Design, Synthesis and Biological Evaluation of Non-Covalent, Ampc Beta-Lactamases Inhibitors

Design, synthesis and biological evaluation of non-covalent, AmpC Beta-Lactamases inhibitors

Filippo Genovese · Sandra Lazzari · Ettore Venturi · Luca Costantino · Jesus Blazquez · Claudia Ibacache-Quiroga · Donatella Tondi · Maria Paola Costi

Supplementary materials

1.Synthetic schemes

Scheme S1: synthesis of methyl (3-aminophenyl)[(tert-butoxycarbonyl)amino]acetate(4): (i) HNO3/H2SO4; (ii) MeOH, SOCl2; (iii) (Boc)2O, TEA, CH2Cl2; (iv) H2, Pd/C.

Scheme S2: synthesis of methyl 3-amino 2,6-difluorophenylacetate (5): (i) HNO3; (ii) H2, Pd/C; (iii) MeOH, SOCl2.

Scheme S3: synthesis of ethyl 2-(3-aminophenyl)-propanoate (8): (i) dimethylcarbonate, K2CO3, 10 bar, 180° C; (ii) EtOH, H2SO4, reflux; (iii) H2, Pd/C.

Scheme S4: synthesis of intermediates 9a-c: (i) pyridine, 50° C, 24h; for the synthesis of 11a-c: (ii) NaOH 2N, 80° C, 3h. a: n = 1, m- substitution; b: n = 1, p- substitution; c: n = 2, m- substitution.

Scheme S5: synthesis of 9d-f: (i) pyridine, CH2Cl2; (ii) only for compound 9f: TFA, CH2Cl2; for the synthesis of 11d-f: (iii) NaOH 2N.

Scheme S6: synthesis of 9g (R = CH3): (i) pyridine; synthesis of 11g (R = H): (ii) NaOH 2N. For intermediate 9g see chemistry section.

Scheme S7: Synthesis of 9h and 9i (R = CH3): (i) TEA, CH2Cl2; synthesis of 11h and 11i (R = H): (ii) NaOH 2N.

Scheme S8: synthesis of 9l-r: (i) carbonate buffer pH 8.5; synthesis of 11l-r: (ii) KOH 2N.

Scheme S9: synthesis of 10a: (i) methanol, Pd/C, H2; synthesis of 10b: (ii) pyridine; synthesis of 11m: (iii): NaN3, NEt3∙HCl, toluene. For intermediate 10a and 10b see chemistry section

Final compounds 11a-11m: atoms numbering.
11a / / Mass: 341,35
Formula: C13H11NO6S2
Composition: C, 45,74; H, 3,27; N, 4,1.
Found: C, 45,78; H, 3,25; N, 4,0
11b / / Mass: 341,35
Anal. Calcd for: C13H11NO6S2
Composition: C, 45,74; H, 3,25; N, 4,1.
Found: C, 45,70; H, 3,22; N, 3,98.
11c / / Mass: 355,38
Anal. Calcd for: C14H13NO6S2
Composition: C, 47,32; H, 3,69; N, 3,94.
Found: C, 47,30; H, 3,70; N, 3,92.
11d / / Mass: 356,37
Anal. Calcd for: C13H12N2O6S2
Composition: C, 43,81; H, 3,39; N, 7,86.
Found: C, 43,84; H, 3,41; N, 7,83.
11e / / Mass: 377,33
Anal. Calcd for: C13H9F2NO6S2
Composition: C, 41,38; H, 2,4; N, 3,71.
Found: C, 41,35; H, 2,39; N , 3,70.
11f / / Mass: 355,38
Anal. Calcd for: C14H13NO6S2
Composition: C, 47,32; H, 3,69; N, 3,94.
Found: C, 47,30; H, 3,71; N, 3,90.
11g / / Mass: 265,25
Anal. Calcd for: C7H7NO6S2
Composition: C, 31,7; H, 2,66; N, 5,28.
Found: C, 31,89; H, 2,62; N, 5,20.
11h-i / / Mass: 279,28
Anal. Calcd for: C8H9NO6S2
Composition: C, 34,41; H, 3,25; N, 5,02.
Found: C, 34,38; H, 3,20; N, 5,00.
11l / / Mass: 279,28
Anal. Calcd for: C8H9NO6S2
Composition: C, 34,41; H, 3,25; N, 5,02.
Found: C, 34,45; H, 3,28; N, 5,06.
11m / / Mass: 365,38
Anal. Calcd for: C13H11N5O4S2
Composition: C, 42,73; H, 3,03; N, 19,17.
Found: C, 42,76; H, 3,00; N, 19,15.

2. Enzymology.

Inhibitors were dissolved in DMSO at a concentration of 10 mM; more diluted stocks were subsequently prepared as necessary. Kinetic measurements were performed using nitrocefin as substrate in 50 mM Tris buffer, pH 7.0, and monitored in an HP8453 UV–vis spectrophotometer. The Km of nitrocefin for AmpC determined in this buffer was 127 μM. The concentration of AmpC was determined spectrophotometrically in concentrated stock solutions and subsequently diluted; this enzyme had been previously expressed and purified.23

The concentration of enzyme in all reactions was 1.75 nM. Inhibition Ki values were obtained from IC50 plots assuming competitive inhibition, an assumption consistent with both previous inhibition patterns in this series and with experiments investigating the effect of increasing substrate concentrations (not shown).24

3. Microbiology

Compounds in the thiophene series were tested for synergy with the beta-lactam ceftazidime CAZ against pathogenic bacteria from clinical isolates (Spain). The bacteria were resistant to β-lactams because of the expression of class C β-lactamases. Strains tested were, Klebsiella pneumoniae 20/013, Escherichia coli 40/012, Enterobacter cloacae 558349, Enterobacter aerogenes 563334, Pseudomonas aeruginosa 12, Pseudomonas aeruginosa 59, Pseudomonas aeruginosa 120, Pseudomonas aeruginosa 148, Pseudomonas aeruginosa 199. (From the collection of the Spanish Network for Research in Infectious Pathology-REIPI).

Minimum inhibitor concentration values were determined with Mueller-Hinton broth using the microdilution method according to NCCLS guidelines. 20 To test the inhibitory activity, compounds were dissolved in DMSO and dilutions were performed using growth medium. An adequate final concentration in which to determine the MIC was obtained where the concentration of DMSO was <5%. The inhibitor/CAZ ratio was 1:1. A control experiment showed that DMSO did not have an effect on the bacteria growth at the concentration used.

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