Spiropyrimidinetrione DNA Gyrase Inhibitors with Potent and Selective Antituberculosis Activity

Abstract

New antibiotics with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new spiropyrimidinetriones (SPTs), DNA gyrase inhibitors having activity against drug-resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB. While the clinical candidate zoliflodacin has progressed to phase 3 trials for the treatment of gonorrhea, compounds herein demonstrated higher inhibitory potency against Mtb DNA gyrase (e.g., compound 42 with IC₅₀ = 2.0) and lower Mtb minimum inhibitor concentrations (0.49 μM for 42). Notably, 42 and analogues showed selective Mtb activity relative to representative Gram-positive and Gram-negative bacteria. DNA gyrase inhibition was shown to involve stabilization of double-cleaved DNA, while on-target activity was supported by hypersensitivity against a gyrA hypomorph. Finally, a docking model for SPTs with Mtb DNA gyrase was developed, and a structural hypothesis was built for structure-activity relationship expansion.

Figure 1.

Known inhibitors of gyrase and Topoisomerase IV - compound class in parentheses.

Figure 2.

(A) A hybrid illustration of the double-stranded DNA break on complementary strands four bases apart with each break creating a FQ/SPT binding site, one showing QPT-1 and the other moxifloxacin. (B) Energy minimized structure of QPT-1 (yellow carbons) translocated into the Mtb gyrase binding site.

Figure 3.

Model of 5 (yellow carbons) docked into Mtb gyrase making the same contacts as the modelled QPT-1 and showing an additional H-bond contact between the methyloxadiazole and R482.

Figure 4.

SAR explorations.

Figure 5.

(A) Model of 10 (yellow) with the dihydroxypyrrolidine forming H-bond interactions with the sidechain functionality of T500 and R482; (B) Model of 42 (yellow) with the CN forming H-bond with the guanidine of R128 and the pyrrolidinone forming H-bonds with the backbone NH and hydroxyl of T500.

Figure 6.

Compound MIC versus gyrase inhibitory potency.

Figure 7.

Stabilization of doubly cleaved DNA complex for gyrase by 23 and moxifloxacin each at 100 μM. Lanes from left to right are 1) negatively supercoiled DNA control, 2) Mtb gyrase + DNA with no drug, 3) Mtb gyrase + DNA + moxifloxacin, 4) Mtb gyrase + DNA + 23, 5) Linear DNA standard. Nick = nicked DNA (single-stranded DNA break), Lin = linear DNA (double-stranded DNA break), (−)SC = negatively supercoiled pBR2322 DNA.

Scheme 1.

Synthesis of 1 via tertiary amino effect reaction (T-reaction) − chiral enablement

Scheme 2.

Synthesis of Compounds 8–33. Reagents and conditions: (a) For cyclic amines: DBU, CH₃CN, 90 °C, 16 h, 30–95%; For alcohols: NaH, DMF, 0–30°C, 1 h, 24–95%; (b) EtOH/2M HCl (10:1), 80 °C, 16 h, 3–65%, (c) LiOH, 1,4-dioxane: H₂O (3:1), 30°C, 1 h, 36%.

Scheme 3.

Synthesis of Compounds 34–38. Reagents and conditions: (a) amine [meso-2,6-dimethylpiperazine (for 50a) or meso-2,6-dimethylpiperazin-1-ol hydrochloride S3 (for 50b)], K₂CO₃, CH₃CN, 80°C (MW), 1–2 h or Et₃N, DMSO, 90–110°C, 2 h, 41–86%; (b) (Ac)₂O, pyridine, CH₂Cl₂, 25°C, 24 h, 80% (for 50c) or BrCH₂CN, K₂CO₃, acetone, 25°C, 16 h, 68% (for 50d) (c) barbituric acid, AcOH:H₂O (4:1) or EtOH, 80°C, 2 h, 2–23%; (d) Br-CN, K₂CO₃, acetone, 25°C, 16 h, 14%.

Scheme 4.

Synthesis of compounds 39–43. Reagents and conditions: (a) meso-2,6-dimethylpiperazine-1-carbonitrile hydrochloride (S5) for 52a or meso-3,5-dimethylpiperidine for 52b, K₂CO₃, CH₃CN, 80–110 °C, 1–16 h, 26–81%; (b) ethylene glycol, p-TSA, toluene, 130 °C, 16 h, 57–87%; (c) 1H-1,2,4-triazol for 54a, [(S)-3-hydroxypyrrolidine for 54b, (R)-3-hydroxypyrrolidine for 54c, [(S)-5-(hydroxymethyl)pyrrolidin-2-one for 54d and 54e, DBU in CH₃CN (for N-linked analogues) or NaH in DMF (for O-linked analogues), 30–110 °C, 1–16 h, 69–95%; (d) barbituric acid, EtOH:2M HCl (10:1), 80 °C, 16 h, 10–27%.

Scheme 5:

Synthesis of compounds 44 and 45. Reagents and conditions: (a) (2R,6R)-2,6-dimethylpiperazine dihydrochloride, K₂CO₃, CH₃CN:water (20:1), 90 °C, 13 h, 92%; (b) Br-CN, K₂CO₃, acetone, 27 °C, 9 h, 77%; (c) barbituric acid, EtOH, 110 °C, 20 h, 4–10%; (d) (meso-3S,4r,5R)-3,5-dimethylpiperidin-4-ol hydrochloride, K₂CO₃, CH₃CN:water (10:1), 90 °C, 1 h, 89%; (e) barbituric acid, EtOH, 80 °C, 16 h, 10–18%.