Development of Some Novel Coumarin-Chalcone Compounds as Anti-proliferative Agents

Introduction: Cancer is the world's second leading cause of death and morbidity, behind only heart failure, which claimed the lives of 18.2 million people in 2020. While massive initiatives to establish newer leads and innovative chemotherapeutic methods for combating different types of cancer, continues to be a major concern around the world. As a result, identifying cell-cycle inhibitors and apoptotic triggers to fight cancer cells is an appealing method for finding and developing new anti-tumor drugs. Materials and Methods: The present study involves the rational development and characterization (both physicochemical and spectroscopy) of coumarin-chalcone compounds (A1–A10) and their antiproliferative potentials against cancer lines of breast cancer origin (MDA-MB468, MDA-MB231, and MCF-7) and non-cancer breast epithelial cell (184B5). Results: The compound A2 exhibited the highest anti-proliferative activity against the cell line MDA-MB-231 as indicated by the GI50 value of 10.06 μM, the compound A6 exhibited the highest anti-proliferative activity against the cell line MDA-MB-468 as indicated by the GI50 value of 17.54 μM, the compound A1 exhibited the highest anti-proliferative activity against the cell line MCF-7 as Original Research Article Patel et al.; JPRI, 33(45B): 105-111, 2021; Article no. JPRI.73746 106 indicated by the GI50 value of 25.86 μM, and the compound A6 exhibited the highest antiproliferative activity against the cell line 184B5 as indicated by the GI50 value of 23.26 μM. Conclusion: Furthermore, the research urges medicinal chemists to choose chalcone prototypes with well-defined pathways and SARs while developing more powerful inhibitors. Furthermore, it opens up new pathways for the discovery of anti-cancer derivatives using low molecular weight ligands.


INTRODUCTION
Cancer is the world's second leading cause of death and morbidity, behind only heart failure, which claimed the lives of 18.2 million people in 2020 [1]. While massive initiatives to establish newer leads and innovative chemotherapeutic methods for combating different types of cancer, continues to be a major concern around the world [2]. The quest for new or unexplored types of substances to combat cancer cells has piqued the interest of scientists all over the world. As a result, identifying cell-cycle inhibitors and apoptotic triggers to fight cancer cells is an appealing method for finding and developing new anti-tumor drugs [3].
Recently, many heterocyclic and nonheterocyclic scaffolds have been investigated for antiproliferative activity against various tumor cell lines [4]. Among them chalcones is one of the most promising classes of compounds that exhibit anticancer activities along with other pharmacological activities. Chalcones are structurally simple compounds of the flavonoid family and are present in a variety of plant species [5]. Chemically, these are 1,3-diphenyl-2-propen-1-one and have reported a wide range of biological activities, including antileishmanial, antiinflammatory, antimitotic, modulation of Pglycoprotein-mediated multidrug resistance, and antimalarial activities, etc [6].
Chalcone, also known as (E)-1,3-diphenyl-2propene-1-one, is a source of flavonoids and isoflavonoids, as well as an open-chain intermediate in the synthesis of flavones by aurones. They can be found in nature in a variety of conjugated ways with a benzylideneacetophenone scaffold, which connects the two aromatic nuclei with a threecarbon, unsaturated carbonyl bridge [6]. Kostanecki was the first to coin the concept after synthesizing a sequence of natural chromophoric compounds. Chalcone and its variants can be produced using a Claisen-Schmidt reaction between benzaldehyde and acetophenone and a 40 percent sodium hydroxide solution as a catalyst [7,8]. Thanks to its basic chemistry, simplicity of synthesis, a large amount of replaceable hydrogen to yield a large number of derivatives, and variety of promising biological activities such as anti-diabetic, anti-neoplastic, anti-hypertensive, anti-retroviral, antiinflammatory, anti-histaminic, anti-oxidant, antimalarial, etc., the chemistry of chalcones has remained a curiosity among researchers in the twenty-first century [9].
The present study involves the rational development and characterization (both physicochemical and spectroscopy) of coumarinchalcone compounds (A1-A10) and their antiproliferative potentials against cancer lines of breast cancer origin (MDA-MB468, MDA-MB231, and MCF7) and non-cancer breast epithelial cell origin (184B5) [10].

Instrumentation
Sigma-Aldrich ® and Merck ® provided chemicals that were used in this synthesis. The other solvents and reagents were purchased from industrial suppliers and were of analytical grade. The melting points were determined using the Perfit melting point apparatus and are unadjusted. On a Win IR FTS 135 instrument, infrared spectra were captured in KBr disks. The JEOL-JMS-DX 303 instrument was used to collect mass spectra. Merck ® silica gel G coated TLC plates were used for thin-layer chromatography.

Synthesis of 3-acetyl-4-hydroxy-2Hchromen-2-one
4-hydroxy coumarin (0.01 M) was combined with 15 mL glacial acetic acid, and then 60 mL phosphorous oxychloride was steadily applied to the mixture and heated for 1.5 hours. The reaction mixture was then cooled and dumped onto crushed ice to extract the solid substance, which was then cleaned with water and crystallized from absolute alcohol (Scheme 1).

Synthesis of coumarin-Chalcones
In 6 ml of chloroform, 4-hydroxy-3-acetyl coumarin (0.031 M) and the substituted aromatic aldehyde (0.03 M) were dissolved. The reaction mixture was refluxed for 3 hours with a catalytic volume of piperidine (0.001 M). The chloroform was removed from the substance, which was then washed with methanol (Scheme 1).

Anti-cancer screening
The cytotoxic effects of all the compounds synthesized were tested on three breast cancer cell lines, MDA-MB468, MDA-MB231, and MCF7, as well as one non-cancer breast epithelial cell line, 184B5. This Anti-cancer activity assay was done according to National Cancer Institute protocol. The results were stated in terms of growth inhibition (GI50 lM) values.
Plates were incubated at standard conditions for 48 h and assay was terminated by the cold TCA. Addition Cells monolayer were fixed with cold TCA (50 ml, 30% (w/v)) and incubated at 4 C for 60 min. Discarded the supernatant, washed the plates and air dried. Prepared the solution of Sulforhodamine B (50 ml) in acetic acid (1%) and poured into wells, then incubated the plates at room temperature for 25 min. After staining, recovered unbound dye and removed residual dye by washing with acetic acid and plates air dried. The protein bound dye afterward eluted by dissolving in trizma base (10 mM) and absorbance was noted via Elisa plate reader at 540 nm with reference wavelength (690 nm) [11][12].

Statistical Analysis
The mean was used to express the outcomes of the tests. The magnitude of the variations between the control and treated groups was determined using an ANOVA accompanied by a Dunnett's t-test, with P < 0.05 found meaningful.

Scheme 1. Overall synthetic scheme
Proposed structure
The mass spectra of all the compounds showed that the molecular ion peak of the compound was as similar as was anticipated. Finally, the structure of compounds was confirmed by FT-IR spectroscopy and mass spectra. All the band frequencies for the molecular weight of synthesized compounds were matching (within limits of variation) with those reported ( Table 2).

Anti-cancer Activity
Compound A6 had the strongest anti-proliferative efficacy against all cell lines, as shown by the GI50 values, in the SRB assay ( Table 3). The compound A2 exhibited the highest antiproliferative activity against the cell line MDA-MB-231 as indicated by the GI50 value of 10.06 μM, the compound A6 exhibited the highest antiproliferative activity against the cell line MDA-MB-468 as indicated by the GI50 value of 17.54 μM, the compound A1 exhibited the highest antiproliferative activity against the cell line MCF-7 as indicated by the GI50 value of 25.86 μM, and the compound A6 exhibited the highest antiproliferative activity against the cell line 184B5 as indicated by the GI50 value of 23.26 μM. As opposed to the traditional anti-cancer medication cisplatin, both of the compounds had higher GI50 levels. The substituents and their locations played an important role in exhibiting antiproliferative behavior by modulating different unknown goals, according to Structure-Activity Relationships (SARs) and logical chalcone scaffold architecture. The electron-withdrawing substituents such as Br results in an improvement in anti-cancer capacity as well as boosts the anti-cancer behavior due to an increase in the log P-value. A compound's lipophilicity (or hydrophobicity) is a critical physical property that influences bilipid membrane permeation, dissolution rate, compound bioavailability, and drug interaction with biological systems [12,13,14]. It was also discovered that analogs with an electronwithdrawing group in the R4 role had higher antiproliferative behavior than analogs with other positions.

CONCLUSION
The research discovered that coumarin-chalcone compounds had the ability to be effective antiproliferative agents. The thesis highlighted the important function and locations of substitution on the system's phenyl moiety. According to the GI50 values, compound A6 had the most antiproliferative effect against all cell lines. It is reasonable to conclude that cytotoxic effects of all of the synthesized derivatives were mediated by various pathways, none of which were evaluated in this study. Furthermore, the research urges medicinal chemists to choose chalcone prototypes with well-defined pathways and SARs while developing more powerful inhibitors. Furthermore, it opens up new pathways for the discovery of anti-cancer derivatives using low molecular weight ligands.

DISCLEMAR
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.