INTRODUCTION:

Over the years, researchers have struggled to understand the complex, multifactorial, and deadly disease known as cancer. One of the leading causes of death globally is cancer. One out of every six deaths across the globe occur due to cancer. Breast cancer has become the most common cancer in the world in both sexes. In India, breast cancer was the most common incident cancer overall amounting to 13.5% of all new cancer cases overall and 26.3% of new female cancer cases.

In 2020 alone, 1,78,361 new breast cancer cases and 90,408 deaths due to breast cancer were reported in India. Hence there is an increased need for finding molecules with anti-proliferative properties to treat diseases like breast cancer in developing countries like India¹.

Recently, many investigators have demonstrated that over-expression of receptors and growth factors, oncogene activations and tumor suppressor gene inactivation are the root causes for the development of an aggressive and resistant cancer phenotype. Dysfunctions in intracellular signaling pathways have also been implicated in the development and progression of cancer². Globally lung, colon, prostate and female breast cancers are the four most prevalent types of cancer³. There are several additional readily accessible cancer therapies, but chemotherapy is the most widely utilized one. Chemotherapeutics operate by either killing cancer cells or preventing them from growing⁴. Angiogenesis is one of the important mechanismsof cancer growth⁵. The process of vascular expansion known as angiogenesis, which results in the growth of pre-existing vessels, may be crucial for growth, wound healing, and embryonic development. Numerous clinical conditions, such as arthritis, neovascular disorders, metastasis, and tumor formation, are associated with abnormal angiogenesis. Zebrafish serve as a great model vertebrate since their circulatory systems are comparable to those of mammals. Zebrafish embryos are an appealing model for the study of angiogenesis because blood vessel creation can be easily observed and blood flow can be scored easily. Because of their small embryo size, zebrafish have been employed extensively in recent years to check for developmental abnormalities.The formation, progression, aggressiveness, and metastasis of numerous solid tumors, including non-small cell lung cancer⁶ and head and neck malignancies, are significantly influenced by these growth factor receptor kinases⁷. The field of research is developing quickly, as evidenced by the identification of several key receptors involved in angiogenesis, such as the EGFR and VEGFR receptors in vascular endothelial growth factor. A key component of the angiogenesis process, which is essential to the survival of cancer cells, is the epidermal growth factor receptor (EGFR)⁸. EGFR and its family members are the major contributors to a complex signaling cascade that modulates the growth, signaling, differentiation, adhesion, migration, and survival of cancer cells. Erlotinib inhibits EGFR which is overexpressed in tumors which promotes cell proliferation, angiogenesis, anti-apoptosis, and metastasis and is an approved antitumor agent⁹. To produce novel therapeutic anticancer drugs, these variables therefore serve as crucial targets¹⁰.

Researchers are still fascinated by the chemistry of chalcones in the twenty-first century because of their abundance of replaceable hydrogens, which enable the generation of numerous derivatives and a wide range of intriguing biological functions¹¹. By cyclizing chalcones with various reagents, heterocyclic compounds with nitrogen, oxygen, and sulfur heteroatoms can have five or seven members. Examples of these compounds are pyrazolines, phenyl pyrazolines, and isoxazoles, which are five-membered heterocyclics; derivatives of amino pyrimidines and cyano pyridines, which are six-membered heterocyclics; and 1,5-benzoxazepines and 1,5-bezothiazepines, which are seven-membered heterocyclics¹². Heterocyclic compounds containing nitrogen, like pyrazolines, have attracted considerable attention in recent years due to their versatile biological and pharmacological activities¹³. Chalcones have been extensively used to synthesize pyrazolines^14,15. Pyrazolines have been studied extensively because of their diverse, broad-spectrum biological activities¹⁶.

MATERIAL AND METHODS:

The solvents and chemicals were all bought from Sigma Aldrich, Research Lab, and S. D. Fine Chem. Without further purification, analytical grade reagents were used. High-grade commercial solvents were utilized. They were used after purification. The completion of the reaction was monitored by means of thin layer chromatography (TLC) on silica gel-coated aluminium sheets obtained from Merck Specialities Ltd., Mumbai. Melting points were determined on a VEEGO (VMP-D) melting point apparatus and are uncorrected. 1H NMR spectra were recorded on AGILANT VARIAN (600 MHz) in CDCl₃ and DMSO-d6 as solvent using TMS as an internal standard at Tata Institute of Fundamental Research (TIFR) Navy Nagar, CST-Mumbai. IR (ATR) spectra were captured using an infrared spectrophotometer, a Shimadzu 8100. In Mumbai, Ambernath Organics Pvt Ltd, mass spectra were recorded.

EXPERIMENTAL:

General procedure for the synthesis of 1,5-bis-(substituted phenyl) penta-1,4-diene-3-one derivatives (3a-3h):

In a conical flask, a mixture of aldehyde (0.113mol), acetone (0.055mol), and lithium hydroxide monohydrate (1.26g) was added, along with 30 mL of ethanol. Using a magnetic stirrer, the reaction mixture was agitated until a solid mass with a yellowish colour precipitated out. The flask was kept for cooling in an ice bath, and the precipitated solid was filtered. In order to obtain pure chalcone, the crude product was extensively washed withcold water-ethanol mixture, allowed to air dry, and then recrystallized.^17-19.

General procedure for synthesis of (5-(substituted phenyl)-3-[(E)-2- (substitutedphenyl)ethenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde derivatives (4a-4h):

A mixture of (1,5-di (substituted aryl) penta-1,4-dien-3-one.) (3a-3h) (15mmol) and hydrazine hydrate (30mmol) in 15ml formic acid was heated under reflux. Completion of the reaction was monitored by TLC using Hexane: ethyl acetate (0.8:0.2) as the mobile phase. The resulting solution was poured on crushed ice. The precipitate obtained was filtered and washed with water and then recrystallized.²⁰

5-(4-methoxyphenyl)-3-[(E)-2-(4-methoxyphenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1- carbaldehyde (4a)

Molecular formula:C₂₀H₂₀N₂O3; Molecular weight: 336.17; R_f: 0.46; Yield (%): 60; M.P (°C): 217; IR (ν, cm^-1): ^{3056 (Ar-C-H stretching),}1653 (C=O ^stretching), ^{1583
(C=N stretching), 1337 (C-N stretching);}1047 (C=C ^stretching), 774(C-Cl); ¹H NMR(DMSO-d6, 600 MHz) (δppm): 7.59-6.80 (m, 10H, Ar-H), 6.93-6.90 (d, H_αJ= 18), 7.15-7.09 (d, H_β J= 18), 3.01-2.97 (dd, H_{a ,}J=15.6 ), 3.70-3.67 (m, H_b), 5.42-5.38 (dd, H_x,J= 11.4), 6.37; MS m/z: 337.1 (M+1)⁺.

5-(4-fluorophenyl)-3-[(E)-2-(4-fluorophenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1- carbaldehyde (4b):

Molecular formula: C₁₈H₁₄F₂N₂O; Molecular weight: 312.15; R_f: 0.63; Yield (%): 55; M.P (°C): 244; IR (ν, cm^-1): ^{3016
(Ar-C-H stretching),}1668 (C=O ^stretching), ^{1601 (C=N stretching), 1211 (C-}N ^stretching), 1014 (C=C ^stretching), 1154 (C-F str)^{; 1}H NMR(DMSO-d6,600 MHz) (δppm): 7.70-6.60 (m, 10H, Ar-H), 6.92-6.90 (d, H_α,J=18), 7.76-7.75 (d, H_βJ=18), 3.07-3.03 (dd, H_{a ,}J=15.6 ), 3.64-3.61 (m, 1H, H_b), 5.40-5.36 (dd, H_x,J= 11.4), MS m/z: 313.1 (M+1)⁺.

5-(3-fluorophenyl)-3-[(E)-2-(4-hydroxyphenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1- carbaldehyde (4c):

Molecular formula:C₁₈H₁₅N₂FO₂; Molecular weight: 310.99; R_f: 0.66; Yield (%): 73; M.P(°C): 202; IR (ν, cm^-1): ^{3362 (Ar-C-Hstretching),} 1669 (C=O ^stretching), ^{1515
(C=Nstretching), 1327 (C-Nstretching}), 966 (C=C ^stretching)^;
1H NMR (DMSO-d6,600 MHz) (δppm): 7.56-6.60 (m, 10H, Ar-H), 6.63-6.64 (d, H_α,J=18), 6.77-6.82 (d,H_β,J=18), 3.01-2.98 (dd, H_{a ,}J=15.6 ), 3.54-3.52 (m, 1H, H_b), 5.50-5.41 (dd, H_x,J= 11.4),9.38 (s, 1H, -CHO), 9.79 (s, 1H, -OH); MS m/z: 311.1 (M+1)⁺.

5-(4-methylphenyl)-3-[(E)-2-(4-methylphenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1- carbaldehyde (4d):

Molecular formula: C₂₀H₂₀N₂O; Molecular weight:304.14; R_f: 0.69; Yield (%): 75; M.P (°C): 167; IR(ν, cm^-1): ^{3025 (Ar-C-H stretching),} 1669 (C=O ^stretching), ^{1515
(C=N stretching), 1327 (C-Nstretching}), 966 (C=C ^stretching)^;
1H NMR (DMSO-d6,600 MHz) (δppm): 7.80-6.80(m, 8H, Ar-H), 6.56-6.55(d, H_α,J=18), 6.97-6.95(d, H_β,J=18), 3.26-3.23(dd, H_{a ,}J=15.6 ), 3.65-3.61(m, 1H, H_b) ,5.36-5.33(dd, H_x,J= 11.4), 8.83 (s, 1H, -CHO); MS m/z: 305.1 (M+1)⁺.

5-(2,6-dichlorophenyl)-3-[(E)-2-(2,6-dichlorophenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde (4e)

Molecular formula: C₁₈H₁₂N₂OCl₄; Molecular weight: 413.97; R_f: 0.80; Yield (%): 85; M.P(°C): 261; IR(ν, cm^-1): ^{3056 (Ar-C-H stretching),}1696 (C=O ^stretching), ^{1583
(C=N stretching), 1225 (C-}N ^stretching), 955 (C=C ^stretching)^;
1H NMR (DMSO-d6,600 MHz) (δppm): 7.40-6.80 (m, 8H, Ar-H), 6.92-6.91(d, H_α,J=18), 6.86-6.81(d, H_β,J=18), 3.26-3.23 (dd, H_{a ,}J=15.6 ), 3.65-3.61 (m, 1H, H_b), 5.51-5.49 (dd, H_x,J= 11.4), 8.80 (s, 1H, -CHO); MS m/z: 201.1. (M+1)⁺.

5-(2,4-dichlorophenyl)-3-[(E)-2-(2,4-dichlorophenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1- Carbaldehyde (4f):

Molecular formula: C₁₈H₁₂N₂OCl₄; Molecular weight: 413.97; R_f: 0.78; Yield (%): 77; M.P (°C): 172; IR(ν, cm^-1): ^{3057(Ar-C-Hstretching),}1668 (C=O ^stretching), ^{1557
(C=Nstretching), 1320(C-Nstretching}), 1010 (C=C ^stretching)^{; 1}H NMR (DMSO-d6,600 MHz) (δppm): 8.80-7.20 (m, 8H, Ar-H), 6.88-6.85(d, H_α,J=18), 7.02 (d, H_β,J=18), 3.01-2.97 (dd, H_{a ,}J=15.6 ), 3.54-3.51 (m, 1H, H_b), 5.51-5.49 (dd, H_x,J= 11.4), 8.80 (s, 1H, -CHO); MS m/z: 101.0 (M+1)⁺.

5-(3,4-dichlorophenyl)-3-[(E)-2-(3,4-dichlorophenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1- Carbaldehyde (4g):

Molecular formula: C₁₈H₁₂N₂OCl₄; Molecular weight: 413.97; R_f: 0.53; Yield (%): 73; M.P (°C): 218; IR(ν, cm^-1): ^{3037 (Ar-C-H stretching),}1670 (C=O ^stretching), ^{1558
(C=N stretching), 1320 (C-Nstretching)}, 1010 (C=C ^stretching) 754(C-Cl)^{; 1}H NMR (DMSO-d6,600 MHz) (δppm): 7.90-7.10 (m, 8H, Ar-H), 6.97-6.96(d, H_α,J=18), 7.03-7.01(d,H_β,J=18), 3.07-3.00(dd, H_{a ,}J=15.6 ), 3.61-3.56 (m, 1H, H_b), 5.51-5.49 (dd, H_x,J= 11.4), 8.80 (s, 1H, --CHO); MS m/z: 101.1 (M+1)⁺.

5-(4-chlorophenyl)-3-[(E)-2-(4-chlorophenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde (4h):

Molecular formula: C₁₈H₁₄Cl₂N₂O; Molecular weight: 345.01; R_f: 0.69; Yield (%): 64; M.P (°C): 202; IR(ν, cm^-1): ^{3087 (Ar-C-H stretching),}1669 (C=O ^stretching), ^{1623
(C=N stretching), 1325 (C-}N ^stretching), 1029 (C=C ^stretching), 815(C-Cl ^stretching)^{; 1}H NMR (DMSO-d6,600 MHz) (δppm): 8.10-7.20 (m, 8H, Ar-H), 6.77-6.74 (d,H_α,J=18),7.08-7.05 (d,H_β,J=18), 3.10-3.03 ((dd, H_{a ,}J=15.6 ), 3.54-3.51 (m, 1H, H_b), 5.51-5.49 (dd, H_x,J= 11.4), 8.80 (s, 1H, -CHO); MS m/z: 157.1 (M+1)⁺.

In-vivo Study:

Zebrafish embryos were obtained from a local supplier for the developmental angiogenesis model. Instruments required for this study were electronic weighing balance, 96 well sterile micro titre plate, Motic Digital Microscope (4X, 10X), and micro syringe. Chemicals used for this study were dimethyl sulfoxide (DMSO) and methylene blue. Motic Image Plus software was used to capture the images of zebrafish embryos.

Procedure for developmental angiogenesis in zebrafish embryos:

Zebrafish embryos were procured from local supplier. Zebrafish embryos are kept in embryonic medium. They were kept into water tank and proper aeration was provided. 12 hrs light and 12 hrs dark light cycles was maintained. Each test compound was divided into 5 groups having 12 zebra fish embryos in each. Antiangiogenic activity was checked at three concentration levels i.e., 0.1, 0.5 and 1µg/ml of standard and test compounds. After treating the zebrafish embryos with the drug solution, the embryos were kept in the wells of cultural medium and observed after 24, 48 and 72 hfp (hours per fertilization) by using Motic digital microscope. The survival rate was recorded upto 72hpf.^21,22

In-vitro study:

Anti-cancer activity using MTT assay:

The cells were seeded into a 96-well flat-bottom microplate, which was then maintained at 37°C, 95% humidity, and 5% CO₂ overnight. The samples were treated at various doses (100, 50, 25, 12.5, 6.25µg/ml). The cells endured an additional 48hours of incubation. Following two PBS washes, each well received 20 µL of the MTT staining solution, and the plate was incubated at 37ºC. Following a 4-hour period, 100µL of DMSO was introduced into each well to dissolve the formazan crystals, and a microplate reader was used to detect the absorbance at 570nm^23-25.

Biological evaluation:

The synthesized compounds (4a-4h) were screened for anti-angiogenic activity i.e., in-vivo study using zebrafish embryo model and five compounds were tested for in-vitroanti-cancer activity against human breast cancer cell line MCF-7 using standardized technique MTT assay^23-25.

Prediction of physicochemical, pharmacokinetic, and ADMET studies:

Computational analysis of the synthesized compounds (4a–4h) was conducted to predict their molecular properties using the SwissADME web platform. Among the synthesized compounds, calculations were made for the molecular weight, molecular volume, log of the partition coefficient, number of hydrogen-bond donors and acceptors, topological polar surface area, number of rotatable bonds, and Lipinski's rule of five.

Molecular Docking Studies:

Procedure:

In the current work, the crystal structures of the tyrosine kinase domain of the Epidermal growth factor receptor were used to assess the effectiveness of the AutoDock 4.2.6 docking tool. The crystal structure of the tyrosine kinase domain of the epidermal growth factor receptor was obtained using the Protein Data Bank (PDB ID 1M17)¹². There is just one chain in it (A). After being removed, the heteroatoms and water molecules were saved as pdb files. Ligand compounds were created, optimized, and saved in pdb format for AutoDock 4.2.6 docking studies with the help of the Discovery Studio tool. The resultant ligand molecules' flexible torsions were all defined and saved in the pdbqt format using AutoDock Tools 1.5.6. The synthesized ligands were input files for a later docking procedure. Docking simulations were performed with AutoDock 4.2.6 and a Lamarckian genetic approach. The conventional technique (for 10 independent runs per ligand) was used to bind a flexible ligand and a rigid protein with established torsion angles. In the x, y, and z axes, a grid with 80, 62, and 66 points was modified according the active pocket. Grid spacing for the energy map computation was set at 0.375 Å. We assessed the produced structural files using the visualization tool, Discovery Studio.

RESULTS AND DISCUSSION:

Chemistry:

A new series of 5-(substituted phenyl)-3-(-2(substituted)ethenyl-4,5-dihydro-1H-pyrazole-1- carbaldehyde derivative (4a-4h) was synthesized by reacting various substituted aryl/heteroaryl aldehydes (1) with acetone (2) in the existance of lithium hydroxide and ethanol as a solvent to give 1,5-bis-(substituted phenyl) penta-1,4-diene-3-one (3a-3h which were then refluxed with hydrazine hydrate in abundance of formic acid using ethanol as a solvent to obtain 5-(substituted phenyl)-3-(-2(substituted)ethenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde derivative (4a-4h). The reaction monitoring was analyzed by performing thin-layer chromatography (TLC) and purified by recrystallization using ethanol.

Fig 1: Scheme for the synthesis of 5-(substituted phenyl)-3-(2(substituted) ethenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde derivatives (4a-4h).

The recently developed compounds were characterized using spectroscopic techniques such as mass spectroscopy, proton nuclear magnetic resonance (1H NMR), and Fourier transform infrared (FT-IR). IR spectra was used to confirm the structure of compound 1,5-bis-(substituted phenyl) penta-1,4-diene-3-one (3a-3h) where aromatic C-H stretching was detected at 3010 cm^-1 and aromatic C=C stretching at 977 cm^-1 in the infrared spectrum. At 1649 cm^-1, C=O stretching vibration was observed for ketone. The structure of compounds 5-(substituted phenyl)-3-(2(substituted) ethenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde derivatives (4a-4h) were analysed by spectra which revealed the presence of C=O stretching vibration at 1651 cm^-1, aromatic C=C stretching vibration at 1593 cm^-1, 1139 cm^-1, stretching vibration of CHO was observed at 1654 cm^-1 and C=N stretching vibration was observed at 1615-1593 cm^-1. The positional elucidation of protons was aided by the diagnostic utility provided by the ¹H-NMR spectra. The aromatic protons of 1,5-bis-(substituted phenyl) penta-1,4-diene-3-one showed a characteristic signal at δ 7.70-7.20 ppm. H_α proton was observed at δ 6.93-6.90 ppm and H_β proton was observed at δ 7.15-7.09 ppm as a doublet ofdoublet.

Fig 2: General structure of 5-(substituted phenyl)-3-(2(substituted) ethenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde derivatives (4a-4h)

5-(substituted phenyl)-3-(2(substituted)ethenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde derivatives (4a-4h) characterized by the formation of prominent doublet of doublet each having integration ^{of one proton. The pyrazoline formed was
confirmed with a doublet of doublet for Ha at δ 2.9-3.0ppm having J value
17.4 , for Hx at δ 5.30-5.32ppm having J value 11.4 and Hb was observed as
a multiplet at δ 3.72-3.73ppm .The distinct multiple signals of aromatic
protons were detected within the range of δ 7.70-7.20ppm.}

Drug Likeness and ADMET Prediction:

The pharmacokinetic parameters and features of drug-like substances can be determined based on their chemical structures using a computer programme called ADMET Predictor. ADMET properties were obtained using online SWISS ADME software for the synthesized compounds. (4a–4h) Molecular weight were found between 304.39 to 414.11g/mol, and their logP values fell within the permissible range of 2.00 to 3.62. Regarding aqueous solubility Compounds 4a and 4c are soluble in water, whereas compounds 4b, 4d, and 4h are moderately soluble, and compounds 4e and 4g are weakly soluble. With respect to absorption parameter, all compounds demonstrated poor GI absorption. TPSA value was <140 Å² which indicates a significant predicted oral bioavailability. The bioavailability score for all compounds was 0.55.

Table 1: Pharmacokinetics and drug-likeness predictions for the synthesized compounds (4a- 4n) by SwissADME.

Code	MW^a	HBA^b	HBD^c	TPSA^d	LogP^e	ESOL^f Class	GIA^g	Bioavailability score	P-gp^h substrate	BBBⁱ Permeability
4a	336.38	4	0	51.13	3.04	Soluble	High	0.55	No	Yes
4b	312.31	4	0	32.67	2.66	Moderately soluble	High	0.55	No	Yes
4c	310.32	4	1	52.9	2.22	Soluble	High	0.55	No	Yes
4d	304.39	2	0	32.67	3	Moderately soluble	High	0.55	No	Yes
4e	414.11	2	0	32.67	3.62	Poorly soluble	High	0.55	No	Yes
4f	414.11	2	0	32.67	3.34	Poorly soluble	High	0.55	No	Yes
4g	414.11	2	0	32.67	3.28	Poorly soluble	High	0.55	No	Yes
4h	345.22	2	0	32.67	3.02	Moderately soluble	High	0.55	No	Yes

Note- a: Molecular weight; b: Hydrogen bond acceptors; c: Hydrogen bond donors; d: Topological surface area; e: octanol-water partition coefficient; f: Solubility class; g: Gastro-intestinal absorption; h: P-glycoprotein; i: Blood-Brain Barrier.

The free binding energies of the designed compounds range from -7.4 to -9.27 kcal/mol, while the free binding energy of the co-crystallized ligand is -7.4 kcal/mol. Among the 8 compounds, the compound 5-(2,6-chlorophenyl)-3-[(E)-2-(2,6-chlorophenyl) ethenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde (4e),exhibited the binding affinity to EGFR in terms of the low binding energy of -9.27 kcal/mol; The compound 4e showedhydrogen bondinteractionswith ASP831, MET769 and hydrophobic interactions with THR766, LYS721, VAL702, LEU694, MET742 amino acids respectively which are comparable withthe interactions of standard.

Fig 4: Hydrogen and hydrophobic interactions of compound 4e. The labeled by heteroatom are blue for N, red for O and green for Cl. Hydrogen bond interactions are shown as yellow dash lines and represent the bond length in angstrom (Å).

Fig 5: Hydrogen and hydrophobic interactions of co-crystallized ligand (Erlotinib). The labeled by heteroatom are blue for N, and red for O. Hydrogen bond interactions are shown as yellow dash lines and represent the bond length in angstrom (Å).

Biological Evaluation:

In-vivo Study:

Zebrafish embryos were observed for a period of 72 hours (at 24,48 and 72 hpf) to evaluate the effect of synthesized compounds [4a-4h] and Doxorubicin on various stages of embryonic development. Phenotypical abnormalities were started developing at 24 hpf (hours per fertilization). Embryos of synthesized compounds [4a-4h] and of Doxorubicin were treated with different concentrations such as 0.1µg/µl ,0.5µg/µl and 1µg/µl. Embryos treated with vehicle control didn’t show any developmental abnormalities. Embryos exposed to test compounds and standard drug (Doxorubicin) displayed a variety of abnormalities, including tail bending, an aberrant yolk sac, abnormal vasculature, haemorrhages, pericardial oedema, and delayed hatching. In some embryos, developmental delay with the absence of vasculature was also seen. Survival rates of the embryos were found to decrease with time. Hence it can be said that synthesized compounds [4a-4h] possess cytotoxic activity against zebrafish embryos. Also, all the phenotypical changes observed were displayed in Fig 8 and Fig 9.Hence it can be predicted that synthesized compounds [4a-4h] possess anti-angiogenic activity.

Fig 6: Survival rate of Test Solution compounds 4a-4h at 72hpf.

Fig 7: Phenotypical changes of control vehicle group at 72 hpf

Fig 8: Phenotypical changes of Test solutions of compounds (4a-4h) at 72 hpf

Fig 9: Phenotypical changes of standard drug doxorubicin 72 hpf

In vitro study:

Results of in vitro anticancer activity against Breast cancer cell line (MCF-7):

In this study, from all synthesized compounds (4a-4h), compounds 4a-4e were screened for in-vitro anticancer activity against human breast cancer cell line (MCF-7) using MTT assay at different concentrations (100, 50, 25, 12.5, 6.25μg/ml) using Paclitaxel as a standard. A graphical representation of results obtained from in-vitro antiproliferative activity of compounds is given below in Fig.10

Fig 10: Graphical representation of result obtained for in-vitro anticancer activity of compounds on Breast cancer cell line.

Cytotoxicity of synthesized compounds (4a-4h) against the MCF-7 cell line was studied. The outcomes demonstrated the cytotoxicity of compounds 4b and 4d against the MCF-7 cell line, with IC₅₀ values of 38.49±0.17 and 35.49±2.44 and the IC₅₀ value of Paclitaxel (standard) is 0.35±0.10. When comparing the IC₅₀ values of the compounds with the standard, compounds 4b and 4d were shown to be moderately active at higher concentrations.

Table 3: IC50 values of the synthesized compounds

Compound code	IC₅₀(µM)
4a	59.53±4.60
4b	38.49±0.17
4c	86.45±9.42
4d	35.49±2.44
4e	42.93±1.57
Paclitaxel	0.35±0.10

CONCLUSION:

In conclusion, a series of novel pyrazoline derivatives (4a–4h) were synthesized successfully using the conventional methods of synthesis presented in the scheme and purified by recrystallization. The identification of compounds was established by single spot TLC, melting point, and spectroscopic techniques involving FT-IR, Mass spectroscopy, and ¹H NMR. The compounds had drug-likeliness and demonstrated good BBB permeability. The compounds were found to be similarly interacting or very good interacting with the target enzyme as with co-crystallized ligand. The binding interaction was observed with the amino acids like ^{ASP831, MET769, THR766, LYS721,
VAL702, LEU694 and MET742.}

ACKNOWLEDGMENT:

The authors are grateful to Dr. Kishor G. Bhat Maratha Mandal’s Central Research Laboratory, Belgaum, for providing biological data for anticancer activity against cancer cell line MCF-7. We also thank Tata Institute of Fundamental Research (TIFR) and Ambernath Organic Pvt. Ltd. Mumbai for providing assistance for spectral characterization of ¹H-NMR and Molecular ion peak respectively. The authors are especially thankful to Dr. V. J. Kadam, Principal, of Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai, for providing us with the facilities during the course of the project.

REFERENCES:

1. Viswanathan N, Gowri V, Punnagai K. Investigation of In-vitro antiproliferative activity of ampelocissus latifolia root extract on MCF-7 breast cancer cells ampelocissus latifolia root extract against MCF-7 breast cancer cells. Research Journal of Pharmacy and Technology. 2023; 16(8): 3595-600.

2. Seshacharyulu P, Ponnusamy MP, Haridas D, Jain M, Ganti AK, Batra SK. Targeting the EGFR signaling pathway in cancer therapy. Expert Opinion on Therapeutic Targets. 2012: 16(1): 15-31.

3. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2021; May; 71(3): 209-49.

4. Abd El-Meguid EA, Moustafa GO, Awad HM, Zaki ER, Nossier ES. Novel benzothiazole hybrids targeting EGFR: Design, synthesis, biological evaluation and molecular docking studies. Journal of Molecular Structure. 2021; Sep 15; 1240: 130595.

5. Wu CL, Ping SY, Yu CP, Yu DS. Tyrosine kinase receptor inhibitor-targeted combined chemotherapy for metastatic bladder cancer. The Kaohsiung Journal of Medical Sciences. 2012; Apr 1; 28(4): 194-203.

6. Sangani CB, Makawana JA, Duan YT, Yin Y, Teraiya SB, Thumar NJ, Zhu HL. Design, synthesis and molecular modeling of biquinoline–pyridine hybrids as a new class of potential EGFR and HER-2 kinase inhibitors. Bioorganic and Medicinal Chemistry Letters. 2014 Sep 15;24(18):4472-6.

7. Tateishi M, Ishida T, Mitsudomi T, Kaneko S, Sugimachi K. Immunohistochemical evidence of autocrine growth factors in adenocarcinoma of the human lung. Cancer Research. 1990 Nov 1;50(21):7077-80.

8. Rao CM, Yejella RP, Rehman RS, Basha SH. Molecular docking based screening of novel designed chalcone series of compounds for their anti-cancer activity targeting EGFR Kinase Domain. Bioinformation. 2015; 11(7): 322.

9. Housman G, Byler S, Heerboth S, Lapinska K, Longacre M, Snyder N, Sarkar S. Drug resistance in cancer: an overview. Cancers. 2014; Sep 5; 6(3): 1769-92.

10. Zhang HJ, Qian Y, Zhu DD, Yang XG, Zhu HL. Synthesis, molecular modeling and biological evaluation of chalcone thiosemicarbazide derivatives as novel anticancer agents. European Journal of Medicinal Chemistry. 2011; Sep 1; 46(9): 4702-8.

11. Rammohan A, Reddy JS, Sravya G, Rao CN, Zyryanov GV. Chalcone synthesis, properties and medicinal applications: a review. Environmental Chemistry Letters. 2020; Mar; 18: 433-58.

12. Morrison, R. T., Boyd, R. N.; Organic chemistry, 7th edition, pg no-860.) (Monograph on green chemistry laboratory experiments, Green Chemistry Task Force, DST, pg no. 13-14.

13. Bhat KI, Kumar A. Synthesis and Biological Evaluation of Some Novel Pyrazoline Derivatives Derived from Chalcones. Research Journal of Pharmacy and Technology. 2017; 10(5): 1344-6.

14. Vikram BS, Rao TS, Ramesh K. Synthesis and in Vitro Antimicrobial Activity of some New 3, 5 di-substituted pyrazoline derivatives. Asian Journal of Research in Chemistry. 2009; 2(3): 285-8.

15. Nikalje AP, Malhotra P, Ghodke M. Ultrasound-promoted Green Synthesis and Pharmacological Screening of Some Novel 4-(3, 5-diaryl substituted)-4, 5-dihydro-1H-pyrazol-1-yl) Benzene Sulfonamide. Asian Journal of Research in Chemistry. 2011; 4(11): 1712-6.

16. Murugan V, Revathi S, Sumathi K, Geetha KM, Divekar K. Synthesis of Some 1-[Bis-N, N-(2-Chloroethyl) Aminoacetyl]-3, 5-Disubstituted-1, 2-Pyrazolines as Possible Alkylating Anticancer Agents. Asian Journal of Research in Chemistry. 2010; 3(2): 496-9.

17. Bajaj SD, Mahodaya OA, Tekade PV. Acoustical study of chalcones synthesized by green protocol using lioh. h2o catalyzed aldol condensation. Int. J. Res. Biosci. Agric. Technol. 2014;2:24-36

18. Bhat KI, Kumar A. Synthesis and Biological Evaluation of Some Novel Pyrazoline Derivatives Derived from Chalcones. Research Journal of Pharmacy and Technology. 2017; 10(5): 1344-6.

19. Vikram BS, Rao TS, Ramesh K. Synthesis and in Vitro Antimicrobial Activity of some New 3, 5 di-substituted pyrazoline derivatives. Asian Journal of Research in Chemistry. 2009; 2(3): 285-8.

20. Singh P, Negi JS, Singh K, Pant GJ, Rawat MS, Joshi GC. Synthesis and structure dependent photophysical properties of novel 2-pyrazolines. Synthetic Metals. 2012; Dec 1; 162(21-22):1977-80.

21. Bakkiyanathan, A., Nathan, J. R., Ravikumar, S., Gopalakrishnan, T. S., Aruldas, F. M. M., Malathi, R. Anti-Angiogenic Effects of Theophylline on Developing Zebrafish (Danio Rerio)Embryos. Biomed. Prev. Nutr. 2012; 2(3): 174–178. https://doi.org/10.1016/j.bionut.2012.03.001.

22. Khedkar PM, Dhande SR and Jagdale DM: Acute toxicity testing of synthesized pyrazoline derivatives in adult zebrafish. Int J Pharm Sci and Res. 2018; 9(1): 277-81. doi: 10.13040/IJPSR.0975-8232.9(1).277-81.

23. Abdelsalam EA, Abd El-Hafeez AA, Eldehna WM, El Hassab MA, Marzouk HM, Elaasser MM, Abou Taleb NA, Amin KM, Abdel-Aziz HA, Ghosh P, Hammad SF. Discovery of novel thiazolyl-pyrazolines as dual EGFR and VEGFR-2 inhibitors endowed with in vitro antitumor activity towards non-small lung cancer. Journal of Enzyme Inhibition and Medicinal Chemistry. 2022; Dec 31; 37(1): 2265-82.

24. Dureja H. Reverse phase high-performance liquid chromatographic estimation and in vitro cytotoxicity of Boswellic acids on a-375 melanoma cancer cell lines. Asian Journal of Pharmaceutical Analysis. 2018; 8(1): 13-9.

25. Shanthi D, Saravanan R. Evaluation of cytotoxicity of normal vero and anticancer activity of human breast cancer cell lines by aqueous unripe fruit extract of Solanum torvum. Research Journal of Pharmacy and Technology. 2021; 14(7): 3504-8.

Received on 17.02.2024 Revised on 12.06.2024

Accepted on 05.08.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):203-211.

DOI: 10.52711/0974-360X.2025.00031

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Compound code	Estimated free energy of binding	Estimated inhibition constant (Ki)	Interactions
Compound code	Estimated free energy of binding	Estimated inhibition constant (Ki)	H-Bonding	Hydrophobic Bonding
4a	-7.4	3.63 µM	THR766, ASP831, GLN767	LEU694, ALA719, THR830, MET742, LEU820
4b	-7.79	1.95 µM	THR766, LYS721	LYS721, LEU764, GLY833, LYS836, ILE735, LEU723
4c	-8.29	836.75 nM	LYS721, THR766	LEU820, MET742, THR766, PRO770, ALA719, LEU764, LYS721, LEU694
4d	-8.18	1.02 µM	MET769, ASP831	LEU768, ALA719, MET742, LEU694, PRO770, MET769, LEU820, CYS751, THR830
4e	-9.27	160.06 nM	ASP831, MET769	THR766, LYS721, VAL702, LEU694, MET742
4f	-8.61	488.08 nM	THR766, GLN767	LEU820, CYS751, ALA719, MET769, LYS721, LEU694
4g	-8.87	314.85 nM	ASP831, THR766	VAL702, LYS721, LEU764, MET742, THR766, ALA719, LEU820
4h	-8.07	1.22 µM	MET769, LYS721, ASP831	THR830, LEU694, MET742, CYS751, LEU820, LYS721, MET769
Co-crystallised ligand/Standard (Erlotinib)	-7.4	3.21 µM	MET769, THR766	MET742, LEU764, ALA719, LEU768, LYS721, LEU820, CYS773, LEU694, THR830