SAR study of piperidine derivatives as inhibitors of 1,4-dihydroxy-2-naphthoate isoprenyltransferase (MenA) from Mycobacterium tuberculosis

Abstract

The electron transport chain (ETC) in the cell membrane consists of a series of redox complexes that transfer electrons from electron donors to acceptors and couples this electron transfer with the transfer of protons (H⁺) across a membrane. This process generates proton motive force which is used to produce ATP and a myriad of other functions and is essential for the long-term survival of Mycobacterium tuberculosis (Mtb), the causative organism of tuberculosis (TB), under the hypoxic conditions present within infected granulomas. Menaquinone (MK), an important carrier molecule within the mycobacterial ETC, is synthesized de novo by a cluster of enzymes known as the classic/canonical MK biosynthetic pathway. MenA (1,4-dihydroxy-2-naphthoate prenyltransferase), the antepenultimate enzyme in this pathway, is a verified target for TB therapy. In this study, we explored structure-activity relationships of a previously discovered MenA inhibitor scaffold, seeking to improve potency and drug disposition properties. Focusing our campaign upon three molecular regions, we identified two novel inhibitors with potent activity against MenA and Mtb (IC₅₀ = 13-22 μM, GIC₅₀ = 8-10 μM). These analogs also displayed substantially improved pharmacokinetic parameters and potent synergy with other ETC-targeting agents, achieving nearly complete sterilization of Mtb in combination therapy within two weeks in vivo. These new inhibitors of MK biosynthesis present a promising new strategy to curb the continued spread of TB.

Keywords: 1,4-dihydroxy-2-naphthoate prenyltransferase; MenA; MenA inhibitors; Menaquinone; Mtb; Mycobacterium tuberculosis; Piperidine derivatives; SAR.

Figure 1:

Promising recent Mtb ETC inhibitors and their molecular targets. *NDH-1 - proton-translocating (type I–NDH-1) Nuo complex, NDH-2 - two nonproton-pumping (type II) Ndh and NdhA complexes, SDH – succinate dehydrogenase, III-IV complex – Cytochrome bc1:aa3 complex (complex III – QcrCAB - cytochrome c reductases C, A and B, IV – QcrCDE – cytochrome c oxidase C, D and E), Cyt bd – cytochrome bd menaquinol oxidase.

Figure 2:

Summary of menaquinone biosynthesis in Mtb. Conversion of phosphoenol phosphate (PEP) and D-erythrose 4-phosphate (E4P) into shikimate and chorismate ultimately leads to synthesis of menaquinone via classical or alternative patway catalysed by MenA-H or MnqA-G enzyme, respectively.

Figure 3:

Function of MenA in MK synthetic pathway.

Figure 4:

MenA inhibitors developed by two research groups, Kurosu et al. (A) and Narayanasamy et al. (B). Lead compound 1 is divided in three fragments, i.e Western site (red), Eastern site (green), Center site (blue). Also illustrated, the two additional modification sites, i.e. oxgyen substitution (black arrow) and methylene bridge (purple arrow) (C); LORA: low oxygen recovery assay; MABA: Microplate alamar blue assay; SI value: in vitro activity/toxicity ratio.

Figure 5:

SAR study for compound 1

Figure 6:

Graphical representation of relationship between antibacterial activity against Mtb and inhibitory potency against MenA.

Figure 7:

Assessing MenA inhibitors against methyltransferase MenG. A) Final steps of the canonical MK biosynthesis pathway. Products of each enzyme are nearly identical, and previously reported MenG inhibitor DG70 shares structural similarities with our MenA inhibitors; B) MenA inhibitors fail to antagonize MenG. TLC autoradiogram showing inhibition of MK8 synthesis by DG70, but no inhibiton of MenG by analogs from this study. Lane 1: DMSO (negative control), Lane 2: DG70 (positive control), Lane 3: 2, Lane 4: 11, Lane 5: 37, Lane 6: 14, Lane 7: 30, Lane 8: 23, Lane 9: 3, 10: 19; C) Inhibition graph of 8 most promising MenA inhibitiors against MenG.

Figure 8:

Bactericidal synergy assay. Synergy study of compound 11 in combination with another known ETC inhibitor, such as imidazopyridine Q203 against Mtb H37Rv-LP strain. Compound 11 showed synergistic kill with Q203. Column 1 includes controls:1A-1D = niclosamide positive control; 1E-1H = DMSO negative control; Compound 2 (across the plate); Key: + : Weak additive/synergistic effect; 1 shade difference from most potent compound alone; ++ : Moderate additive/synergistic effect; 2 shade difference from most potent compound alone; +++: Strong additive/synergistic effect; ≥ 3 shade difference from most potent compound alone; n/a: below LOD (complete kill by one compound alone);

Scheme 1:

Synthetic route for compound 1. Conditions: a) iPrMgCl, THF, −15 °C, 1 h, then 4-chloro-N-methoxy-N-methylbenzamide at −75 °C -> 0 °C, 3 h; b) HBr (48% in H₂O), CH₃COOH, reflux, 38 h; c) Boc-piperidine MeOH, DIAD, TPP, THF, rt, 21 h; d) 50% TFA in DCM, rt, 2 h; e) 4-(methyl(propyl)amino)benzaldehyde, NaBH(OAc)₃, DCM, rt, 30 h.

Scheme 2:

Synthetic route for Western and Eastern site piperidine derivatives. Conditions: f) Boc₂O, Na₂CO₃, THF/H₂O, rt, 18 h; g) Ts-Cl, Et₃N, 0 °C -> rt, 18h; h) Ms-Cl, Et₃N, 0 °C -> rt, 18h; i) Ph-OH, Cs₂CO₃, ACN, reflux, 3h; j) TFA/DCM, rt, 1h; k) aldehyde, NaBH(OAc)₃, DCM, 0 °C -> rt, 16h; l) aldehyde, Et₃SiH, [IrCl(cod)]₂, toluene, 110 °C, 16 h; m) carboxylic acid, EDC·HCl, Et₃N, DCM; n) carbonyl chloride, Et₃N, DCM.

Scheme 4.1.

1Synthetic scheme of benzaldehydes

Scheme 4:

Synthesis of 4-benzyl-1,3-oxazolidin-2-one fragment of compounds 54 and 57.