Structure-activity relationships of antitubercular nitroimidazoles. 3. Exploration of the linker and lipophilic tail of ((s)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (6-amino PA-824)

Abstract

The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 μM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.

Figure 1

Figure 2

Superpositions of PA-824 (1) and some R₃ derivatives that were geometry optimized at the level of B3LYP/61-31G*. In each the head, tail and linker region of 1 is shown with yellow carbons, A the tail is shown in the pseudo-equatorial conformation while in B the tail is shown in the pseudo-axial conformation. A and B show R₃ analogs 32c, 32d, 32f, 41b, and 41f. The inset table shows the difference in enthalpy (ΔH) and Gibbs free energy (ΔG) at 298.15 K calculated for the pseudo-equatorial conformer compared with the pseudo-axial conformer for each compound with a solvent reaction field of cyclohexane. Atoms represented by colors are as follows: green, carbon; dark green, fluorine or chlorine; blue, nitrogen; red, oxygen. Hydrogen atoms are not shown.

Scheme 1

Reaction conditions: i) HCO₂H, Ac₂O, THF, 0 °C, 1h, 55%; ii) R₁COCl, NaH, DMF rt − 70 °C; iii) R₁CHO, NaBH(OAc)₃, MeOH/AcOH; iv) triphosgene, EtNH₂·HCl, Et₃N, THF, 0 °C – rt, 66%.

Scheme 2

Reaction conditions: n-BuLi, ZnCl₂, CuI, THF, −78 °C – rt, 1.5 h, then 4-trifluoromethoxybenzoyl- chloride, rt, 1 h, 40%; ii) diisopropylcarbamyl chloride, DIPEA, 4-trifluoromethoxybenzaldehyde, CH₃CN, reflux, 19 h, 73%; iii) 50 % TFA in water, THF, reflux, 15 h, 82%; iv) H₂, Pd/C, MeOH, 1 atm, 81%; v) MsCl, Et₃N, CH₂Cl₂, rt, 1 h; vi) 7, NaH, THF, rt, 40 h, 15%.; vii) RMgBr, THF, 0 °C – rt when R = Et and nPr; nBuLi, THF, −78 °C – rt when R = nBu; viii) PBr₃, ether, 0 °C – rt; ix) 7, K₂CO₃, DMF, KI, 90 °C.; x) 4-trifluoromethoxybenzaldehyde, neat, 100 °C, 5 min. then TMSCN, 100 °C, 30 min, 50%; xi) EtOH/HCl, −10 °C, 38%; xii) 7, NaCNBH₃, AcOH, EtOH, 5%; xiii) 4-methoxybenzylalcohol, KO^tBu, 60 °C, 2 h, 34%; xiv) PDC, CH₂Cl₂, rt, 24 h, 62%; xv) TBDMSOTf, CH₂Cl₂, rt, 5 min, 86%; xvi) ethylbromoacetate, Zn, CH₂Cl₂, rt, 3 h; xvii) LiAlH4, THF, 0 °C - rt, 2 h, 25% over two steps; xviii) MnO₂, CH₂Cl₂, rt, 6 h, 50%; xix) 7, Ti(iOPr)₄, AcOH, NaBH₃CN, 9%.

Scheme 3

Reaction conditions: i) EtOH/H⁺, 80 °C, 80 %; ii) 1-bromo-2-chloroethane, K₂CO₃, DMF, rt, 15 h, 80%; iii) KO^tBu, THF, 1 h, 20 °C, 78 % over two steps; iv) Et₂Zn, CH₂I₂, ClCH₂CH₂Cl, toluene, rt, 17 h, 51%; v) BH₃-DMS, THF, reflux, 1.5 h; vi) PCC, CH₂Cl₂, rt, 0.5 h, 80 % over two steps; vii) RX, K₂CO₃, DMF, 70 °C, 30 min - 2 h, when R = Bn, cyclopropylmethyl; MOMCl, DIPEA, CH₂Cl₂, rt, 3 h; viii) LiAlH₄, THF, 0 °C – rt, 1 h; ix) 4-fluoronitrobenzene, NaH, DMF, 100 °C, 2 h, 67%; x) H₂, Pd/C, EtOAc, rt, 1.5 h, 87%; xi) NaNO₂, H₃PO₂, 6N HCl, 50 °C, 1 h; xii) NaBH(OAc)₃, AcOH, DMF, 20 h; xiii) 6N HCl, THF, 1h, rt.%.

Scheme 4

Reaction conditions: i) styrene, Pd(OAc)₂, Et₃N, 95 °C, 16 h, 64%; ii) OsO₄, NaIO₄, acetone-water, rt, 16 h, 16%; iii) 7, NaCNBH₃, AcOH, DMF, 20 h; iv) ethylene glycol, p-TSA, benzene, 80 °C, 8 h, 86%; v) Pd(OAc)_2, Cs₂CO₃, Xantphos, dioxane, amine, 90 °C, 8 h; vi) THF, 6 N HCl, 30 min., room temperature; vii) n-BuLi, THF, −78 °C, DMF, 15 min.

Scheme 5

Reaction conditions: i) TBSCl, imidazole, CH₂Cl₂, rt, 60%; ii) s-BuLi, TMEDA, THF, −78 °C, 1 h, then FB(OMe)₂, −78 °C, 30 min followed by alk, H₂O₂, 30 min, 28%; iii) K₂CO₃, MeI, DMF, 70 °C; iv) MOMCl, DIPEA, DMF, rt, 16 h, 82 %; v) TBAF, THF, rt, 1.5 h; vi) PCC, CH₂Cl₂, rt, 1 h; vii) 7, NaCNBH₃, AcOH, DMF; viii) 4-fluoronitrobenzene, NaH, DMF, 100 °C, 2 h, 30 %; ix) Fe/NH₄Cl, EtOAc-water, reflux, 1.5 h; x) NaNO₂, H₃PO₂, 6N HCl, 50 °C, 1 h, 52 % over two steps; xi) ethylene glycol, p-TSA, benzene, 80 °C, 8 h, 72 %; xii) Pd(OAc)_2, Cs₂CO₃, Xantphos, dioxane, amine, 90 °C, 8 h; xiii) THF, 6 N HCl, 30 min., rt; xiv) sec-BuLi, MeOCOCl, THF, −78 °C – rt, 3 h.

Scheme 6

Reaction conditions: i) NaH, DMF, −78 °C – rt; ii) 6N HCl, THF, rt, 4 h.