Microwave-assisted Synthesis of Hybrid Heterocycles as Potential Anticancer Agents

In a one pot procedure, a series of novel hybrid heterocycles 6a–g and 7a–g were prepared by condensation of (3aS,4S,6S,6aS)-6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbaldehyde 5 with mercapto acids and primary amines in the presence of ZnCl2 under both microwave irradiation and conventional heating conditions. Compound 5 was prepared from di-acetone D-mannose via a click reaction, primary acetonide deprotection and oxidative cleavage. Characterization of new compounds has been done by IR, NMR, MS and elemental analysis. Anticancer activity of the compounds has also been evaluated.


Introduction
1,2,3-Triazoles are one of the most important classes of heterocyclic organic compounds, which are reported to be present in a plethora of biologically active compounds, useful for diverse therapeutic areas. 1 The 1,2,3-triazole motif is associated with diverse pharmacological activities, such as antibacterial, antifungal, hypoglycemic, antihypertensive and analgesic properties. Polysubstituted five-membered aza heterocycles rank as the most potent glycosidase inhibitors. 2 Further, this nucleus in combination with or in linking with various other classes of compounds such as amino acids, steroids, aromatic compounds, carbohydrates etc., became prominent in having various pharmacological properties. 3 1,2,3-Triazole modified carbohydrates have became easily available after the discovery of the Cu(I)-catalyzed azide-alkynes 1,3-dipolar cycloaddition reaction 4 and quickly became a prominent class of non-natural sugar derivatives. The chemistry and biology of triazole modified sugars is dominated by triazole glycosides. 5 Therefore, the synthesis and investigation of biological activity of 1,2,3-triazole glycosides is an important objective, which also received a considerable attention by the medicinal chemists.
So called hybrid molecules have been shown to be highly active and effective in medicinal chemistry. Synergistic effects are obtained via hybridization of two different bioactive moieties with complementary pharmacophoric functions, or with different modes of action. 14 The confirmation of this hypothesis has been well established in previous studies of 4-thiazolidinones coupled with other heterocyclic fragments, 15 i.e. resulting in high anti tumor activity.
Microwave irradiation is an alternative heating technique based on the transformation of electromagnetic energy into heat. Often this method increases the rate of chemical reactions 16 and results in higher yields. In recent years, multi component reactions (MCRs) 17 have received interesting attention due to their simplicity, efficiency, atom economy, shortened reaction times, and the possibility for diversity oriented synthesis.
Following the successful introduction of triazoles and thiazolidinones, microwave-assisted MCR reactions, inspired by the biological profile of triazoles, thiazolidinones, and in the continuation of our work on biologically active heterocycles [18][19][20][21][22][23][24][25][26][27][28][29] we have developed a series of novel triazole-linked thiazolidenone derivatives, we have investigated the application of microwave irradiation for the synthesis of our hybrid molecules and evaluated their anticancer activity.

Result and Discussion
Di-acetone D-mannose (1), prepared from D-(+)mannose by treating with acetone in the presence of a catalytic amount of sulphuric acid according to the literature procedure, 30 on subsequent propargylation in DMF in the presence of NaH in 1 h afforded propargyl ether 2 (80%). Next, the propargyl ether was converted into triazole 3 Sheme 2. Reagents and conditions: (a) Propargyl bromide, NaH, DMF, 0 °C → rt; (b)  (82%) by using a 1,3-dipolar cycloaddition with p-chlorophenyl azide, which was carried out at ambient temperature in the presence of CuSO 4 and glucose which reduced CuSO 4 in a mixture of 1:1 t-BuOH-H 2 O. Acid hydrolysis of 5,6-acetonide 3 in 60% AcOH furnished the diol 4 (85%), which on oxidative cleavage with NaIO 4 gave the aldehyde 5 (Scheme 1). Subsequently one pot synthesis of triazole-linked thiazolidinone glycosides was carried out by the condensation reaction between 5, primary aromatic amine and a thioglycolic acid and thiomalic acid in the presence of ZnCl 2 under microwave irradiation or conventional heating (Scheme 2). In the classic method, the reactions were performed in dry toluene at reflux for a long time (2-4 h), often leading to degradation processes and consequent low yields of isolated products, whereas upon the application of microwave-assisted technology the reaction was completed in only 5-10 minutes and the compounds, isolated by conventional work-up, were obtained in satisfactory yields, often higher than those achieved by traditional methods (Table 1). The structures of synthesized compounds were confirmed by IR, NMR, MS and elemental analysis. Further, the compounds were subject to anticancer testing.

Cytotoxicity Evaluation Against Different Cancer Cell Lines
The cytotoxic effect of the compounds was tested by performing a Sulforhodamine B Assay (SRB) on different representative cell lines. Initially, the cell line of interest was seeded in a flat bottom 96-well plate (5000 cells/100 μL) in a medium containing 10% serum, followed by incubation for 18-20 h in an incubator continuously supplied with 5% CO 2 , so as to ensure proper adherence of the cells to the surface bottom of the wells. After 18 h the cells were treated with the compound. Working dilutions of concentration of the compounds were prepared, of which 2 μL aliquot was added to each well, thereby making the final concentration of compound 0 to 100 μM. Each compound was tested in triplicate and the cytotoxicity was determined as the average of that triplicate. DM-SO and doxorubicin (as standard control anti cancer drug) were taken as vehicle and positive controls, respectively. Further, the plates were incubated for another 48 h in an incubator maintained at 37 °C with a constant supply of 5% CO 2 . After the period of 48 h, the cells were fixed using 10% TCA solution and incubated for 1 h at 4°C after which the plate was rinsed carefully with MQ water and air dried; this was followed by addition of 0.057% SRB solution which was kept for approx. 30 min before it was rinsed off using 1% acetic acid. The plates were then air dried and the absorbance was measured using Perkin-Elmer Multimode Reader at 510 nm. To measure the absorbance, 100 μL of 10 mM Tris Base was added to each well to solubilize the SRB. The value of absorbance is directly proportional to cell growth and is thus used to calculate the IC 50 values. In this study for initial screening, four types of cancer cell lines, i.e. human lung cancer (A549), human breast cancer (MCF-7), prostate cancer (DU145) and HeLa cell lines were tested for the cytotoxic effect of the series of compounds. Based on the IC 50 values obtained, the compound 7b was picked for further assays to ascertain its effect on prostrate cancer cell line (DU145).

1. Change in Morphology
Based on the cytotoxic ability of the compound, its effect on the morphology of the cells was also ascertained. To achieve this, a 24-well plate was seeded with cells in a manner previously described and incubated for 18-20 h. Then, the cells were treated with increasing concentra-

2. Colony Formation Assay
The long term effect of the 7b on the anchorage independent nature of cancer cells was further tested in the following experiment. The experiment was a soft agar assay which was conducted as reported previously with minor modifications. In the experiment, base agar was prepared by mixing 1% of agarose (Bacto Agar: Becton, Dickinson, Sparks, MD) with 2 × DMEM along with 20% FBS and 2X antibiotics in 6-well plates in order to achieve final concentration of 0.5% of agar in 1X growth medium with 10% serum concentration. After the solidification of the base agar, 2.5 × 10 4 cells were mixed with cultivation medium containing compound at varying concentrations along with agar solution to obtain a final concentration of 0.35% agar. This was spread on top of the base agar previously solidified. The plate was incubated for 9 days with periodic replenishment every 3 days with medium and compound. Over the period of time, plates were monitored regularly for appearance of colonies. After 9 days of incubation the plates were stained with 0.005% crystal violet solution until colonies turned purple in color. The excessive stain was washed off using MQ water and the colonies were photographed and counted using a microscope.

Determination of Caspase-3 and Caspase-9 Activities
Caspase activity, specifically, caspase-9 and caspase-3 activities were analyzed in the cell lysates obtained from DU 145 cells previously treated with compound 7b. The activity was observed using fluorogenic substrates, namely Ac-DEVD-AMC and Ac-LEHD-AFC for caspase-3 and caspase-9, respectively. After 48 h treatment of cells with compound 7b, harvested cells were lysed directly in caspase lyses buffer (50 mM HEPES, 5 mM CHAPS, 5 mM DTT, pH 7.5). The lysates were incubated with the respective substrate (Ac-DEVD-AFC/Ac-LEHD-AMC) in 20 mM HEPES (pH 7.5), 0.1% CHAPS, 2 mM EDTA and 5 mM DTT at 37 °C for 2 h. The release of AFC and AMC was analyzed by a fluorimeter using an excitation/emission wavelength of 400/505 nm (for AFC) and 380/460 nm (for AMC) which is directly proportional to caspase-9 and caspase-3 activity, respectively. The observed fluorescence values were normalized with total protein concentration estimated by Bradford method and the relative caspase activities were calculated as the ratio of values between mock treated (DMSO) and treated cells.

4. Senescence Assay
Compounds with anti-cancer potential may have the possibility to induce senescence in cells, thus limiting their proliferation. The ability to induce cellular senescence was determined by measuring senescence-associated beta-galactosidase (SA-βgal) activity (pH 6.0) in DU145 cells exposed to compound 7b. A 24-well plate was seeded with DU 145 cells as previously described and treated with the compound and subsequently incubated for 48 h. After 48 h, the media was aspirated and the cells were washed with PBS (2 × 1 min) and fixed by adding enough volume of fixation solution (2% formaldehyde and 0.2% glutaraldehyde in PBS solution) to submerge the cells in solution. After incubation for 5 min at room temperature Table 2. Four representative cell lines were tested with the series of compounds to determine their cytotoxicity. the fixation solution was removed and the cells were washed twice with PBS (2 × 1 min). The resultant fixed cells were then stained with freshly prepared staining solution (40 mM citric acid/Na phosphate buffer, 5 mM K 4 [Fe(CN) 6 ]·3H 2 O, 5 mM K 3 [Fe(CN) 6 ], 150 mM sodium chloride, 2 mM magnesium chloride and 1 mg X-gal in 1 mL distilled water) overnight at 37 °C. The excess stain was removed by repeated washings with PBS and plate was allowed to dry at room temperature. The cells stained with SA-βgal levels were observed and photographed under an Olympus Xi71 microscope.

5. PI Uptake for Analysis of Cell Death
Cell death induced by compound 7b was determined as a measure of PI uptake. Cells were harvested after treatment with compound at desired concentration and fixed in 70% ethanol at -20 °C overnight. The cells were then collected in the form of pellet. All cells in the form of a pellet were then resuspended in PI solution (RNase 0.1 mg/mL, Triton X-100 0.05%, PI 50 μg/mL) and incu-bated for 1 h in dark at room temperature. The excess PI solution was washed away by repeated washings with PBS buffer. The resultant PI uptake was analyzed by fluorescence activated cell sorting (FACS Caliber System; BD Bio-science, Erembodegem, Belgium) in a FL-2 fluorescence detector (10000 events were recorded Table 3. Compound 7b induced G0/G1 phase cell cycle arrest in DU145 cells. Cells were treated with varying concentrations of compound 7b (5, 10, 15, 20 and 25 μM) for 48 h and cell cycle progression was examined by flow cytometry.  for each condition). Flow cytometry data was analyzed using FCS express 4 software (De Novo Software, Los Angeles, CA).

Conclusion
A series of novel triazole linked thiazolidenone derivatives 6a-g and 7a-g was prepared and evaluated for their anticancer activity. The screened compound 7b exhibited potent anticancer activity compared to standard drug at the tested concentrations.