Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure-Activity Relationship and Target Identification Studies

Abstract

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc₁ complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.

Figure 1

New TB drug development candidates identified from whole cell screening.

Figure 2

Compound 1 and biology triage data.

Figure 3

SAR exploration strategy.

Scheme 1. General Route for the Synthesis of 6-Aminopyrimidine-4-carboxamides and Alternative Cores

Reagents and conditions: (i) amine, DIPEA, HATU, DMF, 25 °C; (ii) amine, i-PrOH, DIPEA, 100 °C.

Scheme 2. Synthetic Routes Used To Access Core Modifications

Reagents and conditions: (i) 1-(4-fluorophenyl)-N-methylmethanamine, Et₃N, dioxane, 60 °C; (ii) aniline, Mo(CO)₆, Et₃N, TBAC, toluene, tetraglyme, 150 °C; (iii) (a) POCl₃, 110 °C; (b) aniline, Et₃N; (iv) 4-fluorobenzylamine, Et₃N, dioxane, microwave, 125 °C; (v) 4-fluorobenzylamine, H₂O/EtOH (1.5:1), 25 °C; (vi) LiOH, MeOH; (vii) aniline, HATU, DMF, Et₃N; (viii) 4-fluorobenzaldehyde, NaBH₄, EtOH; (ix) MeI, K₂CO₃, DMF.

Scheme 3. Synthesis of 27 and 28

Reagents and conditions: (i) (a) KOH, EtOH; (b) HCl; (ii) SOCl₂, DMF; (iii) 1-(4-fluorophenyl)-N-methylmethanamine, K₂CO₃, DMF; (iv) LiOH, H₂O:/THF (2:1), 25 °C; (v) HATU, DIPEA, DMF, 25 °C; (vi) (COCl)₂, EtOAc, aniline; (vii) 1-(4-chlorophenyl)-N-methylmethanamine, i-PrOH, DIPEA, 100 °C.

Scheme 4. General Route for the Synthesis of 6-Aminopyrimidine-4-carboxamides and Selected Core Modifications

Reagents and conditions: (i) R-CH₂NHCH₃, THF, 0 °C; (ii) 10% Pd/C, H₂, MeOH; (iii) LiOH, MeOH; (iv) amine, DIPEA, HATU, DMF; (v) LiOH, THF/H₂O (1:1); (vi) NH₃ or cyclopropylamine, i-PrOH, Et₃N, 100 °C.

Scheme 5. Synthetic Schemes Used To Access Reverse Amide and Amide Isosteres

Reagents and conditions: (i) BzCl, DIPEA, THF, rt to 50 °C; (ii) 1-(4-fluorophenyl)-N-methylmethanamine, i-PrOH, Et₃N, 100 °C; (iii) HATU, Et₃N, N,O-dimethylhydroxylamine, DMF, rt; (iv) (a) TMS-CF₃, toluene, 20% CsF; (b) TBAF, H₂O, 50 °C; (v) 4-fluoroaniline, NaCNBH₃, Et₃N, TiCl₄, MeOH, DCM (50:50), N₂; (vi) benzonitrile, K₂CO₃, BuOH, 120 °C.

Figure 4

Summary of SAR.

Figure 5

Affinity chemoproteomics revealed BCG_3193 and BCG_3827, novel TB targets with unknown function, as targets of the 6-dialkylaminopyrimidine carboxamides. (A) BCG_3193 and BCG_3827 show reduced binding to 6-dialkylaminopyrimidine carboxamide analogue beads due to compound 40 binding, but no effect on these proteins was seen by the inactive compound. Shown are relative protein amounts captured by the beads in a log 2 scale. (B) Compound structures used for the affinity chemoproteomics experiments. (C) The apparent dissociation constant of compound 40 for BCG_3193 was determined in two different experiments as 0.61 μM and 0.69 μM and for BCG_3827 as 2.1 μM and 3.8 μM.