Synthesis of indole inhibitors of silent information regulator 1 (SIRT1), and their evaluation as cytotoxic agents

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Highlights

  • We designed and synthesized a series of new achiral indole analogues of EX-527.

  • The most active compounds inhibited the activity of SIRT1 selectively over SIRT2.

  • Indoles bearing bulky substituents at position 3 strongly inhibited SIRT2.

  • Some compounds were more cytotoxic than EX-527 on cancer cell lines.

Abstract

A series of achiral indole analogues of the selective sirtuin inhibitor EX-527 (a racemic, substituted 1,2,3,4 tetrahydrocarbazole) was designed to stabilize the bioactive conformation, and synthesized. These new indoles were evaluated against the isolated sirtuin enzymes SIRT1 and SIRT2, and against a panel of nine human cell lines. Structure-activity relationship studies demonstrated the influence of the substituent at position 3 of the indole. The most potent SIRT1 inhibitor 3h, bearing an isopropyl substituent, was as potent as EX-527, and more selective for SIRT1 over SIRT2. Compound 3g, bearing a benzyl substituent, inhibited both sirtuins at micromolar concentration and was more cytotoxic than EX-527 on several cancer cell lines.

Introduction

Histone deacetylases (HDACs) are enzymes involved in epigenetic regulation, and their inhibition has been successfully employed to develop cancer therapeutics [1]. Sirtuins are NAD+-dependent enzymes belonging to the class III of HDACs, and they regulate the activity and fate of a variety of protein substrates, including histones and transcription factors [2]. There are seven known members in the human sirtuin family of enzymes, SIRT1-7. Due to their broad biological activities, they constitute potential therapeutic targets, and several small molecules able to increase or inhibit their activity have been developed [3,4]. SIRT1 is the deacetylase that has attracted the most attention so far, in particular for the potential treatment of diseases of aging, including cancer and neurodegenerative disorders [5,6]. One of the most described potent and selective SIRT1 inhibitors is the 1,2,3,4 tetrahydrocarbazole EX-527 (Selisistat) [7,8]. Following encouraging animal studies [9], this inhibitor was evaluated in a clinical trial to treat Huntington disease (HD) [10]. The efficacy was not found sufficient to encourage further development of this compound in HD, possibly due to the slow onset of the disease and the comparatively short length (2 weeks) of the clinical trial. Interestingly, in a clinical trial with healthy volunteers, EX-527 exhibited high bioavailability and good tolerability [11].

The role of SIRT1 inhibitors in cancer is complex and depends on the cancer type and phase [12,13]. The overexpression of SIRT1 was correlated with metastasis in pancreatic ductal adenocarcinoma [14]. SIRT1 is also overexpressed in hepatocellular carcinoma (HCC), and has been shown to promote tumorigenicity, metastasis, and chemoresistance [15]. These results suggested testing SIRT1 inhibitors against tumor cell-lines. Some of them are shown in Fig. 1.

The sirtuin inhibitor nicotinamide has been shown to reduce the proliferation of pancreatic cancer cells, to induce the apoptosis in breast cancer cells, and to enhance their sensibility to cytotoxic agents [16,17]. The inhibitor 4bb displayed a cytotoxic activity against colon cancer HCT-116 cells, and a mechanism involving p53 as a substrate of SIRT1 was proposed [18]. A prostaglandin derivative, J11-Cl exhibited antiproliferative activity against SKOV cells through activation of apoptotic or autophagic cell death pathways [19]. Sirtinol and cambinol are β-naphthol containing inhibitors of SIRT1. Sirtinol was shown to induce senescence-like growth arrest in breast cancer MCF-7 and lung cancer H1299 cells. This effect was accompanied by a reduction of the Ras-MAPK pathway activity [20]. Tenovin-6 is another example of sirtuin inhibitor displaying toxicity on mammalian cancer cells, and able to decrease the growth of tumor xenograft in mice [21]. For some compounds like nicotinamide, sirtinol, and tenovin-6, non-specific inhibition and/or dual inhibition of SIRT1 and SIRT2 may explain in part the observed cytotoxic activities. Indeed, SIRT2 is also a potential therapeutic target in several diseases, including cancer [22].

SIRT1 overexpression in several kinds of tumors correlates strongly with attenuated p53 transcription dependent apoptosis upon DNA damage and oxidative stress. p53 can be the target of several covalent modifications including phosphorylation and acetylation, and acetylated p53 is activated and stable. SIRT1 counteracts p53-mediated apoptotic pathways by deacetylating it and decreasing its DNA binding [23]. For example, in HCC cell lines (HepG2 and Huh7), treating by EX-527 caused a decrease of sirtuins activity, a significant increase in the acetyl-p53/p53 ratio, and a decrease in HCC cells survival and migration [24]. Therefore, sirtuins activity blockage could be beneficial to HCC treatment. In this study, the data on p53 acetylation status is consistent with the apoptotic behavior of this tumor suppressor protein [24]. In breast cancer cells MCF-7, EX-527, which is more specific for SIRT1 than SIRT2, induced G1 phase cell cycle arrest, but no increase in p53 acetylation and cytotoxic effects only at high concentration (>50 μM). However, sirtinol, which is not specific, induced acetylation of p53 and cytotoxic effect. Because SIRT2 is also able to deacetylate p53, the Authors concluded that inhibition of both SIRT1 and SIRT2 is required to induce p53 acetylation and cell death in breast cancer cells [25]. We note here that the doses of EX-527 employed in different studies and cell lines have to be compared carefully, because at high concentration it inhibits SIRT2. Ohanna et al. found that SIRT1 was overexpressed in several melanoma cell lines [26]. The use of anti SIRT1 siRNA in melanoma cells increased the level of p53 acetylation and induced G0/G1 cell cycle arrest. Treatment with EX-527 exhibited similar results, which led to cellular senescence rather than a temporary growth arrest [27].

Treatment of two glioma cell lines (U87MG and LN-299) with EX-527 increased the number of apoptotic cells through the induction of caspase [28]. As p53 is a substrate of SIRT1, the Authors proposed that inhibition of SIRT1 by EX-527 increased the activity of p53 [28]. In vivo, EX-527 decreased the tumor growth of xenografted mice with human endometrial and lung cancer cells [29,30]. EX-527 and one of its analogues (S)-35 were described as selective SIRT1 inhibitors, with IC50 values in the range of 100 nM for SIRT1 and 2–20 μM for SIRT2, and no activity against a panel of other HDAC and NAD+-glycohydrolase [7]. They are mixed-type inhibitors against both NAD+ and acetylated peptide, therefore the nature of the peptide substrates and the concentrations are expected to have a large influence on the IC50 values [7,31]. A crystal structure of (S)-35 in the active site of SIRT1 has been described (PDB: 4I5I) [32]. It represents a useful tool for the design of new inhibitors of SIRT1.

Based on these results, we now report the design, synthesis and assay of new indole compounds derived from EX-527. Envisioned structural modifications involved the removal of the asymmetric carbon and the introduction of hydrophobic substituents of increasing steric bulk at positon 3 of the indole. This position was chosen because a suitable small hydrophobic pocket is present in the active site of SIRT1. The new compounds were tested against isolated enzymes SIRT1 and SIRT2, with the objective to identify compounds with better activity and/or selectivity for one of the enzymes. Finally, their cytotoxic activities were determined on a panel of nine cell lines, including cancer cell lines.

Section snippets

Design and synthesis of new EX-527 indole derivatives

The parent indoles EX-527 and analogue (S)-35 possess an asymmetric carbon. Therefore, they have to be used as racemic mixtures or purified by an additional chiral phase preparative chromatography [7]. We designed new indole derivatives in which the aliphatic cycle is opened, consequently removing the asymmetric carbon (Fig. 2). The primary carboxamide group was kept, because it establishes key hydrogen bond interactions with SIRT1. In our new compounds, hydrophobic substituents of varied

Conclusion

During the course of this work, a series of indoles designed from EX-527 was prepared in three steps from easily accessible commercial products. Fischer indole synthesis was followed by functionalization at position 2 via palladium catalyzed C–H activation, and amide formation. The prepared products were first tested against SIRT1. Several molecules prepared have inhibitory activities close to that of the reference molecule. This result validates the strategy employed, which makes it possible

Chemistry

Melting points were determined on a Stuart SMP3 Instrument and are uncorrected. 1H NMR and 13C NMR spectra were recorded on 400 MHz 1H (100 MHz 13C) BRUKER spectrometer in deuterochloroform (with residual chloroform as an internal reference, calibrated at 7.26 ppm for 1H and 77.00 ppm for 13C) or in deuterated dimethylsulfoxide with dimethylsulfoxide as an internal reference (calibrated at 2.50 ppm for 1H and 40.00 ppm for 13C). Chemical shift values are reported in ppm (δ). Coupling constant

Declaration of competing interest

The authors declare no competing financial interest.

Acknowledgments

This work was supported by the Paris Descartes University, the French CNRS and INSERM. We thank Karim Hammad and Regis Guillot respectively for their help with NMR measurements and X-ray diffraction data collection.

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