Structure-activity relationships of potentiators of the antibiotic activity of clarithromycin against Escherichia coli

Abstract

Several studies that have identified agents that potentiate the antimicrobial activity of antibiotics, but there are limited insights into their structure-activity relationships (SAR). The SAR associated with select N-alkylaryl amide derivatives of ornithine was performed to establish those structural features that were associated with potentiation of the antimicrobial activity of clarithromycin against E. coli ATCC 25922. The data indicate that the N-propyl derivative was slightly more active in reducing the effective MIC of clarithromycin against E. coli ATCC 25922. In addition, the S-enantiomer of compound 9 was somewhat more potent than the R-enantiomer in potentiating clarithromycin activity. No significant enhancement in potentiation activity was observed with the conversion of these secondary amides to their N-methyl tertiary amides. Formation of the N-methyl or N,N-dimethyl derivatives of the primary amine of 9 was associated with the loss of potentiation activity. Conversion of this primary amine to a guanidine was also not associated with an increase in potentiation activity. Among the isomeric diamino pentamides, 15 potentiated the antibacterial activity of clarithromycin to the greatest extent. In addition to these amide derivatives, the desoxy derivatives 16 and 18 were the more potent potentiators within this triamine series. The relative location of the primary amines, as indicated by the relative differences in the potentiation observed with 16 compared to 14, appears to be a critical factor in determining potentiation activity. Cell-based membrane permeabilization and efflux inhibition studies in E. coli ATCC 25922 suggest that the potentiation of clarithromycin activity by 16 reflects its ability to inhibit efflux pump activity and to a lesser extent its actions as a permeabilizer of the outer leaflet of the outer cell membrane.

Keywords: Antimicrobial; Clarithromycin; E. coli; Potentiators; Synergy.

Figure 1.

Diaminopentamide derivatives that act as potentiators of levofloxacin activity against Pseudomonas aeruginosa. MPC₈ denotes minimum potentiation concentration, and is defined as the minimum concentration of potentiator required to decrease the minimum inhibitory concentration (MIC) of the antibiotic by 8-fold.

Figure 2.

N-[Bis(arylalkyl)methyl]carboxamides and N-(methyl)-N-[bis(arylalkyl)methyl]carboxamides of (S)-2,5-diaminopentanoic acid selected for synthesis and biological evaluation.

Figure 3.

Membrane permeabilization assays of 16 in E. coli ATCC 25922. Polymyxin B was included as a comparator membrane-targeting agent. Both 16 and polymyxin B were used at concentrations ranging from 1/32X to 1/2X the minimum inhibitory concentration (MIC), with the MIC values of 16 and polymyxin B being 100 and 1.56 μg/ml, respectively. The compound vehicle was DMSO. (A,B) Impact of E. coli treatment with 16 (A) or polymyxin B (B) on the intensity of the fluorophore N-phenyl-1-naphthylamine (NPN) at a concentration of 10 μM. (C,D) Impact of treatment with 16 (C) or polymyxin B (D) on the percentage of E. coli cells stained with the fluorophore propidium iodide (PI) at a concentration of 50 μM.

Figure 4.

Assay for inhibition of the efflux of the fluorophore Hoechst 33342 (H33342) by 16 in E. coli ATCC 25922. When present, 16 was used at concentrations ranging from 1/32X to 1/2X MIC (3.13 to 50 μg/mL). All reactions were initiated by the addition of 50 mM glucose except where indicated. H33342 was used at a concentration of 10 μM. Compound vehicle was DMSO.

Scheme 1.

General method for the preparation of 1-[bis(arylalkyl)methyl]carboxamides of (S)-2,5-diaminopentanoic acid. Reagents and conditions: (a) Peptide coupling agents – EDC, HOBt, DMF (64%) for 5; PyBrop, Et₃N, DCM, for R = Me [6 (68%), 8 (59%) and 10 (74%)]; PyBoP, Et₃N, CH₂Cl₂ for 7 (58%); BoP, Et₃N, CH₂Cl₂ for 9 [9S (45%), 9R (90%); b) TFA, DCM.

Scheme 2.

Synthesis of the requisite arylalkylamines. Reagents and conditions: a) hydroxylamine, ethanol/pyridine; b) LAH; c)methylamine, glacial acetic acid, NaBH(OAc)₃

Scheme 3.

Preparation of 11 and 12. Reagents and conditions: a) 1,5-diphenylpentan-3-amine, PyBoP, Et₃N, CH₂Cl₂ (77%); b) TFA, CH₂Cl₂ (67%); c) i) benzaldehyde, ethanol, 3Å Molecular sieves, NaBH₃CN, d) 37% formaldehyde, NaBH₃CN (43%) for 11_, 20% MeOH/DCM, 37% formaldehyde, NaBH(OAc)₃ (10 eq) (66 %) for 12; e) (i) 20% Pd(OH)₂/C, H₂, Boc₂O, ethanol, (ii) TFA, DCM for 11 (96%); 20% Pd(OH)₂/C, H₂, ethanol for 12 (97%).

Scheme 4.

Preparation of 13. Reagents and conditions: a) 1,5-diphenylpentan-3-amine, PyBoP, Et₃N, CH₂Cl₂ (92%); b) TFA, CH₂Cl₂ (100%).

Scheme 5.

Reagents and conditions: a) N-methylmorpholine, isobutylchloroformate, DME, ii) NaBH₄, DME/H₂O); b) phthalimide, DIAD, PPh₃, THF; c) hydrazine, Methanol; d) 1,5-diphenylpentan-3-one, NaBH₃CN, MeOH, 5Å molecular sieves, 70 °C; e) (i) 20% Pd(OH)₂/C, H₂, ethanol (ii) Boc₂O, DCM, (iii) TFA, DCM to provide 14 (17%) and 20 (28%):

Scheme 6.

Reagents and conditions: a) N-methylmorpholine, isobutylchloroformate, DME, ii) NaBH₄, DME/H₂O); b) phthalimide, DIAD, PPh₃, THF; c) hydrazine, Methanol; d) Boc₂O, Et₃N, CH₂Cl₂ (52%) for Intermediate C, CbzCl, Et₃N, CH₂Cl₂ (65%) for Intermediate D; e) 20% Pd(OH)₂/C, H₂, ethanol for Intermediate C, TFA, CH₂Cl₂ (67%) for Intermediate D: f)) 1,5-diphenylpentan-3-one, NaBH₃CN, MeOH, 5Å molecular sieves, 70 °C; (46–47%) g) 20% Pd(OH)₂/C, H₂, ethanol for 18 (98%), TFA, DCM for 22 (100%).

Scheme 7.

Synthesis of 15, 16 and 19. Reagents and Conditions: a) 1,5-diphenylpentan-3-amine, EDC, HOBt, 2,6-lutidine, DMF (73% for 15 and 16), (76% for 19); b) 20% Pd(OH)₂/C, H₂, ethanol (51%); c) (i) ethyl chloroformate, THF, triethylamine, (ii) TMSCH₂N₂, acetonitrile (64%); d) anhydrous methanol, silver benzoate, Et₃N (60%); e) LiOH.H₂O, THF, water (87%); f) (i) 20% Pd(OH)₂/C, Boc₂O, H₂, ethanol (77%), (ii) TFA, DCM (100%); g) (i) BH₃.THF (1M), THF, (ii) Boc₂O, DCM (16%); (iii) TFA, DCM (100%)

Scheme 8.

Synthesis of 17 and 21. Reagents and Conditions: a) 1,5-diphenylpentan-3-amine, EDC, HOBT, 2,6-Lutidine, DMF (n = 1; 92%), (n = 2; 91%),; b) LiBH₄, ethanol, THF (n = 1;65%), (n = 2; 51%); c) (i) DPPA, THF, DBU MsCl, Et₃N, DCM (n = 1) (n = 2, 100%), , d) NaN₃, DMF, 60 °C (n = 1, 65%) (n = 2, 25%); e) 20% Pd(OH)₂/C, H₂, ethanol (100% for 17); f) (i) Boc₂O, DCM (73%), (ii) TFA, DCM (95%)

Scheme 9.

Synthesis of 23 and 24. Reagents and Conditions: a) NaBH(OAc)₃, DCM (11%); b) NaBH₄, THF/ethanol (1.2:1) (54%); c) MsCl, Et₃N, CH₂Cl₂ (92%); d) K₂CO₃, DMF, 100 C (12%)