Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity

Abstract

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 μM) and in vitro ADMET profiles.

Scheme 1. General Route to 1,2,4-Oxadiazole Carboxamides

Reagents and conditions: (i) triethylamine, methyl 2-chloro-2-oxo-acetate, DCM, 0–40 °C; (ii) triethylamine, amine, MeOH, 60 °C; (iii) methyl iodide, K₂CO₃, DMF, rt; (iv) T3P, amine, triethylamine, DMF, rt, or HATU, amine, triethylamine, DMF, 0 °C to rt, or ammonia HOBt, EDCI HCl, DIPEA, THF, rt; (v) hydroxylamine hydrochloride, DIPEA, ethanol, 80 °C.

Scheme 2. Route to Introduce a Substituted Pyridyl

Reagents and conditions: (i) potassium (2-((tert-butoxycarbonyl)amino)ethyl)trifluoroborate, Pd(dppf)Cl₂, Cs₂CO₃, toluene (3 mL), and H₂O, 25–100 °C; (ii) TFA/DCM, 0–25 °C; (iii) ethyl 3-(3,4-dimethoxyphenyl)-1,2,4-oxadiazole-5-carboxylate, triethylamine, MeOH, 60 °C.

Scheme 3. Route to Linker Chain Substituted with Nitrile

Reagents and conditions: (i) HATU, DIPEA, NH₄Cl, DCM, 25 °C; (ii) CO, Pd(dppf)Cl₂, triethylamine, EtOH, 80 °C; (iii) LiOH, EtOH, water, 25 °C; (iv) HATU, DIPEA, amine, DCM or DMF, 20−25 °C; (v) TFA, DCM, 20–25 °C; (vi) ethyl 3-(3,4-dimethoxyphenyl)-1,2,4-oxadiazole-5-carboxylate, triethylamine, MeOH, 60 °C; (vii) triethylamine, TFAA, THF, N₂, 0–25 °C or 0–20 °C; (viii) lithium 3-(3,4-dimethoxyphenyl)-1,2,4-oxadiazole-5-carboxylic acid, HATU, DIPEA, DMF 0–20 °C; (ix) LiOH·H₂O, MeOH, water, 50 °C.

Scheme 4. Alternative Route to Linker Chain Substituted with Nitrile

Reagents and conditions: (i) amine, triethylamine, DCM, 0 °C, or DMAP, triethylamine, amine, N₂ 0 °C to rt; (ii) 2-(benzhydrylideneamino)acetonitrile, NaOH, benzyltriethylammonium chloride, DCM, rt or 2-(benzhydrylideneamino)acetonitrile, NaOH, THF, 20 °C; (iii) HCl, water, dioxane, 20 °C or HCl, THF rt; (iv) sodium 3-(3,4-dimethoxyphenyl)-1,2,4-oxadiazole-5-carboxylate, HATU, DIPEA, DMF, N₂, rt; (v) ethyl 3-[3-[(1-hydroxycyclopropyl)methoxy]-4- methoxy-phenyl]-1,2,4-oxadiazole-5-carboxylate, triethylamine, MeOH, 40 °C; (vi) NaOH, EtOH, rt; (vii) ethyl 2-chloro-2-oxo-acetate, DIPEA, THF, 0–80 °C; (viii) 1-tetrahydropyran-2-yloxycyclopropyl)methanol, PPh₃, DEAD, THF, 0–25 °C.

Scheme 5. Routes to Amide Modifications

Reagents and conditions: (i) NaH, methyl iodide, DMF rt; (ii) 2-phenylethanamine, triethylamine, DCM, 40 °C.

Scheme 6. Route to Dimethoxyphenyl Ring Modifications

Reagents and conditions: (i) ethyl 2-chloro-2-oxo-acetate, DIPEA, THF, 0–80 °C; (ii) [4-(2-aminoethyl)phenyl](azetidin-1-yl), triethylamine, MeOH, 60 °C; (iii) HATU, DIPEA, amine, DCM, 25 °C; (iv) TFA/DCM 25 °C.

Scheme 7. Routes to Methoxy Modifications

Reagents and conditions: (i) bromide, K₂CO₃, DMF, 40–60 °C, or bromide, KI, CsCO₃, DMSO, 140 °C; (ii) hydroxylamine hydrochloride, DIPEA, EtOH, 70–80 °C; (iii) ethyl 2-chloro-2-oxo-acetate, DIPEA, THF, 0–60 °C; (iv) 4-(2-aminoethyl)phenyl]-(3-methoxyazetidin-1-yl)methanone hydrochloride, triethylamine, MeOH, 60–70 °C; (v) 1-tetrahydropyran-2-yloxycyclopropyl)methanol, PPh₃, DEAD, THF, 0–25 °C; (vi) HATU, DIPEA, amine, DCM, 25 °C; (vii) HCl/dioxane.

Scheme 8. Route to Isoxazole Core with a Reverse Amide

Reagents and conditions: (i) ethylene glycol, 4-methylbenzenesulfonic acid hydrate, toluene, 110 °C; (ii) hydroxylamine hydrochloride, 7 M NH₃/methanol, quinolin-8-ol, MeOH, 70 °C, HCl, EtOH, 120 °C; (iii) 3-(3,4-dimethoxyphenyl)propanoic acid, thionyl chloride, DCM, 40 °C.

Scheme 9. Route to Alternative 1,2,4-Oxadiazole Isomer

Reagents and conditions: (i) ethyl 2-(hydroxyamino)-2-imino-acetate, triethylamine, DCM, 0 °C to rt; (ii) phenethylamine, triethylamine, MeOH, 60 °C.

Scheme 10. Route to Furan Core

Reagents and conditions: (i) (3,4-dimethoxyphenyl)boronic acid, K₃PO₄, Pd(dtbpf)Cl₂, triethylamine, DCM, N₂ 80 °C; (ii) NaOH, water, EtOH, 80 °C; (iii) 2-phenylethanamine, HATU, triethylamine, DMF, rt.

Scheme 11. Route to Phenyl Core

Reagents and conditions: (i) HATU, triethylamine, 2-phenylethanamine, DMF, rt; (ii) (3,4-dimethoxyphenyl)boronic acid, K₃PO₄, Pd(dtbpf)Cl₂, triethylamine, DCM, N₂ 80 °C.

Scheme 12. Routes to Triazole and 1,3,4-Oxadiazole Cores

Reagents and conditions: (i) ethyl 2-amino-2-thioxo-acetate, 180 °C; (ii) AcOH, 100 °C; (iii) amine, triethylamine, MeOH, 60 °C; (iv) ethyl 2-chloro-2-oxoacetate, triethylamine, DCM, 0–25 °C; (v) pTsOH, DCM, 0–25 °C; (vi) HATU, DIPEA, amine, DCM, 25 °C; (vii) HCl/dioxane or TFA/DCM 25 °C.

Figure 1

Dose response curves for compounds 50 and 105 tested against a tet-regulated Pks13 strain. Growth in the presence of anhydrotetracycline (ATc) results in modest transcriptional overexpression of pks13, while removal of ATc results in transcriptional repression of pks13 expression. Growth in the presence of negative control (rifampicin), positive control (TAM16), 50, and 105 is shown relative to DMSO-treated samples. Data are representative of two independent experiments.

Figure 2

Novel binding mode of 50 to M. tuberculosis Pks13 thioesterase domain. Overall view of the structure of the Pks13-TE-50 complex (PDB ID 8Q0T) represented as cartoon. An enlarged L-shaped binding pocket, shown as a gray tunnel, is the binding site for 50 (yellow sticks) (A). 2F_o – F_c map (blue), contoured at 1σ around 50 (B). Close-up views of the interactions formed by binding of 50 show the dimethoxyphenyl and oxadiazole rings bound in the active site (C, D, and E). Residues and side chains within local proximity to the ligand are shown as sticks, while hydrogen bonds are represented by black dashed lines, with water molecules shown as red spheres.

Figure 3

Differences in binding modes between TAM16 and 50 within the Pks13 TE domain, showing that structural rearrangement is required to accommodate different ligands. Cartoon representation of the relative orientations of 50 (yellow) and TAM16 (cyan) binding within the TE domain of Pks13 (A). Superimposition of Pks13-TAM16 (PDB ID 5V3Y: cyan and purple) with the Pks13-50 (PDB ID 8Q0T: yellow and green) complexes, showing that significant structural rearrangement is required to allow the binding of TAM16 compared to 50 (B). 50 enters the tunnel, allowing it closer access to the catalytic triad (Ser1533, Asp1560, and His1699) in comparison to TAM16. Phe1670 “flip-out” from the 50-bound structure (green) is required for TAM16 binding (purple) (C). Rearrangement of the side chain of Arg1563 is also required from the Pks13-TAM16 (purple) to that of Pks13-50 (green), allowing the ligand to fully enter the tunnel, and a stacking interaction forms with the phenyl ring of the bound ligand (D).