To investigate the effects of substituents at positions 3/4/5 on biological activity, we developed two structural modifications to enhance potency: (A) Diacylated derivatives and (B) Monoacylated derivatives followed by acetonide protection of vicinal diols. This study evaluates explicitly the impact of these modified compounds on autophagy-activating activity.

We first used HEP15 as a substrate for acylation (Scheme 2), selecting nicotinoyl and acetyl groups to test the effects of products 1/2 on lysosome biogenesis. We constructed human HeLa cells to estimate autophagy activity systemically. As shown in Fig. 1, compound 2 promotes lysosome biogenesis, indicating that the C1 domain of PKC can accommodate smaller diacylated products, which was also supported by docking experiments. In contrast, compound 1 inhibited this activity. Although the exact mechanism remains unclear, the bulkier nicotinoyl group in 1 likely prevents its entry into the C1B domain of PKCδ, thereby interfering with PKC membrane-binding process and disrupting normal lysosomal turnover.

Reagents and conditions: a ROH (10 eq.), DMAP (10 eq.), EDCI (10 eq.), DCM, r.t. b RCl (10 eq.), DMAP (10 eq.), Et3N, DCM, 40 ℃

Inducing autophagy-flow activity of ester derivatives. The tested concentration was 20 μM. Torin 1 (2 μM) as positive control. Data are representative of three independent experiments. *P < 0.05, ****P < 0.0001 vs the DMSO

Subsequently, to further investigate the effect of acyl chain length on lysosome biogenesis, we acylated substrate 20-deoxyingenol (D1) using acetyl, propionyl, butyryl, pivaloyl, nicotinoyl, and Boc-protected isoleucinyl as acylating agents (Scheme 3). Since both the 3- and 5-positions of D1 can undergo acylation, all mono-acylated products except the pivaloylated derivative underwent migration during isolation and storage, making it impossible to obtain pure mono-acylated products. Therefore, we synthesized 3,5-diacylated 20-deoxyingenol esters. Additionally, we successfully isolated the 3-mono-pivaloylated 20-deoxyingenol esters 7 and 8 (Scheme 4).

Reagents and conditions: a ROH (10 eq.), DMAP (10 eq.), EDCI (10 eq.), DCM, r.t. b RCl (10 eq.), DMAP (10 eq.), Et3N, DCM, 40 ℃

Synthesis of 7, 8. (a) PivCl (10 eq.), DMAP (10 eq.), Et3N, DCM, 40 ℃. (b) DSAT, DCM, 0℃ to r.t

As shown in Fig. 2, activity screening revealed that among these diacylated products, the disubstituted propionylated derivative 4 exhibited the highest activity, surpassing even the positive control HEP14. Additionally, compound 5 has a slightly longer side chain than compound 4, yet exhibits marginally lower activity. While compound 7 demonstrates detectable activity, its potency shows no significant difference compared to the control group and remains substantially lower than that of compound 4. We further derivatized this compound to generate 8 by removing the 5-hydroxyl group and introducing a fluorine atom at position 7 to enhance lipophilicity. However, this structural modification resulted in complete loss of autophagy-activating activity.

Inducing autophagy-flow activity of ester derivatives. The tested concentration was 20 μM. Torin 1 (2 μM) as positive control. Data are representative of three independent experiments. *P < 0.05, ****P < 0.0001 vs the DMSO

If diacylated products are active, can monoacylated derivatives also activate autophagy? Given the propensity for acyl group migration between the 3- and 5-positions, we first protected the adjacent hydroxyl groups with an acetonide moiety. This exclusively yielded the 3,4-acetonide-protected product, with no observed formation of the 4,5-protected analogue. Based on this scaffold, we synthesized a series of acylated derivatives with varying chain lengths (compounds 9–13, Scheme 5). Biological evaluation revealed that compound 12, bearing an acyl group with more than three carbons, retained autophagy activation capability; however, its activity was markedly lower than that of the positive controls Torin 1 and HEP 14 (Fig. 3). Notably, this observation aligns with the previously reported trend in saturated linear esters, where increased ester chain length correlated with improved HIV-1 latency reversal activity [5]. Comparative analysis of inactive compounds (9–11 and 13) suggests that their shared acetonide group is unlikely to act as the catalytically active moiety.

Synthesis of 5-O-acyl-20-ingenol (9–19). Reagents and conditions: a 2,2-dimethoxypropane (4.8 eq.), PTSA (0.05 eq.), acetone, r.t; b ROH or Boc-L-Ile (10 eq.), DMAP (10 eq.), EDCI (10 eq.), DCM, r.t. c RCl (10 eq.), DMAP (10 eq.), Et3N, DCM, 40 ℃

Inducing autophagy-flow activity of ester derivatives. The tested concentration was 20 μM. Torin 1 (2 μM) as positive control. Data are representative of three independent experiments. *P < 0.05, ****P < 0.0001 vs the DMSO

Subsequently, we investigated the effects of different amino acid residues on activation (compounds 14–19). As shown in Fig. 3, the majority of compounds displayed potency of inducing autophagy activity, whereas compound 15 showed almost no biological activity. In contrast, compound 18, the protected derivative of methylglutamic acid, was the best potent compound to promote autophagy-inducing activity at a 2.31-fold than control. Notably, its activity surpassed that of the positive control Torin 1 at 2 μM (2.20-fold) and reached levels comparable to HEP14. In addition, compounds 14, 16, 17, and 19 exhibited weaker activity compared to 18. This suggested that introduction of amino acid with larger size groups such as methyl groups and aromatic ring on the terminal of side chain could decreased the activity.

Our previous study demonstrated that HEP14/15 induces TFEB-dependent lysosome biogenesis. To determine whether 4 and 18 exert similar effects, we treated HeLa and HepG2 cells with these compounds for 3 h. LysoTracker Red staining revealed a significant increase in lysosome numbers following treatment with 4 and 18 (Fig. 4A, B), and this effect was dose-dependent (Fig. 4C). Furthermore, both compounds 4 and 18 promoted the nuclear translocation of TFEB in cells ectopically expressing TFEB-EGFP, whereas the subcellular localization of TFE3 remained largely unchanged (Fig. 4D).

(A and B) Images (left) and quantifications (right) of endogenous Lyso Tracker Red in HeLa (A) and HepG2 (B) cells treated for 3 h with Drugs (20 μM) or Torin1 (1 μM). n = 3 independent experiments. Bars represent 10 μm in all images except. C Images (left) and quantifications (right) of endogenous Lyso Tracker Green in HeLa cells treated for 3 h with Drugs (1–40 μM). n = 3 independent experiments. D Images (left) and quantification (right) of the subcellular locations TFEB-EGFP or EGFP-TFE3 in HeLa cells treated with Drugs (40 μM, 3 h). n = 3 independent experiments. E Images (left) and quantifications (right) of HeLa cells treated with Drugs (20 μM, 3 h) and co-stained with BODIPY-pepstatin A (1 μM). n = 3 independent experiments. Data (mean ± s.e.m) were compared using t-tests or ANOVA ∗∗p < 0.01, ∗∗∗p < 0.001

Given that 4 and 18 induce lysosome biogenesis, we next investigated whether these compounds enhance lysosome-dependent cellular clearance. In HepG2 cells overloaded with oleic acid to induce lipid droplet formation, treatment with 4 and 18 led to a reduction in lipid droplet numbers. However, in the presence of bafilomycin A1 (BFA1), an inhibitor of lysosomal degradation, neither 4 nor 18 reduced lipid droplet numbers. These findings indicate that 4 and 18 promote lysosome-dependent clearance of lipid droplets (Fig. 4E).

To further verify that 4 and 18 enhances autophagy, we also assessed the expression of proteins related to autophagy-lysosome system by Western blot. The human cervical cancer cell line (HeLa cells) was treated with the compounds 4 and 18 for 24 h. Then, the proteins were extracted after cell lysis. We used Dimethyl Sulfoxide (DMSO) as a control and Torin 1, an autophagy inducer, as a positive control. The protein level of lysosomal-associated membrane protein 1 (LAMP1) was increased in a dose-dependent manner in response to 4 (Fig. 5A, B). Moreover, the protein level of cathepsin D (CTSD) which are important protease in lysosomes were also increased (Fig. 5A, C). These results indicate that lysosome function is enhanced. In addition, 4 increased the protein level of the lipidated (PE-conjugated) form of MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3 beta; LC3-II):LC3-I (Fig. 5A, D), which partially indicated that autophagy was enhanced. Collectively, these results suggest that compound 4 can activate autophagy-lysosome system and might be a promising autophagy inducer.

The autophagy-lysosome system was activated by 4. A Representative Western blot result showed the protein levels of LAMP1, CTSD, LC3-II/LC3-I and Ponceau S staining in the HeLa cells treated with 4, 18 or Torin 1. B–F Quantifications of the protein levels in the HeLa cells in a based on two independent experiments. The one-way ANOVA with the post hoc Holm-Sidak test was used to detect the expression differences between groups, and the values were expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****; ns, not significant

Previous study reported that D1 ester promoted autophagy by binding to PKCδ C1B domain [13]. Thus, molecular docking was employed to further analyze the interaction of 18 with PKC (Fig. 6). According to binding result, the methyl eater of L-Glu was anchored to biding site of the activity pocket and protective group located on the surface of protein. Two hydrogen bonds were formed between 18 and δC1B domain: a hydrogen bond between the C = O group of methyl ester and the NH group of THR 242 at 2.50 Å, another between the C = O group of the protective group and NH group of Gly253 at 1.95 Å. The t-butyl ester and methyl groups on the rigid ring skeleton displayed hydrophobic interaction with the hydrophobic residues in present in the upper portion [23]. In addition, it is required for PKC activation that ligand’s hydrophobic segment inserted into cell membrane [24, 25]. The simulation model suggested that the rigid rings exposure outside of protein might be inserted in the phospholipid bilayers and help PKC anchoring to cell membrane [23, 24]. Therefore, supported by docking study and bioassay results, we concluded that 18 might interact with PKC to possessed autophagy-inducing activity might be based on binding with PKCδ.

Predicted binding mode of compound 18 with PKCδ C1B domain (PDB code: 1PTR). The oxygen atoms and nitrogen atoms are shown in red and blue, respectively. The yellow dotted line indicates possible hydrogen bond. The figures were generated using PyMol (https://www.schrodinger.com/products/pymol)