GREEN SYNTHESIS OF PYRAZOLO [3,4]-PYRIMIDINE-THIONES USING IONIC LIQUID 2-METHYL-IMIDAZOLIUM-OXALATE AS POTENT EHRLICH ASCITES CARCINOMA RECEPTOR ANTAGONISTS

Objective: Pyrazolopyrimidines are heterocyclic molecules containing nitrogen as the main composition, and hence, they exhibit pharmacological efficacy. They are analogs of purines so that possessing wide applications in the field of medicinal chemistry. The main objective of this study is to synthesize different derivatives of pyrazole-pyrimidine classes by adopting simple methodology as well as by employing green chemistry. The purpose of the synthesis of these molecules is to study the antitumor activity against Ehrlich ascites carcinoma (EAC) cell lines. Methods: After literature studies, it makes us to involve in the research of synthetic organic chemistry, especially to synthesize new compounds of pyrazolopyrimidines. We are reported solvent-free synthesis of pyrazolo [3,4-d]-pyrimidine-thiones through ethyl acetoacetate, hydrazine hydrate, thiourea, and different benzaldehydes. An ionic liquid 2-methyl-imidazolium-oxalate catalyzed the reactions under ultrasonication bath. Both conventional and ultrasonic methods were employed and comparison studies have been made. It was found that ultrasonic method completed the reaction quicker than the conventional method. All the synthesized compounds were confirmed their structures by 1HNMR, Fourier transform infrared, 13C-NMR, and elemental analysis spectra. The compounds were tested for in vitro anticancer activity against EAC cell lines. Most compounds revealed significant anticancer activity relative to doxorubicin as a positive control with inhibitory concentration (IC50) values. Results: Ultrasonication method is a simple method under which all the reactions were completed at faster time (<7 min) compared to the convention method. Among eight molecules, 8a and 8d completed the reactions at a faster rate. We reported IC50 values of all the molecules, in which 8e and 8g were exhibited excellent potency against EAC cell lines at different concentrations . Conclusions: Ultrasonication method is an excellent method for the organic synthesis. We are herein reported that under this method, all the reactions are completed within 7 min. Hence, it is superior method than the conventional method. All synthesized molecules have shown good inhibitor potency against EAC cell lines. Among them, two molecules 8e and 8g have shown excellent inhibitor potency.


INTRODUCTION
Pyrazolo [3,4-d]-pyrimidines are analogs of most purine-based drugs, having huge scope in the past decade for a consequence of their wide usage in medicinal field [1]. On literature studies, pyrazolopyrimidine derivatives have considerable potential in the field of chemotherapy, as they were found to exhibit their antitumor activity by inhibiting different types of enzymes such as cyclin-dependent kinase [2][3][4], Src and Abl tyrosine kinase [5], glycogen synthase kinase-3 [6][7][8], adenosine deaminase [9], and epidermal growth factor receptor protein tyrosine kinase. We synthesized pyrazolo [3,4-d]-pyrimidinethiones through ethyl acetoacetate, hydrazine hydrate, thiourea, and different benzaldehydes by the ultrasonication under solvent-free condition. This methodology attained green chemistry. Reactions carried under ultrasonication were more effective for the reaction time and also for the high yield of the products than the conventional method [10]. 2-Mim + oxalatewas used as a catalyst and found more efficient due to its ionic nature [11]. All the synthesized compounds were analyzed by Fourier transform infrared (FT-IR), 1 HNMR, 13 CNMR, and elemental analysis. The potency of these compounds was screened for in vitro test of anticancer activity against Ehrlich ascites carcinoma (EAC) cell lines. Most of the compounds showed excellent potency (inhibitory concentration [IC 50 ] values) against EAC cell lines.

METHODS
All chemicals were purchased by SDFCL Company. Reactions were managed by ultrasonic bath and magnetic stirrer (REMI). Melting point was determined by open capillary tubes in Buchi B-540 melting point apparatus. The reaction was monitored by thin-layer chromatography (TLC) using silica gel glass plates. FT-IR (Vertex version from Bruker), 1 HNMR (Bruker, 400 MHz), 13 C-NMR, and elemental analyzer were used. Biological activity (EAC cell lines) has been screened by Cytxon Biosolutions Pvt. Ltd., Hubballi -580031, Karnataka, India.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay protocol
The cells were trypsinized and aspirated into a 5 ml centrifuge tube. Cell pellet was obtained by centrifugation at 300×g. The cell counted was adjusted, using Dulbecco's Modified Eagle's Medium HG medium, such that 200 μl of suspension contained approximately 10,000 cells. To each well of the 96-well microtiter plates, 200 μl of the cell suspension was added and the plate was incubated at 37°C and 5% CO 2 atmosphere for 24 h. After 24 h, the spent medium was aspirated. 200 μl of different test concentrations (100, 200, and 300 μg/ml from stock) of test drugs were added to the respective wells. The plate was then incubated at 37°C and 5% CO 2 atmosphere for 24 h. The plate was removed from the incubator and the drug-containing media were aspirated. 200 μl of Yallappa et al. media containing 10% MTT reagent was then added to each well to get a final concentration of 0.5 mg/ml and the plate was incubated at 37°C and 5% CO 2 atmosphere for 3 h.

General procedure for the synthesis of pyrazolo [3,4-d]pyrimidine-thiones
To a solution of ethyl acetoacetate (10 mmol), add hydrazine hydrate (10 mmol), thiourea (10 mmol), and different benzaldehydes (10 mmol) in a round bottom flask. The reaction mixture along with 2-methylimidazolium-oxalate was kept in an ultrasonic bath to proceed and monitored by TLC. The solid precipitate out in the solution and filtered off. The crude solid was recrystallized by hot water, ethanol, and finally dried.
Preparation of 2-methyl-imidiazolium-oxalate 2-methyl-imidazole (10 mmol) was treated with sodium oxalate (10 mmol) and dissolved in an acetone (30 ml) solvent. The mixture was kept on magnetic stirrer for 30 min. The reaction was monitored by TLC. The cleaned transparent solution was obtained. Anal. calculated (%) for C 13 13

RESULTS AND DISCUSSION
All synthesized molecules are confirmed by FT-IR, 3300-3400 cm −1 (NH group of pyrazole) and 1630-1660 cm −1 (C=S), 1 HNMR by 6.7 ppm-8.5 ppm confirms aromatic hydrogen, and 8.5-10.1 indicated the presence of -NH protons. To follow the green chemistry, solventfree reactions were maintained under ultrasonication Reaction Scheme-1 [12]. Ionic liquids found to be better catalysts for organic synthesis. Bronsted acidic ionic liquid 2-methyl-imidazolium-oxalate catalyzed the reactions for good yield as well as instant completion. Experimental data revealed that ultrasonic method is a more superior method than conventional. Reaction times and yield of the products were studied for both methods (Table 1) [13].
The conventional method was managed at room temperature by stirring. Under this method, the reaction times for all the molecules are higher and yields were lower than ultrasonication method.
From Table 1, under ultrasonication, we found that 8a and 8d molecules completed the reaction at shortest time and yielded the highest quantity (%). Due to electron releasing group present at the 2-position of phenyl substituent, both 8a and 8d are found to be stable molecules. Hence, these molecules yield excellent. Similarly, 8e, 8f, and 8g molecules possess electron-withdrawing group at phenyl substituent and, therefore, yield lower than the molecules possessing electron releasing group.
Under the convention method, 8c and 8d compounds ended the reactions at shortest time. 8e and 8f molecules yield lower than the remaining molecules due to the presence of electron-withdrawing group at phenyl substituent.

Yallappa et al.
the higher anticancer activity of molecules 8e and 8g (Table 1 and Fig. 1) is attributed to the presence of nitro group (2-position) and two OH groups in addition to C=S group that facilitates the C-S bonding with the active sites which increase its reactivity respectively. From Fig. 2, at 200 µg/ml, 8e and 8g molecules have excellent IC 50 values.
It is competing with the substrate, so the concentration of ligand needed to reduce the enzymatic activity (EAC) by 50% depends on the concentration of substrate and how tightly it binds the enzyme. The experimental binding energies (∆Gexp) were calculated from the measured IC 50 by Equation (1), using the gas constant (R) and the temperature (T) [18][19][20].
∆Gexp=−RT ln IC 50 (1) Where, "∆Gexp" is the binding energy of the molecule with cancer cell lines, "R" is the universal gas constant (8.314 J K −1 mol −1 ),     "T" is the temperature at which a molecule binds (298°K) at room temperature, "IC 50 " is the inhibitory concentration of the molecule. ˂100.00

CONCLUSIONS
We reported that organic reactions were brought very good yield by momentarily under ultrasonic waves compared to the conventional method. 2-methyl-imidazolium-oxalate, an ionic liquid was found to be a good catalyst for organic syntheses. Pyrazolopyrimidines proved as potent molecules for cancer treatment. All the synthesized molecules exhibited moderate inhibition against EAC cell lines. 8e and 8g have the most potent IC 50 values.

ACKNOWLEDGMENTS
One of the authors, Ganesh N Yallappa acknowledges the Other Backward Classes Commission, Government of Karnataka, for providing a Research Fellowship during this period.

CONFLICTS OF INTEREST
Authors declare that they have no conflicts of interest.

AUTHORS' CONTRIBUTIONS
All the authors have contributed equally in the design, development, review, and finalization of the contents of the manuscript.