Dibal-h Reaction Class 12

[Mahmod Ohner]

Guidedby computational analysis, herein we report the design, synthesis and evaluation of four novel diazine-based histone deacetylase inhibitors (HDACis). The targets of interest (TOI) are analogues of panobinostat, one of the most potent and versatile HDACi reported. By simply replacing the phenyl core of panobinostat with that of a diazine derivative, docking studies against HDAC2 and HDAC8 revealed that the four analogues exhibit inhibition activities comparable to that of panobinostat. Multistep syntheses afforded the visualized targets TOI1, TOI2, TOI3-rev and TOI4 whose biological evaluation confirmed the strength of HDAC8 inhibition with TOI4 displaying the greatest efficacy at varying concentrations. The results of this study lay the foundation for future design strategies toward more potent HDACis for HDAC8 isozymes and further therapeutic applications for neuroblastoma.

One of the most important posttranslational modifications involve acetylation/deacetylation of histone proteins by histone deacetylases (HDACs) [1]. HDACs belong to an important family of enzymes consisting of 18 isozymes. They control protein acetylation, which is a change that occurs after translation. In addition, they regulate gene transcription, cell differentiation, cell cycle progression and apoptosis by targeting both histone and non-histone proteins. The balance between acetylation and deacetylation is pivotal for typical cell function. Abnormal or increased HDAC expression has been reported in several human tumors and cancer cell lines [2]. As such, the development of novel HDAC inhibitors (HDACis) has become a rapidly evolving area where targeted inhibition has emerged in clinical research as a potential therapeutic approach for the treatment of various cancers as well as neurodegenerative disorders and immune related diseases [3-5]. Of specific interests are Class I HDAC isozymes, HDAC2 and HDAC8, which are important targets in cancer models as both are associated with high risk diseases such as prostate cancer and neuroblastoma [6-8]. Compounds such as vorinostat, givinostat and panobinostat have been successfully applied as HDAC inhibitors [3]. Among these drugs, panobinostat (Farydak, Novartis) an FDA approved drug, has been recognized as a pan-deacetylase inhibitor [9,10]. As a hydroxamic acid pan-HDACi, it is zinc-dependent, capable of binding in a bidentate fashion to the zinc-containing catalytic domain of the HDACs, and classified as highly potent amongst traditional HDACis [11]. According to previous reports, panobinostat not only induces apoptosis in cells, but also stimulates cell growth inhibition, and cell-cycle arrest in a time- and dose-dependent manner. Thus, panobinostat has demonstrated high therapeutic potential in anticancer efforts. Although panobinostat offers a versatile approach for the inhibition of cancer cell growth and survival, a lack of selectivity and bioavailability can cause negative molecular and clinical effects, specifically in combination therapies.

Despite advances in Class I HDAC inhibition, there remains an obvious need to develop compounds having better therapeutic properties as a single-agent therapeutic drug. Our recent research based on computational studies indicated heterocyclic cores as suitable surrogates for the central core of the hydroxamate derivative, panobinostat [12]. It should be noted that TOI3-rev in this article is different from TOI3 in the previous reporting [12]. Here, the 1,2-diazole ring has been replaced with that of a pyrimidine core. Given the abundance of literature regarding analogues having modifications of the indole amine unit and vinylogous hydroxamic moieties [13-15], the non-availability of the central core modification stimulated our interest toward altering the central core to evaluate efficacy. We found particular interest in the replacement of the phenyl ring with diazine cores (pyridazines, pyrimidines and pyrazines) as their docking values were on par with that of the parent molecule, panobinostat (Figure 1). Given that the diazine-containing compounds are considered to be one of the most important classes of heterocycles, their presence in a plethora of pharmacology and drug molecules motivated us to synthesize these analogues and subject them towards biological evaluation [16-19]. In addition, our envisioned dinitrogen heterocycle cores may have increased interactions in the binding pockets, and thus leading to a better therapeutic activity.

Aiming to provide a strategy to address our global objective of developing single agent therapeutic hydroxamate derivatives, we began the synthesis of four leading HDAC8 diazine-based HDACis: TOI1, TOI2, TOI3-rev and TOI4 (Figure 1). Herein, we provide a summary of the design process followed by an outline of the multistep synthesis and preliminary biological evaluation of each target. HDAC8 was selected for testing due to its unique structure and multifaceted functional activities [4,5,20,21]. HDAC8 is also upregulated in neuroblastoma, a childhood pediatric cancer, hence considered a drug target for this cancer subtype [22,23]. Despite a multifaceted array of alternative treatment options for neuroblastoma, some patient cohorts who are considered high risk at the time of diagnosis face poor prognosis [24,25]. Recent studies indicated that HDAC8 inhibition induces differentiated phenotypes and reduces neuroblastoma growth in vitro and in vivo with few adverse effects [22]. However, there are very few effective therapeutic options in neuroblastoma that inhibit HDAC8 [26]. Thus, the design of novel HDAC8 inhibitors as potential neuroblastoma therapeutics could be valuable to expand the treatment options for this patient population [27]. Therefore, we sought to focus on the biological evaluation of our proposed inhibitors in HDAC8. This study aimed at offering additional therapeutic options to be used in conjunction with or in place of panobinostat while providing a rationale design guideline towards HDACis.

In review of the computational results, the inclusion of the nitrogen atoms in the core ring structure of panobinostat produced compounds with predicted binding affinities similar to panobinostat. Thus, aiming to develop improved compounds to effectively target HDAC8, the synthesis of TOI inhibitors and their evaluation was undertaken.

Finally, the ethyl ester 11 was converted to the hydroxamic acid derivative, TOI1 using the bidentate nucleophile hydroxylamine either under neutral or basic conditions [36,37]. We first explored neutral conditions where aqueous hydroxylamine was added to compound 11 in methanol, and a predominant polar spot was observed by TLC. However, the isolated product was not the expected TOI1, as 1H NMR revealed two new peaks at δ 4.53 and 2.66 ppm (Supporting Information File 1, Figure S36) presumably indicating that a favorable Michael addition followed by intramolecular cyclization or vice versa provided compound 12, which was validated by 13C and DEPT NMR studies (Supporting Information File 1 Figures S37and S38).

Marred with these observations, compound 11 was treated with aqueous hydroxylamine in the presence of strong base (i.e., 10 equivalents of methanolic sodium hydroxide or aqueous sodium hydroxide) at 0 C. The reaction was monitored by TLC and it revealed that methanolic sodium hydroxide provided a cleaner reaction than aqueous sodium hydroxide conditions. The reaction mixture was quenched with a saturated ammonium chloride solution at 0 C after 12 h, the solvent was evaporated, and the compound was subjected to reversed-phase column chromatography using C-18 silica gel. After initial unsuccessful purification protocols with water/ACN or water/THF solvent systems, we identified an optimized water/methanol mixture to provide the pure product TOI1 in 55% yield. The isolated compound was thoroughly characterized by spectroscopic techniques.

Having successfully establish reaction conditions for the synthesis of TOI1, we then focused our efforts on the generation of regioisomers TOI2 and TOI3-rev, respectively. Initial attempts to oxidize the methyl group at the benzylic position in starting materials 2 and 3 to provide the corresponding aldehyde compounds 13 and 14 failed, despite using rigorous reaction conditions of SeO2 or alternative strong oxidizing agents (e.g., MnO2 and oxone). Thus, we considered the critical role of the electronic effects of the nitrogen atoms on this cyclic substrate, and then we revised our synthetic strategy by a) tethering an alkene functional group on the aromatic ring and b) then conducting the oxidation of the benzylic group to afford the aldehyde product. Towards this end, we performed a Suzuki coupling reaction between boronic acid 15 with chloro compound 2 (Scheme 2). To the best of our knowledge, there is no report of a Suzuki coupling reaction using boronic acid 15 in the literature. However, we generated this required boronic acid from the corresponding methyl propiolate [38].

The biochemical evaluation of the proposed inhibitors TOI1, TOI2, TOI3-rev, and TOI4 was performed to experimentally determine the potency of HDAC8 inhibition. The obtained results, as shown in Table 1, aligned with the predicted computational studies. In a homogeneous fluorogenic assay, the HDAC activity is quenched with a fluorescent dye that is tethered to an acetyllysine-containing peptide. If the acetyl moiety of the fluorophore is enzymatically hydrolyzed by HDAC8, it will produce a strongly fluorescent signal at 360 nm. Table 1 shows the percentage of HDAC8 inhibition at 100, 10, and 1 M concentration of the designed inhibitors and panobinostat. All compounds displayed HDAC8 inhibition as predicted by our computational studies [12]. Complete inhibition of HDAC8 was observed for panobinostat at 100 M or 10 M concentration, while only 89% inhibition was recorded at 1 M concentration. The in vitro IC50 values for panobinostat against HDAC8 have been shown to be 277 nM [11]. In vivo studies measuring the IC50 values of panobinostat have also been performed, however, since panobinostat is a pan-DAC inhibitor it has been difficult for researchers to specifically correlate its IC50 value for HDAC8 in a physiological system [40]. Theoretical studies predicted TOI1 as the most potent inhibitor over TOI2, TOI3-rev, and TOI4. TOI1 was shown to be the most potent inhibitor designed in this study producing 100% inhibition at a 100 M, 90% inhibition at a 10 M, and 44% at a 1 M concentration. At a 100 M concentration, TOI2, TOI3-rev and TOI4 inhibited 79%, 89%, and 93% of HDAC8 activity, respectively. Strong inhibition was seen at a 10 M concentration for all inhibitors. At a concentration of 1 M all inhibitors showed less inhibition of HDAC8 than panobinostat. It was noted that TOI2 and TOI3-rev produced similar inhibition results against HDAC8. Both of these inhibitors have pyrimidine rings in the core; thus, the data suggests that while this ring structure did produce inhibition, it was not as effective as the inclusion of a pyrazine ring. This finding is consistent with our computational data which demonstrated that TOI1 would slightly outperform TOI2 and TOI3-rev.

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