Axinellamines as broad-spectrum antibacterial agents: scalable synthesis and biology

Abstract

Antibiotic-resistant bacteria present an ongoing challenge to both chemists and biologists as they seek novel compounds and modes of action to out-maneuver continually evolving resistance pathways, especially against Gram-negative strains. The dimeric pyrrole-imidazole alkaloids represent a unique marine natural product class with diverse primary biological activity and chemical architecture. This full account traces the strategy used to develop a second-generation route to key spirocycle 9, culminating in a practical synthesis of the axinellamines and enabling their discovery as broad-spectrum antibacterial agents, with promising activity against both Gram-positive and Gram-negative bacteria. While their detailed mode of antibacterial action remains unclear, the axinellamines appear to cause secondary membrane destabilization and impart an aberrant cellular morphology consistent with the inhibition of normal septum formation. This study serves as a rare example of a natural product initially reported to be devoid of biological activity surfacing as an active antibacterial agent with an intriguing mode of action.

Figure 1

Antibiotic drug discovery. ^aExamples of drug class not necessarily introduced on the exact date depicted (modified from ref (11)).

Figure 2

Representative plots showing the outer-membrane permeabilization of E. coli K-12 MG1655 containing pBR322 as a function of [4], μg/mL, using the cleavage of nitrocefin by periplasmic β-lactamase as the reporter. Melittin was used as the positive control. Other runs are shown in Supporting Information, Figure SI-1.

Figure 3

Cellular morphology of E. coli K-12 MG1655 at 1000× magnification: (A) prior to treatment, (B) treated with DMSO at 20 min, (C,D) treated with Ax A (4) at 20 min. The scale bar (panel A; top right) is equal to 5 μm.

Scheme 1. (A, top) First- and (B, bottom) Second-Generation Retrosynthetic Analysis of Spiroaminoketone (9)

Scheme 2. Validation and Initial Feasibility Studies

Reagents and conditions: (a) i. propargyl alcohol (1.0 equiv), Co₂(CO)₈ (1.0 equiv), CH₂Cl₂, rt, 2 h; ii. (E)-1,4-dimethoxybut-2-ene (3.0 equiv), NMO (6.0 equiv), CH₂Cl₂, rt, 12 h. (b) PPh₃ (1.5 equiv), DIAD (1.5 equiv), bis-Boc-guanidine (1.1 equiv), THF, rt, 2 h. (c) NaBH₄ (4.0 equiv), CeCl₃·7H₂O (1.0 equiv), CH₃OH, 0 °C, 12 h. (d) AcCl (1.2 equiv), DMAP (10 mol%), CH₂Cl₂, rt, 12 h. (e) SOCl₂ (1.4 equiv), CH₂Cl₂, 0 °C, 1 h. (f) Zn (5 equiv), H₂O/NH₄Cl.

Scheme 3. Chemoselective Functionalization Strategy to Chlorospirocyclization Precursor 15

Scheme 4. Synthetic Route Finalization and Chlorospirocyclization of Advanced Allylic Guanidine Intermediate 15

Reagents and conditions: (a) i. BSA (7.0 equiv), CH₂Cl₂, 40 °C, 3h; ii. 45 (1.0 equiv), diol-N-oxide (3.0 equiv), CH₂Cl₂, rt, 12 h. (b) CeCl₃ (1.0 equiv), NaBH₄ (4.0 equiv), MeOH, 0 °C, 12 h. (c) PPh₃ (3.4 equiv), NCS (3.4 equiv), THF, rt, 12 h. (d) 2,2,2-Trifluoro-N-(2-oxoethyl)acetamide (10 equiv), zinc (16 equiv), indium (1.9 equiv), THF, NH₄Cl (6% aqueous), rt, 3 h. (e) NaN₃ (10 equiv), DMF, 85 °C, 16 h. (f) i. TFA (50% in CH₂Cl₂), rt, 23 h then TEA (5.0 equiv), 50 (1.5 equiv), rt, 12 h; ii. tBuOCl (2.0 equiv), CH₂Cl₂, 0 °C, 1 h.

Scheme 5. TfNH₂ Effect on Model Chlorospirocyclization

Addition of 1.0 equiv of TfNH₂ to 53 produced 55 in quantitative yield.

Scheme 6. Invention of Reactive yet Stable CBMG

CBBG decomposition was observed when dissolving in acetone. Chlorinated products were detected from the mixture of CBBG and 2-methylallyl-4-bromobenzonate.

Scheme 7. Scalability of Final Synthetic Route and Reactivity of 2-Aminoimidazole toward DMDO Oxidation

Reagents and conditions: (a) i. TFA/CH₂Cl₂ (1:1, 0.2M), rt, 12 h; ii. CH₂Cl₂, TEA (5.0 equiv), N,N′-bis-Boc-N″-triflylguanidine (1.5 equiv). (b) i. TfNH₂ (0.25 equiv), CH₂Cl₂, tBuOCl (2.0 equiv), 0 °C, 30 min; ii. DMP (1.2 equiv), rt, 12 h. (c) H₂O/TFA (1:1, 0.2 M), 70 °C, 36 h. (d) i. AcOH; ii. NCNH₂ (25 equiv), 0.2 M NaOH (pH ∼5.5), 70 °C, 4 h. (e) i. TFA/CH₂Cl₂ (5:95, 0.1 M), DMDO (1.25 equiv), 0 °C, 1 h; ii. TFA/CH₂Cl₂ (1:1, 0.1 M), rt, 12 h. (f) H₂O/TFA (9:1, 0.07 M), silver(II) picolinate (2.5 equiv), rt, 2 h. (g) i. H₂O/TFA (19:1, 0.1 M), PtO₂ (0.1 equiv), H₂(g); ii. 2,3-dibromo-5-trichloroacetylpyrrole (5.0 equiv), DIPEA (4.5 equiv).