**7. Metabolism of SHetA2**

Using liquid chromatography and tandem mass spectroscopy, four GSH adducts were identified along with four mono- and dihydroxylated SHetA2 metabolites [58]. At least one of these metabolites (**Figure 9**, **25**) has been deemed active against KIT kinase as mentioned earlier. Apart from the hydroxylated metabolites, other metabolites of SHetA2 were also detected *in vivo* [58, 59]. These metabolites may be produced upon formation of GSH adducts. The proposed mechanism for the formation of GSH adduct is shown in **Figure 10**. Subsequently, the GSH adduct could undergo further reactions that may result in the cleavage of the

**Figure 10.** Proposed mechanism for the GSH adduct formation of SHetA2.

and the angiogenic 2-deoxy-D-ribose-1-phosphate, as well as its metabolite, deoxy-D-ribose. Protein levels of the angiogenic vascular endothelial growth factor A (VEGF) was also found to be downregulated despite its up-regulated mRNA level upon SHetA2 treatment. Interestingly, both mRNA and protein levels of the angiogenic basic fibroblast growth factor (bFGF) were also up-regulated, but its effect was deemed limited due to brief up-regulation of the gene only after hours of treatment with high concentration of SHetA2 [12]. Hence, the net effect of SHetA2 on these various proteins is antiangiogenic, and is supported by the decrease of endothelial tube formation observed in a number of cancer cell lines as well as human umbilical

vascular endothelial cells (HUVECs) [12].

84 Anti-cancer Drugs - Nature, Synthesis and Cell

**6.4. Kinase inhibitory activity**

**7. Metabolism of SHetA2**

**Figure 9.** Structural comparison of SHetA2 and its metabolite with sorafenib and linifanib.

observed, making it a versatile chemotherapeutic agent.

SHetA2 is also being evaluated for its ability to inhibit kinase activity based on its structural similarity to sorafenib and linifanib (**Figure 9**) [63]. Sorafenib is an FDA approved diarylurea multikinase inhibitor that inhibits tumor growth, while linifanib is a KIT-3 kinase inhibitor [66, 67]. All three compounds consist of a three atom urea or thiourea linker between two aromatic rings, of which this structural conformation is found to be vital for the formation of key Hbonds within the binding pockets of several kinases, including B-Raf, BCR-ABL, and KIT [63]. Upon evaluation with 442 different human kinases, SHetA2(**Figure 9**) has exhibited good binding affinity for KIT kinase (binding constant, Kd = 820 nM). This indicates that SHetA2 is a potential candidate for kinase inhibitor development. More importantly, one of the metabolites of SHetA2 (**Figure 9**, **25**) has also shown comparable binding affinity for KIT kinase (Kd = 1200 nM) [63]. This suggests that other metabolites of SHetA2 may also be active, which could have acted via different mechanisms of action and contributed to the various anticancer effects

Using liquid chromatography and tandem mass spectroscopy, four GSH adducts were identified along with four mono- and dihydroxylated SHetA2 metabolites [58]. At least one of

**Figure 11.** Proposed mechanism for the formation of metabolites following the formation of GSH adducts.

thiourea linker, yielding two metabolites that were also detected *in vivo* [58]. The proposed mechanism for the formation of these metabolites is shown in **Figure 11**.

Given that ShetA2 has several mechanisms of action involving various cellular targets, these findings suggest the possibility for SHetA2 to be metabolized into several active metabolites, each targeting a different molecular pathway. This is further supported by a recent *in vivo* study where the monohydroxy SHetA2 (**25**) was found to be the major metabolite of SHetA2 in rat plasma, and it was detected at a much higher concentration than the parent compound after oral and intravenous administration [13].
