Esterase-Sensitive Prodrugs of a Potent Bisubstrate Inhibitor of Nicotinamide N-Methyltransferase (NNMT) Display Cellular Activity

Abstract

A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC₅₀ value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.

Keywords: NNMT; cell permeability; cellular activity; esterase; inhibition; prodrug.

Figure 1

(A) Prodrug strategy of compound GYZ-319. The carboxylic acid can be masked as an ester and the amine can be masked as an amide using the esterase-sensitive trimethyl-lock (TML). (B) The mechanism of the trimethyl-lock cleavage. Deacetylation by esterases results in spontaneous lactonization, releasing the free amine.

Scheme 1

Synthesis of the prodrug forms of amino acid building blocks 5b–f and 9a. Reagents and conditions: (a) RI, DMF, K₂CO₃, rt, overnight (65–79%); (b) 10% Pd/C, MeOH, overnight (82–90%); (c) CH₃NHOCH₃·HCl, BOP, Et₃N, CH₂Cl₂, rt, 2 h (77–83%); (d) DIBAL-H (1 M in hexanes), THF, −78 °C, assumed quant; (e) HCl (4N in dioxanes), 0 °C to rt, 2.25 h; (f) TML acid 7, BOP, Et₃N, CH₂Cl₂, rt, overnight, 88% over two steps.

Scheme 2

Synthesis of prodrugs 12b–f and 14a–f. Reagents and conditions: (a) aldehydes 5b–f or 9a, NaBH(OAc)₃, AcOH, DCE, rt, overnight (34–73%); (b) TFA, CH₂Cl₂, H₂O, rt, 2 h (70–93%); (c) TFA, CH₂Cl₂, rt, 2 h; (d) TML acid 7, BOP, Et₃N, CH2Cl₂, rt, 2 h (77–83%).

Figure 2

Esterase-mediated hydrolysis bisubstrate inhibitor prodrugs. (A) Hydrolysis of isopropyl ester prodrug 12e and (B) hydrolysis of isopropyl-TML dual prodrug 14e. The data show clean conversion of the prodrugs to the parent compound 17u. For the dual prodrug 14e, conversion of the TML happens first followed by the hydrolysis of the isopropyl ester moiety.

Figure 3

Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay on HSC-2 human oral cancer (grey), T24 human bladder cancer (blue), and A549 human lung cancer cells (green) with compounds 6-methylaminonicotinamide (6-MANA), parent compound GYZ-319, and prodrugs 12e and 14e. Compounds were incubated for 24, 48, and 72 h at 1, 10, and 100 µM concentrations. As no effects were observed at 1 and 10 µM, only results at 100 µM are depicted. Results are presented as mean values with standard deviation from three independent experiments performed in triplicate.

Figure 4

Concentration of 1-methyl nicotinamide (MNA) in A549 lung carcinoma cells after 24-h treatment with 0.1–100 µM of compounds: (A) 6-MANA; (B) GYZ-319; (C) 12e; and (D) 14e. n = 6, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to untreated control (untr).