Design, Synthesis and Anticancer Properties of Novel Hydrazino-Fused Pyrimidines

Globally widespread cancer causes an urgent need to develop innovative, more potent drugs that can deliver better therapeutic outcomes. The study aimed to synthesize pyazolo(1,5-a) pyrimidines (5a-5j) and test their cytotoxic properties using DPPH and in vitro anticancer activity using MTT assay. The measured bioactivity scores from -0.5 to 0 and the DPPH assay revealed all except 5c, 5b, and 5h exhibited the lowest inhibition observed when compared with the reference ascorbic acid and cytotoxic assay performed with MCF-7 and HepG-2 cell lines and reported that 5b was a potential candidate towards MCF-7 and 5c showed potent cytotoxicity towards HepG-2. Concluding that the mentioned compounds were reported as potent candidates and good agreement was observed between in vitro and in silico studies.


INTRODUCTION
Protein kinases have emerged as a possible therapeutic targets, with nearly 30 distinct kinase targets being developed for the phase-I trials 1 .The cell cycle regulation and proliferation mainly depend on cyclin-dependent kinases (CDKs).The many biochemical targets are deregulated and there are three approved CDKIs like palbociclib, ribociclib and abemaciclib 2 .Because they are crucial in regulating growth factor signaling, tyrosine kinases are a particularly crucial target and many oncogenic tyrosine kinases catalytic domains ATP binding site is the prime site for the inhibitors 3 .Gefitinib 4 , lapatinib 5 is a low molecular weight EGFR/ erbB2-tyrosine kinase dual inhibitor that competes with the ATP for blockade of the catalytic domain.Purine is a heterocyclic nucleus present in various antimetabolites and modified at positions 2,4 and 9 in the creation of protein kinase inhibitors.A quinazoline scaffold with a phenylamino pyrimidine moiety is one of the structural characteristics 6 .Pyrazolo-pyrimidine is a bioisostere of purine 7 was given much consideration while creating anticancer scaffolds, as evidenced by PKI-1668 and erlotinib 9 .Over the past few decades, multifunctional compounds have been employed in treating multifactorial disorders like cancer.These compounds are generically classed as hybrid and chimerical medications comprising two or more merged pharmacophore groups permitting binding to two or more targets 10 .The rational construction of novel "smart" molecules that keep the pre-selected qualities of the original templates is made possible by combining two or more overlapping pharmacophore groups on the same scaffold.This process also has certain advantages, such as a decreased risk of toxicity and unpleasant responses 11,12 .
specifically implicated in the promotion of metastasis and tumor growth.A low molecular weight EGFR/ erbB2-tyrosine kinase dual inhibitor, gefitinib 4 and lapatinib 5 , competes with ATP to block the catalytic domain.Purine is a heterocyclic nucleus found in many antimetabolites and its structural properties include a quinazoline scaffold with a phenylamino pyrimidine moiety and different substituent's at the various positions in the design of protein kinase inhibitors 6 .PKI-166 8 , erlotinib 9 , imatinib 16 , BIRB796 17 and BAY43-9006 18 received significant attention in the development of scaffolds as anticancer medicines.

Fig. 1. The features of antimetabolites' structures
Methotrexate, a competitive inhibitor of DHFR that converts DHF to THF, was the foundation for developing the novel pyrazolo(3,4-d) pyrimidines 13 .Recently reported as cytotoxic on BEAS-2B (lung carcinoma) cells, Pemetrexed (PMX) is a dihydropyrrolo [2,3-d]pyrimidin nucleus with ethylene spacer linkage to PABA moiety and acts as a folate antagonist.Studies revealed that the greater affinity complexation of WP6A and ATP favouring competitive replacement of PMX was confirmed by NMR spectroscopy 5 .A folate antagonist previously reported to be an oseltamivir-comparable prospective drug effective against seasonal influenza viruses suppressed SARS-CoV-2 proliferation in Calu-3 cells 14,15 .
The epidermal growth factor (EGF) which regulates epithelial cells, the origin of all carcinomas, and the corresponding cell surface receptor are over expressed in tumours and overproduced with mutant hyperactive variants of EGFR being

Materials
The target proteins were downloaded from the PDB 19 server and Chemdraw Ultra was used to draw the ligand structures.SwissADME 20 utilized to predict the molecular parameters oand molinspiration was one of the web servers to produce the bioactivity scores 21 .Molecular docking simulations were performed by the tool Schrodinger 20.3 22 and the discovery studio visualized by Biovia 23 .All the chemicals availed from Aldrich & SD Fine Chemicals and M.Ps were found using an open capillary tube (Stuart Scientific SMP1).Ethyl acetate and n-butanol (20:80) were used in TLC using precoated silica plates to ensure the purity of the compounds.The infrared spectra in KBr (Vmax in cm -1 ) were captured using a Shimadzu-FT-IR infrared spectrometer. 13C (100-MHz) & 1 H (400-MHz) NMR spectra using Bruker DPX 300 spectrometers using TMS as an internal standard.Thermo Finnigan LCQ Ion Trap & Agilent-1200 series LC instruments were used to collect high-resolution mass spectra, which were then shown in m/z as molecular ion peaks.In order to perform the elemental analysis, VARIO EL-III was employed.Human cell lines from NCCS, Pune, India, include MCF-7 and Hep-G2.Ninety-six well plates, 25 cm 2 and 75 cm 2 flasks and more from Eppendorf India were purchased.

Evaluation of the molecular descriptive characteristics
In order to anticipate the drug-like properties, which is the key step in the drug development process, the SwissADME online program was used 20 .The main characteristics of a drug candidate are molecular weight, absorption, a limited amount of molecular flexibility and TPSA 24 .It is possible to assess a compound permeability and bioavailability using straight forward molecular information 25 .Based on molecular properties using lipinski's rule of five criteria states that a molecule is a drug like property if it has five hydrogen bond donors, ten hydrogen bond acceptors, five partition coefficients logP, and a molecular weight of 500 daltons 26 .

Molecular docking analysis
It has become possible to get the crystal structures of tyrosine kinase and cyclin-dependent kinases from the PDB (ID: 7QHL & 2SRC).The protein structure was constructed using the Schrodinger suite 20.3 tool for protein construction.The protein preparation wizard enabled the energy minimization of the structure the addition of the missed atoms, deletion of water located less than 5 from the binding pocket and the addition of the missing atoms.The grid was created by selecting the co-crystals active location and placing a grid box in its center.The ligands were sketched and energy was minimized, then ligands were made by Lig-Prep Schrodinger module to produce various conformers.The conformers were docked using glide in normal precision mode.The poses were sorted and the top pose was identified.It was known that previous reports stated that co-crystal of target CDK receptor binding modes that the three essential hydrogen binding interactions at active site besides hydrophobic interactions are GLU81, ASP86, and LEU83, and validation was confirmed by redocking of co-crystallized structure with target kinase.

Synthesis protocol General Procedure for synthesis of pyazolo(3,4-d) pyrimidines Scheme 1. Representative scheme for the synthesis of Pyrazolopyrimidines
Weigh about 12.77 g (0.1 mol) of 4-Chloro aniline (1), transferred to a beaker and 9.8 mL of nitrous acid was added in cool condition the mixture and kept aside for 30 minutes.By adding 100 mL of ice-cold water to the solid material that had precipitated, it was separated and dried to produce 4-chloro benzene diazonium chloride (2).The compound 2 was taken about 8.5 g (0.05 mol), transferred into R.B.flask and was treated with malononitrile in acetic anhydride under reflux for 5 hrs to afford (4-chlorophenyl)carbonohydrazonoyl dicyanide (3).The compound 3 reacts with hydrazine hydrate in ethyl acetate solution under reflux for 4 h to afford the compound 4-[2-(4-chlorophenyl) hydrazinyl]-4,5-dihydro-1H-pyrazole-3,5-diamine (4).In the next step, chalcones were prepared using different aldehydes and ketones.They obtained chalcones by treating compound 4 independently under magnetic agitation for 6 h in ethanol and drying to afford the compounds 5a-5j.

Biological evaluation DPPH radical scavenging assay
The imbalance between free radical production and antioxidant defense systems is known as oxidative stress.It is connected to many pathophysiological conditions, such as cancer and neurological diseases.Antioxidants, whether exogenous or endogenous, enzymatic or nonenzymatic, can reduce or stop cellular damage, primarily through scavenging free radicals.The synthesized compounds were examined for their capacity to scavenge free radicals using the DPPH assay method.In order to create a series of dilutions with concentrations ranging from 100 to 200 µg/ mL in methanol.The methanolic solution of the synthesized compound (2 mL) was added to a 0.003% (w/v) methanolic solution of DPPH (1 mL).The mixture was vigorously shaken for five minutes and kept aside for 30 min and measured absobance and ascorbic acid served as the reference at 517nm.The equation used to get the inhibition ratio (%I) for all substances tested is %I=(Ac-As)/Acx100, where Ac is the absorbance of the control and As is the absorbance of the sample 27,28 .

MTT Assay for Cytotoxic activity
The MTT assay is a colorimetric technique that measures how much mitochondrial succinate dehydrogenase lowers the yellow dye.The study assumed that a certain number of cells existed and that dead cells in the medium did not diminish the amount of tetrazolium dye and the dye penetrates cells and travel to the mitochondria, where it is transformed into formazan and color intensity measured at 570nm.
The MTT assay was done in triplicates of six independent concn's to predict cell viability and evaluate cell viability in the suspension and trypsinization.The trypan blue test was carried out.The cells were seeded in 96 well plates at a density of 5.0X10 3 cells per well in 100 mL culture medium and incubated overnight at 37°C.The hemocytometer was used to count the cells.Old media was removed after incubation and replaced with a new medium containing test samples at 5,10,25,50 and 100 µg/mL concentrations.After 48 h, new tetrazolium dye was added and incubated at 37 o C for 3 h before the solution's absorbance was measured in a DMSO medium at 570nm 29 .Calculating the growth inhibition percentage involved: Y = mx+C, was used to get the IC 50 value.Here, the viability graph's Y = 50, M and C values were calculated.

Evaluation of the molecular descriptors
This section lists physical properties and except for a few exempt characteristics like mass and HBA, all the chemicals were within the allowed ranges; all the information was provided in Table 1.

Bioactivity Score Calculation Using the Molinespiration Toolkit
Calculate the inhibitory activity against various receptor ligands, inhibitors, and enzymes using the molinspiration online toolbox 21 .
Inactivity is expected for scores below -0.50, moderate activity is projected between -0.50 and 0.00 and considerable activity if its bioactivity score is more than 0.00.All data are shown in Table 2.
The compound 5c reported -7.192 (Fig. 3), and 5h reported -7.04K.Cal per mole (Fig. 4) against tyrosine kinase, where the standard doxorubicin and pemetrexed reported -8.814K.Cal per mole showed -7.646 K.Cal per mole respectively (Figure 5 & 6).The reference, doxorubicin scored -8.924 K.Cal per mole against CDKs and pemetrexed which reported -8.34 K.Cal per mole (Fig. 9 & 10), the compounds 5b and 5h exhibited -6.799 (Fig. 7) and -6.915 K.Cal per mole (Figure 8) respectively.4), which was condensed with chalcones to afford the compounds 5a-5j, as shown in the Scheme 1.The compounds 5a-5j were obtained in moderate to good yields (52-82%) and recrystallized from ethanol and ethyl acetate.The analogues IR spectra showed the existence of C=N bonds in the 1525-1685 cm -1 area, two NH bonds in the 2900-3150 cm -1 region, OH groups in the 3200-3500 cm -1 region, C-Cl bonds in the 560-850 cm -1 region, and C-Br bonds in the 525-585 cm -1 region.In 1 H-NMR spectra, a singl et at., d 1.75 to 2.85 ppm assignable for a methyl group, singlet at d 3.78-3.88for methoxy protons, singlet for NH protons d 7.78-8.55for hydrazine protons and multiplet for protons in aromatic region at d 7.08-8.5 in 13 C-NMR spectra aromatic carbons revealed at a range of 120-155 ppm, and the pyrazolopyrimidine scaffold carbons absorb at 90-160 ppm region.Moreover, the HRMS and elemental analysis were carried out to confirm the novel compounds.

Biological Evaluation
The scavenging effect percentage of the compound 5 h at 100, 150, and 200 µg/mL is 51.4,60.2, and 68.3.The scavenging property of 5b at 100, 150, and 200 µg/mL is 51.5, 62 and 67.8 respectively.Ascorbic acid demonstrated a 98.3% scavenging efficiency at a 200 µg/mL concentration.The good inhibition of 5c showed 52.8, 62.5, and 69.4% at 100, 150 and 200 µg/mL respectively.All the compounds except 5c, 5b, and 5h exhibited lowest inhibition.The electron-donating hydroxyl groups in the compounds 5c, 5b, and 5h exhibited the highest antioxidant activities compared to other compounds.Table 4 displays the RSC as a percentage.Using the MTT assay, the cytotoxic effects of each substance were examined against the MCF-7 and HepG-2 cell lines.The cytotoxicity was assessed using various concentrations and 5-FU as the standard drug.The consolidated concentrations of compounds with respect to inhibition percentage as well as cell viability against MCF-7 and Hep G2were given in Table 2.For in vitro anticancer investigations, every chemical underwent screening.The cytotoxic evaluations were done MTT assay revealed that the compounds 5b (Graph-1 & Table 7) reported as potential against MCF 7 with the IC 50 , 16.61 followed by 5d with the IC 50 19.67µg/mL, where the standard reference showed 14.34 µg/m and 5c (Graph-2 & Table 8) reported as potential against Hep G2 with the IC 50 , 14.32 µg/mL.This was followed by 5h with the IC 50 19.24µg/mL, where the standard reported 11.36 µg/mL, and all cell viability studies were reported in Table 5.

CONCLUSION
As a result, many new pyrazolo(1,5-a) pyrimidines (5a-5j) were synthesized with excellent yield and identified using various spectral methods.All the compounds were evaluated for antioxidant studies, which revealed that compounds 5c, 5b and 5h were potential candidates due to the electrondonating hydroxyl groups being crucial for potency.Also evaluated for cytotoxic MTT assay revealed that the compounds 5b and 5d were potent against MCF-7, and 5c and 5h were potential candidates against HepG-2 cell lines.Some compounds showed mild to moderate cell viability compared with reference compounds, and compounds 5c and 5h had potential against tyrosine kinase and CDKs.