Discovery of 5-Benzylidene-2-phenyl-1,3-dioxane-4,6-diones as Highly Potent and Selective SIRT1 Inhibitors

SIRT1, a member of the sirtuin family, catalyzes the deacetylation of proteins with the transformation of NAD+ into nicotinamide and 2′-O-acetyl-ADP-ribose. Selective SIRT1/2 inhibitors have potential application in the chemotherapy of colorectal carcinoma, prostate cancer, and myelogenous leukemia. Here we identified novel SIRT1 inhibitors with the scaffold of 5-benzylidene-2-phenyl-1,3-dioxane-4,6-dione. The most potent inhibitor 12n displayed an IC50 of 460 nM and a selectivity for SIRT1 over SIRT2, SIRT3, and SIRT5 of 113.5-, 254.3-, and 10.83-fold, respectively. It did not affect the activity of SIRT6. To elucidate the inhibitory mechanism, we determined the inhibition type of the inhibitor by enzyme kinetic analysis, showing that the inhibitor was competitive to the acetyl peptide and noncompetitive to NAD+. Further, the interaction of the inhibitor in SIRT1 was studied by using molecular docking, which was validated by the structure–activity relationship analysis of the inhibitors and the site-directed mutagenesis of SIRT1. Consistent with the in vitro assays, the inhibitors increased the acetylation level of p53 in a concentration-dependent manner in cells.

SIRT1, inhibitor, structure−activity relationship, interaction, deacetylase The acetylation state of proteins regulates diverse biological processes and is related with age-related diseases.1 Sirtuins are a family of NAD+-dependent deacetylases that catalyze the deacetylation of proteins with the transformation of NAD+ into nicotinamide and 2′-O-acetyl-ADP-ribose.2 Human sirtuins include seven family members, SIRT1–7. They have different subcellular localizations and functions. SIRT1, mostly present in the nucleus, is associated with a variety of metabolic pathways and involved in diseases including metabolic syndrome and insulin resistance.3,4 Activation of SIRT1 was found to mediate the progesterone resistance, which is believed to contribute to the infertility and progression of endometriosis.5 Besides, SIRT1 suppresses intestinal tumorigenesis and colon cancer growth through deacetylation of β-catenin6 but functions as a tumor promoter in colorectal carcinoma, prostate cancer, and myelogenous leukemia.7−11 Selective SIRT1/2 inhibitors have potential application in the chemotherapy of these cancers.12−16 To date, several SIRT1 inhibitors have been discovered. For instance, Cambinol (1), a SIRT1/2 inhibitor, exhibits antitumor activity in the Daudi Burkitt lymphoma xenograft model (Figure 1).17 EX-527 (Selisistat, 2), a selective SIRT1 inhibitor, was entered into clinical trials for Huntington’s disease and endometrial receptivity.18,19 In a double-blind, randomized phase II clinical trial involving Huntington’s disease patients, EX-527 was found to be safe and well-tolerated.20 However, no clinical benefit was observed after 2 weeks’ treatment.21 Recently, a SAFER trial for EX-527 was started in the clinical setting of in vitro fertilization and embryo transfer (clinical trial number NCT04184323).22 Besides, Lu et al. disclosed a small molecule, Inauhzin (3), that promotes the p53-dependent apoptosis of cancer cells by inhibiting SIRT1.23 Some benzylidene compounds such as 3-benzylideneindolin-2-one (GW 5074, 4) have also been described as sirtuin inhibitors.24−26 A screen for sirtuin activators identified suramin (5) as a SIRT1 inhibitor.27,28 Further, Jung et al. developed a suramin analogue, NF675 (6), with an improved inhibitory potency.29 Even so, the road of SIRT1 inhibitors to the clinical applications remains challenging. Structurally diverse and highly active SIRT1 inhibitors are still in urgent need, which highlights the significance of developing a novel chemotype of selective SIRT1 inhibitors. To identify novel SIRT1 inhibitors, a fluorescent assay that detects the deacetylation of an Abz-labeled acetyl peptide by SIRT1 was employed to screen our in-house compound library (Figure 2A).30 We found that compound 7 (Figure 2B) inhibited the activity of SIRT1 with an IC50 of 13.63 μM (Figure 2C). To confirm the inhibitory effect, we purchased 14 derivatives of the compound from SPECS company (https://www.specs.net) and determined their IC50. The assay showed that 13 derivatives possessed the inhibitory effect with IC50 lower than 20 μM (Figure 2D). Among them, compound 8m is the most active with an IC50 of 1.9 μM. In order to develop more potent SIRT1 inhibitors, we synthesized the derivatives of the compound and studied their structure–activity relationship (SAR). The synthesis of compounds 11a–11j and 12a–12s is outlined in Scheme 1. Benzaldehyde (9) reacts with malonic acid to obtain the 2-phenyl-1,3-dioxane-4,6-dione (10) in moderate yield. The condensation of 2-phenyl-1,3-dioxane-4,6-dione (10) and various substituted benzaldehydes gives the target compounds 11a–11j and 12a–12s. We evaluated the inhibitory effect of the synthesized derivatives. EX-527, a selective SIRT1 inhibitor whose IC50 was originally determined with the substrate of RHKKac-AMC,18 was used as the positive control. Herein, we used the peptide of Abz-GVLKacAYNO2GY-NH2 as the substrate of the enzyme assay. As summarized in Table 1, compound 11a (R1 = F) exhibited a potent effect against SIRT1 in low micromolar concentration (IC50: 3.76 μM). Replacement of fluorine with bromide (11b) maintains the activity. Both compounds displayed better activities than compound 8k, indicating that small substituent at R1 may enhance the interaction of the compound in SIRT1. Similar result may be obtained by comparing the activities of compounds 11c and 11i. Meanwhile, derivatives with various substituents (such as methyl, halogens, alkoxyl) at the ortho position (i.e., R2) of hydroxy (11c–11h) showed the inhibitory potency in the low micromolar range. Interestingly, the chlorine-substituted analogue 11e exhibits a better activity than fluorine-substituted analogue 11d. The bromide-substituted analogue 11f exhibited an improved potency compared with 11e with an IC50 of 1.40 μM. In addition, the atom linked to the ortho position has a marked impact on the activity. Halogens and oxygen seem more favorable than carbon. Beyond that, introducing an alkyl at R2 may improve the activity (comparing compound 11b to 11j, compound 11g to 11h). This suggests that the space in SIRT1 to accommodate R1 and R2 should be limited. Substitution at these positions with appropriate chemical groups would enhance the interaction of the compound with SIRT1. Table 1. Chemical Structures and Inhibitory Activities of Derivatives 11a–11j. In light of the above SAR analysis, 19 new compounds were designed and synthesized to improve the potency of 11f (Table 2). Compounds 12a–12c contain the same alkyl substituents at R1 and R2 on the phenyl group, but the lengths of the substituents are different (methyl, ethyl, and tert-butyl). Clearly, the enzymatic assay showed that the inhibitory activities of compounds with more steric hindrance were decreased (12c vs 12a). Compound 12d was much less potent than 12a likely due to the electro-donating property of the methoxy group. In contrast, introduction of electro-withdrawing groups (such as ester, nitro, bromide) at R1 led to enhanced inhibitory activities (2.4–8.0-fold) compared to compound 12d. Notably, compounds 12i and 12j potently inhibited SIRT1 with IC50 values of 1.31 and 1.14 μM, respectively. In addition, diiodo-substituted analogue 12n exhibited improved potency with IC50 of 0.46 μM, whereas dichloro-substituted and dibromo-substituted analogues 12l and 12m showed slightly weaker potency. The IC50 of the compounds displayed a decreasing trend of F > Cl > Br > I. Besides, compounds 12o–12q containing halogens and benzyloxy/methoxy at the phenol group showed the IC50 of 0.68, 0.61, and 0.89 μM, respectively, suggesting that introduction of halogens at the ortho position of pheno is essential for the activity. Moreover, replacement of hydroxy by amino group reduced the potency dramatically (compounds 12r and 12s), indicating that the hydroxy is important for the interaction of the compounds with SIRT1. Table 2. Chemical Structures and Inhibitory Activities of Derivatives 12a–12s. | Compound | R1 | R2 | X | IC50 (μM) | |---|---|---|---|---| | 12a | Me | Me | OH | 3.07 ± 0.17 | | 12b | Et | Et | OH | 3.10 ± 0.15 | | 12c | t-Bu | t-Bu | OH | 18.96 ± 0.10 | | 12d | OMe | OMe | OH | 11.90 ± 2.62 | | 12e | Bn | OMe | OH | 2.47 ± 0.10 | | 12f | COOBn | OMe | OH | 2.66 ± 0.09 | | 12g | NO2 | OEt | OH | 3.69 ± 0.10 | | 12h | NO2 | OMe | OH | 3.49 ± 0.11 | | 12i | Br | OMe | OH | 1.31 ± 0.26 | | 12j | Br | OEt | OH | 1.14 ± 0.08 | | 12k | F | F | OH | 4.54 ± 0.03 | | 12l | Cl | Cl | OH | 1.20 ± 0.09 | | 12m | Br | Br | OH | 0.70 ± 0.03 | | 12n | I | I | OH | 0.46 ± 0.09 | | 12o | OBn | Br | OH | 0.68 ± 0.07 | | 12p | OBn | I | OH | 0.61 ± 0.06 | | 12q | OMe | I | OH | 0.89 ± 0.01 | | 12r | Br | Br | NH2 | 7.63 ± 0.11 | | 12s | I | I | NH2 | 10.21 ± 0.17 | As each sirtuin has its distinct roles in the physiological process, selectivity among the sirtuins should be considered when developing SIRT1 inhibitors. Thus, we determined the inhibitory potencies of our compounds against SIRT2, SIRT3, SIRT5, and SIRT6. As shown in Table 3, 12i, 12l, 12n, and 12q exhibited significant selectivity for SIRT1 over SIRT2, SIRT3, SIRT5, and SIRT6. The most potent inhibitor 12n displayed a SIRT1 selectivity over SIRT2, SIRT3, or SIRT5 of 113.5-, 254.3-, or 10.83-fold, respectively. All compounds did not affect the activity of SIRT6. In comparison, EX-527 displayed a SIRT1 selectivity over SIRT2 of only 8.78-fold and over SIRT3 of 29.47-fold, indicating that compound 12n is a very potent SIRT1 inhibitor with high selectivity. Table 3. Inhibitory Activities of Our Compounds and EX-527 against SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 (IC50: μM)a. | SIRT1 | SIRT2 | SIRT3 | SIRT5 | SIRT6 | | |---|---|---|---|---|---| | 12i | 1.31 ± 0.26 | 54.04 ± 6.3 | 136.70 ± 14.30 | 19.84 ± 1.24 | NA | | 12l | 1.20 ± 0.09 | 56.44 ± 0.44 | 171.80 ± 13.60 | 37.42 ± 1.47 | NA | | 12n | 0.46 ± 0.09 | 52.24 ± 2.88 | 117.0 ± 1.60 | 4.98 ± 0.88 | NA | | 12q | 0.89 ± 0.01 | 49.45 ± 5.95 | 135.80 ± 14.70 | 22.45 ± 0.78 | NA | | EX-527 | 0.99 ± 0.10 | 8.69 ± 0.08 | 29.18 ± 1.60 | NA | NA | NA means “not active”. As known, the deacetylation catalyzed by SIRT1 is a bisubstrates reaction (i.e., the acetyl lysine and NAD+). The acetyl lysine binds to the D-site whereas NAD+ occupies the A-, B-, and C-sites in SIRT1.31 To unveil the inhibitory mechanism, we determined the inhibition type of compound 12i and examined how the inhibitor affects the binding of acetyl substrate and NAD+ in SIRT1. From the Lineweaver–Burk plot of the acetyl substrate at various concentrations of 12i (Figure 3B), we may see that the lines intersect in the third quadrant, indicating that the initial velocity V0 and the apparent Michaelis–Menten constant Km of the acetyl substrate decrease with the increasing concentration of 12i. Thus, compound 12i is a mix-type competitive inhibitor for the acetyl lysine. It may occupy the D-site, which is the binding site of the acetyl lysine in SIRT1. Meanwhile, the Lineweaver–Burk plot of NAD+ shows that the lines intersect at the horizontal axis (Figure 3D), indicating that the initial velocity V0 decreases but the apparent Michaelis–Menten constant Km of NAD+ remains unchanged with the increasing concentration of 12i. Therefore, compound 12i did not affect the interaction of NAD+ in SIRT1. It is a noncompetitive inhibitor for NAD+ and should not occupy the binding sites of NAD+. To elucidate the inhibitory mechanism, we simulated the interaction of 12i in SIRT1 (PDB ID: 4I5I) by using swissdock (http://www.swissdock.ch).32 SIRT1 contains a catalytic core constituted by a Rossmann-fold domain and a Zn2+-binding domain. The acetyl lysine binds to the cleft enclosed by two domains (i.e., D-site). As shown in Figure 4, compound 12i occupies the binding site of acetyl lysine. A hydrophobic interaction was formed between the phenyl at the C2 position of 12i and the residues Phe413, Phe414, and Val445 of SIRT1. The benzylidene at the C5 position of the inhibitor is in the vicinity of Ile347 and forms an edge-to-face π–π interaction with Phe273. Substitution with bromine atom may cause electron deficiency of the benzene ring of benzylidene and enhance the π–π interaction between compound 12i and Phe273, which may explain the positive correlation between halogen substitution at the benzylidene and the inhibitory potency of inhibitors. In addition, the hydroxy at the benzene ring of benzylidene forms hydrogen-bonds with the side-chains of Asn346 and Asp348. Substitution of the hydroxy with amino dramatically reduced the inhibitory activity (Table 2). To validate the binding mode of 12i in SIRT1, the residues Phe273, Asn346, Ile347, Asp348, and Phe414 of SIRT1 were selected for single-site mutation. The dissociation equilibrium constant Ki of compound 12i with SIRT1, SIRT1F273L, SIRT1N346A, SIRT1I347A, SIRT1D348A, or SIRT1F414A mutant was determined by the secondary plots of the kinetics data of 12i (Figure 5). We may see that the Ki of 12i for wild type SIRT1 is 0.10 μM whereas the Ki values for SIRT1F273L, SIRT1N346A, SIRT1I347A, SIRT1D348A, and SIRT1F414A mutants are 0.65, 1.94, 0.71, 7.06, and 18.17 μM, respectively. These demonstrated that Phe273, Asn346, Ile347, Asp348, and Phe414 did interact with compound 12i, among which Phe414 had the greatest contribution to the interaction with the compound. All-trans retinoic acid (ATRA) had been shown to induce the differentiation of SH-SY5Y cells through activation of SIRT1.33 As p53 is a substrate of SIRT1, modulation of SIRT1 activity may change the acetylation state of p53.34 To examine whether our inhibitors are active in cells, we treated SH-SY5Y cells with ATRA and the inhibitors and detected the expression of acetyl p53 by Western blot. As shown in Figure 6A, treatment of SH-SY5Y cells with ATRA alone (lane: control) decreased the expression level of acetyl p53 (compared to the lane of blank), while incubation of the cells with ATRA and 10 μM of compounds 12i, 12q, 8m, 12l, or 12n for 72 h significantly increased the acetyl-p53 level. To further confirm the result, we examined the inhibitory activity of 12n through a titration assay with the concentrations of 0.1, 1, 10, and 50 μM. The result showed that 12n increased the ratio of acetyl-p53 to p53 in a dose-dependent manner (Figure 6B). It displayed a comparable activity as EX-527 in cells. In summary, here we identified novel SIRT1 inhibitors with the scaffold of 5-benzylidene-2-phenyl-1,3-dioxane-4,6-dione. The derivatives of the inhibitor were synthesized and evaluated for their SIRT1 inhibitory activity. Based on the SAR of the derivatives, we developed a highly potent and selective SIRT1 inhibitor, 12n. The enzyme kinetic analysis showed that the inhibitor was competitive to the acetyl peptide and noncompetitive to NAD+. We also simulated the interaction of the inhibitor in SIRT1 by molecular docking, which was supported by the SAR analysis of the derivatives and the SIRT1 mutagenesis studies. In line with the in vitro assays, the inhibitor significantly increased the acetylation of p53 in cells. Glossary Abbreviations - SIRT1 silent mating type information regulation 2 homologue-1 - NAD+ nicotinamide adenine dinucleotide - Ac2O acetic anhydride - SAR structure–activity relationship - ATRA all-trans retinoic acid Supporting Information Available The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00559. Experimental details, characterization of all compounds, and the Lineweaver–Burke plots of NAD+ for SIRT1 and its mutants (PDF) Author Contributions ⊥ C.L., S.-S.H., and L.Y. contributed equally. The manuscript was written through contributions of all authors. The work was supported by National Natural Science Fund of China (Grants 21672233, 81973239, 81620108027, and 21632008), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12020375 and XDA12050411), SA-SIBS Scholarship Program, and the Youth Innovation Promotion Association CAS. The authors declare no competing financial interest. Supplementary Material

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