Synthesis, Biological Activity and Cytotoxicity of New Fused Pyrazolo[1,5-a]pyrimidine from 5-Aminopyrazole Incorporated with p-Chloroaniline

5-Amino-3-(4-chlorophenylamino)-1H-pyrazole-4-carbonitrile 3 was prepared in high yield from the reaction of hydrazine hydrate with known 2-[(4-chlorophenylamino)(methylthio)methylene]malononitrile 2a (which was prepared from reaction of 2-[bis(methylthio)methylene]malononitrile 1 with p-Chloroaniline) under reflux in ethanol. The compound 3 was utilized as a key intermediate for the synthesis of pyrazolo[1, 5-a]pyrimidines 4a-b, 5a-c and 6 by reactions with some of ketene-S, S-and N, S-acetals. The antibacterial and antifungal activities, as well cytotoxicity against Breast cancer cells (MCF7) of some selected compounds are also reported.


Chemistry
All melting points were determined using a hot stage Gallenkamp melting point apparatus. Infrared spectra were recorded from KBr discs on FT-IR 8300 Shimadzu spectrometer. 1 H NMR and 13 C NMR spectra were recorded on FT-NMR 400 MHz Joel, ECP and FT-NMR 600 MHz Bruker, AVANCE III spectrometer operating at 400 MHz, and 600 MHz for 1 H NMR and at 100 MHz, 150 MHz for 13 C NMR in DMSO-d 6 as solvent and using TMS as internal standard. DIMS spectra were recorded on QP5050A Shimadzu apparatus. X-ray diffraction (XR-D) data were collected at room temperature with Bruker APEXII CCD a spectrometer. General purpose silica gel of Merck No. 5545 with UV indicator were used in TLC experiments to monitor completion of reactions, in which DCM was used as eluent. from 5-Aminopyrazole Incorporated with p-Chloroaniline

Antibacterial and Antifungal Evaluations
Some of the selected synthesized compounds were evaluated for their antibacterial and antifungal activities using the agar diffusion technique [15]  The test is carried out by placing 6 mm diameter of paper disc containing antibiotic onto a plate which microbes are growing. The microbe culture is standardized to 0.5 McFarland standards which is approximately 10 8 cells. Not more than 6 discs should be placed on the same agar plate. Streptomycin standard are used for each bacteria and Nystatin standard are used for fungi. The plates are inverted and incubate at 30-37°C for 18-24 hours, 24-48 or until sufficient growth has occurred. After incubation, each plate is examined. The diameters of the zones of complete inhibition (as judged by the unaided eye) are measured, including the diameter of the disc. Zones are measured to the nearest whole millimeter, using sliding calipers or a ruler, which is held on the back of the inverted Petri plate.

Cytotoxicity Assay
Some of the selected synthesized compounds were also tested against human breast adenocarcinoma (MCF-7) cell lines by using the MTT assay. Human MCF-7 breast adenocarcinoma cell line was procured from ATCC. The cells were cultured in a humid environment at 37°C and 5% CO 2 as a monolayer in DMEM (Dulbecco's Modified Eagles Medium; US Biological) supplemented with 10% FBS (Fetal Bovine Serum; Bioclot) and 1% penicillin/streptomycin (Invitrogen). Cells were grown up to 85-90% confluence and harvested using 0.25% trypsin/EDTA solution before sub-cultured onto 96-well plates. Cells were then treated with different compounds at a final concentration ranging from 0.47-30 µg/mL for 24 hrs. Stock solutions were prepared in dimethylsulfoxide (DMSO) and stored at 20°C until used. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay developed [16] with modification was used to screen the cytotoxic activity of compounds. Briefly, 100µl of the MCF-7 cells (1 x 105 cells/mL) were subculture onto sterile flat-bottomed 96 well plates and exposed to 7 different concentrations (30.00, 15.00, 7.50, 3.75, 1.87, 0.93 and 0.47 µg/mL) of each compound for 24 h. After the completion of Incubation in 37°C, 5% CO 2 incubator, 20 µL of MTT reagent (Invitrogen) in 5.0 mg/mL phosphate buffered saline (PBS) was added to each well and further incubated for 3 h at 37°C, 5% CO 2 incubator. MTT solution was then removed before 100 µL of DMSO (Sigma Aldrich) were added to each well and mix thoroughly to dissolve the blue formazan crystals. Further incubation was carried out for 20 min. Finally, the optical density (OD) of each well was measured on ELISA reader at 570 nm (test wavelength) and 630 nm (reference wavelength). This cytotoxixity test was performed in two independent experiments, each time in triplicate. The percentage of cytotoxicity compared to the untreated cells was determined. The percentage of viability against each compound concentration were plotted to determine the CC 50 value (the concentration at which 50% cell proliferation is inhibited). from 5-Aminopyrazole Incorporated with p-Chloroaniline The percentage of cells viability was calculated in relative with the number of viable cells as a percentage of control by defining the absorbance at 570 nm for the control as 100%.

Chemistry
The  The chemical structure of the compound 3 was established on the basis of its spectral data. IR spectrum of 3 showed bands at ν 3430, 3254 cm -1 for NH and NH 2 groups. The NH 2 protons in 1 H NMR spectrum were at δ 6.30 ppm, whereas the two singlet signals at δ 8.54 and 11.19 ppm assignable to the respective ArNH and cyclo-NH protons. The 13 Figure 2). The starting material ketene-S,S-acetals were prepared according to the previously reported procedure [17].  The proposed mechanism for the formation of pyrazolo[1, 5-a]pyrimidines 4a-b and 5a-c is similar to that for the formation of 5-aminopyrazole 3, except that the final step in the former involves 1, 3-hydrogen migration rather than 1, 5-hydrogen migration in the latter (Figure 4). We also attempted a direct synthesis of pyrazolo[1, 5-a] pyrimidine 6 by treating 5-aminopyrazole 3 with respective pentane-2, 4-dione in refluxing dimethylformamide (DMF) containing a catalytic amount of glacial acetic acid ( Figure  5). The structure of the pyrazolo [1, 5-a] pyrimidine 6 was elucidated on the basis of its spectral data. The characteristic absorption band in the IR spectrum of 6 is at ν 3304 cm -1 for NH stretching vibration. The 1 H NMR spectrum of compound 6 displayed singlet signals at δ 2.50 and 2.66 ppm, which correspond to six protons of two methyl groups of CH 3 and COCH 3 . The structure of the isolated product was supported by its direct infusion mass spectrometry (DIMS) result, which showed molecular ion corresponding to the molecular formula. The DIMS of 2-(4-chlorophenylamino)-5, 7-dimethylpyrazolo [1, 5-a] pyrimidine-3-carbonitrile 6 showed a molecular ion at m/z=297.70 which corresponds to the molecular formula C 15 H 12 ClN 5 (297.74). The mechanism for the formation of 6 is shown in Figure  6. The conversion involves three major steps, as follows: (i) nucleophilic attack of the NH 2 group from 5-aminopyrazole 3 onto the carbonyl group of acetylacetone, by releasing water The structure of compound 6 was identified by X-ray diffraction analysis. The molecular structure and the numbering scheme are presented in Figure 7. Suitable crystals of 6 were grown by slow evaporation from DMSO solution. The crystal data and structure refinement results for 6 are given in Table 1. Compound 6 crystallized in a monoclinic system with space group of P21/n. Selected bond distances and bond angles for 6 are given in Table 2. The bond lengths and angles of the new molecule are within in the normal ranges [18]. The phenyl ring (C1−C6) is essentially planar with a maximum deviation of 0.001 (3) Å, for atom C1. The maximum deviation in the pyrazole ring N2/N3/C7/C8/C10 in 6 is 0.000 (2) Å, for atom N2. The dihedral angle between the mean planes of the pyrazole and the 4, 6-Dimethyl-1, 6-dihydro-pyridazine ring in 6 is 1.52 (14) ° (see Electronic Supplementary Information [19]). In crystal packing of compound 6, the molecules are connected by weak C−H … N and N−H … N intermolecular hydrogen bonds forming one-dimensional chains along the b axis ( Figure 8).

Antibacterial, Antifungal Evaluations and Cytotoxicity Assay
The antibacterial and antifungal activities results are listed in Table 3. The results for the pyrazolo [1, 5-a] pyrimidines from 5-Aminopyrazole Incorporated with p-Chloroaniline showed moderate against tested bacteria and fungi. Compounds 4b and 5b exhibited inhibitory activity against all bacteria tested. Values are mean inhibition zone (mm) ± S. D of results done in triplicate. 6 mm is the diameter of the disc Cytotoxicity results of tested compounds are summarized in Table 4. The CC 50 value was graphically obtained by plotting the percentage growth inhibition against the corresponding different concentrations of the test compound used. The CC 50 values for compound 4a was found to be 7.5 µg/ml (graphically represented in Figuer. 9) while the other three compounds namely 5b, 5c, and 6 CC 50 value of more than 30 µg/ ml. According to Chandrashekar et al [20], CC 50 value of more than 20 µg/ml can be considered as non cytotoxic.