Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth

Abstract

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.

Figure 1

Kill kinetics for 29 against replicating and nonreplicating M. tuberculosis. Bacterial viability in the presence of compound was determined by cfu over 28 days (A) under replicating conditions and (B) under nonreplicating conditions. The dashed lines represent the upper and lower limits of detection.

Figure 2

Decreased MmpL3 expression results in hypersensitivity to the spirocycle series. Removal of anhydrotetracycline (atc) results in transcriptional repression of mmpL3. Growth in the presence of a negative control ethambutol (A) and a positive control SQ109 (B) as well as representative series compound, 4 (C) and 23 (D) are recorded relative to DMSO-treated samples. Data are representative of two independent experiments.

Figure 3

Efficacy in a mouse model of acute TB infection. BALB/c mice were infected with M. tuberculosis H37Rv via a low-dose aerosol exposure. Treatment was started 7 days post-aerosol and continued for 12 consecutive days. Drugs were administered once daily by oral gavage at 100 mg/kg (3) and 300 mg/kg (29).

Scheme 1. General Synthetic Routes for the Synthesis of Compounds 1–21

Reagents and conditions: (i) BH₃.THF, THF, 100 °C, and 1 h; (ii) nBuLi, THF, −78 °C, 1 h, then tert-butyl 4-oxopiperidine-1-carboxylate −78 °C—rt, and 18 h; (iii) MeSO₂Cl, Et₃N, DCM, reflux, and 1.5 h; (iv) TFA, DCM, and 18 h; (v) 1-[(4-methoxyphenyl)methyl]piperidin-4-one, MeSO₃H, PhMe, reflux, Dean–Stark, and 18 h; (vi) 1-chloroethyl carbonochloridate, DCM, 0 °C then MeOH, reflux, and 1 h; (vii) DIPEA, DMSO, rt, and 18 h; (viii) AcOH or EtOH, reflux, and 2 h; (ix) R2B(OH)₂, Cu(OAc)₂, pyridine, DCM, and 2 h; (x) Et₂O, rt, 1 h, then NaOMe, MeOH, rt, 18 h, then H₂SO₄, MeOH, reflux, and 48 h; (xi) PPh₃, DIAD, THF, MeOH, rt, and 18 h; (xii) DIBAL, DCM, −78 °C—rt, then MnO₂, DCM, rt, and 48 h; (xiii) AcOH, DCM, then NaBH(OAc)₃, and 18 h; (xiv) R4B(OH)₂, Cu(OAc)₂, pyridine, DCM, rt, and 3–18 h.

Scheme 2. General Synthetic Routes for the Synthesis of Compounds 22–30 and 35

Reagents and conditions: (i) EtOH, AcOH, reflux, and 1–18 h; (ii) spiro[isochromane-1,4′-piperidine], AcOH, DCM, then NaBH(OAc)₃, and 18 h; (iii) EtOH, H₂O, 35 °C, and 18 h; (iv) SOCl₂, pyridine, 50 °C, 2 h then ethanolamine, DCM, rt, 3 h, then SOCl₂, rt, and 18 h; (v) NaH, THF, 0 °C, and 3 h; (vi) DIBAL, DCM, −78 °C—rt, then MnO₂, DCM, rt, and 48 h; (vii) spiro[isochromane-1,4′-piperidine], AcOH, DCM, then NaBH(OAc)₃, 18 h; and (viii) 3 M HCl, 100 °C, and 18 h.

Scheme 3. General Synthetic Routes for the Synthesis of Compounds 31–34

Reagents and conditions: (i) POCl₃, DMF, 100 °C, and 2 h; (ii) R3H, K₂CO₃, DMF, microwave, 120 °C, and 1 h; (iii) pTsOH, MeOH, microwave, 120 °C, and 1 h; (iv) R3I, K₂CO₃, DMF, 0 °C—rt, and 2 h; (v) DIBAL, DCM, −78 °C—rt, then MnO₂, DCM, rt, and 48 h; (vi) AcOH, DCM, then NaBH(OAc)₃, and 18 h; and (vii) N-formylsaccharin, Pd(OAc)₂, Xantphos, KF, DMF, 80 °C, and 18 h then H₂O.