Design and synthesis of a library of C8-substituted sulfamidoadenosines to probe bacterial permeability

Abstract

Gram-negative bacteria pose a major challenge in antibiotic drug discovery because their cell envelope presents a permeability barrier that affords high intrinsic resistance to small-molecule drugs. The identification of correlations between chemical structure and Gram-negative permeability would thus enable development of predictive tools to facilitate antibiotic discovery. Toward this end, have advanced a library design paradigm in which various chemical scaffolds are functionalized at different regioisomeric positions using a uniform reagent set. This design enables decoupling of scaffold, regiochemistry, and substituent effects upon Gram-negative permeability of these molecules. Building upon our recent synthesis of a library of C2-substituted sulfamidoadenosines, we have now developed an efficient synthetic route to an analogous library of regioisomeric C8-substituted congeners. The C8 library samples a region of antibiotic-relevant chemical space that is similar to that addressed by the C2 library, but distinct from that sampled by a library of analogously substituted oxazolidinones. Selected molecules were tested for accumulation in Escherichia coli in a pilot analysis, setting the stage for full comparative evaluation of these libraries in the future.

Keywords: Antibiotic resistance; Diversity-oriented synthesis; Drug discovery; Gram-negative bacteria; Nucleoside analogue.

Figure 1.

Retrosynthetic analysis of AMSN-C8 library (1).

Figure 2.

Synthesis of 8-Br-AMSN scaffold. DBU = 1,8-diazabicyclo[5.4.0]-undec-7-ene; TFA = trifluoroacetic acid.

Figure 3.

Principal component analysis of enumerated AMSN-C8 library (blue shaded diamonds) compared to regioisomeric AMSN-C2 library (purple shaded squares) and a related oxazolidinone library (green shaded triangles), with a reference set of 120 antibacterial drugs representing 11 classes. See Supplementary Figure S5 for PC1 vs PC3 plots.

Figure 4.

Synthesis of AMSN-C2 library members 13 via Suzuki–Miyaura cross-coupling reactions with boronate reagents B01–B28. Analogues 13j–q were not in the original planned library, but were added to enhance structural diversity further; reagent numbers B14–B21 were not used in this library. a) Boronic acid cross-coupling conditions: bromide scaffold 11 (1.0 equiv), Ar boronic acid (2.0 equiv), Pd(PPh₃)₄ (10 mol%), K₂CO₃ (4.0 equiv), 1,4-dioxane/H₂O (2:1), 90 °C, 16 h; b) Pinacol boronate cross-coupling conditions: bromide scaffold 11 (1.0 equiv), Ar boronate ester (2.0 equiv), Pd(dppf)Cl₂ (30 mol %), Na₂CO₃ (4.0 equiv), 1,4-dioxane/H₂O (2:1), 90 °C, 16 h; c) Intermediate cross-coupling product could not be separated from unreacted starting material and was carried forward directly to deprotection step.

Figure 5.

Synthesis of AMSN-C2 library members 14 via Sonogashira cross-coupling reactions with alkyne reagents Y01–Y31. Analogues 14l–o were not in the original planned library, but 14l–n were added to test the hypothesis that a primary amine would enhance compound accumulation of this scaffold in E. coli, and 14o was added to enhance structural diversity further. Alkyne cross-coupling conditions: bromide scaffold 11 (1.0 equiv), alkyne (2.0 equiv), Pd(PPh₃)₂Cl₂ (10 mol%), CuI (20 mol%), Et₃N/CH₃CN (1:1), 55 °C, 16 h. a) Intermediate cross-coupling product could not be separated from unreacted starting material and was carried forward directly to deprotection step. b) Coupled as corresponding Boc-protected amine, which was cleaved in the final acidic deprotection.

Figure 6.

Venn diagram of successful cross-coupling reactions of the uniform reagent set in AMSN-C8, AMSN-C2, and oxazolidinone (Oxa) libraries. ^aIncludes one Sonogashira reagent that also coupled successfully in the Oxa library, but the product could not be deprotected. ^bIncludes one Sonogashira reagent that also coupled successfully in the AMSN-C2 library, but the product could not be deprotected. See Supplementary Figure S6 for complete details.

Figure 7.

Pilot analysis of compound accumulation in wild-type E. coli (SPE-MS/MS analysis of lysates); see Supporting Information for complete details. Ciprofloxacin and tetracycline are positive controls; vancomycin and daptomycin are negative controls. Concentrations normalized to ciprofloxacin accumulation at 80 μM; mean of n = 4 independent biological replicates; significance at 80 μM relative to vancomycin (bottom) and daptomycin (top) assessed by Welch’s ANOVA with Dunnett’s T3 multiple comparison test: **p<0.01, ns = not significant.