The functionalized thiols (i.e., enethiols and amidothiols) were investigated for activity against the promastigotes of Leishmania major, the causative agent of human cutaneous leishmaniasis. Leishmania is a diverse group of parasites with about 20 causing disease in humans with three major clinical forms including cutaneous, mucocutaneous, and visceral (Akhoundi et al. 2016; Dondji et al. 2008). This latter form of the disease is fatal in the absence of treatment (Dondji et al. 2005). The clinical form of disease is dependent on the Leishmania species, the host’s genetic makeup, and the type of immune response generated by the host (McMahon-Pratt and Alexander 2004; Tacchini-Cottier 2008; Awasthi et al. 2004; Sakthianandeswaren et al. 2009). Currently, the drugs used for the treatment of leishmaniasis include pentavalent antimonial compounds, amphotericin B and miltefosine. However, these compounds have several serious side effects, including kidney failure and electrolyte abnormalities (McGwire & Satoskar, 2013). In addition, these drugs are expensive and treatment with them requires long duration of hospitalization. Therefore, there is an urgent need for new compounds that are active against Leishmania, less toxic and affordable.
In our quest to develop new antileishmanial therapeutics, we screened a total of 21 thiol compounds in vitro using the Alamar Blue colorimetric assay. All the molecules were tested at a concentration of 100 µg/mL. The biological activity of the compounds was compared to that of Amphotericin B (Amp B), a drug of choice for the treatment of leishmaniasis (Pieroni et al., 2012). Of the thiols tested, 13 were classified as active namely compounds 1, 2, 3, 4, 5, 7, 12, 13, 14, 15, 16, 17, and 18 (Table 1). Eight of these compounds failed to decrease the survival of the parasites in our assay and were labelled inactive. Overall, 62% of the compounds tested showed leishmanicidal activity while 38% did not.
Table 1
Activity of thiols against Leishmania major. Compounds are numbered 1–21. Forty-eight hours after incubation with the compounds, the anti-Leishmania activity was evaluated using a colorimetric assay. The optical density recorded of test wells was compared to that of Amphotericin B, one of the drugs of choice used in the treatment of leishmaniasis. Based on this comparison, compounds were classified as either active or inactive.
Compounds | Activity against Leishmania parasites |
---|
1 | Active |
2 | Active |
3 | Active |
4 | Active |
5 | Active |
6 | Inactive |
7 | Active |
8 | Inactive |
9 | Inactive |
10 | Inactive |
11 | Inactive |
12 | Active |
13 | Active |
14 | Active |
15 | Active |
16 | Active |
17 | Active |
18 | Active |
19 | Inactive |
20 | Inactive |
21 | Inactive |
In drug discovery, one of the core principles is the identification of the lowest concentration at which compounds retain desired activity (Khadir et al. 2018). Consequently, some representative functionalized thiols that had shown leishmanicidal activities were tested for dose dependency response at 2.5, 5, 10, 25, 50 and 100 µg/mL. These compounds included 13 and 15. Interestingly, compound 13 showed the same level of activity for 50 and 100 ug/mL preparations at 48 and 72 hours post-incubation with the parasites (Fig. 3A). Activity better or equal to that of Amphotericin B was maintained at 10 µg/mL at both post-incubation periods. The lowest concentrations of compound 13 (2.5 and 5 µg/mL) had leishmanicidal effects but lower than that of Amp B. Similar to compound 13, the same level of activity was recorded for 50 and 100 µg/mL preparations of compound 15 at 48 and 72 hours post-incubation with the parasites (Fig. 3B). However, at 25 µg/mL, compound 15 activity was lower than that of Amp B unlike similar concentration of compound 13.
In order to further evaluate thiols as potential therapeutics that might be utilized to control leishmaniasis, we assessed the relationship between compounds with “similar” basic molecular structure and their leishmanicidal activity. As described earlier, compounds 1–12 share the enethiol substructure whereas compounds 13–21 share the amidothiol motif. A structure-activity relationship analysis was conducted for the 21 compounds. For compounds 1–12, the first five compounds differ only at the nitrogen bearing one hydrogen atom (i.e., the secondary amino nitrogen highlighted in red in Fig. 1). These five compounds contain cycloheptyl, cyclopentyl, cyclopropyl, tert-butyl, and benzyl substituents, respectively. All five compounds were biologically active with 4 and 5 displaying potency at levels similar to or above Amp-B. The results suggest that noncyclic substituents have a beneficial effect than the cyclic substituents. When the aromatic substituent resident in these enethiols is modified, significant changes are observed. For example, there is a complete loss in activity when the phenyl group in 3 is replaced by a para-nitrophenyl group (see 6). However, it is foolhardy to conclude that the nitro group alone has a deleterious effect on the biological activity since potency is regained when the cyclopropyl group in 6 is replaced by the allyl group in 7. When the isopropyl group on the tertiary amine (highlighted in blue) is replaced by the α-methylbenzyl group, there is a complete loss of potency (see 8–11). Activity is once again restored when both nitrogen atoms have an isopropyl group (see 12). These results clearly indicate that minor changes in structure can lead to profound changes in the biological activity.
A separate structure-activity relationship analysis was conducted using compounds 16–21, which all harbour the amidothiol substructure (Figure 4A). Compounds 16‒18 differ at the nitrogen-substituted aromatic ring (highlighted in red). These three compounds contain 4-methoxyphenyl, 3-trifluoromethylphenyl, and 3-methoxyphenyl substituents, respectively. All three compounds were biologically active and displayed potency at levels marginally better than Amp-B though not statistically significant. In vitro biological activity of these amidothiol compounds is shown in Figure 4B. Compounds 19-21 have the same nitrogen substituent as 16 but differ in the substitution of the other aromatic ring (highlighted in blue). Notably, replacement of a hydrogen atom with a nitro group (19), a methoxy group (20), or methoxy and acetoxy groups (21) led to complete loss of biological activity unlike compound 16. This loss of activity was statistically significant (16 vs. 19, p<0.01; 16 vs. 20, p<0.01; 16 vs. 21, p<0.05). The biological activity linked to structure observed here is frequently reported in studies evaluating chemical compounds for antileishmanial activities (Anderson et al. 2020; Pieroni et al. 2012).