Synthesis and Antiproliferative Activity of Nitric Oxide-Donor Largazole Prodrugs

Abstract

The marine natural product Largazole is the most potent Class I HDAC inhibitor identified to date. Since its discovery, many research groups have been attracted by the structural complexity and the peculiar anticancer activity, due to its capability to discriminate between tumor cells and normal cells. Herein, we discuss the synthesis and the in vitro biological profile of hybrid analogues of Largazole, as dual HDAC inhibitor and nitric oxide (NO) donors, potentially useful as anticancer agents. In particular, the metabolic stability of the modified thioester moiety of Largazole, bearing the NO-donor function/s, the in vitro release of NO, and the antiproliferative activity in tumor cell lines are presented.

Figure 1

Chemical structures of Largazole, Largazole-SH, and Vorinostat (SAHA).

Figure 2

Chemical structures of Largazole analogues 1 and 2.

Figure 3

Retrosynthetic approach for the synthesis of NO-donor Largazole derivatives 1 and 2.

Scheme 1. Optimized Synthesis of Fragment A

Reagents and conditions: (a) isobutyl chloroformate, N-methyl morpholine, NH₄OH, THF, −20 °C to r.t., 1 h; (b) Lawesson’s reagent, CH₂Cl₂, r.t., 15 h; (c) 3-bromopyruvic acid, THF, 50 °C, 1 h; (d) Boc₂O, 1N NaOH, dioxane, r.t., 40 min; (e) TFAA, diisopropylethyl amine, CH₂Cl₂, 0 °C, 1 h; (f) TEA, MeOH, reflux, 7 h.

Scheme 2. Synthesis of the Mono-nitrate Olefin Derivative 14

Reagents and conditions: (a) AgNO₃, dry CH₃CN, 70 °C, 2 h. (b) Lawesson’s reagent, CH₂Cl₂, 60 °C, 10 min, MW. (c) 4-bromo-1-butene, K₂CO₃, acetone, 0 to 20 °C, 30 min.

Scheme 3. Cross-metathesis Reaction

Reagent and conditions: Grubbs II, dry CH₂Cl₂, 90 °C, 16 h.

Scheme 4. Synthesis of Bis-nitrate Olefin 21

Reagents and conditions: (a) DIC, DMAP, CH₂Cl₂, 0 °C to r.t., 1 h; (b) Br₂, CCl₄, 0 °C to r.t., 10 min; (c) AgNO₃, dry CH₃CN, 70 °C; 24 h. (d) NaOH 2 N, THF/EtOH, 0 °C, 30 min; (e) Lawesson’s reagent, CH₂Cl₂, 60 °C, 15 min, MW; (f) 4-bromo-1-butene, K₂CO₃, THF/MeOH, 0 °C to r.t., 1 h.

Scheme 5. Synthesis of Compound 2

Reagents and conditions: (a) triphenylmethanethiol, DIC, DMAP, dry CH₂Cl₂, 0 °C to r.t., 4 h; (b) TFA, triethylsilane, dry CH₂Cl₂, 0 °C to r.t., 1 h; (c) 4-bromo-1-butene, Grubbs II, dry CH₂Cl₂, 90 °C, 16 h; (d) K₂CO₃, acetone, 0 °C to r.t., 5 h.

Figure 4

NO release assay. Compounds 1 (A) and 2 (B) were incubated at 0.1 mM concentration in 50 mM phosphate buffer (pH = 7.4) at 37 °C, both in the absence and in the presence of l-cysteine (0.5 mM and 5 mM). The yield of NO₂^– is expressed as % with respect to the initial concentration of compound at 1 h, 5 h, and 24 h, respectively. The reported values are the average of three independent experiments.