1. INTRODUCTION:

Cancer is a complex group of diseases characterized by abnormal and uncontrolled growth of cells, which can invade and abolish healthy tissues, organs and potentially spread to other parts of the body. It is a leading cause of death globally and has been responsible for a considerable burden of morbidity and mortality over the past few decades. It is a significant public health issue worldwide and a foremost cause of morbidity and mortality, accounting for an estimated 14 million deaths in 2024¹. Various factors, including genetic mutations, environmental factors, lifestyle choices, and infections, cause cancer. The development and progression of cancer are governed by a wide range of cellular and molecular mechanisms that disrupt average cell growth, differentiation, and death. It arises from the uncontrolled growth of abnormal cells that have the potential to invade and spread to other parts of the body, compromising the function of critical organs and tissues. The etiology of cancer is multi factorial and involves a complex interaction between genetic, environmental, and lifestyle factors².

As per WHO, in 2023, there were approximately 2.3 million new cases of breast cancer worldwide. Breast cancer is the most common cancer among women globally, representing a significant public health issue, with over 2.3 million cases occurring each year, making it the most common cancer among adults. In 95% of countries, breast cancer is the first or second leading cause of female cancer deaths. In the United States, breast cancer accounts for about 30% of all new cancer cases in women each year. Survival from breast cancer is widely inequitable between and within countries; nearly 80% of deaths from breast and cervical cancer occur in low- and middle-income countries, underscoring the ongoing need for effective prevention, early detection, and treatment strategies.

Cancer treatment options include surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. Chemotherapy, which involves cytotoxic drugs to kill cancer cells, is a cornerstone of cancer treatment⁴. However, the emergence of drug resistance is a significant challenge in chemotherapy, leading to the development of additional potent and selective drugs with fewer side effects. Cancer is a complex and multifaceted disease that remains a significant public health challenge worldwide. Identifying risk factors, early detection, and advances in cancer treatment have improved patient outcomes, but more research is needed to develop better therapies and ultimately find a cure. Novel therapeutic strategies targeting specific molecular pathways and immune cells are currently under investigation and offer promising results⁵. The future of cancer treatment lies in personalized medicine, combining genomic and molecular profiling with innovative therapies tailored to individual patients. Multiple cancer treatment options have been developed as a result of improvements in healthcare systems. The sort of treatment will depend on the cancer's type and stage, the patient's general health, and personal preferences. There are numerous different therapy options^6-11.

Benzopyrones, a chemical class of compounds containing coumarin, have a flexible nucleus and a wide range of biological functions. Coumarin is a phenolic plant compound that comprises fused benzene and pyrone rings. The first natural product discovered in 1820 was coumarin, found in 150 species in 30 different plant families, including Umbelliferae, Clusiaceae, Guttiferae, Oleaceae, and Rutaceae, Coumarin is present in all members of the plant kingdom, including essential oils found in cinnamon bark oil, cassia leaf oil, fruits, green tea, and foods like chicory¹².Ithas several pharmacological effects, such as anti- HIV, anti-bacterial, anti-fungal, anticonvulsant, antihypertensive, anti-tubercular, scavenging of reactive oxygen species (ROS), anti-inflammatory, and anti-cancer effects^13-16.Coumarin-based nuclei are found in several potent medicines available on the market. This study aims to create coumarin fused/tethered heterocyclic compounds.

The Coumarin nucleus is one of the most potent heterocyclic scaffolds with various pharmacological effects, including anti-cancer efficacy¹⁷. Therefore, researchers are now concentrating on determining the effectiveness of the coumarin nucleus in improving therapeutic agents for breast cancer therapy¹⁸.

Henceforth, there is a need for extra research to develop personalized and effective therapies that can improve patient outcomes. The proposed research works aims to contribute to the current knowledge on breast cancer by investigating the molecular mechanisms underlying the pathogenesis of breast cancer and exploring novel therapeutic targets¹⁰.

2. MATERIAL AND METHODS:

2.1 Chemicals

All AR grade chemicals were purchased from Merck, Alfa, Aesar, CDH, HI media, Sigma Aldrich.

2.2 Molecular Docking studies

Various types of coumarin derivatives were virtually screened to explore their anticancer potency. The Molecular docking study was carried out to investigate the possible interactions of the various designed coumarin derivatives. Studies of designed molecules were carried out against human epidermal growth receptor-2 in order to evaluate the binding patterns of the molecules with the receptor. Various docking scores of designed molecules RB1-RB30 calculated by LEAD software against the protein ^24-28.

2.3 Ligand Preparation

2- Dimensional structure of different coumarin derivatives and reference to the standard compounds i.e. Exemestane and trastuzumab were generated utilizing Chem Draw Ultra (version 12.0). The generated structure was converted into 3- Dimensional Structure, energetically reduced and later saved as MDL Mol file.

3.1 General Procedures ^11-20

3.1.1 Preparation of 7-Hydroxy-4-Methyl-2H-Chromene-2-One

Added 3.7g of resorcinol to 4.5g of ethyl acetate stirred the reaction mixture until a complete solution is obtained. Now added this solution slowly to the sulphuric acid so that the temperature of mixture did not rise above 0°C then continued the stirring for 30 minutes. Poured the mixture on the crushed ice the solid 7-hydroxy-4-methyl-2h-chromene-2-one. Filtered off the coumarin at the pump. For purification fist dissolve the coumarin on cold 10% aqueous sodium Hydroxide solution and reprecipitate it by addition of dilute hydrochloric acid and the recrystallised it from ethanol or methylene spirit.

3.1.2. Preparation of 3-Acetyl-7-Hydroxy-4-Methyl-2H-Chromene-2-One

3g of 7-hydroxy-4-methyl-2H-chromene-2-one was dissolve in 16ml of acetic acid and 5.6ml of phosphorous oxychloride was added. The mixture was heated to reflux for 30 min. After cooling the precipitate were collected and recrystallised from ethanol.

3.1.3 Preparation of 7-Hydroxy-4-Methyl-3(3-Phenyl Acryloyl) – 2H-Chromene -2-One (Sa1-30)

Place a solution of 0.03mol of 3-acetyl-7-hydroxy-4-methyl-2H-chromene-2-one and the appropriate substitution 0.003mol aromatic aldehyde was dissolve in chloroform. The catalytic amount of 0.02mol piperidine was added and the reaction mixture was refluxed for 5hrs. The chloroform was distilled out and the residue was washed with methanol.

3.1.4 Preparation of 7-Hydroxy -4-Methyl-3(1-Hydroxy- Imino)-3-Phenyl Allyl)-2H-Chromene -2-One (Sb1-30)

A mixture of 1.5g of compound 6 added 0.9g of hydroxyl amine hydrochloric acid, 3.1ml of 95% ethyl alcohol and 0.6ml of water in two round neck flasks. To this added in portion with shaking 1.7g of sodium hydroxide. If the reaction become vigorous cooling with tap water may be necessary. After all the sodium hydroxide has been added the cool the mixture on ice bath. After cooling the content are poured into a solution of 4.6ml concentration hydrochloric acid in 2.6ml of water. The precipitate is filtered and washed thoroughly with water and recrystallised from ethanol or methylated spirit.

2.2.5 Preparation of 7-Hydroxy-4-Methyl-3(3-(2-oxo-3-yl)-4, 5-dihydroisoxazol -5-yl) benzaldehyde)-2H-chromene-2-one (Sc1-30)

The compound 7(1mol), 1M hydroxylamine hydrochloric acid and 5 ml water was added to 25 ml round bottom flask. The mixture was then stirred at 50 °C. For 24 hrs. After completion of reaction the mixture was then cooled at room temperature. The precipitate was collected.

2.2 Characterization of the synthesized compound

The reaction was observed on precoated TLC aluminium plates (Merck Germany) silica gel 60 F₂₅₄ thin layer plates using UV lamp for visualization of spots. TLC on a silica gel plate was used to verify the purity of the compound using varied proportion of n-hexane, ethylene acetate^8-9. The open capillary method on a Digital melting point device was used to determine the melting points of synthetic analogues. The IR spectra of the synthesized derivatives were recorded by KBr pellet method (λmax in cm^-1) using Shimadzu Fourier Transform Infrared Spectrophotometer using software version 2.27. 1H- NMR (Nuclear Magnetic Resonance) spectra and 13C-NMR spectra were obtained using JEOL JNM ECX-500 (13C-NMR at 125 MHz and 1H-NMR at 500 MHz) spectrometer. CDCl₃ with TMS was used as an internal standard. The assigned splitting pattern was: s- singlet, d doublet, dd-doublet of doublets, t- triplet, and m- multiplet. Coupling constants (J) are given in Hz (Hertz). The chemical shifts (δ) were recorded in ppm. Mass spectra (HRMS) were acquired by means of Bruker Impact HD spectrometer. UV/Visible spectra were recorded on UV/VIS Spectrophotometry LABINDIA® 3000+ and data was analyzed by using software UV-Win^8-9.

MTT Assay for MCF-7 Cell line and COLO320DM

MTT (3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) was employed to conduct cytotoxicity of synthesized coumarin derivatives on selected cancer cell lines, i.e., MCF-7, procured from the National Center for Cell Sciences, Maharashtra, Pune, INDIA. The procedure was carried out in a biosafety cabinet (class II) in the aseptic zone with laminar flow. Over the next 24 h at 37°C, MCF-7 cells were incubated in a culture medium at a concentration of 1x10⁴ cells mL^-1 under CO₂⁴⁷.

Then, 100 μL solutions of each synthesized derivatives at strengths of 10, 20, 40, and 80 μg mL^-1 were dropped onto tissue culture grade micro-96-well plates, and cells were cultured for continuous 24 h in an incubator at 37±2°C. Simultaneously, MTT dye was dissolved in buffer solution at a concentration of 1000 µg mL^-1 and then passed through membrane filter paper (0.2 μm). An additional 20 μL of MTT dye solution was introduced to each well, followed by re-incubation for 4 h at the same temperature. Consequently, 200 μL of DMSO solution was introduced into each well, placing the plate in a CO₂ incubator for 10 min. At 550–570 nm, the plate absorbance was determined using an Elisa microplate plate reader^48-51

Table 1: Anticancer coumarin derivatives showing Docking Score, Interaction Data along with protein residues


Compound	R	Mol Dock Score	Protein residue
Sc-1		-8.5	ALA 751, ASP 863, LEU 796, LYS 753, THR 862, VAL 734, LEU 852, LEU 726, PHE, 1004, CYS 805
Sc-2		-8.27	LEU 852, VAL 734, THR 862, ARG 784, SER 783, LEU 785, ASP 863, PHE 864, LEU 796, ALA 751, LEU 800, MET 801, GLY 804, PHE 1004, LEU 726, CYS 805
Sc-3		-7.3	VAL 734, LYS 753, PHE 864, LEU 796, SER 783, THR 862, ALA 751, LEU 852,
Sc-4		-7.7	LYS 753, LEU 852, VAL 734, ALA 751, ASP 863, LEU 796, LEU 785, PHE 864, MET 774
Sc-5		-7.5	VAL 734, LYS 753, ASP 863, ALA 751, LEU 852
Sc-6		-7.5	ARG 849, ASP 845, LYS 753, ASP 863, VAL 734, ALA 751, LEU 852, LEU 800, MET 801
Sc-7		-7.5	VAL 734, GLY 804, PHE 1004, LEU 852, ALA 751, SER 783, PHE 864, LEU 785
Sc-8		-7.49	LYS 753, VAL 734, LEU 852, ALA 751, ASP 863
Sc-9		-7.4	SER 783, LYS 753, VAL 734, THR 862, LEU 796, ASP 863, ALA 751, LEU 852, CYS 805
Sc-10		-8.02	VAL 734, GLU 770, LEU 796, LYS 753, THR 862, ALA 751, LEU 852, LEU 726, PHE 1004, CYS 805
Sc-11		-7.3	PHE 864, ASP 863, SER 783, LYS 753, VAL 734, ALA 751, LEU 852, CYS 805
Sc-12		-7.3	PHE 1004, LEU 852, ALA 751, ASP 863, LYS 753, LEU 796, PHE 864, LEU 785, SER 783, THR 862, VAL 734
Sc-13		-7.2	LYS 753, THR 862, LEU 796, VAL 734, ALA 751, LEU 852
Sc-14		-7.2	LEU 852, ALA 751, LEU 796, MET 774, LEU 785, PHE 864, THR 862, LYS 753, VAL 734
Sc-15		-7.1	LYS 753, THR 862, PHE 864, LEU 785, MET 774, LEU 796, SER 783, ALA 751, LEU 852
Sc-16		-6.8	VAL 734, GLY 804, LEU 852, ALA 751, ASP 863, LEU 785, PHE 864
Sc-17		-6.8	VAL 734, LYS 753, THR 862, SER 783, ASP 863, ALA 751, LEU 852, CYS 805
Sc-18		-6.65	ALA 751, SER 783, LYS 753, LEU 796, ASP 863, LEU 852, VAL 734, LEU 726, PHE 1004
Sc-19		-6.6	CYS 805, PHE 1004, LEU 726, LEU 800, ALA 751, SER 783, THR 862, LYS 753, LEU 796, GLU 770, VAL 734, LEU 852, CYS 805
Sc-20		-6.6	LYS 753, LEU 796, ASP 863, PHE 864, THR 862, SER 783, ALA 751, VAL 734, LEU 852

Table 2: Physical Data of the Synthesized Coumarin Derivatives

Comp	R	Mol. Formula	Mol. Weight (g/mol)	M.P(^oc)	Yield (%)	Colour
Sa-1		C₁₉H₁₄O₄	306.32	202-203	86.89	Cream
Sa-2		C₁₉H₁₃ClO₄	340.76	212-213	81.47	Yellow
Sa-3		C₁₉H₁₃ClO₄	340.76	214-215	76.95	Pale yellow
Sa-4		C₁₉H₁₃ClO₄	340.76	213-214	80.58	Yellow
Sa-5		C₁₉H₁₃ClO₄	340.76	212-213	82.57	Light Orange
Sa-6		C₁₉H₁₃BrO₄	385.21	218-219	86.49	Yellow
Sa-7		C₁₉H₁₃BrO₄	385.21	219-220	77.69	Yellow
Sa-8		C₁₉H₁₃BrO₄	385.21	218-219	84.38	Light brown
Sa-9		C₁₉H₁₃BrO₄	385.21	220-221	78.39	Dark yellow
Sa-10		C₁₉H₁₃NO₆	351.31	206-207	81.58	Light Brown
Sa-11		C₁₉H₁₃NO₆	351.31	207-208	80.68	Brown
Sa-12		C₁₉H₁₃NO₆	351.31	208-209	76.87	Reddish Brown
Sa-13		C₁₉H₁₃NO₆	351.31	206-207	82.69	Brown
Sa-14		C₁₉H₁₁NO₄	317.30	217-218	79.96	Light orange
Sa-15		C₁₉H₁₁NO₄	317.30	217-218	82.67	Orange
Sa-16		C₁₉H₁₁NO₄	317.30	217-218	79.31	Light yellow
Sa-17		C₁₉H₁₁NO₄	317.30	217-218	80.87	Yellow
Sa-18		C₂₁H₁₈O₆	366.37	223-224	78.69	Yellow
Sa-19		C₂₁H₁₈O₆	366.37	224-225	81.79	Cream
Sa-20		C₂₁H₁₈O₆	366.37	225-226	83.87	Light brown
Sa-21		C₂₁H₁₈O₆	366.37	227-228	76.89	Cream
Sa-22		C₂₂H₂₀O₇	396.40	229-230	81.79	Light brown
Sa-23		C₂₂H₂₀O₇	396.40	228-229	76.85	Brown
Sa-24		C₂₂H₂₀O₇	396.40	229-230	86.96	Cream
Sa-25		C₂₂H₂₀O₇	396.40	230-231	87.31	Yellowish brown
Sa-26		C₂₂H₂₀O₇	396.40	228-229	89.59	Cream
Sa-27		C₂₀H₁₆O₅	336.34	221-222	78.96	Cream
Sa-28		C₂₀H₁₆O₅	336.34	222-223	84.69	Light Brown
Sa-29		C₂₀H₁₆O₅	336.34	221-222	77.96	Cream
Sa-30		C₂₀H₁₆O₆	352.34	224-225	85.78	Light Brown

Figure 2: 3D images of the Docking Study

Table 3: Physical Data of the Synthesized Coumarin Derivatives

Comp	R	Mol. Formula	Mol. Weight (g/mol)	M.P(^oc)	Yield (%)	Colour
Sb-1		C₁₉H₁₅NO₄	321.33	206-207	76.33	Light brown
Sb-2		C₁₉H₁₄ClNO₄	355.77	218-219	73.19	Brown
Sb-3		C₁₉H₁₄ClNO₄	355.77	219-220	74.26	Light yellow
Sb-4		C₁₉H₁₄ClNO₄	355.77	218-219	74.12	Yellow
Sb-5		C₁₉H₁₄ClNO₄	355.77	220-221	73.96	Cream
Sb-6		C₁₉H₁₄BrNO₄	400.23	220-221	74.36	Yellow
Sb-7		C₁₉H₁₄BrNO₄	400.23	221-222	73.12	Pale orange
Sb-8		C₁₉H₁₄BrNO₄	400.23	220-221	74.88	Brown
Sb-9		C₁₉H₁₄BrNO₄	400.23	222-223	73.29	Light yellow
Sb-10		C₁₉H₁₄N₂O₄	366.33	213-214	73.96	Yellow
Sb-11		C₁₉H₁₄N₂O₄	366.33	215-216	74.31	Cream
Sb-12		C₁₉H₁₄N₂O₄	366.33	217-218	74.22	Cream
Sb-13		C₁₉H₁₄N₂O₄	366.33	214-215	73.21	Yellow
Sb-14		C₂₀H₁₄N₂O₄	346.34	215-216	73.98	Pale orange
Sb-15		C₂₀H₁₄N₂O₄	346.34	216-217	73.86	Yellow
Sb-16		C₂₀H₁₄N₂O₄	346.34	215-216	74.32	Cream
Sb-17		C₂₀H₁₄N₂O₄	346.34	217-218	73.27	Yellow
Sb-18		C₂₁H₁₉NO₆	381.38	217-218	74.66	Pale yellow
Sb-19		C₂₁H₁₉NO₆	381.38	217-218	73.68	Yellow
Sb-20		C₂₁H₁₉NO₆	381.38	219-220	74.89	Light Orange
Sb-21		C₂₁H₁₉NO₆	381.38	218-219	73.89	Yellow
Sb-22		C₂₂H₂₁NO₇	411.41	226-227	75.62	Yellow
Sb-23		C₂₂H₂₁NO₇	411.41	227-228	75.31	Light brown
Sb-24		C₂₂H₂₁NO₇	411.41	229-230	74.26	Dark yellow
Sb-25		C₂₂H₂₁NO₇	411.41	227-228	76.32	Light Brown
Sb-26		C₂₂H₂₁NO₇	411.41	226-227	74.21	Brown
Sb-27		C₂₀H₁₇NO₅	351.36	225-226	76.93	Reddish Brown
Sb-28		C₂₀H₁₇NO₅	351.36	224-225	77.41	Brown
Sb-29		C₂₀H₁₇NO₅	351.36	223-224	76.36	Light orange
Sb-30		C₂₀H₁₇NO₆	351.36	222-223	76.81	Orange

4.2 Chemistry

The present study aimed to synthesize biologically active coumarin derivatives. As per the proposed reaction scheme, in the initial step 7-hydroxy-4-methyl-2chromene -2-one was synthesized via the base catalyzed Claisen –Schmidt condensation of 3-acetyl-7-hydroxy-4-methyl-2H-chromene. The ring closure reaction of the 7-hydroxy-4-methyl-2H-chromene-2-one with sulphuric acid and sodium hydroxide afforded 3-acetyl-7-hydroxy-4-methyl-2H-chromene-2-one derivatives Sa (1-30). After the completion of the reaction the product were purified by suitable method.

Characteristics of the synthesized coumarin derivativesof step 6 Physical Characteristics of the all coumarin derivatives of the step 6 were represented in Table 1.2

7-Hydroxy-4-Methyl-3-(3-Phenyl Acryloyl)-2H-Chromene-2-One (Sa-1)

Rf: 0.50 (Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 1495(Ar C=C); 1720 (C=O); 1083(C-O-C); 3327(O-H); 2953(CH₃). ¹HNMR (500 MHz, DMSO) δppm: 2.39-2.40(d, 3H, J=1.24Hz, CH₃); 6.13(d, 1H, J=1.36Hz, C-H); 6.82-6.4(m, 7H, Ar-H); 7.47 (s, 1H, C-H); 7.49(S, 1H, Ar-H); 8.30(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 154.7(C-1); 159.8 (C-2); 126.4(C-3); 169.5 (C-4); 126.2 (C-5); 158.1(C-6); 153.4(C-7); 183.7(C-1'); 125.4(C-2'); 142.2(C-3'); 128.5(C-1"); 128.6 (C-2"); 127.9(C-3"); 128.6(C-4"); 128.5 (C-5"); MS(ESI)m/z: 329.09 [M+Na].

(E)-3-(3-(2-Chlorophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-2)

Rf: 0.51 (Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 1446(C=C); 1673 (C=O); 1059(C-O-C); 3428(O-H); 2950(CH₃); 740(C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.39(d, 3H, J=1.24Hz, CH₃); 6.13-6.19(d, 1H, J=1.2Hz, C-H); 6.80-6.88(m, 4H, Ar-H); 6.98 (s, 1H, C-H); 7.24-7.31(m, 3H, Ar-H); 8.20(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 134.7 (C-1); 159.4 (C-2); 126.8(C-3); 169.5 (C-4); 126.2 (C-5); 112.6 (C-6); 158.1 (C-7); 183.7(C-1'); 125.4(C-2'); 153.7 (C-3'); 127.8(C-1"); 126.7 (C-2"); 129.3(C-3"); 129.9(C-4"); 134.0(C-5"); MS(ESI)m/z: 364.07 [M+Na].”ECGUE

(E)-3-(3-(3-Chlorophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-Ones (Sa-3) Rf: 0.53 (Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 1472(C=C); 1664(C=O); 1074(C-O-C); 3329(O-H); 2846(CH₃); 754(C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.31(d, 3H, J=1.20Hz, CH₃); 6.19-6.20(d, 1H, J=1.2Hz, C-H); 6.73-6.79(m, 4H, Ar-H); 6.94 (s, 1H, C-H); 7.31-7.47 (m, 3H, Ar-H); 8.27(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 138.3 (C-1); 151.3 (C-2); 121.3(C-3); 161.3 (C-4); 123.6 (C-5); 110.2 (C-6); 143.5(C-7); 187.2(C-1'); 128.4(C-2'); 157.1 (C-3'); 126.1(C-1"); 132.6 (C-2"); 122.9(C-3"); 124.8(C-4"); 127.8(C-5"); MS(ESI)m/z: 364.07 [M+Na].”

(E)-3-(3-(4-Chlorophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-4)

Rf: 0.55 (Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 1467(C=C); 1710(C=O); 1024(C-O-C); 3470(O-H); 2912(CH₃); 722(C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.27(d, 3H, J=1.31Hz, CH₃); 6.10-6.12(d, 1H, J=1.10Hz, C-H); 6.62-6.70(m, 4H, Ar-H); 6.74 (s, 1H, C-H); 7.21-7.45 (m, 3H, Ar-H); 8.41(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 127.4 (C-1); 156.5 (C-2); 120.4(C-3); 163.2 (C-4); 124.1(C-5); 109.3 (C-6); 141.4(C-7); 181.8(C-1'); 123.4(C-2'); 151.2 (C-3'); 124.6(C-1"); 129.2 (C-2"); 131.7 (C-3"); 123.4 (C-4"); 128.9 (C-5"); MS(ESI)m/z: 364.07 [M+Na].

(E)-3-(3-(5-Chlorophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-5)

Rf: 0.51 (Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 1415(C=C); 1684 (C=O); 1046 (C-O-C); 3340 (O-H); 2947 (CH₃); 734 (C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.34(d, 3H, J=1.27 Hz, CH₃); 6.13-6.14(d, 1H, J=1.2Hz, C-H); 6.72-6.78(m, 4H, Ar-H); 6.84 (s, 1H, C-H); 7.31-7.47 (m, 3H, Ar-H); 8.37(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 137.4(C-1); 159.4 (C-2); 121.3 (C-3); 167.9(C-4); 128.5(C-5); 113.9 (C-6); 159.1(C-7); 184.6(C-1'); 124.4(C-2'); 158.7 (C-3'); 128.4(C-1"); 121.9 (C-2"); 139.3(C-3"); 127.3(C-4"); 128.9 (C-5"); MS(ESI)m/z: 364.07 [M+Na].

(E)-3-(3-(2-Bromophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-6)

Rf: 0.47(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 1444(C=C); 1671 (C=O); 1119 (C-O-C); 3119 (O-H); 2802 (CH₃); 633 (C-Br). ¹HNMR (500 MHz, DMSO) δppm:

2.39(d, 1H, J=1.24Hz, CH₃); 6.13(s, 2H, C-H); 6.81-6.85(m, 3H, Ar-H); 7.46-7.48(m, 3H, Ar-H); 8.24-8.32(d, 2H, J=3.46 O-H); ¹³CNMR (125 MHZ DMSO) δppm:154.7(C-1); 126.8 (C-2); 126.8 (C-3); 159.5(C-4); 126.4(C-5); 112.6(C-6); 158.1(C-7); 183.7(C-1'); 125.4(C-2'); 152.6(C-3'); 127.5(C-1''); 126.3(C-2''); 127.8(C-3''); 132.6(C-4''); 122.4(C-5''); MS (ESI) m/z: 408.06 [M+Na].”

(E)-3-(3-(3-Bromophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-7)

Rf: 0.41 (Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1):3270(O-H); 1480(C=C); 1694(C=O); 1047(C-O-C); 2896(CH₃); 670(C-Br).¹HNMR (500 MHz, DMSO) δppm: 2.47 (d,1H,J=1.28Hz, CH₃); 6.24(s, 2H,C-H); 6.79-6.83(m,3H, Ar-H); 7.42-7.45(m,3H, Ar-H); 8.41-8.52(d,2H,J=3.86Hz,O-H). ¹³CNMR (125 MHZ DMSO) δppm: 158.3(C-1); 157.2(C-2); 129.4(C-3); 153.4(C-4); 123.5(C-5); 110.9(C-6); 159.7(C-7); 181.4(C-1'); 123.7(C-2'); 151.9(C-3'); 121.3(C-1''); 121.9(C-2''); 133.7(C-3''); 120.4(C-4''); 127.3(C-5''). MS (ESI) m/z: 408.06[M+Na].

(E)-3-(3-(4-Bromophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-8)

Rf: 0.43(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3410(O-H); 1520(C=C); 1721(C=O); 1012(C-O-C); 1934(CH₃); 654(C-Br). ¹HNMR (500 MHz, DMSO) δppm: 2.53(d, 1H, J=1.34Hz, CH₃); 6.18(s, 2H, C-H); 6.80-6.84(m, 3H, Ar-H); 7.51-7.54(m, 3H, Ar-H); 8.63-8.71(d, 2H, J=3.42Hz, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 151.2(C-1); 153.4(C-2); 123.7(C-3); 153.4(C-4); 123.5(C-5); 110.9(C-6); 159.3(C-7); 185.2(C-1'); 122.9(C-2'); 154.3(C-3'); 125.4(C-1''); 122.3(C-2'');123.4(C-3''); 138.4(C-4''); 125.9(C-5'')MS (ESI) m/z:408.06 [M+Na].

(E)-3-(3-(5-Bromophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-9)

Rf: 0.48(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1):3329(O-H), 1374(C=C); 1666(C=O); 1139(C-O-C); 2813(CH₃); 698(C-Br).¹HNMR (500 MHz, DMSO)δppm: 2.74(d, 1H, J=1.31Hz, CH₃); 6.20(s, 2H, C-H); 6.71-6.76(m, 3H, Ar-H); 7.48-7.52(m, 3H, Ar-H); 8.31-8.40(d, 2H, J=4.32Hz, Ar-H, O-H).¹³CNMR (125 MHZ DMSO) δppm: 156.2(C-1); 155.4(C-2); 125.8(C-3); 155.9(C-4); 121.9(C-5); 114.7(C-6); 155.3(C-7); 187.3(C-1'); 128.2(C-2'); 157.8(C-3'); 123.5(C-1''); 127.8(C-2''); 125.9(C-3''); 131.4(C-4''); 120.4(C-5''). MS (ESI) m/z: 408.06[M+Na].

(E)-3-(3-(2-Nitrophenyl) Acryloyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sa-10)

Rf: 0.61(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 1382(N=O); 3487(O-H); 1668(C=C); 1063(C-O-C); 1483(C=O). ¹HNMR (500 MHz, DMSO) δppm: 2.39(d, 3H, J=1.24Hz, CH₃); 3.15(s, 1H, C-H); 6.13(s, 1H, C-H); 6.82-7.48(m, 7H, Ar-H); 8.31(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 154.7(C-1); 159.4(C-2); 126.8(C-3); 169.5 (C-4); 126.2(C-5); 112.6(C-6); 158.1(C-7); 183.7(C-1'); 125.4(C-2'); 152.6(C-3'); 127.3(C-1''); 134.7(C-2''); 128.8(C-3''); 123.8(C-4''); 147.7(C-5''). MS (ESI) m/z: 389.17 [M+Na].”

Various characteristics of the synthesized coumarin were represented in Table 4.3.

Table 4: Physical Data of the Synthesized Coumarin Derivatives


Comp	R	Mol. Formula	Mol. Weight (g/mol)	M.P (^oC)	Yield (%)	Color
Sc-1		C₁₉H₁₅NO₄	321.33	212-213	76.48	Pale brown
Sc-2		C₁₉H₁₄ClNO₄	355.77	225-226	75.88	Yellow
Sc-3		C₁₉H₁₄ClNO₄	355.77	227-228	75.12	Cream
Sc-4		C₁₉H₁₄ClNO₄	355.77	226-227	76.87	Yellow
Sc-5		C₁₉H₁₄ClNO₄	355.77	225-226	74.37	Light brown
Sc-6		C₁₉H₁₄BrNO₄	400.23	227-228	74.89	Yellow
Sc -7		C₁₉H₁₄BrNO₄	400.23	228-229	74.12	Cream
Sc-8		C₁₉H₁₄BrNO₄	400.23	229-230	74.69	Pale orange
Sc-9		C₁₉H₁₄BrNO₄	400.23	227-228	71.67	Cream
Sc-10		C₁₉H₁₄N₂O₄	366.33	214-215	76.21	Light brown
Sc-11		C₁₉H₁₄N₂O₄	366.33	215-216	74.31	Yellow
Sc-12		C₁₉H₁₄N₂O₄	366.33	216-217	75.37	Yellow
Sc-13		C₁₉H₁₄N₂O₄	366.33	215-216	74.86	Cream
Sc-14		C₂₀H₁₄N₂O₄	346.34	222-223	75.34	Cream
Sc-15		C₂₀H₁₄N₂O₄	346.34	225-226	75.13	Light brown
Sc-16		C₂₀H₁₄N₂O₄	346.34	223-224	76.47	Brown
Sc-17		C₂₀H₁₄N₂O₄	346.34	221-222	75.98	Light yellow
Sc-18		C₂₁H₁₉NO₆	381.38	224-225	76.98	Yellow
Sc-19		C₂₁H₁₉NO₆	381.38	225-226	73.78	Cream
Sc-20		C₂₁H₁₉NO₆	381.38	224-225	74.98	Yellow
Sc-21		C₂₁H₁₉NO₆	381.38	227-228	76.66	Pale orange
Sc-22		C₂₂H₂₁NO₇	411.41	228-229	71.22	Brown
Sc-23		C₂₂H₂₁NO₇	411.41	229-230	75.98	Light yellow
Sc-24		C₂₂H₂₁NO₇	411.41	228-229	78.74	Yellow
Sc-25		C₂₂H₂₁NO₇	411.41	230-231	76.35	Cream
Sc-26		C₂₂H₂₁NO₇	411.41	228-229	76.59	Cream
Sc-27		C₂₀H₁₇NO₅	351.36	231-231	71.23	Yellow
Sc-28		C₂₀H₁₇NO₅	351.36	233-234	73.67	Pale orange
Sc-29		C₂₀H₁₇NO₅	351.36	234-235	77.49	Yellow
Sc-30		C₂₀H₁₇NO₆	351.36	227-228	72.89	Yellow

3-((1E,2E)-3-(2-Chlorophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-2)

Rf: 0.41(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3192(Ar-H); 1741(C=O); 1451(C=C); 2810(Ar-H); 3498(O-H); 1274(C-O-C); 1682(C=N); 742(C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.31(d, 3H, J=1.25Hz, CH₃); 6.41(d, 2H, J=1.41Hz, Ar-H); 6.95(m, 3H, Ar-H); 7.25(d, 1H, J=1.14Hz, Ar-H); 7.57(s, 3H, Ar-H); 8.14(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: : 103.14(C-1); 152.36(C-2); 114.2(C-3); 124.5(C-4); 172.4(C-5); 112.4(C-6); 164.3(C-7); 134.7(C-1'); 34.72(C-2'); 31.33(C-3'); 124.7(C-1''); 134.8(C-2''); 149.3(C-3''); 126.4(C-4''); 129.4(C-5''). MS (ESI) m/z: 379.14[M+Na].

3-((1E,2E)-3-(3-Chlorophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-3)

Rf: 0.43(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3142(Ar-H); 1793(C=O); 1473(C=C); 2847(Ar-H); 3474(O-H); 1264(C-O-C); 1629(C=N); 752(C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.31(d, 3H, J=1.25Hz, CH₃); 6.41(d, 2H, J=1.41Hz, Ar-H); 6.95(m, 3H, Ar-H); 7.25(d, 1H, J=1.14Hz, Ar-H); 7.57(s, 3H, Ar-H); 8.14(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 104.5(C-1); 152.3(C-2); 112.4(C-3); 126.4(C-4); 171.5(C-5); 110.4(C-6); 154.7(C-7); 135.4(C-1'); 37.2(C-2'); 32.4(C-3'); 126.4(C-1''); 148.2(C-2''); 136.3(C-3''); 124.1(C-4''); 125.4(C-5''). MS (ESI) m/z: 379.14 [M+Na].

3-((1E,2E)-3-(4-Chlorophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-4)

Rf: 0.53(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3172(Ar C-H); 1720(C=O); 1473(C=C); 2874(Ar-H); 3494(O-H); 1234(C-O-C); 1647(C=N); 762(C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.31(d, 3H, J=1.31Hz, CH₃); 6.42(d, 2H, J=1.32Hz, Ar-H); 6.91(m, 3H, Ar-H); 7.23(d, 1H, J=1.14Hz, Ar-H); 7.59(s, 3H, Ar-H); 8.12(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 102.4(C-1); 154.3(C-2); 113.5(C-3); 128.4(C-4); 173.5(C-5); 112.5(C-6); 132.5(C-7); 137.4(C-1'); 34.9(C-2'); 33.4(C-3'); 127.52(C-1''); 149.2(C-2''); 174.3(C-3''); 139.4(C-4''); 126.3(C-5''). MS (ESI) m/z: 379.14[M+Na].

3-((1E,2E)-3-(5-Chlorophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-5)

Rf: 0.57(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3111(Ar C-H); 1749(C=O); 1460(C=C); 2849(Ar-H); 3448(O-H); 1214(C-O-C); 1680(C=N); 744(C-Cl). ¹HNMR (500 MHz, DMSO) δppm: 2.41(d, 3H, J=1.24Hz, CH₃); 6.47(d, 2H, J=1.32Hz, Ar-H); 6.93(m, 3H, Ar-H); 7.21(d, 1H, J=1.14Hz, Ar-H); 7.55(s, 3H, Ar-H); 8.04(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 104.3(C-1); 164.4(C-2); 111.4(C-3); 129.3(C-4); 164.4(C-5); 110.4(C-6); 134.4(C-7); 139.4(C-1'); 35.2(C-2'); 38.4(C-3'); 126.4(C-1''); 148.2(C-2''); 169.3(C-3''); 144.3(C-4''); 131.4(C-5''). MS (ESI) m/z: 379.14[M+Na].

3-((1E,2E)-3-(2-Bromophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-6)

Rf: 0.61(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3192(ArC-H); 1741(C=O); 1451(C=C); 2810(Ar-H); 3498(O-H); 1274(C-O-C); 1682(C=N); 640(C-Br). ¹HNMR (500 MHz, DMSO) δppm: 2.23(d, 3H, J= 1.24Hz, CH₃); 6.32(d, 2H, J=1.36Hz, Ar-H); 6.78-6.80(m, 3H, Ar-H); 6.90 (m, 2H, C-H); 7.21(d, 1H, J-1.24Hz, Ar-H); 8.32(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 104.3(C-1); 154.9(C-2); 112.4(C-3); 126.4(C-4); 162.4(C-5); 113.4(C-6); 160.7(C-7); 151.4(C-1'); 39.4(C-2'); 34.9(C-3'); 127.4(C-1''); 132.4(C-2''); 147.8(C-3''); 126.4(C-4''); 147.5(C-5''). MS (ESI) m/z: 423.15[M+Na].

3-((1E,2E)-3-(3-Bromophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-7)

Rf: 0.41(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3247(ArC-H); 1741(C=O); 1474(C=C); 2871(Ar-H); 3444(O-H); 1211(C-O-C); 1684(C=N); 650(C-Br). ¹HNMR (500 MHz, DMSO) δppm: 2.34(d, 3H, J= 1.24Hz, CH₃); 6.32(d, 2H, J=1.41Hz, Ar-H); 6.21-6.83(m, 3H, Ar-H); 6.94 (m, 2H, C-H); 7.61(d, 1H, J-1.24Hz, Ar-H); 8.13(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 105.3(C-1); 134.7(C-2); 112.3(C-3); 126.4(C-4); 153.5(C-5); 113.2(C-6); 146.4(C-7); 132.3(C-1'); 39.4(C-2'); 36.3(C-3'); 123.3(C-1''); 126.2(C-2''); 136.9(C-3''); 129.3(C-4''); 149.5(C-5''). MS (ESI) m/z: 423.15[M+Na].

3-((1E,2E)-3-(4-Bromophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-8)

Rf: 0.47(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3180(ArC-H); 1732(C=O); 1482(C=C); 2873(Ar-H); 3494(O-H); 1241(C-O-C); 1643(C=N); 643(C-Br). ¹HNMR (500 MHz, DMSO) δppm: 2.41(d, 3H, J= 1.21Hz, CH₃); 6.42(d, 2H, J=1.34Hz, Ar-H); 6.74-6.81(m, 3H, Ar-H); 6.93(m, 2H, C-H); 7.30(d, 1H, J-1.31Hz, Ar-H); 8.10(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 107.2(C-1); 131.5(C-2); 113.4(C-3); 127.5(C-4); 146.4(C-5); 113.4(C-6); 140.4(C-7); 131.4(C-1'); 38,3(C-2'); 34.5(C-3'); 124.1(C-1''); 127.4(C-2''); 139.4(C-3''); 130.5(C-4''); 134.5(C-5''). MS (ESI) m/z: 423.15[M+Na].

3-((1E,2E)-3-(5-Bromophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-9)

Rf: 0.59(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3170(ArC-H); 1720(C=O); 1432(C=C); 2894(Ar-H); 3324(O-H); 1214(C-O-C); 1671(C=N); 643(C-Br). ¹HNMR (500 MHz, DMSO) δppm: 2.31(d, 3H, J= 1.17Hz, CH₃); 6.31(d, 2H, J=1.31Hz, Ar-H); 6.72-6.83(m, 3H, Ar-H); 6.92 (m, 2H, C-H); 7.29(d, 1H, J-1.24Hz, Ar-H); 8.17(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 107.4(C-1); 152.4(C-2); 114.3(C-3); 128.3(C-4); 152.4(C-5); 117.3(C-6); 157.4(C-7); 152.9(C-1'); 38,3(C-2'); 37.4(C-3'); 126.4(C-1''); 127.4(C-2''); 133.3(C-3''); 128.4(C-4''); 143.4(C-5''). MS (ESI) m/z: 423.15[M+Na].

3-((1E,2E)-3-(2-Nitrophenyl)-1-(Hydroxyimino) allyl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sb-10)

Rf: 0.52(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3144(Ar C-H); 1729(C=O); 1453(C=C); 2849(Ar-H); 3400(O-H); 1213(C-O-C); 1648(C=N); 1344(N=O). ¹HNMR (500 MHz, DMSO) δppm: 2.41(d, 3H, J=1.21Hz, CH₃); 6.21(d, 2H, J=1.31Hz, Ar-H); 6.79-6.84(m, 3H, Ar-H); 6.92(m, 1H, Ar-H); 7.41(s, 3H, Ar-H); 8.21(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm : 104.2(C-1); 153.8(C-2); 156.3(C-3); 114.9(C-4); 147.3(C-5); 114.6(C-6); 159.4(C-7); 161.4(C-1'); 31.4(C-2'); 79.4(C-3'); 127.3(C-1''); 149.4(C-2''); 104.3(C-3''); 129.3(C-4''); 121.4(C-5''). MS (ESI) m/z: 389.09[M+Na].

Various characteristics of the synthesized coumarin were represented in Table 4.4.

7-Hydroxy-4-Methyl-3-(5-Phenyl-4,5-Dihydroisoxazol-3-Yl)-2h-Chromene-2- One (Sc-1)

Rf: 0.47(Ethyl Acetate: n-Hexane: 2.5:7.5). IR (KBr) λmax (cm-1): 3195(Ar C-H); 1739(C=O); 1455(C=C); 2899(Ar-H); 3493(O-H); 1278(C-O-C); 1694(C=N). ¹HNMR (500 MHz, DMSO) δppm: 2.35-2.37(d, 3H, J=1.24Hz, CH₃); 6.21(d, 2H, J=1.33Hz, C-H); 6.52(m, 2H, J=1.36Hz, C-H); 6.83(m, 7H, Ar-H); 7.56(s, 1H, Ar-H); 8.5(s, 1H, O-H). ¹³CNMR (125 MHZ DMSO) δppm: 102.1(C-1); 153.4(C-2); 111.0(C-3); 126.4(C-4); 160.2(C-5); 112.2(C-6); 161.9(C-7); 154.7(C-1'); 39.1(C-2'); 30.9(C-3'); 126.4(C-1''); 123.7(C-2''); 148.9(C-3''); 125.7(C-4''); 129.8(C-5''). MS (ESI) m/z: 344.10[M+Na].

3-(5-(2-Chloropheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-2)

3-(5-(3-Chloropheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-3)

3-(5-(4-Chloropheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-4)

3-(5-(5-Chloropheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-5)

3-(5-(2-Bromopheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-6)

3-(5-(3-Bromopheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-7)

3-(5-(4-Bromopheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-8)

3-(5-(5-Bromopheny)-4, 5-Dihydroisoxazol-3-yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-9)

3-(5-(2-Nitropheny)-4,5-Dihydroisoxazol-3-Yl)-7-Hydroxy-4-Methyl-2H-Chromene-2-One (Sc-10)

In vitro antiproliferative evaluation of the synthesized coumarin hybrids

All the synthesized compounds were screened against MCF-7 cell line to determine their cell death. In vitro testing was done using MDA assay protocol each derivative was tested at 4 dose level (10, 20, 40, 80 µg mL^-1).

Ten synthesized coumarin molecules were evaluated for anticancer activity against breast cancer (MCF-7) cell line. The compound exhibited the IC50 value at micromolar range. Exemestane and Trastuzumab were used as reference drug against breast cancer cell line.

Compound 10 and compound 1 display maximum potency against all the cell lines which was almost comparable to reference to drugs exemestane and trastuzumab against breast cancer cell line. Rest of the compound revealed good to moderate activity against breast cancer cell line.

Thus, the observation suggests that the compounds are safer to normal cells. The overall study suggested that the synthesized molecules can serves as potential lead for drugs development against cancer.

The cell viability of the best ten compounds was also assessed using all the cell lines. By employing the MTT assay cell viability was determined by using optical density of the control 100% viability.

The cancer cell lines were treated with the synthesized compound and were further incubated for 4hrs followed by measurement of the optical density for all the treated cell line.

The cell viability against breast cancer cell line MCF-7 varied after four hour of incubation.

4. CONCLUSION:

The study reported synthesis, characterization, docking studies and biological investigation different series of coumarin derivatives.Different series of coumarin derivatives were synthesized by based catalyzed Claisen –Schmidt condensation resulting in formation of coumarin derivatives respectively. Futher the structure of all the synthesized coumarin derivatives were confirmed by IR, NMR and HRMS spectral analysis. Docking studies of all the synthesized coumarin derivatives were done by using LEAD software. Enzyme was targeted for docking studies for anticancer activity. Similarly Trastuzumab was targeted for docking studies for anticancer activity.By considering the anti-cancer potentials of coumarin moieties, a series of coumarin hybrids was designed and screened for their HER2 binding properties through molecular docking approaches. HER2 are the promising targets associated with the breast cancer. Best thirty molecules were screened out on the basis of the docking scores which were compared to the reference drugs trastuzumab. The interaction patterns of these molecules were also studies with the help of docking poses. Further in silico drug likeliness and ADME properties of these compounds were also evaluated using online tools. Also the in silico screening of the compounds, these best thirty compounds were synthesized and the structures of synthesized derivatives were evaluated by 1HNMR, 13C-NMR and HRMS. The synthesized coumarin hybrids were then subjected to their antiproliferative potentials against various cancer cell lines using the MTT assay. The antiproliferative evaluation was done against breast cancer cell line. This study has provided a new path for the utilization of coumarin derivatives as promising anticancer. Futher preclinical and clinical studies can provide the platform for the management of anticancer.

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Received on 12.02.2025 Revised on 18.02.2025

Accepted on 18.03.2025 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):957-979.

DOI: 10.52711/0974-360X.2025.00140

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

Compound	% viability (Mean± St. Dev.)
Compound	10 µg mL^-1	20 µg mL^-1	40 µg mL^-1	80 µg mL^-1
Compound 1	93.47±5.9079	82.33±1.3142	80.76±1.2950	68.14±1.5964
Compound 2	96.34±2.9540	92.60±1.8310	85.11±1.7860	74.23±1.7090
Compound 3	96.99±6.4618	94.16±1.5738	89.20±1.0800	74.23±1.8557
Compound 4	103.66±0.7380	102.70±1.7340	97.38±1.5570	83.89±1.1580
Compound 5	104.05±0.9231	103.31±2.1104	97.73±1.1250	83.89±1.6153
Compound 6	96.99±6.4618	93.90±1.7867	89.29±0.6900	84.16±1.2681
Compound 7	98.69±2.5847	89.73±1.1773	77.45±1.0470	74.93±1.3054
Compound 8	101.43±2.7693	95.47±2.7673	86.07±1.2250	75.19±1.1965
Compound 9	99.34±0.5539	96.43±0.8393	88.59±0.9160	72.32±1.1965
Compound 10	98.82±1.2924	91.64±2.3499	86.59±1.4260	66.92±1.2206