Synthesis of potent and selective HDAC6 inhibitors led to unexpected opening of a quinazoline ring

Histone deacetylase (HDAC) inhibitors are highly involved in the regulation of many pharmacological responses, which results in anti-inflammatory and anti-cancer effects. In the present work, chemoinformatic analyses were performed to obtain two potent and selective aminotriazoloquinazoline-based HDAC6 inhibitors. We unexpectedly obtained an aminotriazole from a water-driven ring opening of the triazoloquinazoline scaffold. Both compounds were evaluated as HDAC6 inhibitors, resulting in subnanomolar inhibitory activity and high selectivity with respect to class I HDAC1 and HDAC8. Importantly, the compounds were about 3- and 15-fold more potent compared to the reference compound trichostatin A. Additionally, the predicted binding modes were investigated with docking. Considering that the aminotriazole scaffold has never been embedded into the chemical structure of HDAC6 inhibitors, the present study suggests that both the aminotriazoloquinazoline and aminotriazole classes of compounds could be excellent starting points for further optimization of potential anticancer compounds, introducing such novel groups into a relevant and new area of investigation.


Introduction
Epigenetic regulation is generally dened as the change in gene function deriving from DNA modication operated through chromatin remodeling, RNA regulation and histone modication. Several enzymes are involved in epigenetic regulation. 1 Histone deacetylases (HDACs) play a crucial role in this process, catalyzing the removal of acetyl groups from lysine residues in histone and several non-histone proteins. [2][3][4] Currently, eighteen HDACs have been identied and grouped into four different classes, according to their cellular localization and enzymatic activity. Class I HDACs includes four isoforms (i.e., HDAC1, 2, 3,8), which are mainly located in the nucleus and can act on histones and transcription factors. Class II HDACs consists of two subclasses, i.e., IIa (HDAC4, 5,7,9) and IIb (HDAC 6 and 10). Of note, several members of class II HDACs can shuttle between the nucleus and the cell cytoplasm, where they can modulate the activity of various non-histone proteins. Class III histone deacetylases includes seven isoforms named sirtuins (SIRT1-7), which present several differences with respect to the other HDACs. Moreover, the activity of sirtuins is mainly involved in the regulation of metabolic processes as insulin secretion, ammonia detoxication and metabolic inammation. 5 Class IV includes only HDAC11, which is involved in the regulation of many biological processes in cells. 6 Classes I, II and IV require Zn 2+ for catalysis, while sirtuins employ NAD + (nicotinamide adenine dinucleotide) as a cofactor. 3 Recent studies have shown that Zn-dependent HDACs, and especially class I and class IIb isoforms, are overexpressed in many types of tumors, such as breast and liver cancer, multiple myeloma and neuroblastoma. 7 HDAC6 plays a crucial role in protein degradation, cell shape, migration and regulation of immunomodulatory factors 8 and is a well-established target for the development of anticancer compounds. Interestingly, the HDAC6 inhibitors reported so far share recurring features: (i) a zinc binding group (ZBG), which forms a stable coordination complex with the catalytic Zn 2+ ion; (ii) a hydrophobic linker that ts into the catalytic tunnel of the active site, and; (iii) a CAP moiety interacting with the residues that line the entrance of the pocket. 9,10 This latter region has been extensively probed for the design of HDAC6 selective inhibitors. Indeed, HDAC6 presents a CAP region signicantly different in terms of shape and volume with respect to that of the other Zndependent histone deacetylases. As for the ZBG, the most relevant and explored one is the hydroxamic acid (HA), which is a powerful chelator of Zn 2+ . The formation of HA-Zn 2+ complexes generally occur via a bidentate mechanism, resulting in signicant enzyme inhibition. 10 Several structurally different CAP groups have been explored for the design of HDAC6 inhibitors, the quinazoline motif demonstrating to be a valuable scaffold to obtain potent inhibitors. A few key examples of HDAC6 inhibitors bearing the quinazoline scaffold are reported in Fig. 1. [11][12][13][14] For example, compounds 1, 2 and 3 consist of a quinazoline core decorated at position 4 with a substituted aniline, which in turn is connected to an aliphatic chain linked to the ZBG through ethereal (1) or amidic (2 and 3) bonds. Changing the position of the ZBG led to 4, a potent cinnamyl derivative. The most signicant structural modication was obtained by switching the ZBG to position 2, affording 5, which, to the best of our knowledge, is the most active quinazoline inhibitor reported so far (IC 50 ¼ 12 nM). 13 Based on these premises and relying on our previous work on HDAC6 inhibitors, 2,[15][16][17][18][19] in this study we report the computational design and the synthesis of two structurally novel derivatives containing an aminotriazoloquinazoline (11a) and aminotriazole scaffold (18). The two synthesized compounds were tested in vitro to evaluate their inhibitory activity against HDAC6. Docking investigations were performed to provide structural explanations of the observed inhibitory activity. Of note, this is the rst study that explores the aminotriazoloquinazoline and aminotriazole scaffolds as CAP groups of HDAC6 inhibitors. Moreover, for the rst time we observed the opening of the triazoloquinazoline ring in water, which was previously investigated only in the presence of carbon nucleophiles. The results are particularly appealing in light of the potent sub-nanomolar HDAC6 inhibitory activity displayed by the two compounds.

Results and discussion
Design of aminotriazoloquinazoline compounds based on chemoinformatics analyses Compound 11a (Fig. 2) was assembled from a set of chemical scaffolds selected among those more frequently occurring in potent HDAC6 inhibitors, by adopting an approach similar to that of our previous studies. 20 To this aim, HDAC6 inhibitors collected from ChEMBL were rstly fragmented as detailed in the ESI, † identifying a set of substructures frequently present in the active and inactive compounds. Then, the identied chemical fragments were ranked according to their frequency of occurrence in active over inactive compounds. This allowed us to identify the benzohydroxamate, the 1,3-benzodioxole and the quinazoline moieties as valuable building blocks for the design and assembly of novel HDAC6 inhibitors (see Table S1 † and Fig. 2). Interestingly, the benzohydroxamate moiety is frequently present in HDAC6 inhibitors. The hydroxamate coordinates the Zn 2+ , 10 while the phenyl ring establishes favorable interactions with two phenylalanine residues of HDAC6 anking the catalytic tunnel. Moreover, the 1,3-benzodioxole moiety has been explored for the optimization of structurally diverse classes of HDAC6 inhibitors, with promising results. [21][22][23] The three chemical moieties emerging from these analyses were assembled into compound 11a, a substituted aminotriazoloquinazoline derivative. The connectivity between the three fragments was inspired by information available from HDAC6 crystal structures in complex with ligands and then veried by means of extensive similarity estimations (see ESI †) and docking (see below). Interestingly, this is the rst time that the aminotriazoloquinazoline group is explored for the design of HDAC6 inhibitors. Of note, a visual inspection of the iden-tied molecular fragments suggested that the benzohydroxamate moiety could be integrated in either position 8 (11a, Fig. 2) or 9 (11b, Fig. 2) of the aminotriazoloquinazoline group, without potentially altering the HDAC6 inhibitory activity.

Chemistry
The synthetic route to compound 11a was designed following a linear approach based on the construction of the aminotriazoloquinazoline core, a subsequent Suzuki-Miyaura crosscoupling for insertion of the benzylic linker and a nal functional group interconversion to introduce the hydroxamic acid moiety (Scheme 1).
Starting from the commercially available 4-iodoanthranilic acid and 2-(benzo[d] [1,3]dioxol-5-yl)acetyl chloride, the amide 6  was achieved in excellent yield, aer precipitation with HCl. Treatment of 6 with acetic anhydride at reux for 10 min gave quantitatively benzoxazinone intermediate 7, which was aerward reacted with aminoguanidine bicarbonate under microwave irradiation to provide the aminotriazoloquinazoline 8 in high yield. 24 Several conditions for the conversion of the iodo derivative 8 into the corresponding pinacolate boronic ester were evaluated, as reported in Table 1. The use of Pd(PPh 3 ) 4 and the combination of Pd(OAc) 2 -PPh 3 in dry toluene led to starting material recovery (entries 1-2), as well as the use of Pd(PPh 3 ) 4 in 1,4-dioxane/water 1 : 1 (v/v) or PdCl 2 (PPh 3 ) 2 in dry 1,4-dioxane (entries [3][4]. The formation of the desired product was observed using Pd(dppf)Cl 2 in 1,4-dioxane (Entry 5), while switching the solvent to dry DMF improved the conversion up to 63%, as calculated by 1 H NMR (Entry 6). The crude mixture was analysed by LC-MS, which highlighted the predominance of the desired pinacolate boronic ester, together with a low percentage of the free boronic acid. The mixture was ltered off, the solvent was evaporated under reduced pressure and the crude was used in the following step without further purication. The Suzuki-Miyaura cross-coupling with methyl 4-bromomethyl benzoate, using Pd(PPh 3 ) 4 and K 2 CO 3 in toluene/EtOH 3 : 1 (v/v), afforded the desired product 9 in good yield, aer column chromatography purication. Methyl ester 9 was hydrolysed under basic conditions by aqueous LiOH and coupled with O-trimethylsilylhydroxylamine. To our advantage, coupling and -OH deprotection occurred in a single step operation, providing the nal product 11a, aer reverse phase column chromatography purication, with good yield.

In vitro inhibitory activity
The synthetized compounds 11a and 18 were tested in vitro to assess their inhibitory activity on puried recombinant HDAC6 enzyme. The results are reported in Table 2.
Both 11a and 18 displayed potent, subnanomolar inhibitory activity towards HDAC6, resulting about 3-and 15-fold more potent than the reference compound trichostatin A (Table 2). Intriguingly, the unplanned compound 18 showed potent HDAC6 inhibitory activity. This result is particularly appealing, considering that aminotriazole compounds have never been reported as HDAC6 inhibitors. Furthermore, 11a and 18 were tested in vitro against puried recombinant HDAC1 and HDAC8 to evaluate their selectivity prole. Compound 11a resulted more than 12 000-fold and 1000-fold selective for HDAC6 with respect to HDAC1 and HDAC8, respectively, while 18 was even more selective, being than 18 000-fold and 15 000-fold more active on HDAC6 than HDAC1 and HDAC8, respectively (Table  2). Consequently, our study suggests that both the aminotriazoloquinazoline and aminotriazole classes of compounds are novel and excellent starting points for further optimization for the development of highly potent and selective HDAC6 inhibitors.

Molecular docking in the HDAC6 active site
The newly synthesized compounds were docked into a representative crystal structure of HDAC6 as detailed in the ESI, † to evaluate whether they provide favorable docking scores and a binding mode consistent with those reported in crystal structure complexes. The complementarity of 11a and 18 with the HDAC6 binding site was thus evaluated through docking calculations performed on the 5EDU crystal structure of the human HDAC6 enzyme. 28 For both ligands, we found that the ionized hydroxamate group coordinates the Zn 2+ in bidentate mode, 28 and it accepts two hydrogen bonds from Y782 and H610 through the carbonyl and the deprotonated hydroxyl, respectively (Fig. 3). Moreover, the phenyl ring of 11a and 18 establishes favourable p-p stacking interactions with the side chains of F620 and F680. Interestingly, the CAP groups of 11a and 18 are oriented differently (Fig. 3). The aminotriazoloquinazoline moiety of compound 11a stretches over F620 to donate a hydrogen bond to the side chain of D497, and the 1,3-benzodioxole accommodates in proximity of S564 and S568. On the contrary, the CAP group of 18 is directed towards F680, the amide carbonyl hydrogen bonds with the backbone nitrogen of F680, the 1,3-benzodioxole moiety forms a T-shaped stacking with the phenyl ring of F679, and the NH 2 of the triazole hydrogen bonds with the side chain of D567. Compounds 11a and 18 achieved favourable docking scores of À10.4 and À11.0 kcal mol À1 , respectively, compared to the self-docking result of À8.6 kcal mol À1 .

Conclusions
In conclusion, a series of chemoinformatic analyses performed on reported histone deacetylase 6 inhibitors allowed the iden-tication of the benzohydroxamate, 1,3-benzodioxole and quinazoline scaffolds as key moieties which were combined for the design of novel aminotriazoloquinazoline-based HDAC6 inhibitors. Two compounds were synthesized and tested in vitro to assess their inhibitory activity on puried recombinant HDAC6 enzyme. Aminotriazoloquinazoline 11a inhibited the enzyme (IC 50 ¼ 0.5 nM) 3 times more potently than the reference compound, while the unexpected ring-opened derivative 18 was even more active (IC 50 ¼ 0.1 nM), being about 15 times more active compared to thricostatin A. In addition, both 11a and 18 displayed high selectivity towards HDAC6 compared with the class I isoforms HDAC1 and HDAC8. The results indicate that the aminotriazoloquinazoline and aminotriazole scaffolds stand out as new starting points for the development of HDAC6 inhibitors, ranking in a promising area for future investigations.

Chemoinformatic analyses
Histone deacetylase 6 (HDAC6) inhibitors with activity data reported as IC 50 , K i , K d , EC 50 were rstly downloaded from the ChEMBL database (https://www.ebi.ac.uk/chembl/, accessed on: September 28 th , 2021). 29 Then, duplicate records deriving from multiple assays on the same target were removed, retaining those with the best activity value. This allowed to obtain 3582 unique ligands, 2304 and 1278 compounds of which have reported activity data below 1 mM (herein labeled as "actives") and higher than 1 mM (classied as "inactives"), respectively. Aerwards, an analysis of the molecular fragments composition was performed for the collected HDAC6 inhibitors. To this aim, the ligands were fragmented according to the BRICS, 30 Bemis-Murcko 31 and Recap 32 algorithms implemented in the RDKit 33 and OpenEye python toolkits, 34 and by using Chomp (version 3.1.1.2, OpenEye) 35 with default settings. Fragments whose substructure was not included in at least three active molecules and duplicates derived by the different fragmentation algorithms were discarded. Moreover, chemical moieties with a number of atoms lower than 4 or higher than 12 were also removed, obtaining a total of 544 unique fragments. The generated fragments were then used as queries for the identication of the substructures that are more frequently present in already reported HDAC6 compounds. The most interesting substructures emerging from the analysis and their related fragments (Table S2, in ESI †) were used as building blocks for the design of novel candidate compounds. The similarity degree between compound 11a, 11b and HDAC6 inhibitors extracted from ChEMBL was then evaluated by means of 2D ngerprints-based estimations. Similarity estimations were performed with MACCS and ECFP4 ngerprints (fp) implemented in RDKit python toolkits (https://www.rdkit.org), 33 with settings consistent to those employed in our previous studies. 36,37 Similarity records with Tanimoto coefficients below 0.8 and 0.3 for MACCSfp and ECFP4fp, respectively, were discarded. A visual inspection of the best-ranking ligandbased records (Table S1, in ESI †) was nally performed to evaluate whether compound 11a and 11b present key structural features and connectivity characterizing potent HDAC6 inhibitors, while showing a reasonable degree of chemical novelty.

Docking
All the analyses were conducted on the 5EDU crystal structure of the human HDAC6 protein. 28 The crystal structure of the protein underwent an optimization process using the Protein Preparation Wizard tool, implemented in Maestro of the Schrödinger Suite (release 2021-1). 38 Missing hydrogen atoms were added, and bond orders were assigned. The prediction of protonation states for the protein residues was accomplished by using PROPKA, with the pH set to 7.4. Docking studies were performed by using XP protocol of the Glide program, 39 keeping the ligands exible. Default settings were used for the analyses and metal coordination constraint was applied during docking procedure to allow the ligands to coordinate to the catalytic Zn 2+ ion.

Conflicts of interest
There are no conicts to declare.