Improvement of the antimicrobial potency, pharmacokinetic and pharmacodynamic properties of albicidin by incorporation of nitrogen atoms

Abstract

The worrisome development and spread of multidrug-resistant bacteria demands new antibacterial agents with strong bioactivities particularly against Gram-negative bacteria. Albicidins were recently structurally characterized as highly active antibacterial natural products from the bacterium Xanthomonas albilineans. Albicidin, which effectively targets the bacterial DNA-gyrase, is a lipophilic hexapeptide mostly consisting of para amino benzoic acid units and only one α-amino acid. In this study, we report on the design and synthesis of new albicidins, containing N-atoms on each of the 5 different phenyl rings. We systematically introduced N-atoms into the aromatic backbone to monitor intramolecular H-bonds and for one derivative correlated them with a significant enhancement of the antibacterial activity and activity spectrum, particularly also towards Gram-positive bacteria. In parallel we conducted DFT calculations to find the most stable conformation of each derivative. A drastic angle-change was observed for the lead compound and shows a preferred planarity through H-bonding with the introduced N-atom at the D-fragment of albicidin. Finally, we went to the next level and conducted the first in vivo experiments with an albicidin analogue. Our lead compound was evaluated in two different mouse experiments: In the first we show a promising PK profile and the absence of toxicity and in the second very good efficiency and reduction of the bacterial titre in an E. coli infection model with FQ-resistant clinically relevant strains. These results qualify albicidins as active antibacterial substances with the potential to be developed as a drug for treatment of infections caused by Gram-negative and Gram-positive bacteria.

Fig. 1. Molecular structure of albicidin (1) and AzaHis-albicidin (2).

Fig. 2. Molecular structure of albicidin derivatives 2,11 & 14 (ref. 28) and aimed derivatives 3–10, 12, 13 and 15.

Scheme 1. Synthesis of the key building blocks for A-derivatives. Conditions: a LiCl, DBU, MeCN, 25 °C, b aq. KOH, THF, 25 °C, 71% (18a), 80% (18b).

Scheme 2. Synthesis of AB-fragments with modified A or B building block. Conditions: a19, 18a–b, SOCl₂, 95 °C, then (20c or 20d to 19) or (20 to 18a or 18b) and NEt₃ or Na₂CO₃ (for 20d), THF, 0 °C → 25 °C; bi product of 19 and 20c or 20d, KOH, THF, 25 °C, then Boc₂O, DMAP, THF, 25 °C, 75% (21c, 3 steps), 59% (21d, 3 steps); bii product of 20 and 19a or 19b, TFA/CH₂Cl₂ (1 : 1), 0 °C, 57% (21a), 68% (21b), c [(21a or 21b), SOCl₂, 95 °C] or [(21c or 21d), DIPEA, DMAP, EDC, THF], then pentachlorophenol (PCP), THF, 25 °C, 51% (22a), 51% (22b), 12% (22c), 18% (22d).

Scheme 3. Synthesis of the EF-building block for F-variations. Conditions: a SOCl₂, 95 °C, then amine 25a (80%) or 25b (79%), NEt₃, THF, 0 °C → 25 °C; bi Zn, EtOH/AcOH (4 : 1), 0 °C → 25 °C, 25a (30%), 25b (96%), 26 (32%); bii Zn, CHCl₃/AcOH (9 : 1), 0 °C → 25 °C, 25a (96%).

Scheme 4. Synthesis of the EF-building block for E-variations. Conditions: ai27a, SOCl₂, 95 °C, then 28a, NEt₃, THF, 0 °C → 25 °C, 89% (29a); aii27b, SOCl₂, 95 °C, then 28b, NEt₃, THF, 0 °C → 25 °C, 11% (29b), 25% (30); b Zn, AcOH/EtOH (1 : 4), 0 °C, 92% (33a); c (COCl)₂, THF, cat. DMF, 0 °C → 25 °C, then 28b, NEt₃, THF, 0 °C → 25 °C, 89%; d HCl 4 N in dioxane; 0 °C; e TFA/CH₂Cl₂ (1 : 4), 0 °C; f TBAF, THF, 120 °C, 79% (33b).

Scheme 5. Synthesis of final derivatives by assembling of the building blocks. Conditions: a (SOCl₂, 90 °C 27a then 35b, DIPEA) or [27c and (25a, 25b, 33a, 33b or 35a), NEt₃], THF, 0 °C → 25 °C; b Zn, EtOH/AcOH (4 : 1) or CHCl₃/AcOH (9 : 1), 0 °C, 2 steps: 82% (36a), 54% (36b), 41% (36c), 32% (36d), 76% (36e), 75% (36f); c EEDQ, 38, THF, 25 °C; d Pd(PPh₃)₄, morpholine, THF or Pd/C, H₂, EtOH/THF/MeOH (1 : 1 : 1); e 4N HCl, dioxane, 25 °C, 3 steps: 65% (37a), 66% (37b), 20% (37c), 53% (37d), 51% (37e), 37% (37f); f (37a–f, 39) or (37g, 22a–b) or (first 22a or 22b, TFA/CH₂Cl₂ (1 : 1), 0 °C, then 37g), NEt₃, DMF, 25 °C, g 3N KOH, DMF, 25 °C, 2–3 steps: 15% (3), 25% (4), 8% (5), 12% (6), 23% (7), 16% (9), 12% (10), 5% (12), 8% (13), 15% (15).

Fig. 3. D–F fragments of derivatives 2 (a), 7–14 (b–i) and A–B fragments of derivatives 2 (j), 5 (k), 6 (l), extracted from optimized geometries of the corresponding full albicidin derivatives.

Fig. 4. Blood concentration of 2 and 7 after 50 mg kg⁻¹ i.v. administration (a). Murine septicaemia model for the i.v. administration of 7 to CD-1 mice infected with a fluoroquinolone-resistant (FQR) E. coli isolate infected (b).