Oxidative Reactivities of 2-Furylquinolines: Ubiquitous Scaffolds in Common High-Throughput Screening Libraries

Abstract

High-throughput screening (HTS) was employed to discover APOBEC3G inhibitors, and multiple 2-furylquinolines (e.g., 1) were found. Dose-response assays with 1 from the HTS sample, as well as commercial material, yielded similar confirmatory results. Interestingly, freshly synthesized and DMSO-solubilized 1 was inactive. Repeated screening of the DMSO aliquot of synthesized 1 revealed increasing APOBEC3G inhibitory activity with age, suggesting that 1 decomposes into an active inhibitor. Laboratory aging of 1 followed by analysis revealed that 1 undergoes oxidative decomposition in air, resulting from a [4 + 2] cycloaddition between the furan of 1 and (1)O2. The resulting endoperoxide then undergoes additional transformations, highlighted by Baeyer-Villager rearrangements, to deliver lactam, carboxylic acid, and aldehyde products. The endoperoxide also undergoes hydrolytic opening followed by further transformations to a bis-enone. Eight structurally related analogues from HTS libraries were similarly reactive. This study constitutes a cautionary tale to validate 2-furylquinolines for structure and stability prior to chemical optimization campaigns.

Figure 1

Chemical structure of 1 and the frequency with which the 2-(5-methylfuran-2-yl)quinoline-4-carbonyl (red) and the 2-(furan-2-yl)quinoline-4-carbonyl (blue) substructures occur in the University of Minnesota and the NIH MLPCN libraries (521,185 total compounds when combined). The boxed text represents the overall frequency of these chemotypes in each individual library. Overlap between the University of Minnesota and NIH MLPCN libraries is <10%.

Figure 2

Dose response assays for A3G inhibition by freshly synthesized and solubilized 1 at 0, 3, 21, 38 and 72 days. Data indicates that the stock solution of 1 gains A3G inhibitory activity over time. Assays were performed in triplicate and deaminase activity was quantified as previously reported. Means ± standard deviations are indicated.

Figure 3

Analytical HPLC analyses of 10 mM DMSO stocks of 1. (A) Fresh stock solutions of 1 are >99% pure as determined by HPLC (See SI: Section III). (B) After 21 days of gentle shaking in ambient atmosphere at 25 °C, HPLC analysis shows evidence of decomposition through the appearance of multiple new peaks. (C) Compounds 6, 7a-b, and 8 can be assigned to the three prominent peaks in the decomposition mixutre. These compounds were isolated by HPLC and characterized by LC-MS, ¹H NMR, HRMS, and co-injection of the isolated standards with aged samples of 1 (t = 21 d) (See SI: Sections II, V-VII, and IX for characterization data). (D) DMSO stocks of 1 aged under inert conditions (N₂) exhibit drastically reduced decomposition. (E, F) DMSO stocks of 1 aged in the dark or in the presence of NaN₃ exhibit no decomposition.

Figure 4

(A) Proposed mechanistic pathways for oxygen-mediated decomposition of 1. Intermediates highlighted in yellow exhibit m/z [M+H]⁺ = 446.1, which is observed in the LC-MS analysis.

Figure 5

(A) The decomposition of 1 can be achieved in short reactions times (9h vs. 21d) by irradiating DMSO stocks of 1 with visible light (300W) in an oxygen saturated atmosphere. (B) Decomposition readily occurs in the absense of photosensitizer. (C) No decomposition occurs under an N₂ atmosphere. (D) Decomposition in the presence of [¹⁸O]-O₂. Rose bengal was not added to reactions B – D. LCMS traces can be found in the SI (Sections XI-XV).

Scheme 1

Reagents and conditions: (a) 5-acetyl-2-methylfuran, KOH, EtOH, 65 °C to reflux, 79%; (b) EDCI·HCl, HOBt, NMM, DMF, 22%.