Synthesis of new dithiolethione and methanethiosulfonate systems endowed with pharmaceutical interest

Here we report synthetic methodology affording in the most efficient way the rapid preparation of new dithiolethiones (DTTs) and methanethiosulfonates (MTSs). These were evaluated as STAT3 inhibitors since these electrophilic systems could react with thiol groups of STAT3-SH2 domain. The results showed that MTSs strongly interacted with the SH2 domain, whereas the corresponding DTTs possessed lower affinity, independently from the nature of the linked heterocyclic scaffold


Introduction
Recent literature 1,2 shows considerable interest in the chemistry and bioactivity of 1,2-dithiole-3-thiones (DTTs) and methanethiosulfonates (MTSs).4][5][6][7] Since these electrophilic systems are able to react with thiol groups, and therefore, hypothetically, with cysteines in biologically important peptides and proteins, 8,9 we considered that the introduction of these moieties on a suitable scaffold could lead to new inhibitors able to covalently link the active site of several transcription factors.A similar behavior was observed between a DTT derivative and NFkB (Nuclear Factor Kappa-light-chain-enhancer of activated B cells). 10 As STAT3 (Signal Transducer and Activator of Transcription 3) occupies a noteworthy position in cancer biology, 11 we decided to explore the relevance of these chemical systems against this target.Among the recognized STAT3 direct inhibitors reported in the literature many are sulfur-containing compounds, such as sulfones (Stattic), 12 sulfonates (S31-201) 13 or sulfonamides (SF-1-066 and BP-1-102), 13 but no DTTs or MTSs have been so far considered.As we recently published our results on several oxadiazole derivatives able to interfere with the STAT3 signaling pathway, [14][15][16][17] we thought it of interest to link this heterocyclic scaffold to DTT and MTS moieties.In this way, we aimed to promote the targeting of the new compounds towards the SH2 (Src Homology 2) domain.In this paper, we describe synthetic methodology affording in the most efficient way the rapid preparation of new inhibitors having DTT or MTS systems linked to differently substituted 1,2,5-oxadiazole rings through an ester (1, 4 and 7) or an amidic bond (3, 6 and 8) (Figure 1).In addition, we synthesized the bioisosteric analogues of 1 and 4, by replacing the oxadiazole with a substituted N-methylimidazole (2 and 5).
The synthesis of 12, bearing a suitable 3-hydroxymethyl substituent, was optimized starting from the commercially available para-chlorocinnamic acid to give 11, 20 the N-oxide moiety of which was removed by treatment with trimethyl phosphite 21 (Scheme 1a).Compound 15 was prepared from 1H-imidazole-4-carbaldehyde, protected as N-methyl derivative by iodomethane 22  (13), then transformed into 14 through a selective palladium-catalyzed C-H activation in position 5. 23 The subsequent reduction step afforded the desired product 15 in good yield (Scheme 1b).
The intermediates 23, 25 and 27 were synthesized from the appropriate benzaldehyde with some variations on the multi-step procedure previously reported 16 (Scheme 2).Since prolonged (12 hours) treatment in refluxing aqueous NaOH of 20a caused an approximately 50% removal of the Boc (tertbutyloxycarbonyl) group, an additional treatment with tert-butyloxycarbonyl anhydride (Boc 2 O) was required to give 21a.The amides 22 and 24 were obtained by coupling reactions of intermediates 21a,b with 4-(trifluoromethyl)benzoyl chloride, respectively, while the reaction of compound 21c with 4-nitrobenzoyl chloride gave 26.
Deprotection of 22 in acidic conditions led to 23; compound 24 was debenzylated by a catalytic hydrogenation over Pd/C to 25; compound 26 was reduced in the presence of tin(II) chloride to 27.The DTT products 1-3 were obtained in good yields (59-78%) and were easily isolated and purified, thanks to their stability and low solubility.

Biology
The effects of the final compounds on STAT3 dimerization were determined by the AlphaScreen-based assay, 24 an in vitro competitive binding test used to identify compounds able to directly inhibit the binding of SH2containing proteins to their correspondent phosphopeptides.To check the selectivity on STAT3, the new products were tested also on the highly homologous (78%) STAT1.
In addition, their cytotoxic activity (MTT assay) 25,26 was tested on HCT116, a human colon carcinoma cell line which expresses high levels of STAT3. 27Among the assayed compounds, 6 was the most active (IC 50 = 84.5±9.8 µM).Since STAT3 inhibition was tested in a cell-free assay, the low correspondence between STAT3 inhibition and cytotoxicity could be related to the physicochemical properties of the compounds, which will require optimization.
Based on these data, the MTS moiety appears worth of further investigation for targeting STAT3-SH2 domain.

Conclusion
In our paper, we describe an easy and direct synthetic approach for the preparation of DTT and MTS derivatives, bearing a 1,2,5-oxadiazole ring, which has been reported as a promising scaffold for STAT3 inhibitors.The bioisosteric replacement of the oxadiazole with an N-methylimidazole ring was also considered.Due to their better stability, the DTTs (1-3) were obtained in higher yields than the MTSs (4-8).
The binding efficiency towards STAT3 and STAT1 SH2 domain was evaluated by means of AlphaScreenbased experiments.In addition, their antiproliferative activity was investigated on HCT116 cancer cell line.The biological results showed that MTSs interacted more strongly with SH2 domain with respect to the corresponding DTTs.Although we presume that MTS derivatives can react more easily than DTTs with the free thiol group of cysteine residues present in the protein, further studies are required to elucidate the real mechanism of inhibition and the different reactivities of MTSs and DTTs.
In particular, compound 6 showed a higher affinity and selectivity (STAT3 versus STAT1), together with a moderate cytotoxic activity.These data suggest that MTS derivatives are promising ligands of the STAT3-SH2 domain.Optimization of their physicochemical properties is still needed to increase their cytotoxicity.

Experimental Section
General.Reagents and solvents were purchased from Sigma-Aldrich and used without further purification.Reactions involving air-sensitive reagents were performed under nitrogen atmosphere and anhydrous solvents were used when necessary.Reactions were monitored by thin layer chromatography analysis on aluminumbacked Silica Gel 60 plates (70-230 mesh, Merck), using an ultraviolet fluorescent lamp at 254 nm and 365 nm.Visualization was aided by appropriate staining reagents.Purification of intermediates and final compounds was performed by flash chromatography using Geduran® Si 60 (40-63 µm, Merck).DMEM (Dulbecco's Modified Eagle Medium), trypsin-EDTA, penicillin, streptomycin, non-essential amino acid solution, fetal calf serum (FCS), disposable culture flasks and petri dishes were purchased from Euroclone S.p.A. (Pero, Milan, Italy). 1 H and 13 C NMR spectra were recorded in CDCl 3 , CD 3 OD, D 2 O, DMSO-d 6 or acetone-d 6 on Bruker DRX Avance 300 MHz or on a Varian 300 MHz Oxford equipped with a non-reverse probe at 25 o C. Chemical shifts are expressed as δ (ppm) and were referenced to residual solvent proton/carbon peak.Multiplicity is reported as s (singlet), broad s (broad singlet), d (double), t (triplet), q (quartet), m (multiplet), dd (double of doublets), dt (doublet of triplets).The coupling constants (J-values) are given in Hertz (Hz).All spectroscopic data match the assigned structures.ESI-MS analyses were performed by using a Thermo Finnigan (MA, USA) LCQ Advantage system MS spectrometer with an electronspray ionization source and an 'Ion Trap' mass analyzer.The MS spectra were obtained by direct infusion of a sample solution in methanol under ionization, ESI positive.Highresolution mass spectra (HRMS) were performed by FT-Orbitrap mass spectrometer in positive electrospray ionization (ESI).The melting points were determined on a Buchi Melting Point B540 instrument.
General procedures for the synthesis of dithiolethiones (1-3) and methanethiosulfonates (4-8) Procedure A. Compound 12, 15 or 27 (0.1 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC*HCl, 0.15 mmol) and 4-dimethylaminopyridine (DMAP, 0.02 mmol) were mixed together in anhydrous N,N-dimethylformamide (1 mL) and the relevant sulfurated compound (9 18 or 10 19 , 0.11 mmol) was added.The reaction mixture was stirred for 24 h, at rt under an inert atmosphere.The solvent was stripped off and the obtained residue was diluted with EtOAc and washed with a cold solution of 0.5 N HCl and then with a cold solution of 5% NaHCO 3 , cold water and brine.The organic layer was dried with anhydrous Na 2 SO 4 , filtered and evaporated to dryness to get a residue that was purified by flash chromatography (eluent as indicated for each compound).Procedure B. The appropriate alcohol (12, 15 and 25, 0.13 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 0.14 mmol) and DMAP (0.01 mmol) were mixed in anhydrous DMF and 5-(methylsulfonylthio)pentanoic acid (10, 19 0.14 mmol) was added.The reaction mixture was stirred for 4 or 20 h depending on the involved alcohol derivative, at rt under inert atmosphere.After completion, the formed N,N'-dicyclohexylurea was filtered off and the solvent was evaporated.The obtained residue was than dissolved in EtOAc or CH 2 Cl 2 and washed with a cold solution of 0.5 N HCl, afterward with a cold solution of 5% NaHCO 3 , then with cold water and brine.The organic layer was dried with anhydrous Na 2 SO 4 , filtered and evaporated to dryness to get a residue that was purified by flash chromatography or preparative silica-TLC (eluent as indicated for each compound).Procedure C. Compound 23 (0.08 mmol) and the correspondent sulfurated compound (9 18 or 10 19 , 0.09 mmol) were mixed together in anhydrous N,N-dimethylformamide (1 mL) under inert atmosphere.After cooling to 0 °C, N,N,N',N'-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU, 0.1 mmol) and Nmethylmorpholine (NMM, 0.08 mmol) were added and the reaction mixture was stirred for 24 h at rt.After evaporation of the solvent, the residue was taken up with CH 2 Cl 2 or EtOAc and with a cold solution of 0.5 N HCl, a cold solution of 5% NaHCO 3 , cold water and brine.The organic layer was dried with anhydrous Na 2 SO 4 , filtered and evaporated to dryness.The obtained solid residue was then rinsed and recrystallized with the solvent indicated for each compound.

Figure 1 .
Figure 1.Structures of the new synthesized compounds.