Insilico Design and Synthesis, Evaluation of Anti- Colon Cancer Activity of Novel Stilbene Hybrids

Various biologically important Stilbene analogues were competently synthesized using inexpensive, non-toxic, and readily available amino acids and Stilbene; the systematic study was carried out to characterize parameters such as TLC, melting point, IR, H NMR and mass spectral studies. The synthesized compounds were screened for anticancer activities. The molecular docking studies have been performed by using software Autodock 4.2, Autodock vina. The targeted proteins are P450a2 & Estrogen. The reaction of phenylacetic acid substituted Benzaldehyde, and triethylamine in acetic anhydride was irradiated in a microwave oven for 3 minutes at 700W afforded (2E)-3-(substituted phenyl)-2phenylacrylic acid. The above compound, after irradiating with hydrazine provided (2E)-3(substituted phenyl)-2-phenyl acrylic acid hydrazide. Anti-Cancer activity for synthesized compounds was evaluated using the MTT assay technique against colon cancer. The results were obtained as a percentage in cell lysis data. The IC50 value of the compounds was between 0.0370.0257 μM/lt. Among all the compounds, tyrosine derivatives exhibited the more potent activity. Insilico studies PCB-arg having more binding affinity with the receptor Cytochrome P450 A2 and PCB-try having more binding affinity with the receptor estrogen beta when compared to other derivatives. Original Research Article Meka et al.; JPRI, 33(39B): 128-139, 2021; Article no.JPRI.71898 129


INTRODUCTION
Drugs are the greatest contributions of the 20 th century to therapeutics. Their advent changed the outlook of diseases. They are one of the few curative drugs. Their importance is magnified in developing countries, where infectious diseases predominate. As a class, they are one of the most frequently used as well as misused drugs.
Early types of anti-microbial substances were not specifically anti-microbial but were usually toxic to all living cells [1]. They were of value only in so far as they could be employed without severe damage to the host. The search for more suitable anti-microbial agents resulted in the preparation and testing of many substances of the widely different constitution. From the systematic empirical investigations of one such class of chrysoidine dyestuff, knowledge of the activity of sulphanilamide arises. The growing evidence that the anti-microbial action of sulphonyl drugs [2] was due to competition with an enzymic metabolic process suggested that Fildes (1940) approach the problem. stilbene A common name for trans-1,2-diphenyl ethylene is stilbene, or more specifically, stilbene is the alkene, ethene with two phenyl groups on other carbon of parent chain. The name was derived from the Greek word stilbos, which means shinning (Cis-trans isomerism E/Z notation/Z)stilbene, which is sterically hindered and less stable. It is noticed that (Z)-stilbene has a melting point of 5 0 C to 6 0 C while the melting point of (E)stilbene is 125 0 C. This shows the significant difference between the two because (E)-stilbene is more common than (Z)-stilbene.
(E)-1,2diphenylethylene(or) (E)-Stilbene , trans -Stilbene (Z)-Stilbene , Cis -Stilbene (Z)-1,2diphenylethylene Stilbenes, produced by several plants in response to pathogen attacks, regulate many biological functions. In agreement with their role as antifungal [3], antibacterial [4], and cytotoxic activities [5] have been reported. Some of them, such as resveratrol, exert antioxidant activity, which modulates the synthesis of lipids, inhibit ribonucleotide reductase and DNA polymerase increase the activity of Map-kinase, an enzyme potentially related to neurodegenerative diseases such as Alzheimer's and Parkinson's, inhibit platelet aggregation and after the eicosanoid synthesis, both effects probably related the inhibition of the cyclooxygenase and hydroperoxides activities. These findings have stimulated the study of these compounds as antiinflammatory [6] cardiotonic and anti-platelet aggregating agents, anti-convulsant [7].
Also, many scientists have carried out the synthesis and biological activities of stilbene derivatives, which revealed that stilbene plays a significant role in the biomedical field. Pharmacological Studies have established that stilbene inhibits the synthesis of eicosanoids by platelets, reducing the incidence of coronary heart disease. It has also shown significant anticancer [8] antifungal [3], antimicrobial [9], antioxidant [8], anti-inflammatory [6], anticonvulsant activity, and synthesized stilbene derivatives using Wittig reaction [10], But the yield was inferior (10%). Inhibitory activities of stilbene from medicinal plants on the expression of cell adhesion molecules on THP1 cells [11]. there is a new synthesis of trans stilbenes [12,13] and chemoprotective properties resveratrol [14-15] and hydroxy stilbenoids [16] and cytotoxicity activity of hydroxylated resveratrol analogues [17]. synthesis of diaryl ethylene's [18].

Types of Cancer in the Colon and Rectum
Adenocarcinomas: More than 95% of colorectal cancers are a type of cancer known as adenocarcinomas. These cancers start in cells that form glands that make mucus lubricate inside the colon and rectum. Other, less common types of tumours may also begin in the colon and rectum.

Microwave Technology in the Synthesis of Organic Drugs
Several synthetic transformations in pharmaceutical and organic chemistry require prolonged heating and reflux of several hours. However, microwave-induced organic reaction enhancement chemistry is gaining popularity as a non-conventional technique for rapid organic synthesis, where the microwave oven is the source of heating. Experiments involving heating time up to 6 hours in conventional methods, namely preparation of Benzocaine, Butamben, Phenytoin, 3-methyl-1-Phenyl pyrazole-5-one and Fluorescein. All these compounds were prepared easily in high yields in less than 10 minutes of microwave heating, and Purification, recrystallization, and drying of products were completed one day. This method is economical due to the savings of energy, fuel, and chemicals.
Significant limitations of classical chemistry practical are long time, elaborate and tedious apparatus setup, higher cost, longer reaction time and environmental pollution due to large quantities of solvents/reagents. On the other hand, microwave-induced organic reaction enhancement chemistry provides a nonconventional technique for the rapid synthesis of organic molecules.
The application of microwave irradiation is used for carrying out chemical transformations which are pollution-free and eco-friendly [10]. A commercial microwave oven is used as a convenient source of heat in the laboratory. The microwave-assisted organic reactions occur more rapidly, safely and with higher chemical yields, render the microwave method superior to the conventional method [19].

METHODS
TLC was employed for the confirmation of reaction progress and product formation. The melting point and its range were determined by the open capillary tube method. While functional nature of carbons and protons of the structure was assigned by IR (Jasco-510 FT-IR Spectrophotometer at 4 cm-1 using KBr pellet disc), 1H -NMR (Varian VXR Unity at 400MHz using TMS as internal standard and DMSOd6/ CHCl 3 as solvent), and mass spectral (Agilent LC/MS, positive mode, ESI) studies. All chemicals used for synthesis were AR grade purchased from Sd fine chemicals, Mumbai. All six compounds are soluble in the chloroform and the mobile phase for TLC ethyl acetate and petroleum ether in ratio 6:4 ratio. The purification of products was carried out by recrystallization, TLC and column chromatography. The synthesized compounds were confirmed by FTIR, 1H -NMR, MASS spectroscopy.

Synthesis of (2E)-3-(substituted phenyl)-2-phenyl acrylic acid :
A mixture of phenylacetic acid (2 millimoles) substituted benzaldehyde (2 millimoles) and triethylamine (0.5milli moles) in acetic anhydride (5ml) was irradiated in a microwave oven for 3 minutes at 700W. The product obtained was poured into a hot saturated sodium carbonate solution (50ml) and left overnight. The mixture was extracted with ethanol, and the ether extracts were discharged, the aqueous solution was acidified with dilute HCl and the precipitated product was filtered and dried.
The resultant solid was filtered& washed with water & recrystallized with 90% ethanol.

Biological Evaluation
In vitro Anticancer activity of synthesized stilbene derivatives: MTT Assay:

MTT solution preparation (stock solution):
5 mg in 1 ml of PBS.

Cytotoxicity Assay:
Invitro growth inhibition effect of test compound was assessed by colorimetric or spectrophotometric determination of conversion of MTT into "Formazan blue" by living cells.
A) Remove the supernatant from the plate and add fresh MEM solution and treat with different concentrations of extract or compound appropriately diluted with DMSO. The Control group contains only DMSO. In your study, 10, 20, 25, 30 and 50ul of the stock solution (10 mg/ml prepared in DMSO) were added to respective wells containing 100ul of the medium. So, the final concentrations were 10, 20, 25, 30 and 50ug / ml. B) After 48hrs incubation at 37ºC in a humidified atmosphere of 5% Co2, a stock solution of MTT was added to each well (20µl, 5mg per ml in sterile PBS) for further 4 hr incubation.
C) The supernatant was carefully aspirated. The precipitated crystals of "Formazan blue' were solubilised by adding DMSO (100µl), and optical density was measured at a wavelength of 570nm using LISA plus. 4. The results represent the mean of five readings. The concentration at which the OD of treated cells was reduced by 50% with respect to the untreated control. 5. Formula:

Mean OD of test compound × 100
Mean OD at control

Principle of the Assay
This is a colourimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria, reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilized with an organic solvent (e.g., DMSO, Isopropanol) and the released, solubilized formazan reagent is measured spectrophotometrically. Since the reduction of MTT can only occur in metabolically active cells, the activity level is a measure of the viability of the cells.

Anti -Cancer Activity of Novel Stilbene Derivatives
There was a total of six compounds synthesized in this study. Separate studies established all the compounds' structure. The TLC technique authenticated the purity of the compounds. The Anti -Cancer activity for the five synthesized compounds was carried out using the MTT assay technique. The results were obtained as a percentage in cell lysis data. The µg/ml concentration IC50 was converted into µM/lt concentration using a molecular weight of the compounds, and the results are presented in the table above. The IC50 value of the compounds was between 0.037-0.0257 µM/lt. Among all the compounds, tyrosine derivatives exhibited the more potent activity. The critical difference (CD 5%) value was found to be 0.01 or less.
However, the difference in cytotoxicity among the synthesized compounds was significantly less. This indicated the presence and consideration of amino acids for Anti Cancer activity.

Cell line -HT 29
Least concentration to show 50% inhibition of cell line was found to be 10µg.

Docking Protocol
Procedure for molecular docking

Fig. 2. photographs showing Anti-cancer activity of compound PCB-tyr
The ligand has shown the attractive charge with the amino acids arginine, glutamic acid and conventional hydrogen bond interactions with the amino acid proline, unfavorable donor-donor interactions with the amino acid valine, pi-cation and pi-anion interactions with the amino acid glutamic acid and histidine and pi-alkyl interactions with the amino acid leucine.

Binding energy (Kcal/mol) Estrogen receptor beta
The ligand has shown the salt and attractive charge with the amino acid asparagine and conventional hydrogen bond and pi-sulfur interactions with the amino acid cysteine, alkyl and pi-alkyl interactions with the amino acid leucine.

DISCUSSION
Results are described above and can be discussed as follows. The Stilbene derivatives interfered with prostaglandin E2 (PGE2) generation in murine intestinal mucosa in vivo and colon derivative cells in-vitro. The ability of Stilbene derivatives to interfere with Adenomatous polyposis coli (APC). Adenoma development was paralleled by inhibition of PGE2 production, which might be involved as an antitumour promotional mechanism. The superior inhibition of colon cell growth in-vitro by (methylated derivative of resveratrol) DMU-212 compared to resveratrol is not reflected by a corresponding difference in potency as far as inhibition of PGE2 production invitro is concerned. Resveratrol and other Stilbene derivatives bind and activate (Estrogen Receptor Alpha and beta) ER-α as well as ER-β. Resveratrol acts as a selective estrogen receptor modulator, and the effects of revesterol depend on cell type and target organs and the presence of endogenous estrogens.
ER-β is present in colon. Stilbenes prevent colon cancer via estrogenic action and interaction with ER-β in the colon. Stilbene derivatives show the growth-inhibiting action through cell-cycle arrest induced by upregulation of P21, P27, P53 and downregulation of cyclin D 1 and cyclin E. Apoptosis action by upregulating the expression of PUMA (p53 upregulated modulator of apoptosis), downregulating (tumour necrosis factor) TNF related apoptosis-inducing ligand [20].
Two targeted proteins were selected for docking subsequently, the studies were carried out. Docking studies carried out using autodock 4.2 reveals that the PCB-arg has a more binding affinity towards cytochrome p450 A2 receptor when compared with the other synthesized derivatives. Compared with the other synthesized products, the PCB-try has shown a more binding affinity with the estrogen receptor beta. The PCB-try, PCB-tyr, PCB-ala has shown the medium binding affinity with the cytochrome p450 A2 receptor. The PCB-arg, PCB-tyr, and PCB-ala have shown medium binding affinity with the estrogen receptor beta. Cytochrome p450 plays an important role in the metabolism of endogenous and exogenous substances, especially drugs. Moreover, many p450 can serve as targets for disease therapy [11,21].

CONCLUSION
The synthesized stilbene derivatives are considered a promising molecules for fighting cancer. Some of these newly synthesized compounds have stimulating effects. Stilbene derivatives of tyrosine and alanine are the most active, while other compounds have moderate activity. Comparing the compounds indicates that protecting groups like hydroxyl and amine groups lead to better action. Moreover, the absence of the methoxy groups provides a better lipophilic property.
In most cases, the efficacy of the other synthesized compounds has less activity compared to tyrosine and alanine, which have the most potent activity. Furthermore, the lack of effect on non-tumour cells demonstrates the selectivity of these molecules for tumour cells, which is an essential aspect for therapeutic applications. In vitro cell culture experiments and preclinical animal studies with stilbene derivatives suggest a multitude of mechanisms for the pharmacological activity of this group of compounds. Elucidation of mechanisms of action and in-vivo efficacy of stilbenes may lead to new approaches for the treatment and prevention of various neoplasms, including colon cancer. Docking studies revealed that PCB-arg has a more binding affinity with the receptor Cytochrome P450 A2. PCB-try has a more binding relationship with the receptor Estrogen beta compared to other derivatives. [22][23] This study showed that the ligands might have high tumour suppressing properties and explain about medium tumour suppressing properties of other derivatives.

DISCLAIMER
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.

CONSENT AND ETHICS APPROVAL
It is not applicable.