Slide 1: Title

Slide 2: Introduction

• Uncontrollable maturation of a human cell is known as cancer. 
• Affected body parts by cancer: liver, lungs, brain, blood, prostate etc. 
• Normal human cells and Cancer cells have distinct characteristics. 
• Therapy for cancer: chemotherapy, radiation therapy, immunotherapy etc.

Figure 1: Cancer cell vs Normal cell

(Source: https://www.cancerfoundation.org.au/what-is-cancer-.html)

Speaker Notes: It was found that when the growth of human cells become uncontrollable, that can be an indication of the cancer disease. Cancer condition can affect a vast variety of human body parts, for example, the liver, lungs, brain, blood, prostate etc. It was identified that the development of cancer cells and a normal cell of human being contain distinct characteristics. Recent medical science has developed different types of therapies and medicines such as chemotherapy, radiation therapy, immunotherapy etc.     

Slide 3: Structural complexity of Benzamine

• “Structure activity relationship” = high level of binding affinities S-enantiomers.
• Benzamine is derived from “amide derivative” of benzoic acid.
• Benzamine solid state = White Powder.
• Solubility = Partial in Water. 

Figure 2: Chemical Structure 

(Source: https://www.indiamart.com/proddetail/n-n-diethyl-benzamide-deba-2849390412388.html)

Figure 3: Physical Property

(Source: https://www.researchgate.net/figure/Chidamide-and-the-structure-activity-relationship-of-benzamide-class-HDACs-inhibitors_fig1_334943543)

Speaker Notes: It was identified that the formation of “structure activity relationship” of the Benzamine as an anticancer agent is related with the S-enantiomers. It was found that the S-enantiomers of the Benzamine have shown a high level of binding affinities. Also, it was identified that the Benzamine is derived from the “amide derivative” of benzoic acid. It sometimes appears as a white solid powder in its solid state (Ghasemi et al. 2020). However, it sometimes appears as a colorless crystal in its crystalline form. In many cases, it was discovered that it could be partially soluble in water.  

Slide 4: Backgrounds of Benzamine as an Anticancer Agent

• Use of benzamide in the development of antitumor agents.
• Reaction mechanism of the Benzamine analogues. 

Figure 4: Benzamine as an Anticancer Agent

(Source: https://www.sciencedirect.com/science/article/abs/pii/S022352342300209X) 

Speaker Notes: The use of benzamide become very significant in the development of antitumor agents in the field of medical science (Wang et al. 2021). It was found from previously completed research papers that a wide variety of cancer thematic drugs could be created with the help of the reaction mechanism of Benzamine. On the other hand, it was also found that the formation of benzylamide analogues acts very well as an inhibitor agent.   

Slide 5: Aims and Hypothesis of Experiment

• Aim: Formation of reactions and methods for the development of production of benzamides. 
• Hypothesis: Development of new reaction pathways for Benzamine that can reduce the effectiveness of cancer. 

Speaker Notes: The primary aim of this research is the formation of reactions and methods for the development of production of benzamides. On the other hand, this study also aims to identify the results of Benzamine as an anticancer agent. 

The hypothesis of this research includes development of new reaction pathways for Benzamine that can vitally decrease the effectiveness of cancer.  

Slide 6: Mechanism of Reaction

Figure 7: Mechanism of Reaction

(Source: Self-created in ChemDraw)

Speaker Notes: The reaction mechanism in this particuler research includes two primary compounds, which are 4-methoxybenzylamine and benzyl chloride. From the given reaction mechanism, it can be identified that the chemical reaction between these two components has created “N-benzyl-4-methoxybenzylamine” (Dang, 2021). The reaction, which have created this particuler compounds, was a “Nucleophilic Addition”. On the other hand, the percentage of product yield was calculated as 82.36 % in this particuler case.   

Slide 7: Mechanism of Reaction (Continue)

• Mechanism of Reaction holds 5 different steps. 
• Activation of acyl chloride was the starting of this experiment. 
• It occurs by the creation of a “nucleophilic catalysis mechanism”.

Figure 8: Steps of the Reaction Mechanism

(Source: Self-created in MS Word) 

Speaker Notes: The formation of this particuler reaction mechanism have followed five different steps. These reaction steps include, “Activation of Acyl chloride”, “Nucleophilic Addition”, “transfer of Proton”, “Elimination of Chloride Ion” and lastly formation of the benzylamide (Pintor et al. 2020). It was identified that the formation of reaction between 4-methoxybenzylamine and benzyl chloride started with the help of activation of acyl chloride. Previous research examples have shown that this particuler process happens with the creation of a “nucleophilic catalysis mechanism”.  

Slide 8: Methodologies MTT assay

• The MTT assay technique was accomplished in this particuler research.
• It was used for the determination of products’ cytotoxicity.
• In the first step, the “MTT solution” and “MTT solvents” were prepared.

Speaker Notes: The MTT assay method in this particuler study was conducted in this research to examine the formation of cytotoxicity of the formed product. The steps and procedures of the MTT assay experiment were performed by following all the required guidelines. The “MTT solution” and “MTT solvents” were prepared with the use of ethanol, water, 4 mm Hall and “0.1% NP40 in isopropanol”, in the first section (Nga et al. 2020). After that, the plate of the MTT assay was incubated at 37°C temperature for 3 hours.    

Slide 9: Methodologies MTT assay (Continue)

• 150 µL of MTT solvent was mixed with plate 1’s wells.
• The MTT assay plate was shaken for 15 minutes for the proper mix in an orbital shaker.
• The absorbance of the product was checked in 540 nm and 690 nm wavelengths. 

Speaker Notes: In the following part of this process, 150 µL of MTT solvent was combined with each well of plate used in the MTT assay. After that, the MTT assay plate was shaken for 15 minutes for the proper mix in an orbital shaker (Carreño et al. 2021). When the complete dissolving of this distinct mixture was completed, the absorbance was checked in two diverse wavelengths, which were 540 nm and 690 nm.   

Slide 10: NMR results

• This graph displays the NMR measurements of the “N-benzyl-4-methoxybenzylamine”.
• The acquisition time of the experiment was 6.55 seconds.
• The peak value was specified at 8.20. 

Figure 9: Results of the NMR measurements

(Source: Provided)

Speaker Notes: This particuler graph holds the formation of the NMR measurement results, which were completed, with the use of ““N-benzyl-4-methoxybenzylamine””. This particuler result was observed by using the solvent compound chloroform (Hu et al. 2021). It can be identified with the help of this graph, that the acquisition time of this experiment was 6.55 seconds with a peak of 8.20.    

Slide 11: FTIR graph

• The FTIR measurement results are present in this particular graph.
• At 92.56%, the identified wavenumber was 2064 (peak value).
• At 17.43%, the identified wavenumber was 1243 (lowest value).

Figure 10: Results of the FTIR measurements

(Source: Provided)

Speaker Notes: This above-mentioned graph holds the results of the FTIR measurements, which were conducted on the “N-benzyl-4-methoxybenzylamine” induced samples of cells (Magalhães et al. 2021). The peak value of the graph was observed at 92.56%, and the peak value was 2064. On the other hand, the lowest value was observed at 17.43% and the identified value was 1243.    

Slide 12: MTT Assay 

Figure 11: MTT assay 

(Source: https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/cell-proliferation-kit-i-mtt)

• The MTT assay experiment was completed to find out about the cytotoxicity of the anticancer agent.
• The well plates was used for the measure of cell viability.

Speaker Notes: In this particular experiment the MTT assay experiment was completed to find out about the cytotoxicity of the anticancer agent (Carreño et al. 2021). On the other hand, by using the well plates the measure of cell viability was conducted in this study. 

Slide 13: IC50 Graph

• IC50 values are less than 50.
• “N-benzyl-4-methoxybenzylamine” holds a significant impact on decreasing the growth of cancer cells.

Figure 12: IC50 Graph with concentration and viability

(Source: Self-created in MS Excel) 

Speaker Notes: The results of the IC50 graph shows that the IC50 value is lower than 50 in both of the cases. Also, the results of this particuler graph shows that this drug “N-benzyl-4-methoxybenzylamine”  holds a significant impact on decreasing the growth of cancer cells. 

Slide 14: Follow-up Studies

• Follow Up Study Aim: Increase production of Benzylamide with effective reaction mechanism.

• Industrial production of benzylamide as anticancer agent. 

Speaker Notes: The follow up study of this particuler research will include the testing and examining of reaction mechanism which can increase the production of benzylamide. The aim of follow up studies will be the identification and implementation of these pathways. 

Slide 15: Follow-up Studies Approach and procedure

Figure 13: Follow up Study Approaches

(Source: Self-created)

Speaker Notes: The increase production of  benzylamide research will include three different steps which are the “adjusting settings of the reactions”, “effective pathways to increase production” and “purification of products”.

Slide 16: Increase Yield within Scalability

Figure 14: Increase Yield within Scalability

(Source: Self-created)

Speaker Notes: The scalability and effectiveness of increased benzylamide production needs to be done by N-deprotonation and Benzoyl chloride reaction pathways (Nga et al. 2020). It is estimated that this process will be essential for increasing the yield by approximately 70%.

Slide 17: Purification Methods

Figure 15: Supercritical fluid chromatography

(Source: https://www.sciencedirect.com/science/article/abs/pii/S0039914016306610) 

Speaker Notes: The purification process of the products will include the methods of non active chromatography. For example Column Chromatography, Supercritical fluid chromatography and HPLC Chromatography etc. Among these methods Supercritical fluid chromatography can be an effective method for the follow up studies. The method of this purification process is similar to gas or liquid chromatography (Si-Hung and Bamba, 2020). However, instead of using a gas or liquid as a mobile phase, it includes the use of supercritical fluid as mobile phase for example, pressurized carbon dioxide (CO2) and a solvent. Increased flow rates of this method provides higher effectiveness because of these reason it widely used in pharmaceutical areas.       

Slide 18: Conclusion

• Identification of the anticancer agent “N-benzyln-methylamine”.
• Used Methods: NMR, FTIR, IC50, and MTT Assay.
• Analysis of “N-benzyln-methylamine” as effective anticancer agent.   

Speaker Notes: Throughout this entire presentation, the reaction of two compounds identified which have anticancer characteristics. The reaction of “4-methoxybenzylamine” and “benzyl chloride” were examined for the production of “N-benzyln-methylamine” which is a very effective anticancer agent.     

Slide 19: Reference List

Journals

Carreño, E. A., Alberto, A. V. P., de Souza, C. A. M., de Mello, H. L., Henriques-Pons, A., & Anastacio Alves, L. (2021). Considerations and technical pitfalls in the employment of the MTT assay to evaluate photosensitizers for photodynamic therapy. Applied Sciences, 11(6), 2603. [Retrieved From: https://www.mdpi.com/2076-3417/11/6/2603][Retrieved On: 25.06.24]

Dang, T. H. (2022). New Catalytic Approaches to Efficient Functionalization of CX and CC Bonds (Doctoral dissertation, The University of Texas at San Antonio). [Retrieved From: https://search.proquest.com/openview/d4f4dd69e590b89c9c48f979a405ff38/1?pq-origsite=gscholar&cbl=18750&diss=y][Retrieved On: 25.06.24]

Ghasemi, M., Turnbull, T., Sebastian, S., & Kempson, I. (2021). The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. International journal of molecular sciences, 22(23), 12827. [Retrieved From: https://www.mdpi.com/1422-0067/22/23/12827][Retrieved On: 25.06.24]

Gomis-Tena, J., Brown, B. M., Cano, J., Trenor, B., Yang, P. C., Saiz, J., … & Romero, L. (2020). When does the IC50 accurately assess the blocking potency of a drug?. Journal of chemical information and modeling, 60(3), 1779-1790. [Retrieved From: https://pubs.acs.org/doi/abs/10.1021/acs.jcim.9b01085][Retrieved On: 25.06.24]

Hu, Y., Cheng, K., He, L., Zhang, X., Jiang, B., Jiang, L., … & Liu, M. (2021). NMR-based methods for protein analysis. Analytical chemistry, 93(4), 1866-1879. [Retrieved From: https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c03830][Retrieved On: 25.06.24]

Magalhães, S., Goodfellow, B. J., & Nunes, A. (2021). FTIR spectroscopy in biomedical research: How to get the most out of its potential. Applied Spectroscopy Reviews, 56(8-10), 869-907. [Retrieved From: https://www.tandfonline.com/doi/abs/10.1080/05704928.2021.1946822][Retrieved On: 25.06.24]

Nga, N. T. H., Ngoc, T. T. B., Trinh, N. T. M., Thuoc, T. L., & Thao, D. T. P. (2020). Optimization and application of MTT assay in determining density of suspension cells. Analytical biochemistry, 610, 113937. [Retrieved From: https://www.sciencedirect.com/science/article/pii/S0003269720304693][Retrieved On: 25.06.24]

Pintor, A. V. B., Queiroz, L. D., Barcelos, R., Primo, L. S. G., Maia, L. C., & Alves, G. G. (2020). MTT versus other cell viability assays to evaluate the biocompatibility of root canal filling materials: a systematic review. International Endodontic Journal, 53(10), 1348-1373. [Retrieved From: https://onlinelibrary.wiley.com/doi/abs/10.1111/iej.13353][Retrieved On: 25.06.24]

Wang, Z., Yue, G., Ji, X., Song, H., Yan, P., Zhao, J., & Jia, X. (2021). Tandem Michael Addition–Cyclization of Nitroalkenes with 1, 3-Dicarbonyl Compounds Accompanied by Removal of Nitro Group. The Journal of Organic Chemistry, 86(20), 14131-14143. [Retrieved From: https://pubs.acs.org/doi/abs/10.1021/acs.joc.1c01586][Retrieved On: 25.06.24]

Si-Hung, L. and Bamba, T., 2022. Current state and future perspectives of supercritical fluid chromatography. TrAC Trends in Analytical Chemistry, 149, p.116550. [Retrieved From: https://www.sciencedirect.com/science/article/pii/S0165993622000334][Retrieved On: 25.06.24]