Novel anti-cancer compounds and process for synthesising thereof

Abstract

ABSTRACT “NOVEL ANTI-CANCER COMPOUNDS AND PROCESS FOR SYNTHESISING THEREOF” The present invention provides novel anti-cancer compound / s. Most particularly, it provides substituted acetic 2-(1,3- dioxo-1,3-dihydro-2H-isoindol-2-yl) anhydride derivatives as anti-cancer drugs which are useful for the treatment of breast cancer. Anti-cancer compounds are represented by following formula Wherein ‘R’ is independently selected from , , ,

Description

FIELD OF INVENTIONThe present invention relates to the design and synthesis of novel anti-cancer agents / compounds. Specifically, the invention pertains to compounds, compositions, or formulations exhibiting potent cytotoxic activity against human breast cancer cell lines, especially MCF-7 (Michigan Cancer Foundation-7) cells, and their use in the treatment and / or prevention of breast cancer.BACKGROUND OF INVENTIONBreast cancer is one of the most prevalent forms of cancer affecting women worldwide, representing a significant public health challenge. Despite advances in early detection and therapeutic strategies, a substantial number of patients continue to experience disease progression, metastasis, or recurrence. The complexity and heterogeneity of breast cancer, including hormone receptor-positive (HR+), HER2-positive, and triple-negative breast cancers (TNBC), necessitate the development of novel targeted therapies with improved efficacy and reduced toxicity.Among the in vitro models widely used for evaluating the anticancer potential of new chemical entities is the MCF-7 cell line, derived from human breast adenocarcinoma. The MCF-7 line is estrogen receptor-positive (ER+), making it a valuable model for hormoneresponsive breast cancer research. Compounds that exhibit cytotoxicity or growth inhibition in MCF-7 cells have the potential to act as leads for drug development against estrogen receptor-positive breast cancers.Several natural and synthetic compounds have been reported in the prior art for their antiproliferative activity against breast cancer cell lines, including tamoxifen, doxorubicin, paclitaxel, and newer small molecules targeting specific pathways such as PI3K / Akt / mTOR, CDK4 / 6, and PARP. However, the therapeutic use of many existing chemotherapeutic agents is limited by poor selectivity, multidrug resistance (MDR), doselimiting toxicities, and suboptimal pharmacokinetic profiles.Therefore, there is a continuing need to discover and develop novel anticancer agents that are not only effective against breast cancer cells, particularly MCF-7, but also exhibit improved safety and pharmacological properties.The present invention addresses this long-felt need by providing novel compounds / compositions / formulations that exhibit significant cytotoxic and / or apoptotic effects against MCF-7 cells, indicating their potential utility in the treatment of breastcancer.OBJECTS OF THE INVENTIONThe main objective of this invention is to provide novel anti-cancer agents that exhibit potent cytotoxic activity against human breast cancer cell lines, particularly MCF-7 (Michigan Cancer Foundation-7) cell lines.Another objective of this invention is to provide novel chemical compounds, pharmaceutical compositions, or formulations that are effective in the treatment and / or prevention of breast cancer, having improved efficacy, selectivity, and safety profile compared to conventional treatments.Another object of the present invention is to provide a process for synthesising such anticancer agents.Yet another object of the present invention is to provide compounds and compositions that induce apoptosis and / or inhibit proliferation of estrogen receptor-positive (ER+) breast cancer cells such as MCF-7 by targeting specific molecular pathways involved in tumor growth and survival.Another objective of this invention is to develop anti-cancer agents with favorable pharmacokinetic and physicochemical properties such as increased solubility, metabolic stability, and vascular permeability.Another object of the invention is to overcome the limitations of existing anti-cancer drugs, including low selectivity, off-target toxicity, multidrug resistance (MDR), and limited bioavailability.It is also an object of the invention to provide a method of treating or preventing breast cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the disclosed compounds or pharmaceutical compositions.It is an additional object of the invention to provide a method for inducing cytotoxicity or apoptosis in MCF-7 breast cancer cells using the novel compounds or compositions disclosed herein.BRIEF DESCRIPTION OF FIGURESFigure 1 shows the 1H NMR (CDCl3, 400 MHz, ppm) spectrum of (Acetic (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic anhydride) (AA-I)Figure 2 depicts 1H NMR (CDCl3, 400 MHz, ppm) spectrum of Acetic 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoic anhydride (AA-II)Figure 3 shows 1H NMR (CDCl3, 400 MHz, ppm) spectrum of Acetic 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic anhydride (AA-III)Figure 4 illustrates 1H NMR (CDCl3, 400 MHz, ppm) spectrum of Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic anhydride (AA-IV)Figure 5 shows 1H NMR (CDCl3, 400 MHz, ppm) spectrum of Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic anhydride (AA-V)Figure 6 shows molecular docking poses of (A) Acetic (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic anhydride) (AA-I), (B) Acetic 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoic anhydride (AA-II), (C) Acetic 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic anhydride (AA-III), (D) Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic anhydride (AA-IV), and (E) Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic anhydride (AA-V)Figure 7 shows MD simulation analysis of AA-III-MMP-9 complex (A) RMSD (Protein RMSD is shown in grey while RMSD of compound AA-III is shown in red), (B) Protein RMSF, and (C) Protein-ligand contact analysis of MD trajectory.SUMMARY OF THE INVENTIONMain aspect of the present invention provides a compound of formula (I) represented byWherein 'R' is independently selected fromAnother aspect of the present invention provides a process for synthesising the compound of formula (I), the process comprising:a. triturating a mixture comprising a dione compound and an amino acid to obtain a triturated mixture;b. dissolving the triturated mixture in a solvent and refluxing for a time period in the range of 8 hrs to 10 hrs to obtain a compound of formula (II);c. contacting the compound of formula (II) with an acid halide at a reflux temperature for a time period in the range of 30 mins to 90 mins to obtain the compound of formula (I);d. optionally, purifying the compound of formula (I) in a solvent to obtain a pure compound of formula (I) with a yield in the range of 46% to 65%.DETAILED DESCRIPTION OF THE INVENTIONIn the specification, different terms are used for describing the invention. The definitions of the terms are provided below.The term 'the compound of formula (I)' or 'anti-cancer drug / compound' used herein refers to compounds that inhibit the activity of an anti-cancer drug.The terms 'the compound of formula (I)', 'anti-cancer drug / compound' can be used interchangeably throughout the specification.The term 'mixture' used herein refers to a combination of two or more substances that are physically combined. In the present invention, the mixture comprises a dione compound and an amino acid.The term 'triturated mixture' used herein refers to mixing the dione compound and amino acid into the appropriate physical form so that it will dissolve easily in the solvent or react with other components.The term 'compound of formula (II)' used herein refers to an acid derivative of dione compounds obtained by coupling of 2-benzofuran-1, 3-dione and amino acids inappropriate reaction conditions which is further processed in the synthesis of compound of formula (I).The term 'pure compound of formula (I)' used herein refers to a pure compound of formula (I) obtained by any conventional purification technique, such as but is not limited to crystallisation, recrystallisation, precipitation, etc.The term 'solvent' used herein refers to a substance that can dissolve another substance, or in which another substance is dissolved, forming a solution. The solvent used in the present invention can be a polar or a non-polar solvent. The said solvent may be used in anhydrous form. The solvent may include a deuterated form of the above-mentioned solvent(s).One of the embodiments of the present invention provides a compound of formula (I) represented byWherein 'R' is independently selected fromAnother embodiment of the present invention provides a compound of formula (I), wherein the compound of formula (I) is selected fromAnother embodiment of the present invention provides a process for synthesising the compound of formula (I), the process comprising:a. triturating a mixture comprising a dione compound and an amino acid to obtain a triturated mixture;b. dissolving the triturated mixture in a solvent and refluxing for a time period in the range of 8 hrs to 10 hrs to obtain a compound of formula (II);c. contacting the compound of formula (II) with an acid halide at a reflux temperature for a time period in the range of 30 mins to 90 mins to obtain the compound of formula (I);d. optionally, purifying the compound of formula (I) in a solvent to obtain a pure compound of formula (I) with a yield in the range of 46% to 65%.Another embodiment of the present invention provides a process for synthesising the compound of formula (I), wherein the dione compound is 2-benzofuran-1, 3-dione.Another embodiment of the present invention provides a process for synthesising the compound of formula (I), wherein amino acid is selected from amino acetic acid (glycine), 2-aminopropionic acid (alanine), 2-Amino-3-phenylpropanoic acid (phenylalanine), 2-amino-4-methylpentanoic acid (leucine), and 2-amino-3-methylbutanoic acid (Valine).Another embodiment of the present invention provides a process for synthesising the compound of formula (I), wherein R1 is selected fromAnother embodiment of the present invention provides a process for synthesising compound of formula (I), wherein solvent is selected from water, methanol, ethanol, isopropanol, glacial acetic acid, acetone, acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), N-methyl pyrrolidone (NMP), toluene, chloroform, diethyl ether, carbon tetrachloride, and dichloromethane (DCM).Another embodiment of the present invention provides a process for synthesising a compound of formula (I), wherein the acid halide is selected from formyl chloride, acetyl chloride, propionyl chloride, butyryl chloride, iso-butyryl chloride, valeryl chloride, and p-toluoyl chloride.Another embodiment of the present invention provides a process for synthesising a compound of formula (I), wherein the acid halide is acetyl chloride.EXAMPLEA. SYNTHESIS OF SUBSTITUTED ACETIC 2-(1,3- DIOXO-1,3-DIHYDRO-2H-ISOINDOL-2-YL) ANHYDRIDE DERIVATIVES AS ANTI-CANCER DRUG (COMPOUND OF FORMULA (I))I. Synthesis of (1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic acid and its conversion to Acetic (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic anhydridea. Synthesis of (1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic acid (A-I)A solid mixture of 2-benzofuran-1, 3-dione (0.01 mol) and amino acetic acid (0.01 mol) was triturated and fused by heating at 100°C. The melt was allowed to cool in a desiccator, washed with ample cold water, and allowed to dry. The dried compound was crystallized from rec. spirit and its melting point was determined. The yield obtained was 56%.b. Synthesis of Acetic (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic anhydride (AA-I) from (1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic acid (A-I)Freshly dried and weighed 1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic acid was mixed with acetyl chloride and the mixture was refluxed for 1 hr (TLC monitored). The cooled reaction mixture was poured into ice-cold water to get a white coloured solid precipitate, which was washed with water and dried in a desiccator. The dried solid anhydride was crystallized from chloroform and its melting point was determined. The yield collected was 50%.II. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) propanoic acid and its conversion to Acetic 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoic anhydridea. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) propanoic acid (A-II)1. A solid mixture of 2-benzofuran-1, 3-dione (0.01 mol) and 2-aminopropionic acid (0.01 mol) was triturated and dissolved in glacial acetic acid (10 ml) and was refluxed for 3 hrs (TLC monitored). The heating was continued for one more hour. The reaction mixture was cooled and allowed to stand as it is. Crystalline compound separated at the bottom of the flask was filtered, washed with cold acetic acid, and then with water and dried in a desiccator. The yield was 26%.2. The procedure given above was repeated with refluxing the reaction mixture for 10 hrs (TLC monitored). The reaction mixture was cooled to get a crystalline compound, which was filtered, washed with ample cold water, and recrystallized with methanol. The yield achieved was 75%.b. Synthesis of Acetic 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoic anhydride (AA-II) from 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) propanoic acid (A-II)Freshly dried and weighed 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) propanoic acid (0.01 mol) was mixed with acetyl chloride and the mixture was refluxed for 1 hr (TLC monitored). The cooled reaction mixture was poured into ice-cold water to get a white coloured solid precipitate, which was washed with water and dried in a desiccator. The dried solid anhydride was crystallized from chloroform and its melting point was determined. The yield collected was 46%.III. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic acid and its conversion to Acetic 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic anhydridea. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic acid (A-III)A solid mixture of 2-benzofuran-1, 3-dione (0.01 mol) and 2-Amino-3-phenylpropanoic acid (0.01 mol) was triturated and dissolved in glacial acetic acid (10 ml) and was refluxed for 3 hrs (TLC monitored). The heating was continued for one more hour. The reaction mixture was cooled and maintained under stirring. Crystalline compound separated at the bottom of the flask which were filtered, washed with cold acetic acid, and then with water, and dried in a desiccator. The yield was 33%.The procedure given above was repeated with refluxing the reaction mixture for 10 hrs (TLC monitored). The reaction mixture was cooled to get a crystalline compound, which was filtered further, washed with ample cold water, and recrystallized with methanol. The yield achieved was 82%.b. Synthesis of Acetic 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic anhydride (AA-III)Freshly dried and weighed 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic acid (0.01 mol) was mixed with acetyl chloride and the mixture was refluxed for 1 hr (TLC monitored). The cooled reaction mixture was poured into ice-cold water to get a white coloured solid precipitate, which was washed with water and dried ina desiccator. The dried solid anhydride was crystallized from chloroform and its melting point was determined. The yield collected was 65%.IV. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic acid and its conversion to Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic anhydridea. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic acid (A-IV)A solid mixture of 2-benzofuran-1, 3-dione (0.01 mol) and 2-amino-4-methylpentanoic acid (0.01 mol) was triturated and dissolved in glacial acetic acid (10 ml) and was refluxed for 9 hr (TLC monitored). The heating was continued for one more hour (to get a positive TLC report with no sign of starting materials). The reaction mixture was cooled and allowed to stand as it is. Crystalline compound separated at the bottom of the flask, which was filtered, washed with cold acetic acid, and then with water, and dried in a desiccator. The yield was 60%.b. Synthesis of Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic anhydride (AA-IV)Freshly dried and weighed 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic acid (0.01 mol) was mixed with acetyl chloride and the mixture was refluxed for 1 hr (TLC monitored). The cooled reaction mixture was poured into ice-cold water to get a white coloured solid precipitate, which was further washed with water and dried in a desiccator. The dried solid anhydride was crystallized from chloroform and its melting point was determined. The yield collected was 60%.V. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic acid and its conversion to Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic anhydridea. Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic acid (A-V)A solid mixture of 2-benzofuran-1, 3-dione (0.01 mol) and 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic acid (0.01 mol) was triturated and dissolved in glacial acetic acid (10 ml) and was refluxed for 10 hrs (TLC monitored). The heating was continued for one more hour. The reaction mixture was cooled and maintained under stirring. Crystalline compound separated at the bottom of the flask which were filtered,washed with cold acetic acid, and then with water and dried in a desiccator. The yield was 74%.b. Synthesis of Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic anhydride (AA-V) from Synthesis of 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic acid (A-V)Freshly dried and weighed 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic acid (0.01 mol) was mixed with acetyl chloride and the mixture was refluxed for 1 hr (TLC monitored). The cooled reaction mixture was poured into ice-cold water to get a white coloured solid precipitate, which was further washed with water and dried in a desiccator. The dried solid anhydride was crystallized from chloroform and its melting point was determined. The yield collected was 60%.B. Characterisation of substituted acetic 2-(1,3- dioxo-1,3-dihydro-2H-isoindol-2-yl) anhydride derivatives(COMPOUNDS OF FORMULA (I))(Acetic (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic anhydride) (AA-I); (Acetic 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoic anhydride (AA-II); Acetic 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic anhydride (AA-III); Acetic 2(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic anhydride (AA-IV); and Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic anhydride (AA-V) were characterized techniques such as FTIR spectroscopy, 1H-NMR spectroscopy, and Mass spectroscopy.C. CELL LINE STUDY:Principle of assay :This Colorimetric assay is based on the capacity of Mitochondrial succinate dehydrogenase enzymes in living cells to reduce the yellow water-soluble substrate 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into an insoluble, purple coloured formazan product which is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells.Material required:MCF-7 (Human Breast Cancer Cell Line); Media Required- MEM with high glucose (Cat No. 11965-092), FBS (Gibco, Invitrogen, Cat No. 10270106), Antibiotic- Antimycotic100X solution (Thermo-Fischer Scientific, Cat No. 15240062); 96 Well plate; CO2 Incubator (Thermo- fisher Scientific); Bio Safety Cabinet (SAS Filtration Technologies, Pvt Ltd., Pune.); Elisa Plate Reader (Benesphera E21)Procedure:Experimental procedure:a. MCF-7 Human Breast cancer cell line was procured from National Cancer Centre for cell sciences (NCCS), Pune and maintained in MEM medium supplemented with 10 % fetal bovine serum.b. Cells were incubated at a concentration of 1 x 104 cells / mL in culture medium for 24 hr at 37±1°C and 5% CO2.c. Cells were seeded at a concentration of 70 pl in 104 cells / well at 100 pl culture medium and 100 pl Sample B, C (100-12.5 pg / mL) into microplates (tissue culture grade and 96 wells), respectively.d. Control wells were incubated with DMSO (0.2% in PBS) and the cell line.e. All samples were incubated in triplicate. Controls were maintained to determine the control cell survival and the percentage of live cells after culture.f. Cell cultures were incubated for 24 hr at 37±1°C and 5% CO2 in CO2 incubator (Thermo-Fischer scientific BB150).g. After incubation, the medium was completely removed and 20 pl of MTT reagent (5 mg / min PBS) was added.h. After addition of MTT, cells are incubated for 4 hrs at 37±1°C in CO2 incubator.i. After removing the medium completely. 200 pl of DMSO was added, kept for 10 min, and incubated at 37±1°C (wrapped with aluminium foil).j. Triplicate samples were analyzed by measuring the absorbance of each sample by ELISA reader (Benesphera E21) at 570 nm.Table No.1: Effects of 5-Flurouracil against MCF-7 (Breast cancer cell line) byMTT assayTable No.2: Effects of Samples against MCF-7 (Breast cancer cell line) by MTTassay Conclusion:At the different doses (100-12.5 μg / ml) of Sample was carried out for anticancer activity against MCF-7 cell line. Sample-AAIII-337 showed good activity, Sample-AAI-247, Sample-AAII-261, Sample-AAIV-303, and Sample-AAV-289 showed moderate activity at a concentration of 100-12.5 μg / ml against the breast cancer cell line when compared to the standard drug 5 FU.a. Flow Cytometry:Sample code -AA-IIIPrinciple:Annexin Apoptosis is a form of programmed cell death to remove unwanted, damaged, or senescent cells from tissues. In normal cells, the negative phospholipids reside on the inner side of the cellular membrane, while the outer surface of the membrane is occupied by uncharged phospholipids (PS). After a cell has entered apoptosis, the negatively charged PS is transported from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment. The human anticoagulant, Annexin V, is a 35-36 kDa Ca2+-dependent phospholipid-binding protein that has a high affinity for PS. Annexin V labeled with a fluorophore or biotin can identify apoptotic cells by binding to PS exposed on the outer leaflet. Propidium iodide (PI) is a fluorescent nucleus dye, impermeant to live cells and apoptotic cells, but stains dead cells with red fluorescence,binding tightly to the nucleic acids in the cell. Annexin V-AbFlour TM 488 Apoptosis Detection Kit provides a rapid and convenient assay for apoptosis. After staining a cell population with AbFlour TM 488 annexin V and PI in the provided binding buffer, early apoptotic cells show green fluorescence of the cellular membrane, dead cells show red fluorescence of the nucleus and green fluorescence of the cellular membrane, and live cells show little or no fluorescence. Detection can be analyzed by flow cytometry.Materials:1. MCF-7 (Human Breast cancer) IC50 Value-27.89 μg / mL; 2. Dulbecco's Modified Eagle Media (DMEM) with low glucose -Cat No-11965-092 (Gibco, Invitrogen); 3. Fetal bovine serum (FBS) - Cat No -10270106 (Gibco, Invitrogen); 4. Antibiotic- Antimycotic 100X solution (Thermofisher Scientific)-Cat No-15240062; 5. Annexin V- AbFlour TM488 Apoptosis Detection Kit (KTA0002), Abbkine, Inc.; 6. AbFlour 488 Annexin V: Use 5 d per test; 7. Propidium Iodide (PI) is a convenient, ready-to-use nucleic acid dye. Use 2 μl per test; 8. 10X Annexin V Binding Buffer: 0.1 M Hepes / NaOH (pH 7.4), 1.4 M NaCl; 9. 25 mM CaCl2. For a 1x working solution, dilute 1 part of the 10x Annexin VBinding Buffer to 9 parts of distilled water; 6.5 ml sterile centrifuge tubes and FACS tubes - Tarsons, India; 10. 1x phosphate-buffered saline (PBS) - HiMedia; 11. Cytomics FC500 Flow cytometer, Beckman Coulter, USAProtocol - Apoptosis by Flow CytometryThe cells were seeded in a 6-well flat-bottom microplate and maintained at 37±1°C in CO2 incubator for overnight. The 30 μg / ml concentrations of the sample were treated for 24 hrs. After the incubation, cells were washed with PBS twice. Centrifuge for 5 mins at 500x g at 4±1°C. Discard supernatant and resuspend the cell pellets in ice-cold 1x Binding Buffer to 1 x 106 per mL. Keep tubes on ice. Then add 5 μl of AbFlour 488 Annexin V and 2 PI and mix gently. Keep tubes on ice and incubate for 15 mins in the dark. Add 400 of ice-cold 1x binding buffer and mix gently. Analyze cell preparations within 30 mins by flow cytometry. The analysis was done using FlowJo X 10.0.7 software (v.2.0).Results:FL2-A:: PIPE-AFL1 -A :: ANNEXINE V FITC-AFL1-A :: ANNEXINE V FITC-ANegative control- Treated with Sample AIII Apoptosis by Flow cytometerStages NC AA-IIILive 90.7 67.4Early Apoptosis 09.30 30.6Late Apoptosis 00.00 0.80Dead 0.025 1.25Conclusion:In the negative control, the live cell 90.7% and early apoptosis 9.30% exist, whereas the late apoptosis showed 0.00% and death 0.025%, respectively. When the AIII-compound was treated with the MCF-7 cell line and measured under flow cytometry, the AIII-compound showed 67.4% cells were live and showed 30.6% in early apoptosis, 0.80% in late apoptosis, and 1.25% cells died. As a point of late apoptosis, AIII-compound showed moderate activity when compared to the negative control.C. MOLECULAR DOCKINGMaterials and MethodsThe two-dimensional structure of Acetic (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) acetic anhydride) (AA-I); Acetic 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoic anhydride (AA-II); Acetic 2-(1, 3-Dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-phenylpropanoic anhydride (AA-III); Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-4-methylpentanoic anhydride (AA-IV); and Acetic 2-(1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-3-methylbutanoic anhydride (AA-V) were constructed using the 2D-sketcher module of the Schrodinger Software Suite 2021-2. Subsequent Docking simulations were carried out utilizing the Maestro environment, v.12.8 (Schrodinger,LLC, New York, NY, USA). The LigPrep programme was utilised to generate the stereoisomers of these ligands. Then, using the Epik ioniser, a maximum of four poses for the ligand was produced at a target pH of 7.0 to assure proper protonation states. An optimised low-energy 3D ligand was produced by tautomerizing and desalting using the OPLS 2005 force field while maintaining the required chirality in the input files. The RCSB Protein Data Bank was used to obtain the crystal structure of MMP-9 in complex with inhibitor BE4 (PDB ID: 6ESM). Schrodinger Maestro 9.1's Protein Preparation Wizard module (Schrodinger, LLC, New York, NY, USA) was utilized to create protein structures. Disulfide bonds, bond orders, and formal charges were assigned, and hydrogens were added using the "impref" utility with all heavy atoms fixed. The hydrogen-bonding network was optimized using the "H-bond assignment" tool. Crystallographic water molecules beyond 5 Å from the protein, co-factors, metal ions, and heteroatoms were removed. Computational docking was conducted to explore the binding modes between drug and the receptor. The receptor grids were constructed using a Van der Waals scaling factor of 1.00 and a charge cutoff of 0.25, centered on the ligand's centroid to encompass the entire molecule within a cubic grid. Docking simulations were conducted in Glide XP mode, and only lowest energy poses were retained. After determining the target protein's three-dimensional structure, docking simulations were performed using low-energy candidate ligand. Van der Waals radii for receptor and ligand side chains were reduced by 30% and 50%, respectively. The top 20 poses per ligand were selected based on an RMSD threshold of 0.18. Based on the information gathered, the ligand's Glide Score was calculated.Molecular Docking - ResultsThe results of the docking study of the compounds AA-I to AA-V indicate different binding scores with varied interaction modes, whereas, AA-I has the highest docking score of -5.9 kcal / mol and forms interaction with GLN-227 and HIS-226, and is further stabilized by coordinating with zinc 301 (Figure 6A). In comparison, AA-II displays weaker binding affinity (-3.685 kcal / mol) while maintaining interactions with GLN227 and HIS226 (Figure 6B), though it lacks zinc coordination. Meanwhile, AA-III demonstrates good binding energy (-4.632 kcal / mol), interacting exclusively with TYR240 while also coordinating with Zn 301 (Figure 6C). In the meantime, AA-IV (Figure 6D) and AA-V (Figure 6E) have docking scores of -4.953 kcal / mol and -3.796 kcal / mol, respectively, neither of which form direct amino-acid interactions but both of which bind to Zn 301, which may indicate that metal-coordination plays a role in theirbinding even though there are no specific residue interactions. Altogether, it can be stated that AA-I is the most promising candidate since the bounding energy is quite high and the factor of interaction is twofold, involving both key amino acids and zinc coordination.CC* Docking Score Interaction with Amino Acids OthersAA -I -5.9 GLN227, HIS226 Zn 301AA-II -3.685 GLN227, HIS226 -AA-III -4.632 TYR 240 Zn 301AA-IV -4.953 - Zn 301AA-V -3.796 - Zn 301D. MOLECULAR DYNAMICS (MD) STUDYMaterials and MethodsThe molecular dynamics study was conducted for 100 ns for the potential compound AA-III [Acetic 2-(1,3-dioxo-1,3-dihydro-2H-isoindolin-2-yl)-3-phenylpropanoic anhydride] with the crystal structure of MMP-9 in complex with inhibitor BE4 (PDB ID: 6ESM) using the Desmond tool. The system was immersed in a SPC orthorhombic box with a size of 10 Å x 10 Å x 10 Å, which was then neutralized by adding counter ions. Energy minimization was carried out using the OPLS3e force field. The NPT (isothermal-isobaric) ensemble was used at a temperature of 300 K and a pressure of 1.01325 bars that were maintained by the Nose-Hoover thermostat and the Martyna Tobias-Klein algorithms, respectively. Lastly, Desmond's Simulation Interaction Diagram (SID) was utilized to examine MD trajectories and anticipate the ligand binding orientation for the whole MD simulation by plotting root mean square deviation (RMSD), root mean square fluctuation (RMSF), and receptor-ligand interactions.Molecular dynamic (MD) simulation - ResultsThe binding between inhibitor and receptor is a dynamic process; therefore, the result of docking is not conclusive. The results obtained by docking may be an immediate binding state, and the affinity of the ligand in various conformations may be evaluated by MDsimulation. It also provides crucial details regarding the numerous non-bonded interactions and the resulting energetics of the solvated system. It also captures protein folding events, the impact of loop flexibility, and binding site adaptability. This overcomes the constraints of docking studies and offers precise estimates of binding affinity. Thus, the most active compound AA-III in complex with MMP-9 enzyme was subjected to MD to examine its binding stability and protein-ligand interactions in detail. The metrics RMSD and RMSF, as well as protein ligand interactions were examined to better understand the system's stability across the MD duration.The RMSD is an accurate indicator of protein and ligand conformational stability, and it is a measure of the magnitude of atom position divergence from the initial (native) position. Throughout the simulations, the RMSD of MMP-9 enzyme Ca showed a small gradual increase in the first 30 ns up to 2.0 Å, followed by small fluctuations, and it was maintained in the range of 1.75-2.3 to the end of the simulation (Figure 7A). The determination of the RMSD of the ligand with respect to the protein is also useful in determining the overall stability of the protein-ligand complex. Except for a minor fluctuation around 0-38 ns, the RMSD for ligand (compound AA-III) remains constant with a mean RMSD of 2.1 Å throughout the simulation time.The stability of a protein-ligand complex may be measured in terms of RMSF in protein residue atoms. It is a useful indicator of the flexibility of protein residues and highlights binding site adaptation and other phenomena. The peaks in the RMSF graph represent the oscillation of each residue across the entire simulation. It implies that higher RMSF values indicate more residue flexibility, while lower RMSF values indicate less residue flexibility and stronger system stability. The highest fluctuations were observed in the cluster of C-terminal residues, which included Gly112 (2.96 Å) and Asp113 (1.52 Å). According to the AA-III-MMP-9 enzyme complex RMSF plot, Compound AA-III was identified to contact with 24 amino acids of the MMP-9 enzyme, including Tyr160 (0.7Å), Tyr179 (0.7Å), Phe181 (0.8Å), Asp185 (1.8Å), Gly186 (1.2Å), Leu187 (0.8Å),Leu188 (0.8Å), Ala189 (0.4Å), His190 (0.4Å), Ala191 (0.4Å), Pro193 (0.5Å), Lys214(2.4Å), Tyr218 (1.0Å), Leu222 (0.5Å), Val223 (0.4Å), His226 (0.4Å), Gln227 (0.4Å),His230 (0.4Å), His236 (0.5Å), Tyr245 (0.6Å), Pro246 (0.8Å), Met247 (0.9Å), Tyr248(0.8Å), and Arg249 (1.1Å) (Figure 7B). All of these interacting residues have RMSF values in the range of 0.4 Å - 2.4 Å, as highlighted by the green vertical bars. It reveals that the conformational alteration was minimal, implying that the reported lead compound was tightly bound inside the cavity of the MMP-9 enzyme binding site. As for theprotein-ligand interaction during the simulation, AA-III bound to the MMP-9 enzyme exhibited direct and water-mediated hydrogen bonds during the simulations. His230 and His236 interacted with compound AA-III via a n-n interaction with 77% and 31%, respectively, while Ala191, Gln277, and Leu1880 demonstrated hydrogen bonds (92%, 48%, and 84%, respectively). In this complex, major hydrophobic interactions are shown with Leu187, His190, His230, and His236 (Figure 7C).

Claims

1. A compound of formula (I) represented by Wherein 'R' is independently selected from2. The compound of formula (I) is chosen from3. A process for synthesising the compound of formula (I) as claimed in claim 1, the process comprising: a. triturating a mixture comprising a dione compound and amino acid to obtain a triturated mixture; b. dissolving the triturated mixture in a solvent and refluxing for a time period in the range of 8 hrs to 10 hrs to obtain a compound of formula (II); c. contacting the compound of formula (II) with acid halide at a reflux temperature for a time period in the range of 30 mins to 90 mins to obtain compound of formula (I); d. optionally, purifying the compound of formula (I) in a solvent to obtain a pure compound of formula (I) with yield in the range of 46% to 65%.

4. The process as claimed in claim 3, wherein dione compound is 2-benzofuran-1, 3-dione; and herein amino acid is selected from amino acetic acid (glycine), 2-aminopropionic acid (alanine), 2-Amino-3-phenylpropanoic acid (phenylalanine), 2-amino-4-methylpentanoic acid (leucine), 2-amino-3-methylbutanoic acid (Valine).

5. The process as claimed in claim 3, wherein R1 is selected from6. The process as claimed in claim 3, wherein solvent is selected from Water, Methanol, Ethanol, Isopropanol, Acetic acid, Acetone, Acetonitrile, Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), Tetrahydrofuran (THF), N-Methyl pyrrolidone (NMP), Toluene, Chloroform, Diethyl ether, Carbon tetrachloride, Dichloromethane (DCM).

7. The process as claimed in claim 3, wherein acid halide is selected from Formyl chloride, Acetyl chloride, Propionyl chloride, Butyryl chloride, Isobutyryl chloride, Valeryl chloride, p-Toluoyl chloride.

8. The process as claimed in claim 3, wherein acid halide is acetyl chloride.