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Synthesis and Investigations into the Cytotoxicity of Garlic-Inspired Benzyl Tri- and Tetrasulfides in Cancer Cells
Michael J. Pachinger1, Roger Hunter2, Doaa M. A. Ali,2 Catherine H. Kaschula1
1Department of Chemistry and Polymer Science, Stellenbosch University, Stellenbosch, 7600, South Africa
2Department of Chemistry, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa
Presenter email: 24958557@sun.ac.za
Introduction and Aims
Allium sativum (also known as garlic), contains more than 30 organosulfur compounds (OSCs) that are produced after crushing and heating of the clove. Many of these compounds possess anticancer activity.1 The OSC of interest in this project are diallyl trisulfide (DATS) and diallyl tetrasulfide (DATTS), owing to their superior anticancer activity. Previous structure-activity relationship studies in our lab on DATS against WHCO1 cancer cells showed that analogues containing a benzyl side group are more active than the parent DATS. Benzyl groups modulated for their electronic effects were also investigated, and it was found that a methoxy para-substituted benzyl substituent was the most active, returning a nanomolar IC50 value, approximately 200-fold more active than the parent DATS.2
AIM
To further investigate other methoxy benzyl trisulfide regioisomers and to expand this library to include tetrasulfide analogues.
RESEARCH QUESTION
What are the contributing roles of the modified S-thiolated proteins, perthiol generation and H2S release to the overall cell cytotoxicity of tri- and tetrasulfides in cancer cells?
Synthesis – Via a Common Thiotosylate Intermediate
Scheme 1: The synthetic route to (A) the benzyl trisulfides and (B) the benzyl tetrasulfides via a common intermediate, 2. Procedure B performed by Prof Roger Hunter at UCT. The key steps (iii) and (iv) involve low temperature deprotection of the disulfanyl acetate (4) or dibenzoyl disulfide (5) with sodium methoxide which is rapidly quenched with the thiotosylate (2) to generate the . trisulfide in 30 s to 2 min, and 20 min for the tetrasulfides.3 Characterisation: 1H . and 13C NMR and HPLC. a Over two steps, b determined by rp-HPLC.
Anticancer Effects in Trisulfides: The Effect of The Substituent on the Cytotoxicity
Figure 2: (a) Cancer cell cytotoxicity of para-substituted benzyl trisulfides against WHCO1 cancer cells and (b) the relationship between the cytotoxicity IC50 and the para-Hammett constant of the substituent. 2
It has been found that the structure-activity relationship of several symmetrical para-substituted benzyl trisulfides is modulated by the electronic effects of the substituent.2 The data shows that ring activating groups, such as OMe, with a negative Hammett constant shows enhanced cytotoxicity towards WHCO1 cancer cells. Conversely, ring deactivating groups with positive Hammett constants, such as CF3 and Br, showed reduced cytotoxicity.2
Proposed Cytotoxic Mechanism of Action: is More Sulfur Really…More?
Figure 3: The proposed S-thyolysis cytotoxic mechanism of action of DATS (top) and DATTS (bottom, simplified) in cancer cells. A biological thiol (cysSH, GSH or proteinSH) attacks the terminal sulfur of the trisulfide moiety to produce two equivalents of a mixed disulfide (RSSallyl) and one equivalent of H2S.4 DATTS, on the other hand, has the potential to produce two equivalents of H2S and RSSallyl and a disulfide following attack at the internal sulfur.5
The mixed disulfide RSSAllyl has been shown to exert cancer cell cytotoxicity by inducing ER stress, leading to the unfolded protein response (UPR).
H2S is a gasotransmitter and its anticancer effects in this context are not so clear.6 Thus, the two equivalents of H2S released by the tetrasulfide moiety could influence the extent of cytotoxicity towards cancer cells.
Future Work – Biological Investigations
Determine tetrasulfide IC50 values.
Untangle the mechanism of action with in vitro investigations (Figure 3) using a probe to capture the H2S produced (Figure 4).
Acknowledgments
I would like to extend my greatest appreciation towards all my family and friends that have been supporting me in this journey. Prof Kaschula and Prof Hunter you have been and continue to be wonderful supervisors and I am eternally thankful. And finally, the Kaschula kids, GOMOC and PSP for financial support, thank you.
References
1. D. De Greef and E. M. Barton, et al., Semin. Cancer Biol., 2021, 73, 219–264.
2. Doaa Ali, PhD Thesis, University of Cape Town, 2024.
3. D. Ali., C. H. Kaschula and R. Hunter, et al., J. Org. Chem., 2019, 84, 2862–2869.
4. K. G. Fosnacht, M. D. Pluth. et al., J. Am. Chem. Soc., 2024, 146, 18689−18698.
5 U. Münchberg, C. Jacob, et al., Org. Biomol. Chem., 2007, 5, 1505–1518.
6. A. Sufian, D. Bhattacherjee, et al., Chem. Commun., 2025, 61, 4647-4661.