Novel quinolone-based potent and selective HDAC6 inhibitors: Synthesis, molecular modeling studies and biological investigation
Introduction
Histone deacetylases (HDACs) are enzymes involved in the removal of acetyl groups from histones, thus modulating gene transcription. Their over-expression has been ascertained in solid cancers, hematological malignancies, and parasitic disorders, making them a viable target for developing novel chemotherapeutic agents [1,2], as well as in rare diseases [3]. Of the 18 identified HDAC isoforms (subdivided into 4 classes, I-IV), HDAC6 is unique owing to the presence of a zinc finger ubiquitin binding domain and its localization. Structurally, it contains two catalytic domains, DD1 and DD2, present at the N-terminal and the central region, respectively. With regard to its localization, HDAC6 is a cytoplasmic enzyme (unlike HDAC1, 2 and 3, that are nuclear localized and HDAC8, that can show both nuclear and cytoplasmic spreading) which can shuttle between cytoplasm and the nucleus in response to cellular signaling [4]. Apart from its action on histones it also exerts deacetylase activity on non-histone substrates such as α-tubulin, heat-shock protein 90 (Hsp90), peroxiredoxin, cortactin, and tau [3]. This specific feature of HDAC6 is responsible for its unique role in the pathogenesis of cancer and a number of rare diseases such as idiopathic pulmonary fibrosis, inherited retinal disorders, Rett syndrome, and Charcot-Marie-Tooth disease [3,5]. Comparatively, HDAC10 that has a closer structural correlation with HDAC6 has a very weak activity as a lysine deacetylase and its role has so far been described mostly for autophagy promotion [6].
Several HDAC inhibitors (HDACi) have successfully reached the clinic and are being used as therapeutic tools to treat different malignances. Representative HDACi include: vorinostat (1, SAHA, 1) [7] and romidepsin (2) [8] for the treatment of cutaneous T-cell lymphomas, panobinostat (3) [9] for multiple myeloma, and belinostat (4) [10] for relapsed or refractory peripheral T-cell lymphoma. These compounds behave as pan-HDACi since they inhibit several HDAC isoforms, thus causing many side effects. The discovery of Tubastatin A (5) a tetrahydropyrido[4,3-b]indole hydroxamic acid, as a selective HDAC6i has paved the way for the development of isoform-selective HDACi. However, a potent inhibition of HDAC10 isoform has been reported after exposure with 5 [[11], [12], [13]]. Identification of isoform-selective compounds is vital for dissecting the biological roles of specific HDACs and for developing safer therapeutic tools. The general pharmacophoric model of HDACi encompasses three key components namely: the surface recognition group (cap), the zinc binding group (ZBG) and a linker bridging the two portions. Based on this model several selective HDAC6 inhibitors have been developed mainly by modifying the cap and the linker portions, while retaining hydroxamate as the ZBG [2,14,15].
Many natural products have been identified possessing potent anticancer properties, which have inspired chemists to use them as building blocks in the synthesis of novel chemotherapeutics [[16], [17], [18], [19]]. In this context, functionalized 4-arylquinolin-2(1H)-ones represent an attractive scaffold for developing biologically active molecules, including the orally active anticancer agent tipifarnib, that is currently undergoing clinical investigation [20].
In our pursuit to identify novel cap groups for potent and selective HDAC6i [14,15,24], we exploited the potential of a naturally occurring compound containing the quinolone skeleton as an effective versatile and hindered cap-group suitable for a broad variety of scaffold decorations (as shown in the general structure of Fig. 1). By a virtual screening approach, we identified several cap-groups; a preliminary computational investigation, using compound 6a, characterized by a viridicatin-based cap-group, showed that it perfectly fits into the hydrophobic sub-pocket delimited by L712 and F642 (Danio rerio HDAC6 numbering, corresponding to residues L749 and F679 in hHDAC6). Moreover, by our computational approach, we observed that the cap-group of Tubastatin A and 6a into zfHDAC6 (PDB ID: 6THV) target the same region of the enzyme as highlighted in Fig. 2. Therefore, we selected this interesting quinolone-based cap-group to rationally design and synthesize a new focused library of HDAC6 selective inhibitors as described below.
According to our preliminary in silico analysis and aiming at obtaining compounds with high selectivity index towards HDAC6 (over HDAC1) to be screened against different cancer cell lines we synthesized a library of quinolone-based hydroxamates. The synthesis of the 3-hydroxy-4-arylquinolin-2(1H)-ones moiety was carried out by the application of a multicomponent ring-expansion recently reported by Tangella et al. This protocol permits the conversion of isatin to viridicatin alkaloids after the in situ generation of α-aryldiazomethanes in presence of an aldehyde [25]. This protocol enabled us to develop novel HDAC6i (compounds 6a-d, 7a-k and 8, Table 1) bearing N- or O-appended linker moieties, and a variety of focused modifications at both their heterocyclic core and the aromatic portions.
Section snippets
Chemistry
The structures of final compounds 6a-d, 7a-k and 8 are shown in Table 1. In order to achieve the synthesis of the quinolone core of the compounds, the ring expansion procedure described by Tangella et al. was applied [25]. It is a multicomponent reaction involving: an aldehyde, activated with p-toluenesulfonyl hydrazide (PTSH), and a N-substituted isatin in the presence of K2CO3 as base.
In Scheme 1 the synthesis of the final compounds 6a-d is described. Isatin (9) was alkylated with methyl
Results and discussion
The compounds herein developed were firstly tested in enzymatic assay to evaluate their HDAC6 inhibitory profile compared with the HDAC1 and 8 isoforms. Successively, the ability of the best performing compounds to induce the acetylation of histone, tubulin and HSP90 was evaluated, followed by assessment of their anticancer properties in two cell lines (HCT-116 and U937). We have also determined key pharmacokinetic parameters (solubility and chemical stability), toxicity and mutagenicity of
Conclusion
In this work we describe the synthesis of quinolone-based HDAC inhibitors preferentially targeting the isoform 6. The synthesis of the cap group was achieved by applying a multicomponent reaction involving several substituted isatins, PTSH and a suitable aldehyde. This versatile synthetic strategy could be applied for future scaffold modifications and for the development of optimized analogues. The design of the described molecules was facilitated by applying a computational chemistry approach.
General information
Unless otherwise specified, materials were purchased from commercial suppliers and used without further purification. Reaction progress was monitored by TLC using silica gel 60 F254 (0.040–0.063 mm) with detection by UV. Silica gel 60 (0.040–0.063 mm) was used for column chromatography. 1H NMR and 13C NMR spectra were recorded on a Varian 300 MHz spectrometer or a Bruker 400 MHz spectrometer by using the residual signal of the deuterated solvent as internal standard. Splitting patterns are
Author contributions
The manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
Authors thank Progetto Dipartimento di Eccellenza DBCF-UNISI 2018–2022. A.P.S. Support from the European Union’s Horizon 2020 (EU) Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 721906-TRACT is acknowledged. N.R. Tuscany strategic project POR-FSE 2014–2020, ‘Medicina di Precisione e Malattie Rare’(MePreMaRe), (ACE-ESCC). G.R. acknowledges the CNR-CNCCS “Rare, Neglected and Poverty Related Diseases - Schistodiscovery Project” (DSB.011 CE.001.003). L.A.
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Cited by (0)
- 1
These authors equally contributed.
- 2
Present address: Wellcome Centre for Anti-Infectives Research, Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, DD1 5 EH Dundee, United Kingdom.
- 3
Present address: Department of Pharmacy, University of Napoli Federico II, DoE Department of Excellence 2018–2022, Via D. Montesano 49, 80131, Napoli, Italy.