SelSA, Selenium Analogs of SAHA As Potent Histone Deacetylase Inhibitors

Several etiological pathways like genetic, epigenetic, and cytogenetic process play a role in the transformation of normal cells to cancer cells. Regulation of transcription factors, as well as modification of chromatin structures (also known as epigenetic regulation) are critical regulatory steps in the cellular response to trauma and inflammation. ^{1, 2} The reversible acetylation of the side chain of specific histone lysine residues by histone deacetylase (HDACs) and histone acetyl transferase (HATs) is one of the most widely studied chromatin modifications. ³ The HDACs can be divided into two families, (1) the Zn⁺²-dependent HDAC family composed of Class I, Class IIa/b, and Class IV and (2) Zn⁺²-independent NAD-dependent class III enzymes. Class I comprises HDAC1, 2, 3, and 8 which are located in the nuclei of the cells. Class IIa/b contains HDAC4, 5, 6, 7, 9, and 10 primarily localized to the cytoplasm. HDAC11 has a conserved domain in the catalytic region of both Class I and Class II enzymes and it has been grouped to Class IV. Class III HDACs are NAD+-dependent deacetylase with non-histone protein as substrate and have been linked to regulation of caloric utilization of cells. ^{4, 5}

HDAC catalyzes deacetylation of ε-amino group in lysines located near the N-terminal of core histone proteins. ^{6, 7} Specific HDAC activity results in hypoacetylation that is associated with subsequent gene silencing, whereas histone hyperacetylation is associated with unwinding of the DNA and transcriptional activation. ^{8, 9} Studies have shown that inhibition of HDAC elicits anticancer effects in several tumor cells by inhibition of cell growth, and induction of terminal differentiation in tumor cells. This has led to the development of HDAC inhibitors for anti-cancer chemotherapy ¹⁰ mainly directed at Zn²⁺-dependent Class I and II HDACs. Structural-activity relationships (SAR) and reviews of different HDAC inhibitors and analogs have been previously published. ^{2, 11–20} Most of these HDAC inhibitors were designed to have a hydrophobic cap that blocks the entrance to the active site, a polar site, and a hydroxamic acid type zinc-binding active site. ¹⁵

Hydroxamic acids are the broadest class of inhibitors with high affinity for HDAC that has been shown to inhibit both Class I and Class II HDACs. Trichostatin A (TSA) belonging to hydroximates is one of the first natural product possess HDAC inhibitory activity and it is widely used as reference compound. ^{21– 23} TSA blocks proliferation, inhibits cell growth, decreases differentiation in ovarian cancer cells, and suppresses growth of pancreatic adenocarcinoma cells at naonmolar concentrations. ^{24, 25} A second generation HDAC inhibitor, Suberoylanilide hydroxamic acid (SAHA) inhibits secretion of TNF-α, IL-1β, IL-6, and IFN-γ in LPS-induced PBMC cells, inhibits there in vivo production as shown in an LPS induced animal model, as well as prevents formation of tumors in mice and rats. ^{26 – 28} SAHA (Vorinostat) is under clinical trials in both hematological and nonhematological malignancies and is approved for treatment of cutaneous T-cell lymphoma. ^{29, 30} Another class of HDAC inhibitors includes a group of synthetic benzamide derivatives such as MS-275 and CI-994 that are effective inhibitors of solid tumors in a murine model, but did not inhibit HDAC directly. ³¹ This class of compounds inhibits both histone deacetylation and cellular proliferation at the G1-S phase. ³² MS-275 and CCI-994 are undergoing clinical trials. ^{33, 34} Another class, a cyclic peptide natural product include Trapoxin, having epoxide group may act by chemically modifying an active site nucleophile with the epoxide group and forming hydrogen bonds through the ketone. ³⁵ Trapoxin is supposed to trap HDACs through the reaction of the epoxide moiety with the zinc cation or an amino acid in the binding pocket. ^{36 – 38} FK228 (also referred as depsipeptide) is a natural product derived from Chromobacterium violaceum, inhibit HDACs at nanomolar concentrations, and exhibits potent antitumor activity. ³⁹ The mechanism of action of FK228 is unknown; however, according to one hypothesis, a disulfide bond is reduced inside the cell or organism and the mercaptobutyenyl residue then fits inside the HDAC catalytic pocket. ³⁵ FK228 is currently undergoing evaluation in clinical trials. ^{40 – 42} Thus HDACs have been suggested to be a potential targets for anticancer drug development and many other non-malignant diseases such as rheumatoid arthritis and osteoporosis. ⁴³ Therefore, demand for new HDAC inhibitors having strong inhibitory action is increasing. In this communication, we report the syntheses of two newly developed selenium-based HDAC inhibitors (namely, SelSA-1 and SelSA-2) and evaluation of their HDAC activity compare to SAHA and TSA, a known HDAC inhibitors.

Several structurally diverse HDAC inhibitors have been reported and many of them belong to the family of hydroxamic acid derivatives. ⁴⁴ Metabolic instability and pharmacokinetic problems such as glucuronide and sulfate conjugates that could result in short half life of the drug in biological systems. Therefore, several new non-hydroxamic HDAC inhibitors have been reported in the literature. ⁹ However, they have a reduced potency compared to hydroxamate inhibitors. A cyclic peptide HDAC inhibitor FK228 is a potent HDAC inhibitor having a disulfide bond in the molecule that is reduced in the cellular environment releasing the free thiol analog as the active species. ⁴⁵ Therefore, in a similar manner we hypothesize that in the cellular environment, the selenium dimer (SelSA-1) and selenocyanide (SelSA-2) will be reduced and free SeH will be release as the active species that will bind to the acetate group and cause the potent HDAC inhibitory activities. Based on our hypothesis, we have synthesized two selenium compounds. Synthesis of SelSA-1 was accomplished as illustrated in Scheme 1.

Scheme 1.

Synthesis of SelSA-1. Reagents and conditions: (a) TEA, RT, 18h, 84%. (b) KOH, Adogen, Se powder, NH₂NH₂, RT, 60%.

The amino group of aniline was acetylated with the appropriate acid chloride to give the amide 1 in quantitative yield. ^{46, 47} Amide 1 was treated with selenium powder under basic condition in a biphase system using phase transfer catalyst to give the desired dimer SelSA-1 in 60% yield ⁴⁸.

Synthesis of SelSA-2 was accomplished by reacting of amide 1 with KSeCN in CH₃CN as shown in Scheme 2. ⁴⁹ Acetonitrile was the solvent of choice for the reaction to avoid side products as reported earlier with other solvent. ⁵⁰

Scheme 2.

Synthesis of SelSA-2. Reagents and conditions: (a) KSeCN, CH₃CN, RT, 18h, 64%.

HeLa cell nuclear extract was used as the source of the HDAC activity with HDAC1 and HDAC2 being the major contributors.^{51, 52} We found that SelSA-1 and SelSA-2 inhibited HDAC activity approximately by 81% and 95% at 50nM, respectively. The inhibitory activity of SelSA-2 was statistically significant higher than TSA (90%) at the same concentrations (50nM). ⁵³

Both SelSA-2 and TSA showed higher inhibitory activity than SAHA (77%) at 50nM which was not different than SelSA-1 at 50nM. Based on these findings, we assessed the IC₅₀ of SelSA-2, TSA and SAHA. The IC₅₀ concentrations of SelSA-2, TSA and SAHA were 8.9, 28.9 and 196nM, respectively.

In summary, we have developed novel selenium based HDAC inhibitors and evaluated their inhibitory effect on HDACs. Both selenium compounds are superior in their inhibitory effect (more than 20 fold) on HDAC than the known inhibitor, SAHA. Indeed, SAHA is currently in clinical use for lymphoma and under active evaluation for other indications. ⁵ However, these selenium- based small molecules may play an important role in the fight against cancer. Currently, we are pursuing the structural activity relationship (SAR) studies with analogs of SelSA as HDAC inhibitors.

Figure 1.

Structures of HDAC inhibitors

Figure 2.

% Inhibition of HDAC in HeLa Nuclear Extract

Figure 3.

IC50 value of HDAC inhibitors