Design and synthesis of a library of C2-substituted sulfamidoadenosines to probe bacterial permeability

Abstract

Antibiotic resistance is a major threat to public health, and Gram-negative bacteria pose a particular challenge due to their combination of a low permeability cell envelope and efflux pumps. Our limited understanding of the chemical rules for overcoming these barriers represents a major obstacle in antibacterial drug discovery. Several recent efforts to address this problem have involved screening compound libraries for accumulation in bacteria in order to understand the structural properties required for Gram-negative permeability. Toward this end, we used cheminformatic analysis to design a library of sulfamidoadenosines (AMSN) having diverse substituents at the adenine C2 position. An efficient synthetic route was developed with installation of a uniform cross-coupling reagent set using Sonogashira and Suzuki reactions of a C2-iodide. The potential utility of these compounds was demonstrated by pilot analysis of selected analogues for accumulation in Escherichia coli.

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

Figure 1.

Synthesis of 2-iodo-AMSN scaffolds 6 and 7.

Figure 2.

Histograms of selected physicochemical properties of AMSN-C2 (blue, left axis) and oxazolidinone (green, left axis) libraries enumerated with a uniform cross-coupling reagent set, in comparison to 120 antibiotic drugs (red line, right axis). See Supporting Information for histograms of all 20 parameters analyzed.

Figure 3.

Principal component analysis of AMSN-C2 (blue diamonds) and oxazolidinone (green inverted triangles) libraries enumerated with the uniform cross-coupling reagent set, compared to 120 antibiotic drugs representing 11 classes, using 20 structural and physicochemical parameters. Complete PC1 vs PC2 plot (upper left) is expanded to highlight the region occupied by the synthetic libraries (right), with parameter loadings (lower left) as shown. See Supplementary Figure S2 for additional plots and see Supporting Information for complete PCA data.

Figure 4.

Suzuki–Miyaura cross-coupling reactions. a) Cross-coupling conditions: 6 (1.0 equiv), Ar boronic acid (2.0 equiv), Pd₂dba₃ (10 mol%), XPhos (22 mol%), 0.5 M aq K₃PO₄/THF (1:1), rt, 24–48 h; b) Cross-coupling conditions: 6 (1.0 equiv), Ar boronate ester (2.0 equiv), Pd(dppf)Cl₂ (30 mol%), K₂CO₃ (4.0 equiv), 1,4-dioxane/H₂O (2:1), 40–50 °C, 1–2 h; c) Initially isolated as TFA salt, then converted to HCl salt by two cycles of treatment with HCl in EtOH/CH₃CN/H₂O and lyophilization (¹³C-NMR).

Figure 5.

Sonogashira cross-coupling reactions. a) Coupled as the corresponding Boc-protected amine, which was cleaved in the final deprotection; b) Deprotected 2-iodo-AMSN scaffold 7 was used in the coupling reaction; c) Initially isolated as TFA salt, then converted to HCl salt by two cycles of treatment with HCl in EtOH/CH₃CN/H₂O and lyophilization (¹³C-NMR); d) Originally selected reagent was the free acid, which did not couple effectively; e) Analogue 9u was not part of the original library design and was added to test the hypothesis that a primary amine would enhance compound accumulation of this scaffold in E. coli.

Figure 6.

Venn diagram of successful couplings from the uniform cross-coupling reagent set across AMSN-C2 and oxazolidinone (Oxa) synthetic libraries. ^aOne coupling listed only under the AMSN-C2 library was also achieved in the Oxa library, but could not be deprotected (reagent Y12). ^bOne coupling listed under the Oxa library was also achieved in the AMSN-C2 library, but only in protected form (reagent Y17). See Supplementary Figure S3 for identities of reagents in each category.

Figure 7.

Pilot analysis of compound accumulation in wild-type E. coli (SPE-MS/MS analysis of lysates). Ciprofloxacin and tetracycline are positive controls; vancomycin and daptomycin are negative controls. Concentrations normalized to ciprofloxacin (80 μM); mean of n = 3 independent biological replicates; significance at 80 μM relative to ciprofloxacin assessed by ANOVA: ***p < 0.001, **p < 0.01, *p < 0.05.