Studying the Anti-Cancer Activity of Resveratrol 1,3,4-Thiadiazol Derivatives

Shahlaa Zuhair Abdul-Majeed*, Sarah A. Hamood, Ashour H. Dawood

Al-Esraa University College, Baghdad, Iraq.

*Corresponding Author E-mail: shahla@esraa.edu.iq, sara.ashour@esraa.edu.iq, dr.ashouralsaedi@gmail.com

ABSTRACT:

The used Resveratrol-Thiadiazol derivatives (A, B, C) were prepared and characterized by ¹H-¹³C NMR and FT-IR spectroscopies. This study used two cell lines for cytotoxicity testing: MCF-7 (Human Breast Cancer Cells) and WRL-68 (Human Normal Liver Cells), The result of anticancer study showed that derivative C had significant selectivity on MCF-7 used in this study even in very low concentration used to evaluate the anticancer activity while derivatives A and B showed non-significant selectivity on MCF-7 in low concentration while in high concentration showed small selectivity which may be negligible, this selectivity determined according to the comparison between: MCF-7 (Human Breast Cancer Cells) and WRL-68 (Human Normal Liver Cells).

KEYWORDS: Anti-Cancer activity, 1,3,4-Thiadiazol, Resveratrol derivatives, Thiadiazol, Cytotoxicity Assay, human breast cancer MCF-7.

INTRODUCTION:

Resveratrol is a stilbenoid polyphenol with two phenol rings linked by an ethylene bridge. The molecular structure of resveratrol has two isomeric forms: cis- and trans-resveratrol. Transformation is extremely important in terms of its prevalence, and it is linked to several biological functions, including stimulating cellular processes including cell cycle arrest, differentiation, apoptosis, and cancer cell anti-proliferation^1-3. In addition to these biological properties, resveratrol has been researched for its ability to suppress the reproduction of certain harmful bacteria^4,5.

Thiadiazol is a five-membered organic heterocyclic molecule that contains one sulphur atom and two nitrogen atoms^6,7. Based on the position of the sulfur and nitrogen atoms in the ring, that molecule has four structural isomers^8-10. It also present in various compound and had different pharmacological actions^{11, 12}.

Resveratrol has anti-cancer properties by interfering with all three stages of tumor development: initiation, promotion, and progression¹³. Human cancers such as breast, prostate, stomach, colon, pancreatic, and thyroid carcinoma have been shown to be inhibited by resveratrol in various layers^14,15. Resveratrol inhibits COXs, which produce pro-inflammatory chemicals that promote tumor proliferation, and down-regulates the signaling pathways for AKT, MAPK, and NF-B, all of which reduce inflammation and limit carcinogenesis, according to studies in vitro and in vivo^16,17.

MATERIALS AND METHODS:

All the chemicals purchased from Hyper chem (China), were used without further purification. Reactions were monitored by thin-layer chromatography (TLC) on precoated Merck silica gel 60F254, and the spots were visualized either under UV light orby iodine vapor. Melting points were determined on an electro-thermal melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded on FT-IR spectrophotometer, Shimadzu (Japanas). The¹H NMR and¹³ C NMR spectra were recorded on VARIAN (Japan) using tetramethylsilane TMS as an internal standard.

Preparation of resveratrol 1,3,4-Thiadiazol derivatives:

Table (1): structures and names of prepared compounds (A, B, and C) and there starting compound (1).

Compound	Chemical name	Structure
A	5,5'-(((5-(4-((5-(heptylthio)-1,3,4-thiadiazol-2-yl)methoxy)styryl)-1,3-phenylene)bis(oxy))bis(methylene))bis(2-(heptylthio)-1,3,4-thiadiazole).
B	5,5'-(((5-(4-((5-(butylthio)-1,3,4-thiadiazol-2-yl)methoxy)styryl)-1,3-phenylene)bis(oxy))bis(methylene))bis(2-(butylthio)-1,3,4-thiadiazole).
C	5,5'-(((5-(4-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)methoxy)styryl)-1,3-phenylene)bis(oxy))bis(methylene))bis(2-(phenylthio)-1,3,4-thiadiazole).
1	5,5'-(((5-(4-((5-mercapto-1,3,4-thiadiazol-2yl)methoxy)styryl)1,3phenylene)bis(oxy)) bis(methylene))bis(1,3,4-thiadiazole-2-thiol)

Synthesis of compound A^12,18,19

(1.00g, 1.61mmol.) of 5,5'-(((5-(4-((5-mercapto-1,3,4-thiadiazol-2-yl)methoxy)styryl)1,3phenylene)bis(oxy)) bis(methylene))bis(1,3,4-thiadiazole-2-thiol) compound (1) was added to 30mL of DCM (dichloro methane) in 100mL size round bottom flask and stirred for 10 minutes to ensure that all compound particles being dissolved in solvent.

Potassium hydroxide (0.20mL, 4.83mmol,) has been added drop wise to mixture via the dropper (dissolved in ethanol), solution stirred for 15minutes. The heptyl bromide (C₆H₁₃CH₂Br) (0.75mL, 4.83mmol) was added and let the mixture heated at 30^°C with stirring for two hours. Yellow precipitate was formed, filtered with pump suction, washed with acetone and dried to get the desired product.

Synthesis of compound B^12,18,19

About 1.0gm (1.61mmol) of 5,5'-(((5-(4-((5-mercapto-1,3,4-thiadiazol-2-yl)methoxy)styryl)1,3phenylene)bis(oxy)) bis(methylene))bis(1,3,4-thiadiazole-2-thiol) compound (1)  was poured into round bottom flask 100mL in size and 30mL of DCM (dichloro methane) used as solvent, stirred for 10 minutes and dissolve all components. (0.20 mL, 4.83mmol,) Potassium hydroxide was added drop wise (dissolved in ethanol) and let to for further stirring for about 15 minutes. The butyl bromide (C₃H₇CH₂Br) (0.31mL, 4.83mmoL) added. The mixture heated at 30°C, with stirring for two hours. Solid Precipitate was formed, filtered with pump section, washed with acetone and dried to get the final product.

Synthesis of Compound C^12,18,19

About 1.0gm (1.61 mmol) of 5,5'-(((5-(4-((5-mercapto-1,3,4-thiadiazol-2-yl)methoxy)styryl)1,3phenylene)bis(oxy)) bis(methylene))bis(1,3,4-thiadiazole-2-thiol) compound (1) was let to be dissolved in 100mL in size round bottom flask with 30mL of DCM (dichloro methane) and stirred for 10 minutes. After the compound being completely dissolved in solvent, potassium hydroxide (0.2mL, 4.83mmol,) was added drop wise with continuous stirring (dissolved in ethanol). Benzyl chloride (C₆H₅CH₂Cl) (0.49mL, 4.83mmol) was added after 15 minutes and let the mixture heated at 30^°C with stirring for two hours until precipitate was obtained. The mixture was filtered by pump suction, precipitate washed with acetone and dried to have the desired product.

Cytotoxicity assay on human breast cancer MCF-7 and human hepatic WRL-68 cell lines

Cancer Cell Line

This study used two cell lines for cytotoxicity testing: MCF-7 (Human Breast Cancer Cells) and WRL-68 (Human Normal Liver Cells). The cells were obtained from Pharmacology Department of Malaya University Medicine College. The cell lines were kept alive at (Roswell Park Memorial Institute- 1640Medium, Sigma Aldrich). 450ml of medium was mixed with 50ml of 10% Fetal Bovine Serum (Sigma Aldrich, USA) and 0.5 ml Penicillin/Streptomycin stock solution (10,000U/ml penicillin, 10g/ml streptomycin; Thermo Fisher Scientific, USA). The prepared media were cooled down using refrigeration (Thermo Scientific, United States)²⁰.

Maintenance of cell lines:

Cryopreserved cells were thawed for (1-2) minutes in a sterilized Laminar Air Flow Hood with a water route at 37^oC. (Sanoy, Japan). The container was cleaned with 70% ethanol (BDH, England), then transferred to a blue cap centrifuge tube and stored in a 37°C water bath for 5 minutes to remove any possible outside contamination. Centrifugation at 200X for 5 minutes was used to remove the cryomedia from the cell stock solution (BIO-RAD, Korea). The supernatant was discarded, and the cell pellets were re-suspended in fresh media before being cultured in a CO₂ incubator at 37°C and 5% CO₂ (Sanoy, Japan). The cell lines were sub-cultured once the monolayer was confluent. After the growth medium was decanted, the cell sheet was washed once with 2ml trypsin-versene solution. A part of the solution was decanted after adding 2 to 3ml of trypsin-versene to the cell sheet and gently moving the flask to get roughly one milliliter of trypsinversene solution covering the cell surface. The cells were incubated for 1-2 minutes at 37°C until they detached from the flask. After that, the flask was replaced with full medium and incubated at 37°C for 24-48 hours before the viability test was completed. The procedure was carried out, according to Jennie and Penelope^20.

Cell viability:

The vitality of the cells was assessed by trypan blue exclusion before analyzing the extract's cytotoxic effect on the cell lines. The number of viable cells per well should be in the range of (10⁴–10⁵). Trypsinized and suspended cells for the cell lines (MCF-7 and WRL-68) were sown in a microtiter plate with a 1:10 dilution (3.6 ml medium +0.4ml stock) for the cytotoxicity test. 0.2 ml of cell suspension was mixed with 0.2ml of trypan-blue in 1.6ml of PBS, then 20 microliters of the mixture was added to the haemocytometer to count the diluted cells. Every 1-2 minutes, live and dead cells were counted, and cell concentration (cell/ml), total cell count, and cell viability (%) were calculated using the following equations²⁰:

"c=n x d x 10,000"

“Where C (Cell concentration (cell/ml)), n (number of counted cells), d (dilution factor=10)”

Total viable cells unstained

"Cell viability (%) = ----------------------------------------- x 100"

                        Total cells counted stained and unstained

MTT (Methyl Thiazolyl Tetrazolium) Cytotoxicity Assay:

After the exposure period, the produced derivatives (A, B, C) were exposed to growing cells on a microtiter plate (96wells) at various doses to determine their cytotoxic effect. 200μL/10⁴-10⁵ cells/well were added to all 96 wells of the microtiter plates, shaken, and incubated at 37°C, 5% CO₂ until the cells reached 70% confluence. For MCF-7 and WRL-68 cell lines, five different concentrations of prepared derivatives (1000, 750, 500, 250, 125μg/ml) were added to each well (three replicates for each concentration), as well as 200 microliters of maintenance medium for the control group. After 24 hours of incubation, the cytotoxic activity of the two cell lines was evaluated.

After 24 hours of exposure, cell viability was determined by removing the medium, adding a 20 microliter/well MTT solution, and incubation for 4 hours at 37°C. The crystals were solubilized by adding 40 microliters of Dimethyl Sulphoxide (DMSO) to each well, followed by a 15-minute incubation at 37°C with shaking. The optical density of each 96-well plate sample was then evaluated using a microplate reader at 620 nm^21,22.

STATISTICAL ANALYSIS:

The study employed the Graphed 6 prism program with one–way ANOVA and Dennett's multiple comparison test to collect data reflecting the mean±SD for the cytotoxicity experiment (25). The optical density data from the plate reader was then used to compute the concentration of chemicals required to elicit a 50% reduction (IC₅₀) for each cell line using non-linear regression analysis.

RESULTS AND DISCUSSION:

Characterization of compounds²³

Compound A, B and C prepared successfully according to the method provided by dhiaa et al, compound A showed the following characteristics Yellow powder, yield 79%, M.P = 291-293°C. IR KBr (cm^-1): appearance of 3174 (Aliphatic C=C-H Stretching), 1604 (C=N), 1072 (C-S-S) and disappearance of 2590 (S-H str). ¹H-NMR (DMSO): δ3.12-3.44 (Trip, 6H, CH₂) neighbour to S atom, δ1.05-2.04 (Mult, 30H, CH₂), δ 0.85 (Trip, 9H, CH₃). ¹³C-NMR (DMSO): δ 5.96-19.10 (aliphatic carbons that replace the hydrogen atom from binding to sulfur atom), δ 151.08 (aromatic carbon alpha to SR group), δ161.48 (aromatic C=N alpha to oxygen of thiadiazol ring).

Compound B showed the following characteristics Yellow powder, yield 83%, M.P = 285-287°C. IR KBr (cm^-1): appearance of 3140 (Aliphatic C=C-H Stretching), 1604 (C=N), 1070 (C-S-S) and disappearance of 2590 (S-H str). ¹H-NMR (DMSO): δ 3.52, 3.76 (Trip, 6H, CH2) neighbour to S atom, 0.86-2.09 (mult, 12H, CH₂), δ 0.73 (Trip, 9H, CH3). ¹³C-NMR (DMSO): δ15.55-26.79 (Aliphatic carbon), δ160.27 (aromatic carbon alpha to SR group), δ 161.53 (aromatic C=N alpha to oxygen of thiadiazol ring).

Compound C showed the following characteristics Yellow powder, yield 81%, M.P = 298-299°C. IR KBr (cm^-1): appearance of 3178 (Aliphatic C=C-H Stretching), 1593 (C=N str), 1022 (C-S-C) and disappearance of 2590 (S-H str). ¹³C-NMR (DMSO): δ 157.64 (aromatic C alpha to SR group), δ158.92 (aromatic C=N alpha to oxygen of thiadiazol ring). Table 1 and 2, and (Fig 1-3)

Table (2) Percent Yield, Melting Point, Physical appearance and percent of yield of the prepared Compounds

Comp. No.	Molecular formula	Molecular weight	Description	Yield%	Melting point
A	C44H60N6O3S6	913.36	Yellow powder	79	291-293
B	C41H30N6O3S6	847.09	Yellow powder	81	298-299
C	C35H42N6O3S6	787.12	Yellow powder	83	285-287

IC₅₀ values of prepared compound on human breast cancer (MCF-7) and human hepatic cell lines (WRL68):

The study results showed that prepared compound A and B has non-significant selectivity on MCF-7 in low concentration while in high concentration showed small selectivity which may be negligible, this selectivity determined according to the comparison between: MCF-7 (Human Breast Cancer Cells) and WRL-68 (Human Normal Liver Cells).

Compound C showed remarkable significant selectivity on MCF-7 in high and low concentration when using the normal and cancer cell lines in this study depending on IC₅₀ as shown in table 3 and fig (1-3).

Table (3): the viability of human breast cancer cell line and human hepatic cell line after exposure to the prepared compounds.

Compounds		MCF-7		WRL-68
	Concentration	Mean	SD	Mean	SD
A	1000	64.49	0.64	72.37	1.58
	750	71.76	0.56	82.75	2.84
	500	85.03	3.26	92.44	2.71
	250	92.63	1.66	95.33	1.18
	125	95.72	0.81	95.22	0.82
B	1000	62.57	2.41	73.49	1.64
	750	72.91	0.57	81.92	1.82
	500	85.38	2.94	89.51	2.60
	250	94.06	1.38	94.17	0.77
	125	95.64	1.19	94.64	0.71
C	1000	35.71	3.41	64.62	3.85
	750	42.66	3.33	75.54	1.46
	500	61.25	5.88	85.88	5.17
	250	81.14	0.82	94.17	1.57
	125	94.90	0.96	94.83	0.97

Fig.1: viability% of compound A

Fig.2: viability% of compound B

Fig.3: viability% of compound C

CONCLUSION:

Compound C showed high selectivity against cancer cells of human breast cancer MCF-7 and no selectivity towards the normal human liver cells WRL-68, such result is a good indication for further investigation and studying of this compound against different type of cancer cells and studying different activities.

LIST OF SYMBOLS AND ABBREVIATIONS:

Human Breast Cancer Cells: MCF-7, Human Normal Liver Cells: WRL-68, analysis of variant: ANOVA, dichloromethane: DCM, dimethyl sulfoxide: DMSO, thin layer chromatography: TLC, tetramethylsilane: TMS.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

Team of work would thank Dr. Dhiaa Ali Abdulkader for his valuable help during the preparation of compounds, Al-Esraa University College for their kind assessment which help in completing this work, Pharmacology Department of Malaya University Medicine College, and CAC laboratory for the help to study the anticancer activity of the prepared compound.

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Received on 03.10.2021            Modified on 01.02.2022

Accepted on 12.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(10):4377-4381.

DOI: 10.52711/0974-360X.2022.00734