylsemicarbazide derivatives (10b and 10c), in comparison to the effect presented by the derivative (10a), seems to indicate that the presence of an halogen atom at the para-position of the phenyl ring of the semicarbazide moiety could improve the anti-platelet activity, in the assay induced by U-46619, in this series of derivatives.

Conclusions
As concluding remarks, the synthetic route described herein for access to these new aryl-sulfonylsemicarbazide and phenylsulfonylthiosemicarbazide derivatives (10a-d), structurally planned as hybrids of known TPant compounds, possessing the semicarbazide group (e.g.SQ-29548, 4), represent a useful, efficient and high yield

Chemistry
Melting points were determined with a Thomas-Hoover apparatus and are uncorrected.Proton magnetic resonance ( 1 H-NMR) spectra were determined in deuterated solvents, using tetramethylsilane as an internal standard with a Brucker AC 200 spectrometer.Splitting patterns were as follows: s, singlet; d, doublet; m, multiplet.The carbon magnetic resonance spectra ( 13 C-NMR) were determined in the same spectrometer 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 pellets.The mass spectra (MS) were obtained on a GC/VG Micromass 12 at 70 eV.
The progress of all reactions was monitored by TLC which was performed on 2.0 cm x 5.0 cm aluminum sheets precoated with silica gel 60 (HF-254, Merck) to a thickness of 0.25 mm.The developed chromatograms were visualized 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 Å molecular sieves.

Platelet Aggregation
Blood was collected from rabbits by puncture of the central ear artery into 3.8% sodium citrate (9:1 v/v).Platelet-rich plasma (PRP) was prepared by centrifugation, 500 x g for 9 min, at room temperature, and platelet count was adjusted to 5 x 10 8 platelets/mL.
Test compounds (10a-d) and the vehicle (0.5% DMSO, 2 µL) were added to the PRP samples 5 min.before addition of the aggregating agent.The DMSO used as vehicle did not have either pro-or antiplatelet aggregation activity.Indomethacin (9 µM), a classical cyclooxygenase inhibitor, was used as standard.
The platelet aggregation was expressed as percentage of aggregation for ADP, AA, U-46619 and as the 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.
a ca.60 mg of compound in 0.7 mL of DMSO-d6.
n -number of independent experiments carried out in triplicate.* p < 0.05 compared to appropriate control (Student's "t" test).