Properly Substituted Benzimidazoles as a New Promising Class of Nicotinate Phosphoribosyltransferase (NAPRT) Modulators

Abstract

The prevention of nicotinamide adenine dinucleotide (NAD) biosynthesis is considered an attractive therapeutic approach against cancer, considering that tumor cells are characterized by an increased need for NAD to fuel their reprogrammed metabolism. On the other hand, the decline of NAD is a hallmark of some pathological conditions, including neurodegeneration and metabolic diseases, and boosting NAD biosynthesis has proven to be of therapeutic relevance. Therefore, targeting the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), which regulate NAD biosynthesis from nicotinamide (NAM) and nicotinic acid (NA), respectively, is considered a promising strategy to modulate intracellular NAD pool. While potent NAMPT inhibitors and activators have been developed, the search for NAPRT modulators is still in its infancy. In this work, we report on the identification of a new class of NAPRT modulators bearing the 1,2-dimethylbenzimidazole scaffold properly substituted in position 5. In particular, compounds 24, 31, and 32 emerged as the first NAPRT activators reported so far, while 18 behaved as a noncompetitive inhibitor toward NA (K_i = 338 µM) and a mixed inhibitor toward phosphoribosyl pyrophosphate (PRPP) (K_i = 134 µM). From in vitro pharmacokinetic studies, compound 18 showed an overall good ADME profile. To rationalize the obtained results, docking studies were performed on the NAPRT structure. Moreover, a preliminary pharmacophore model was built to shed light on the shift from inhibitors to activators.

Keywords: NAD biosynthesis; NAPRT activators; NAPRT inhibitors; nicotinate phosphoribosyltransferase; pharmacokinetics; substituted benzimidazoles.

Figure 1

NAD biosynthesis in mammalian cells. NR, nicotinamide riboside; NRK, nicotinamide riboside kinase; NAM, nicotinamide; NAMPT, nicotinamide phosphoribosyltransferase; NMN, nicotinamide mononucleotide; NMNAT, nicotinamide mononucleotide adenylyltransferase; NAD, nicotinamide adenine dinucleotide; NA, nicotinic acid; NAPRT, nicotinate phosphoribosyltransferase; NAMN, nicotinic acid mononucleotide; NAAD, nicotinic acid adenine dinucleotide; NADSYN, nicotinamide adenine dinucleotide synthetase; NAR, nicotinic acid riboside; Trp, tryptophan; QA, quinolinic acid; QPRT, quinolinate phosphoribosyltransferase; PRPP, phosphoribosyl pyrophosphate; PPi, inorganic pyrophosphate. Known inhibitors (in red) or activators (in green) of NAD metabolism are highlighted in the figure.

Figure 2

Structures and K_i or IC₅₀ values of the NAPRT inhibitors reported so far. ^a Taken from reference [14]. ^b Taken from reference [15]. ^c Taken from reference [16]. ^d Taken from reference [17].

Figure 3

Chemical structure of the new 1,2-dimethylbenzimidazole derivatives 17–33.

Scheme 1

Reagents: (a) (CH₃CO)₂O, CHCl₃; (b) (i) toluene, Δ; (ii) NaBH₃CN, ClCH₂CH₂Cl; (c) HCHO, NaBH₃CN, ClCH₂CH₂Cl; (d) 2.5N HCl in MeOH.

Scheme 2

Reagents: (a) (i) toluene, Δ; (ii) NaBH₃CN, ClCH₂CH₂Cl; (b) HCHO, NaBH₃CN, ClCH₂CH₂Cl; (c) H₂/Raney Nickel, CH₃OH; (d) 2.5N HCl in MeOH; (e) (CH₃CO)₂O, CHCl₃.

Scheme 3

Reagents: (a) EDCI·HCl, HOBt, N-methylmorpholine, DMF; (b) BH₃ (CH₃)₂S, THF, Δ; (c) H₂/Raney Nickel, CH₃OH; (d) NaH, CH₃I, DMF; (e) (CH₃CO)₂O, CHCl₃.

Figure 4

Inhibition of NAPRT by 18. Double-reciprocal plots and slope replots (in the inserts) of the inhibition exerted by 18 at (A) NA concentrations ranging from 25 µM to 100 µM, at 0.4 mM PRPP, and (B) PRPP concentrations ranging from 25 µM to 100 µM, at 0.4 mM NA. Each point is the mean of duplicate determinations.

Figure 5

(A) Binding modes of inhibitors 17 and 18. Human dimeric NAPRT protein is represented in ribbon. The two chains are differently labeled and colored (chain A in yellow and chain B in cyan). The two inhibitors (17, in green; 18, in pink) and the residues that interact with the ligands are represented as sticks. H-bonds are indicated as red dotted lines. A modeled PRPP substrate is shown as black wires. (B) A simple pharmacophore model of the inhibitory scaffold. The blobs are color-coded: yellow for hydrophobic; white for aromatic; blue for H-bond donor; red for H-bond acceptor.