Synthesis of Thieno[2,3-b]thiophene Containing Bis-Heterocycles-Novel Pharmacophores

Thioenethiophene derivatives represent an important class of compounds with diverse biological activities. We describe here the synthesis of a new series of thieno[2,3-b]thiophene containing bis-heterocyclic compounds 3–7. All the compounds were evaluated for their in vitro antioxidant potential, α-glucosidase and β-glucuronidase inhibiton and anticancer activity against PC-3 cell lines. Compounds 2b (IC50 = 1.3 ± 0.2 μM), 5a (IC50 = 2.3 ± 0.4 μM) and 5b (IC50 = 8.7 ± 0.1 μM) showed a potent inhibition of β-glucuronidase enzyme, more active than the standard d-saccharic acid 1,4-lactone (IC50 = 45.8 ± 2.5 μM). Compounds 5a (IC50 = 22.0 ± 0.3 μM) and 5b (IC50 = 58.4 ± 1.2 μM) were also found to be potent α-glucosidase inhibitors as compared to standard drug (acarbose, IC50 = 841 ± 1.7 μM).

As part of our continuing efforts toward the synthesis of bisheterocycle system and convenient access to the diverse frameworks present in them, we have explored the synthetic potential and generality of a cyclization strategy to gain facile entry into the thieno [2,3-b]thiophene framework from a conveniently accessible precursor as shown in Scheme 1. Our new results on the thieno[2,3-b]thiophene derivatives, including chemical synthesis, their in vitro antioxidant potential, α-glucosidase and β-glucuronidase inhibition, and anticancer activity against PC-3 cell lines are presented herein.

Chemistry
The synthesis of the pyrimidine, pyrazole, triazolopyrimidine and imidazopyrimidine derivatives 4-7 were carried out by reacting commercially available benzoylacetone 1, dimethylformamide dimethylacetal (DMF-DMA), 4-amino-1,2,4-triazole and 2-aminobenzimidazole. Previously 1,1′-(3-methyl-4-phenylthieno[2,3-b]thiophene-2,5-diyl)diethanone 2 was synthesized starting from benzoylacetone 1. The method consists of sequential directed nucleophilic addition, side chain deprotonation, nucleophilic addition, and cyclization using a nitrogen or sulfur moiety as internal nucleophile. Condensation of 2 with dimethylformamide dimethyl acetal (DMF-DMA) under reflux for ten hours in the presence of xylene furnished enaminone 3. Reaction of enaminone 3 with N-nucleophiles, such as urea derivatives in dioxane or EtOH/DMF mixture under reflux for four to six hours in the presence of a catalytic amount of ZnCl 2 as a Lewis acid afforded 4a-c. The formation of compounds 4a-c would involve an initial addition of the amino group in urea to the activated double bond in enaminone derivative 3, followed by deamination to an intermediate, which then undergoes cyclization and aromatization via loss of water, affording the final isolable product. Similarly, enaminone derivative 3 cyclized with hydrazine compounds by refluxing with absolute ethanol for six hours. The novel bispyrazole 5a was assumed to be formed via addition of the amino group in the hydrazine to the activated double bond of the enamine derivative, followed by deamination, dehydration, and subsequently nucleophilic cyclization to afford the final product. The utility of enaminone 3 in the synthesis of annelated heterocycles was further explored via its reaction with 4-amino-1,2,4-triazole in absolute ethanol under reflux for seven hours in the presence of a catalytic amount of ZnCl 2 affording compound 6. The study was extended to investigate the behavior of enaminone derivatives 3 with different nucleophiles such as 2-aminobenzimidazole in order to synthesize various heterocyclic ring systems. Thus, the reaction of 3 with 2-iminobenzimidazole in refluxing ethanol in the presence of catalytic amount of ZnCl 2 furnished the corresponding product 7. The structures of these compounds were determined by 1 H-NMR, EI, IR and UV spectroscopic, and micro analyses for carbon, hydrogen and nitrogen [22]. Scheme 1. Synthesis of compounds 2-7.

Biological Activity Evaluation
Compounds 3, 4b, 5a, 5b and 7 were evaluated for a variety of biological activities including β-glucuronidase and α-glucosidase inhibiton, antioxidant, and anticancer properties by using non-physiologica and in vitro biochemical and mechanism-based assays. The results of the assays are presented in Table 1. Table 1. Results of various biological assays on compounds 3-7.
Compounds 3-7 were also evaluated for their inhibitory effects against other enzymes such as carbonic anhydrase, α-chymotrypsin, xanthine oxidase, and phosphodiesterase. However, no activity was observed. The results indicate that compounds 5a and 5b have specific and signifcant biological potential and deserve to be further investigated.

Experimental Section
All melting points were measured on a Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. IR Spectra were measured as KBr pellets on a Perkin Elmer FT 1000 spectrophotometer. The NMR spectra were recorded on a Varian Mercury Jeol-400 NMR spectrometer (400 MHz) and 13 C-NMR (100 MHz) spectra were measured in deuterated dimethylsulphoxide (DMSO-d 6 ). Chemical shifts (δ) are referred in terms of ppm and J-coupling constants are given in Hz. Abbreviations for multiplicities are as follows: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer at 70 eV. Elemental analysis was carried out on an Elementar Vario EL analyzer. (2) A mixture of benzoylacetone 1 (16.2 g, 0.1 mol) and anhydrous potassium carbonate (25 g) in DMF (30-40 mL) was stirred vigorously at room temperature for 5 min then carbon disulfide (7.6 mL, 0.1 mol) was added with continued stirring for 30 min. The resulting mixture was cooled in ice bath, then chloroacetone (18.5 mL, 0.2 mol) was added with continued stirring for 15 min, then cooling bath subsequently removed, and the mixture was stirred for further 30 min. The solid product was collected by filtration and washed with water, dried, and the crude product was recrystallized from glacial acetic acid to obtain pale green crystals. Yield

General Procedure for the Synthesis of Compounds 4a-c
A mixture of compound 3 (0.212 g, 0.5 mmol), urea dervitives (2 equiv., 1 mmol) refluxed in dioxane (20 mL) for 4-6 h after in the presence of 0.5 mL of TEA and catalytic amount of ZnCl 2 . After cooling, the resulting solid products were filtered off, washed with ethanol, dried, and recrystallized from DMF/EtOH, afford the corresponding derivatives 4a-c.

General Procedure for the Synthesis of Compounds 5a,b
A mixture of compound 3 (1 mmol) and an excess of hydrazine derivatives (1 mL) were refluxed in EtOH (20 mL) for 6 h. After cooling, the resulting solid products were filtered off, washed with ethanol, dried, and recrystallized from MeOH, to obtain corresponding derivatives 5a,b. growing cells were harvested, counted with a haemocytometer, and diluted with a particular medium with 5% FBS. Cell culture with the concentration of 1 × 10 5 cells/mL was prepared and introduced (100 μL/well) into 96-well plates. After overnight incubation, medium was removed and 200 μL of fresh medium was added with different concentrations of compounds (1-30 μM). Stock solution, 20 mM of compounds were prepared in 100% DMSO, and the final concentration of DMSO at 30 μM is 0.15%. After 48 h, 200 μL MTT (0.5 mg/mL) was added to each well and incubated further for four hours. Subsequently, 100 μL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 570 nm, using a microplate reader (SpectraMax plus, Molecular Devices, Sunnyvale, CA, USA). The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC 50 ) for PC3 cells. The percent inhibition was calculated by using the following formula: % Inhibition = 100 − ((mean of OD of test compound − mean of OD of negative control)/ (mean of OD of positive control − mean of OD of negative control) × 100).
The results (% inhibition) were processed by using SoftMax Pro software (Molecular Device, USA).

In Vitro Antioxidant Activity
Test samples were allowed to react with stable free radical, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH, Wako Chemicals, Shanghai, China) for half an hour at 37 °C. Various concentrations of test samples (prepared in DMSO) were incubated with DPPH (300 μM; prepared in ethanol). After incubation, decrease in absorption was measured at 515 nm by using a microplate reader (SpectraMax plus 384). Percentage radical scavenging activity (% RSA) of samples was determined in comparison with a DMSO-treated control group. Percentage of radical scavenging activity was calculated by using the formula given in statistical analysis section [23].
3.6.3. In Vitro β-Glucuronidase Inhibition Assay β-Glucuronidase inhibitory activity was determined by the spectrophotometric method by measuring the absorbance at 405 nm of p-nitrophenol formed from the substrate p-nitrophenyl-β-D-glucuronide N1627-250 mg (Sigma Aldrich Co., 3050 spruce street, St. Louis, MO, USA). The total reaction volume was 250 μL. The compound (5 μL) was dissolved in DMSO (100%), which became 2% in the ultimate assay (250 μL) and similar conditions were used for standard (D-saccharic acid 1,4-lactone, Sigma Aldrich Co.). The reaction mixture contained 185 μL of 0.1 M acetate buffer, 5 μL of test compound solution, 10 μL of (1U) enzyme solution (G7396-25KU, Sigma Aldrich) was incubated at 37 °C for 30 min. The plates were read on a multiplate reader (SpectraMax plus 384) at 405 nm after the addition of 50 μL of 0.4 mM p-nitrophenyl-β-D-glucuronide. All assays were performed in triplicate. IC 50 Values were calculated by using EZ-Fit software (Perrella Scientific Inc., Amherst, MA, USA). These values are the mean of three independent readings [24].

Statistical Analysis
All reactions were performed in triplicate in a final volume of 200 μL. The results were processed using SoftMax Pro 4.8 software (Molecular Devices, Sunnyvale, CA, USA) and then using MS Excel. The percentage of inhibition was calculated by the following formula: % Inhibition = 100 − (OD of test sample/OD of the control) × 100

Conclusions
In summary, the present investigation describes an efficient method for the synthesis of novel bisheterocycles, many of which may display interesting biological activities in the field of medicinal chemistry. Compounds 4b, 5a,b showed the inhibitory effect against β-glucuronidase enzyme, which is highly expressed in cancer, rheumatoid arthritis, and AIDS. Compounds 5a and 5b also showed a potent inhibition of α-glucosidase enzyme and can serve as potential lead molecules for further investigation.