Ilamycin F has two types of functional groups for modification, C-33 carboxyl group and C-44 hydroxyl group. With the aim of synthesizing new derivatives, several modifications in ilamycin F were introduced by acylation and esterification (Scheme 1). Ilamycin NJL1–NJL12 (1–12) were concisely synthesized by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/1-hydroxybenzotriazole (HOBt) assisted amidation at C-33 carboxyl group using l-amino acid methyl esters and benzylamine derivatives (Figure 2). The yields ranged from 74 to 83%. Moreover, according to the twin drug strategy (Contreras et al., 2008; Ibacache et al., 2018), isoniazid and N-deacetyl-linezolid, two anti-TB substrates, were respectively coupled with ilamycin F to generate ilamycin NJL13–NJL14 (13–14) using the aforementioned amidation method in 76–77% yield (Figure 2). The strategy was proposed to produce synergic effect by binding two targets.

The C-44 hydroxyl group of ilamycin F is another position for derivatization. Although various ether and aliphatic sidechains at C-44 were created through etherification or esterification by Eli Lilly and Company, all derivatives displayed low anti-TB activities (Lambooy, 2000). To further study SAR of ilamycin F, heteroaromatic rings, p-fluorophenylacetic acid and 3-(methylthio) propionic acid were introduced at the C-44 hydroxyl group (Scheme 1). Ilamycin NJL15–NJL18 (15–18) could be successfully obtained in the presence of EDC and 4-dimethylaminopyridine (DMAP) (Figure 3), but owing to low nucleophilicity of the C-44 hydroxyl group, the yields of the reaction were only given to 32–40%.

The anti-TB activities of ilamycin NJL1–NJL18 (1–18) were evaluated against M. tuberculosis H37Rv, which has pathogenic and still popularly used in virulent laboratory (Camus et al., 2002; Målen et al., 2011). As depicted in Table 1, although basic amino acid derivatives (compounds 4–5) at the C-33 had weak activities, the modification of neutral amino acid derivatives, in comparison with ilamycin F, displayed potent anti-TB activities with fast-onset effect. Compounds 1–3, 6, and 8, showed efficient activities on day 3rd, which was higher than that of ilamycin F with 4–19 folds. Importantly, compounds 1, 6, and 8 had slightly stronger activities than that of ilamycin F on day 14th (MIC, 1.6–1.7 μM), speculating that their modification did not change the interaction with targets, but would obviously facilitate to promoting penetration of membranes in Mtb. Moreover, benzylamine derivatives of ilamycin F, compounds 9–11, were also exhibited the fast-onset effect on day 3rd except compound 12. This finding indicated that benzylamine modified with a larger substituent significantly affected anti-TB activity of ilamycin derivatives. Although showed a similar activity with that of ilamycin F on day 14th, compound 10 was 2-fold more potent MIC value than that of 9 and 11, which might result from the promotion of its lipophilicity by fluorine substituent and improved the penetration to cell membranes (Smart, 2001; Purser et al., 2008). However, the activities of compounds 13–14 were significantly decreased under the twin drug strategy, when a carboxyl group at the C-33 was replaced by isoniazid or N-deacetyl-linezolid. This result indicated that the construction of compounds 13–14 affected the binding to their targets, and exhibited no synergistic effect on anti-TB activity. Additionally, compounds 15–18 modified at the C-44 also showed lower activities. The similar groups coupling at the C-33 with beneficial effects could not produce the same promotion at the C-44 hydroxyl group of ilamycin F. The results suggested that the hydroxyl group at C-44 might serve as a pharmacophore, which was critical in achieving anti-TB activity and also consistence with our previous discoveries (Sun et al., 2020).

To evaluate the application potential of these compounds, the cytotoxicity of ilamycin NJLs (1–18) was evaluated in vitro using five human cancer cell lines, including breast adenocarcinoma (MCF-7), cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2), lung cancer (A549), colon cancer (HCT116); two normal cell lines including human hepatic cell line (L02) and human umbilical vein endothelial cell line (Huvec-12). Although most compounds (2–18) showed no or weak cytotoxicity, compound 1 exhibited a moderate IC₅₀ value (5.7–9.0 μM) against MCF-7, A549, HCT116, and L02, which had a 3–8-fold promotion compared with that of ilamyicn F (Table 2). The result indicated that threonine methyl ester modified in C-33 carboxyl group of ilamycin F was favorable for the cytotoxic activity.