Design, synthesis, crystal structures and antimicrobial activity of sulfonamide boronic acids as β-lactamase inhibitors

Design

The geometry and electronic properties of a sulfonamide are quite different from a carboxamide and we were interested in how this substitution would affect affinity (Scheme 1). First, we explored the sulfonamide substitution with a methyl alone as the R group. Since this compound was unexpectedly potent against AmpC, we then synthesized and tested sulfonamide boronic acids with seven more elaborate R groups (Table 1). Finally, we synthesized molecules with a benzyl and m-carboxybenzyl groups as R₂ substituents.

Synthesis

The sulfonamidomethaneboronic acids 3–9 and 11 were synthesized through sulfonylation of the aminomethaneboronate 2 (Scheme 2). The key intermediate 2 was obtained by treating the chloromethylboronic acid pinacol ester 1 with lithium bis(trimethylsilyl)amide.¹⁹In situ deprotection of 2 with equimolar methanol, followed by condensation with suitable sulfonyl chlorides, afforded the sulfonamidomethaneboronic acids 3–9 and 11.

Compound 10 (Table 1), bearing the cephalothin side chain, could not be obtained because of instability of the suitable sulfonyl chloride. In fact, several attempts to obtain the (thiophene-2-yl)-methanesulfonylchloride lead to the corresponding 2-chloromethyl thiophene instead, as reported for other electron rich phenylmethanesulfonyl chlorides.²⁶

According to recent literature reports, related sulfonylaminomethaneboronate compounds^{27, 28} can also be prepared by alkylation of suitable N-tert-butylsulfonamides with pinacol chloromethaneboronate 1, followed by acidic tert-butyl removal. However, this reaction sequence proved to be less efficient and the method was therefore abandoned.

Following our previous approach with carboxamides,²¹ we attempted to synthesize compound 18 (Table 1), but the synthesis failed because of protodeboronation during sulfonylation reaction occurred. The loss of the boronic moiety may occur due to stabilization of the intermediate carbanion formed when a base coordinates to boron.²⁹ Because the presence of the m-carboxyphenyl ring in the present case can account for this stabilization, we decided to add an extra carbon between the boron atom and the phenyl ring (Scheme 3). Therefore, to build the more elaborated inhibitors 16 and 17, boronates 12a,b were first homologated with chloromethyllithium.³⁰ Subsequent Matteson stereoselective homologation with dichloromethyllithium,^{29, 31–33} followed by substitution with bis(trimethylsilyl)amide and sulfonylation afforded compounds 15a,b. The use of (+)-pinanediol as chiral auxiliary allowed the formation of the final R configuration, which mimics the stereochemistry of carbon atom C6/7 of penicillin/cephalosporin. The conversion of pinanediol esters 15a,b to free boronic acids 16 and 17 was achieved through transesterification with phenylboronic acid in a biphasic acetonitrile/water system.³⁴

Enzymology and binding affinities

Except for those binding in the low nanomolar range, all previously studied boronic acids inhibitors of AmpC β-lactamase have been reversible, competitive inhibitors with fast-on, fast-off rates.^{17, 18} The same was found for the new sulfonamide boronic acid series as no incubation effect were detected for any of the compounds.

To investigate the effect of the carboxamide replacement with a sulfonamide we first measured the potency of methanesulfonamide boronic acid 3 (Table 1). Compound 3 inhibits AmpC with a K_i of 789 nM, a 23-fold improvement of potency compared to the carboxamide analog 3c (K_i 18.5 μM). The high affinity of this relatively small molecule prompted us to study larger derivatives. Compound 4, bearing the penicillin G side chain has a K_i of 70 nM, a 10-fold improvement over the initial sulfonamide 3 and an eight-fold improvement compared to its exact carboxamide analog 4c (K_i 570 nM) (Table 1).¹⁹ Because compound 5, isomer of compound 4, has a K_i of 210 nM, we decided to focus on benzylic derivatives and synthesized compounds 6–9, for which required sulfonylchlorides were commercially available or the final products easily accessible. Although most of those derivatives have a lower potency than 4, compound 9 has a K_i of 25 nM, a three-fold improvement versus compound 4. We tried to synthesize compound 10, the exact analog of the carboxamide 10c, but found that the required (thiophene-2-yl)-methanesulfonylchloride was unstable. Unexpectedly, compound 11, bearing the nafcillin side chain, inhibits AmpC with a K_i of 670 nM, 20-fold weaker than the carboxamide analog 11c (K_i 33 nM). This was another indicator that the SAR changes substantially in the sulfonamide series.

In the carboxamide series, addition of a m-carboxybenzyl group to 10c had resulted in a seven-fold improvement of potency for compound 17c. We decided to investigate the same modification for the sulfonamide 4 and synthesized the chiral compound 17. This latter has a K_i of 430 nM, thus rather than a seven-fold improvement, adding a m-carboxybenzyl to compound 4 lead to a six-fold decrease in inhibition. Compound 16, only with a benzyl as R₂ group, has an even worse K_i of 3.7 μM. The weaker potency of compounds 16 and 17 further demonstrate the changed SAR resulting from the carboxamide to sulfonamide conversion.

Structural analysis

To investigate the structural basis of the carboxamide to sulfonamide conversion, we determined co-crystal structures of AmpC in complex with 4, 9 and 17 (Table 2). All complexes were obtained from the same crystallization condition yielding centered monoclinic C2 crystals with two AmpC complexes in the asymmetric unit. The positions of the inhibitors in the active site were unambiguously identified in both chains in the initial F_o-F_c difference density maps (Figure 4). With exception of the complex with compound 4, the conformations of the inhibitors were very similar in both monomers.

For all three inhibitors, electron density connected the O_υ of the catalytic Ser64 to the boron atoms of the inhibitors. The boron geometry was tetrahedral, as expected, and key hydrogen bond interactions in the active site closely resembled those typically observed in β-lactamase structures with transition-state analogs and with β-lactams (Figure 5A–C).^{14, 19, 21, 22} One oxygen atom of the boronic acid is placed in the “oxyanion hole” formed by the backbone amide groups of Ser64 and Ala318. The other oxygen atom of the boronic acid interacts with the conserved Tyr150, which contributes to activating the deacylating water Wat402 for attack on the acyl-intermediate.^{15, 35–39} Both oxygen atoms are also within hydrogen bonding distance to the conserved water molecule Wat402.

The crystal structure of compound 4 (K_i 70 nM) in complex with AmpC was determined at 1.60 Å (Figure 5A). Whereas there were several differences between the two monomers in the asymmetric unit, such as the rotation of the benzyl ring to stack with Tyr221 in either a parallel or herringbone conformation, we will focus on the interaction in chain A. Here, one oxygen atom of the sulfonamide hydrogen bonds to Asn152 (nitrogen-oxygen distance: 2.6 Å). The other sulfonamide oxygen atom hydrogen bonds with a water molecule bridging to Gln120. The benzyl group makes a T-shaped π-π interaction (90° angle) with Tyr221. The sulfonamide nitrogen interacts neither with the protein nor with a well-ordered water molecule.

To understand the influence of the p-carboxylate group, responsible for a three-fold increase affinity of compound 9 (K_i 25 nM) compared to compound 4, we determined the crystal structure of the AmpC/9 complex to 1.78 Å resolution (Figure 5B). The conformation of 9 is similar in both monomers and resembles the conformation of 4 observed in chain A, except that the plane of its phenyl ring is slightly off-set relative to that of 4 (Figure 5D). The slightly altered conformation allows the p-carboxylate group to hydrogen bond with the side chain hydroxyl and amide nitrogen of Ser212. The other carboxylate oxygen atom hydrogen bonds with an ordered water molecule.

To understand the reduced activity of 17 (K_i 430 nM) against AmpC relative to compounds 4 and 9, we determined its crystal structure in complex with AmpC at 1.64 Å resolution (Figure 5C). In this complex, the boron-carbon bond is rotated by 90° compared to the conformation adopted by compound 4. This is necessary to accommodate the additional m-carboxybenzyl R₂ group of 17. Consequently, the sulfonamide is reoriented in the active site placing the benzyl sulfonamide in a totally different location relative to compounds 4 and 9 (Figure 5E). One sulfonamide oxygen of compound 17 makes stretched hydrogen bonds with Asn152 and Lys67 (3.2 and 3.1 Å, respectively) and the nitrogen atom of the sulfonamide hydrogen bonds with the backbone amide of Ala318 (3.1 Å), an interaction missed in the other two structures. Conversely, the second oxygen atom now abuts the backbone carbonyl of Ala318 (2.8 Å) and the quality of the other sulfonamide hydrogen bonds, as judged by distance and angle, has deteriorated substantially. The R phenyl group makes a T-shaped π-π interaction with Tyr221, but in a different conformation than observed in the AmpC/4 and AmpC/9 complex structures.

Antimicrobial activity

The anti-resistance activity of inhibitors 4 and 9 was investigated against clinical bacteria exhibiting high level of resistance against third-generation cephalosporins, such as ceftazidime, via expression of class A or class C β-lactamases. When the compounds were administrated alone in a disk diffusion assay they had little or no detectable activity on bacteria growth, as expected. However, in combination with ceftazidime the compounds produced a large inhibition halo surrounding the disk (Figure 6A). This inhibition of the bacterial growth revealed clear synergy between these compounds. The size of the inhibition zone was similar for both compounds and showed improved inhibition of bacterial growth compared to ceftazidime alone.

Antimicrobial activity was investigated quantitatively to determine the minimum inhibitory concentrations (MICs) of the β-lactam/inhibitor combination necessary to inhibit the bacterial growth (Table 3). The MICs of β-lactams alone against the strains corresponded to a high level of resistance according to Clinical and Laboratory Standards Institute (CLSI) standards.⁴⁰ The compounds had no measurable antibiotic activity when used alone. In combination with β-lactams, the compounds decreased MIC values by eight-fold on average. Interestingly, even larger relative decreases in MIC values were observed for an E. coli strain producing the plasmid-mediated class A β-lactamase CTX-M-14 (16 to 32-fold). This offers preliminary evidence that the sulfonamide boronic acids may inhibit both class C β-lactamases and class A enzymes, consistent with studies revealing activity against this class of enzymes in the low μM range described in this study (Table 4, below) and results from Tan and colleagues.²⁵ More detailed studies will be necessary before inhibition of the class A ß-lactamases can be shown to be broad or robust.

A traditional β-lactam resistance mechanism is the upregulation of β-lactamase-encoding gene transcription caused by β-lactams such as cefoxitin and the β-lactamase inhibitor clavulanate. To test the role of compounds on the induction of AmpC expression, we investigated their ability to potentiate the action of the β-lactam ceftazidime. We compared the potentiation effect of compounds with that of cefoxitin (Figure 6B and 6C). Agar plates were inoculated with K. pneumoniae and E. cloacae strains in which AmpC expression is inducible. A ceftazidime-containing disk and a disk containing the inhibitors (cefoxitin or compounds 4 and 9) were placed on each plate. As ceftazidime diffuses from disks into the agar, a clear zone is created around the ceftazidime-containing disk, indicating where bacteria are unable to grow. The shape of this halo in the vicinity of the inhibitor-containing disk indicated the effect of these compounds on the induction of AmpC. The inhibition halos normally surrounding ceftazidime were substantially diminished in the regions nearest to the cefoxitin disk. This well-known antagonist picture is characteristic of the ampC gene induction by cefoxitin. The antagonist picture was replaced by a synergic picture with compounds 4 and 9; the inhibition halos of ceftazidime were dramatically increased in the region near the compound disks. This result indicates that the compounds inhibit AmpC and do not induce significant upregulation of AmpC-encoding genes in contrast to β-lactams such as cefoxitin.

Selectivity

To investigate the selectivity of sulfonamide boronic acids, compounds 4 and 9 were tested against the serine protease α-chymotrypsin, the cysteine protease cruzain, and against the class A β-lactamase CTX-M-9, that is structurally different from AmpC (Table 4). Compounds 4 and 9 showed no measurable inhibition at 100 μM against cruzain and α-chymotrypsin. Interestingly, the compounds are active against CTX-M-9 with IC₅₀s in the low micromolar range. The K_i values corresponding to the IC₅₀ values are 2.529 and 0.552 μM, respectively, indicating 36-fold and 22-fold selectivity towards AmpC versus CTX-M-9 β-lactamase.

Synthesis and analysis

All reactions were performed under argon using oven-dried glassware and dry solvents. Anhydrous tetrahydrofuran (THF) and diethyl ether were obtained by standard methods and freshly distilled under argon from sodium benzophenone ketyl prior to use. All reagents were purchased from Sigma-Aldrich and Fluka. The −100 °C bath was prepared by addition of liquid nitrogen to a pre-cooled (−80 °C) mixture of ethanol/methanol (1:1). Reactions were monitored by TLC, which were visualized by UV fluorescence and by Hanessian's cerium molybdate stain. Chromatographic purification of the compounds was performed on silica gel (particle size 0.05–0.20 mm). Melting points were measured on a Büchi 510 apparatus. Optical rotations were recorded at +20 °C on a Perkin-Elmer 241 polarimeter and are expressed in 10⁻¹ deg cm² g⁻¹. ¹H- and ¹³C-NMR spectra were recorded on a Bruker DPX-200 or Avance-400 spectrometer; chemical shifts (δ) are reported in ppm downfield from tetramethylsilane (TMS) as internal standard (s singlet, d doublet, t triplet, q quartet, m multiplet, br broad signal); coupling constants (J) are given in Hz. Two-dimensional NMR techniques (COSY, HMBC, HSQC) were used to aid in the assignment of signals in ¹H and ¹³C spectra. Mass spectra were determined on a gas chromatograph HP 5890 with mass spectrometer detector HP 5972 (EI, 70 eV) or on an Agilent Technologies LC-MS(n) Ion Trap 6310A. The purity of all tested compounds was above 95%, determined by elemental analysis performed on a Carlo Erba Elemental Analyzer 1110; elemental analyses for the compounds were within ± 0.3% of the theoretical values. MS fragmentation and elemental analyses of free boronic acids were not obtainable, because of the formation of dehydration products. Nevertheless, these boronic acids could be converted into analytically-pure pinacol/pinanediol esters by simple exposure to equimolar amount of pinacol/pinanediol in anhydrous THF.

General Procedure for the Synthesis of Sulfonylaminomethaneboronates (3–6, 8, 11)⁴²

A solution of 2¹⁹ (1.00 mmol) in THF (3 mL) was added to a solution of anhydrous methanol in THF (2.5 M, 1.00 mmol) at −10 °C under argon flow. After stirring for 10 min at −10 °C, the cooling bath was removed, and the reaction mixture stirred for 1 h at room temperature. Thereafter, the reaction mixture was cooled again to −10 °C and a solution of the selected sulfonyl chloride (1.10 mmol) in THF (2 mL) was slowly added; the resulting mixture was allowed to warm to room temperature overnight. The solvent was evaporated in vacuo and the residue purified by chromatography (dichloromethane/diethyl ether 8:2 to methanol) affording the expected sulfonamides.

Pinacol (methanesulfonylamino)methaneboronate (3)

According to the general procedure described before, the compound was recovered as a colorless oil (78% yield). ¹H-NMR (200 MHz, CDCl₃): δ 1.26 (12H, s, pinacol protons), 2.79 (2H, d, J 4.8, BCH₂), 2.91 (3H, s, CH₃S), 4.45 (1H, br, SO₂NH). ¹³C-NMR (100 MHz, CDCl₃): δ 24.8, 28.0 (CB), 38.6, 84.8. EI-MS, m/z: 220 (M⁺ −15, 7%), 171 (29), 156 (24), 143 (21), 136 (39), 119 (14), 104 (100), 103 (44), 83 (37), 74 (6). Anal. Calcd. for C₈H₁₈BNO₄S: C, 40.87; H, 7.72; N, 5.96; S, 13.64. Found: C, 41.02; H, 7.55; N, 5.77; S, 13.51.

Pinacol (phenylmethanesulfonylamino)methaneboronate (4)

White solid (84% yield). Mp 138–141 °C. ¹H-NMR (200 MHz, CDCl₃): δ 1.27 (12H, s, pinacol protons), 2.71 (2H, s, BCH₂), 4.40 (1H, br, SO₂NH), 4.27 (2H, s, PhCH₂), 7.39 (5H, s, H_Arom). ¹³C-NMR (50 MHz, CDCl₃): δ 24.7, 29.6 (CB), 57.3, 84.7, 128.6, 128.7, 129.6, 130.6. EI-MS, m/z: 311 (M⁺, 2%), 156 (18), 120 (5), 106 (8), 104 (17), 92 (9), 91 (100), 74 (17), 65 (11), 59 (16). Anal. Calcd. for C₁₄H₂₂BNO₄S: C, 54.03; H, 7.13; N, 4.50; S, 10.30. Found: C, 54.32; H, 7.30; N, 4.29; S, 10.55.

Pinacol (4-methylbenzenesulfonylamino)methaneboronate (5)

White solid (84% yield). Mp 103–105 °C. ¹H-NMR (200 MHz, DMSO): δ 1.17 (12H, s, pinacol protons), 2.25 (2H, d, J 5.5, BCH₂), 2.38 (3H, s, CH₃Ph), 7.06 (1H, t, J 5.5, SO₂NH), 7.38 (2H, d, J 8.2, H₃, H₅), 7.65 (2H, d, J 8.2, H₂, H₆). ¹³C-NMR (50 MHz, CDCl₃): δ 21.4, 24.7, 27.6 (CB), 84.6, 127.5, 129.6, 135.8, 143.2. EI-MS, m/z: 296 (M⁺ −15, 15%), 282 (55), 253 (31), 212 (19), 180 (78), 166 (26), 156 (100), 155 (39), 151 (46), 143 (20), 140 (30), 139 (89), 137 (23), 91 (80), 85 (19), 83 (25), 74 (98), 73 (30), 65 (31), 59 (20), 55 (23). Anal. Calcd. for C₁₄H₂₂BNO₄S: C, 54.03; H, 7.13; N, 4.50; S, 10.30. Found: C, 54.26; H, 7.24; N, 4.22; S, 10.38.

Pinacol (3-nitrophenylmethanesulfonylamino)methaneboronate (6)

Yellowish viscous oil (84% yield). ¹H-NMR (400 MHz, CDCl₃): δ 1.29 (12H, s, pinacol protons), 2.80 (2H, d, J 4.4, BCH₂), 4.39 (2H, s, PhCH₂), 4.44 (1H, t, J 4.4, SO₂NH), 7.59 (1H, t, J 8.0, H₅), 7.80 (1H, d, J 8.0, H₆), 8.23 (1H, d, J 8.0, H₄), 8.29 (1H, s, H₂). ¹³C-NMR (100 MHz, CDCl₃): δ 24.8, 28.3 (CB), 56.6, 84.9, 123.5, 125.5, 129.8, 131.8, 136.9, 148.3; EI-MS, m/z: 341 (M⁺ −15, 2%), 327 (2), 240 (8), 165 (8), 156 (24), 137 (11), 136 (100), 129 (8), 104 (54), 103 (14), 90 (52), 89 (32), 83 (36), 74 (34), 59 (38), 43 (15), 41 (19). Anal. Calcd. for C₁₄H₂₁BN₂O₆S: C, 47.21; H, 5.94; N, 7.86; S, 9.00. Found: C, 47.35; H, 6.05; N, 7.73; S, 8.74.

Pinacol (3-aminophenylmethanesulfonylamino)methaneboronate hydrochloride (7)

Palladium 10 wt. % on activated carbon (51 mg, 40% w/w) was added to a solution of 6 (128 mg, 0.36 mmol) in ethyl acetate (4 mL), and allowed to react under hydrogen atmosphere for 16 h. The catalyst was filtered off and the solvent was evaporated in vacuo. The crude residue was dissolved in HCl (0.1 M soln in MeOH, 3.6 mL, 0.36 mmol). After 45 min the mixture was concentrated under reduced pressure and the residue repeatedly washed with diethyl ether, affording 7 as a yellowish solid (118 mg, 90% yield). Mp 193–196 °C. ¹H-NMR (400 MHz, DMSO): δ 1.23 (12H, s, pinacol protons), 2.59 (2H, d, J 3.82, BCH₂), 4.37 (2H, s, PhCH₂), 6.76 (1H, br, SO₂NH), 7.30–7.36 (3H, m, H_Arom), 7.46 (1H, t, J 7.60, H_Arom), 9.60–10.60 (3H, br, NH₃⁺). ¹³C-NMR (100 MHz, DMSO): δ 25.0, 27.5 (CB), 55.9, 84.2, 122.8, 125.3, 130.1, 130.5, 132.7, 133.0. EI-MS, m/z: 326 (M⁺, 5%), 156 (8), 107 (64), 106 (100), 104 (15), 77 (11), 59 (11), 41 (8). Anal. Calcd. for C₁₄H₂₄BClN₂O₄S: C, 46.36; H, 6.67; N, 7.72; S, 8.84. Found: C, 46.41; H, 6.50; N, 7.55; S, 8.68.

Pinacol [4-(methoxycarbonyl)phenylmethanesufonylamino]methaneboronate (8)

According to the general procedure described above, the compound was recovered as a white solid (84% yield). Mp 104#x2013;107 °C. ¹H-NMR (400 MHz, CDCl₃): δ 1.27 (12H, s, pinacol protons), 2.73 (2H, d, J 4.1, BCH₂), 3.93 (3H, s, COOCH₃), 4.33 (2H, s, PhCH₂), 4.37 (1H, br, SO₂NH), 7.49 (2H, d, J 8.3, H₂, H₆), 8.04 (2H, d, J 8.3, H₃, H₅); ¹³C-NMR (100 MHz, CDCl₃): δ 24.8, 28.4 (CB), 52.2, 57.2, 84.8, 129.9, 130.3, 130.7, 134.6, 166.6; EI-MS, m/z: 354 (M⁺ −15, < 2%), 338 (2), 311(2), 269 (3), 156 (25), 150 (24), 149 (100), 121 (24), 118 (13), 104 (19), 90 (19), 74 (26), 59 (13), 41 (9). Anal. Calcd. for C₁₆H₂₄BNO₆S: C, 52.04; H, 6.55; N, 3.79; S, 8.68. Found: C, 51.88; H, 6.74; N, 3.56; S, 8.51.

[(4-carboxyphenyl)methanesulfonylamino]methaneboronic acid (9)

A mixture of 8 (67 mg, 0.18 mmol) and HCl 3 N degassed (3 mL) was allowed to react at reflux for 1 h 30 min. After cooling, the reaction mixture was diluted with water (15 mL) and washed twice with diethyl ether (2 × 20 mL). The aqueous phase was concentrated in vacuo, affording 9 as a yellow solid (42 mg, 86% yield). Mp 118–122 °C (dec). ¹H-NMR (400 MHz, DMSO): δ 2.5 (2H, d, J 4.9, BCH₂), 4.42 (2H, s, PhCH₂), 6.22 (1H, t, J 4.9, SO₂NH), 7.49 (2H, d, J 8.6, H₂, H₆), 7.93 (2H, d, J 8.6, H₃, H₅), 7.94 (1H, s, COOH). ¹³C-NMR (100 MHz, DMSO): δ 31.3 (CB), 55.7, 129.7, 130.7, 131.4, 136.0, 167.6. EI-MS and elemental analysis were not obtainable, but exposure of 9 to an equimolar amount of pinacol in anhydrous THF afforded the corresponding pinacol boronate in quantitative yield and satisfactory elemental analysis; Anal. Calcd. for C₁₅H₂₂BNO₆S: C, 50.72; H, 6.24; N, 3.94; S, 9.03. Found: C, 50.88; H, 6.41; N, 3.80; S, 8.77.

(2-Ethoxy-naphthalene-1-sulfonylamino)methaneboronic acid (11)

According to the general procedure described above, reaction of 2 with 2-ethoxy-naphthalene-1-sulfonyl chloride, obtained from 2-ethoxy-naphtalene,⁴³ afforded 11 as white solid free boronic acid (80% yield). ¹H-NMR (200 MHz, MeOD): δ 1.46 (3H, t, J 6.9, OCH₂CH₃), 1.94 (2H, s, BCH₂), 4.19 (2H, q, J 6.9, OCH₂CH₃), 7.24 (1H, dd, J 9.0, 2.0, H_Arom), 7.29 (1H, s, H_Arom), 7.70–7.91 (3H, m, H_Arom), 8.32 (1H, s, H_Arom). C-NMR (50 MHz, MeOD): δ 13.6, 20.7 (CB), 63.4, 106.2, 120.1, 123.2, 127.3, 127.4, 127.9, 130.1, 133.8, 136.4, 159.0. EI-MS and elemental analysis were not obtainable, but exposure of 11 to an equimolar amount of pinacol in anhydrous THF afforded the corresponding pinacol boronate in quantitative yield and satisfactory elemental analysis; Anal. Calcd. for C₁₉H₂₆BNO₅S: C, 58.32; H, 6.70; N, 3.58; S, 8.19. Found: C, 58.19; H, 6.84; N, 3.40; S, 7.96.

(+)-Pinanediol 3-(tert-butoxycarbonyl)benzeneboronate (12b)

A solution of tert-butyl 3-bromobenzoate (1.70 g, 6.61 mmol), obtained from 3-bromobenzoic acid,⁴⁴ and freshly distilled triisopropyl borate (1.53 mL, 6.61 mmol) in THF (17 mL) was cooled to −100 °C under argon flow and n-butyllithium (2.5 M soln in hexane, 2.91 mL, 7.27 mmol) was added dropwise over 15 min, during which the solution turned cherry red. After 1 h at −100 °C, trimethylsilyl chloride (0.84 mL, 6.61 mmol) was dropped into the reactor and the resulting colorless solution was allowed to warm to room temperature and stirred overnight. Finally, (+)-pinanediol (1.12 g, 6.61 mmol) was added and the solution stirred 1 h at room temperature. The mixture was partitioned between ethyl acetate (100 mL) and water (40 mL) and the aqueous phase was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with brine, dried over MgSO₄, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (light petroleum/ethyl ether 95:5), affording 12b as a yellowish solid (2.10 g, 89% yield). Mp 70–71 °C. [a]_D +7.3 (c 1.1, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ 0.94 (3H, s, pinanyl CH₃), 1.24 (1H, d, J 10.9, pinanyl H_endo), 1.36 (3H, s, pinanyl CH₃), 1.53 (3H, s, pinanyl CH₃), 1.64 (9H, s, t-Bu), 1.98–2.30 (5H, m, pinanyl protons), 4.51 (1H, dd, J 8.5, 2.0, CHOB), 7.46 (1H, t, J 7.6, H₅), 8.0 (1H, d, J 7.6, H₄), 8.12 (1H, d, J 7.6, H₆), 8.45 (1H, s, H₂). ¹³C-NMR (100 MHz, CDCl₃): δ 24.0, 26.5, 27.1, 28.2, 28.7, 35.5, 38.2, 39.5, 51.4, 78.4, 80.9, 86.5, 127.6, 131.5, 132.1, 135.7, 138.7, 169.5, CB not seen. EI-MS: m/z 356 (M⁺, 10%), 300 (36), 283 (41), 231 (50), 204 (38), 83 (65), 67 (59), 57 (100). Anal. Calcd. for C₂₁H₂₉BO₄: C, 70.80; H, 8.20. Found: C, 70.55; H, 8.22.

(+)-Pinanediol phenylmethaneboronate (13a)

n-Butyllithium (2.5 M soln in hexane, 4.66 mL, 11.64 mmol) was added dropwise to a stirred solution of (+)-pinanediol benzeneboronate 12a²¹ (2.48 g, 9.70 mmol) and bromochloromethane (0.95 mL, 14.55 mmol) in THF (25 mL) at −80 °C under argon atmosphere. The mixture was allowed to reach room temperature overnight. Then the solution was partitioned between light petroleum (80 mL) and water (25 mL), the organic phase was washed with saturated ammonium chloride (20 mL) and the combined aqueous phases were extracted with petroleum ether (2 × 40 mL). The organic phases were dried, filtered and concentrated and the crude residue was purified by gradient chromatography (light petroleum to light petroleum/ethyl ether 95:5), affording 13a as a colorless oil (2.33 g, 89% yield). [α]_D +14.2 (c 2.0, CHCl₃). ¹H-NMR (200 MHz, CDCl₃): δ 0.87 (3H, s, pinanyl CH₃), 1.10 (1H, d, J 10.8, pinanyl H_endo), 1.30 (3H, s, pinanyl CH₃), 1.41 (3H, s, pinanyl CH₃), 1.62–2.39 (5H, m, pinanyl protons), 2.37 (2H, s, BCH₂), 4.30 (1H, dd, J 8.8, 1.9, CHOB), 7.10–7.31 (5H, m, H_Ar). ¹³C-NMR (50 MHz, CDCl₃): δ 19.3 (CB), 24.0, 26.4, 27.1, 28.6, 35.5, 38.1, 39.5, 51.3, 78.0, 85.8, 124.8, 128.3, 128.9, 138.8. EI-MS: m/z 217 (M⁺+1, 2%), 270 (M⁺, 81%), 269 (M⁺−1, 21%), 255 (32), 229 (24), 214 (32), 201 (88), 200 (33), 187 (30), 179 (28), 174 (100), 135 (58), 134 (38), 119 (32), 118 (26), 117 (28), 109 (24), 93 (44), 91 (74), 83 (75), 82 (32), 81 (34), 79 (24), 77 (26), 67 (66), 65 (27), 55 (47), 53 (29). Anal. Calcd. for C₁₇H₂₃BO₂: C, 75.57; H, 8.58. Found: C, 75.39; H, 8.44.

(+)-Pinanediol [3-(tert-butoxycarbonyl)phenyl]methaneboronate (13b)

Following the procedure described for the synthesis of 13a, compound 13b was recovered from 12b as a yellow oil (88% yield). [α]_D +8.7 (c 1.4, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ 0.87 (3H, s, pinanyl CH₃), 1.11 (1H, d, J 11.0, pinanyl H_endo), 1.32 (3H, s, pinanyl CH₃), 1.43 (3H, s, pinanyl CH₃), 1.63 (9H, s, t-Bu), 1.84–2.39 (5H, m, pinanyl protons), 2.42 (2H, s, BCH₂), 4.32 (1H, dd, J 8.8, 2.0, CHOB), 7.33 (1H, t, J 7.7, H₅), 7.40 (1H, d, J 7.7, H₆), 7.80 (1H, d, J 7.7, H₄), 7.86 (1H, s, H₂). ¹³C-NMR (100 MHz, CDCl₃): δ 19.3 (CB), 24.0, 26.5, 27.1, 28.2, 28.6, 35.4, 38.2, 39.5, 51.3, 78.0, 80.7, 86.0, 126.2, 128.1, 129.9, 132.0, 133.2, 139.0, 166.0; EI-MS: m/z 370 (M⁺, 18%), 314 (71), 297 (59), 135 (55), 57 (100). Anal. Calcd. for C₂₂H₃₁BO₄: C, 71.36; H, 8.44. Found: C, 71.60; H, 8.27.

(+)-Pinanediol (1R)-1-(N-bis(trimethylsilyl)amino)-2-phenylethaneboronate (14a)

n-Butyllithium (2.5 M soln in hexane, 1.78 mL, 4.44 mmol) was added dropwise to a stirred solution of dichloromethane (0.36 mL, 5.55 mmol) in THF (8 mL) at −100 °C under argon atmosphere; at the end of the butyllithium addition, a white microcrystalline precipitate (LiCHCl₂) became evident. After 30 min, a solution of 13a (1.00 g, 3.70 mmol) in THF (8 mL) was slowly added at the same temperature. The white precipitate disappeared and the mixture allowed to gradually reach room temperature overnight. The resulting solution was concentrated under reduced pressure. The crude was treated with petroleum ether (50 mL), and the resulting white inorganic precipitate was filtered off and washed with abundant petroleum ether. Solvent evaporation in vacuo afforded (+)-pinanediol (1S)-1-chloro-2-phenylethaneboronate, used as such for the subsequent reaction.

The above product was dissolved in THF (10 mL) and lithium bis(trimethylsilyl)amide (1.0 M soln in tetrahydrofuran, 3.70 mL, 3.70 mmol) was added dropwise at −100 °C under argon flow. The reaction mixture was allowed to reach room temperature overnight. The resulting solution was concentrated under reduced pressure and the crude was treated with petroleum ether (50 mL); the white inorganic precipitate (LiCl) was filtered off and washed with abundant petroleum ether. The solvent was evaporated in vacuo to give a residue which was subjected to column chromatography (light petroleum/ethyl ether/triethylamine 98:2:5) recovering 14a as a pale yellow oil (837 mg, 51% yield). [α]_D −14.8 (c 2.3, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ 0.12 (18H, s, 2Si(CH₃)₃), 0.87 (3H, s, pinanyl CH₃), 0.99 (1H, d, J 10.8, pinanyl H_endo), 1.31 (3H, s, pinanyl CH₃), 1.41 (3H, s, pinanyl CH₃), 1.78–2.36 (5H, m, pinanyl protons), 2.71 (1H, dd, J 13.1, 7.6, CHCH₂), 2.87 (1H, t, J 7.4, BCH), 3.08 (1H, dd, J 13.1, 7.3, CHCH₂), 4.31 (1H, dd, J 8.7, 2.0, CHOB), 7.17–7.31 (5H, m, H_Arom). ¹³C-NMR (100 MHz, CDCl₃): δ 3.0, 24.0, 26.0, 27.1, 28.4, 35.2, 38.1, 39.5, 42.0, 45.0 (CB), 51.4, 78.2, 85.6, 125.7, 127.8, 129.7, 141.7. Anal. Calcd. for C₂₄H₄₂BNO₂Si₂: C, 64.98; H, 9.54; N, 3.16. Found: C, 65.19; H, 9.46; N, 2.95.

(+)-Pinanediol (1R)-2-(3(tert-butoxycarbonyl)phenyl)-1-(N-bis(trimethylsilyl)amino)ethaneboro nate (14b)

Following the procedure described for the synthesis of 14a, compound 14b was recovered as a colorless oil (28% yield). [α]_D −12.6 (c 3.9, CHCl₃). ¹H-NMR (200 MHz, CDCl₃): δ 0.10 (18H, s, 2Si(CH₃)₃), 0.83 (3H, s, pinanyl CH₃), 0.94 (1H, d, J 11.1, pinanyl H_endo), 1.28 (3H, s, pinanyl CH₃), 1.38 (3H, s, pinanyl CH₃), 1.61 (9H, s, t-Bu), 1.78–2.35 (5H, m, pinanyl protons), 2.66–2.87 (2H, m, BCH, CHCH₂), 3.06–3.15 (1H, m, CHCH₂), 4.29 (1H, d, J 8.7, CHOB), 7.29–7.43 (2H, m, H₅, H₆), 7.82–7.91 (2H, m, H₂, H₄). ¹³C-NMR (50 MHz, CDCl₃): δ 3.0, 23.9, 26.2, 27.0, 28.2, 28.4, 35.1, 38.1, 39.5, 41.8, 44.8 (CB), 51.4, 78.3, 80.5, 85.6, 126.9, 127.7, 130.8, 131.5, 133.8, 141.7, 166.0. Anal. Calcd. for C₂₉H₅₀BNO₄Si₂: C, 64.06; H, 9.27; N, 2.58. Found: C, 63.84; H, 9.06; N, 2.77.

(+)-Pinanediol (1R)-1-(phenylmethanesulfonylamino)-2-phenylethaneboronate (15a)

A solution of 14a (840 mg, 1.89 mmol) in THF (2 mL) was added to a THF solution of anhydrous methanol (2.5 M, 0.76 mL, 1.89 mmol) at −10 °C under nitrogen and magnetically stirred for 10 min at −10 °C. The cooling bath was removed and the solution was stirred at room temperature for an additional hour. Thereafter, the temperature was lowered to −40 °C and phenylmethanesulfonyl chloride (469 mg, 2.46 mmol) in THF (2 mL) was slowly dropped in. The resulting colorless solution was allowed to gradually reach room temperature and stirred overnight. The solution was partitioned between diethyl ether (30 mL) and water (10 mL), the aqueous phase extracted with diethyl ether (2 × 20 mL) and the combined organic phases were dried (Na₂SO₄), filtered and concentrated. The crude residue was purified by column chromatography (dichloromethane/diethyl ether 6:4), affording 15a as a thick yellowish oil (343 mg, 40% yield). [α]_D +9.7 (c 1.6, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ 0.85 (3H, s, pinanyl CH₃), 1.10 (1H, d, J 10.8, pinanyl H_endo), 1.31 (3H, s, pinanyl CH₃), 1.32 (3H, s, pinanyl CH₃), 1.82–2.42 (5H, m, pinanyl protons), 2.90 (1H, dd, J 13.7, 7.1, CHCH₂), 3.08 (1H, dd, J 13.7, 5.5, CHCH₂), 3.27–3.39 (1H, m, BCH), 4.18 (1H, br, SO₂NH), 4.21 (2H, s, CH₂SO₂), 4.35 (1H, dd, J 8.8, 2.0, CHOB), 7.20–7.34 (10H, m, H_Arom). ¹³C-NMR (100 MHz, CDCl₃): δ 23.9, 26.3, 27.0, 28.3, 35.1, 38.1, 38.8, 39.4, 42.5 (CB), 51.2, 59.1, 78.6, 86.9, 126.6, 128.4, 128.5, 128.6, 129.6, 129.7, 130.7, 138.4. Anal. Calcd. for C₂₅H₃₂BNO₄S: C, 66.23; H, 7.11; N, 3.09; S 7.07. Found: C, 66.04; H, 6.97; N, 2.88; S, 6.91.

Pinanediol (1R)-2-(3(tert-butoxycarbonyl)phenyl)-1-(phenylmethanesulfonylamino)ethaneboron ate (15b)

Following the procedure described for 15a, compound 15b was recovered as a yellowish oil (35% yield). [α]_D +18.7 (c 0.7, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ 0.82 (3H, s, pinanyl CH₃), 1.00 (1H, d, J 10.9, pinanyl H_endo), 1.26 (3H, s, pinanyl CH₃), 1.28 (3H, s, pinanyl CH₃), 1.59 (9H, s, t-Bu), 1.83–2.36 (5H, m, pinanyl protons), 2.95 (1H, dd, J 13.3, 6.4, CHCH₂), 3.10 (1H, dd, J 13.3, 5.6, CHCH₂), 3.31 (1H, q, J 5.9, BCH), 4.17 (1H, d, J 5.9, SO₂NH), 4.23 (2H, s, CH₂SO₂), 4.37 (1H, d, J 8.7, 2.0, CHOB), 7.32–7.44 (7H, m, H_Arom), 7.83–7.85 (2H, m, H_Arom). ¹³C-NMR (100 MHz, CDCl₃): δ 24.0, 26.3, 27.0, 28.2, 29.7, 35.0, 38.2, 38.7, 39.4, 42.5 (CB), 51.1, 59.1, 78.7, 81.0, 87.1, 127.8, 128.5, 129.0, 129.1, 129.6, 130.4, 130.8, 132.1, 134.1, 138.5, 165.7. Anal. Calcd. for C₃₀H₄₀BNO₆S: C, 65.10; H, 7.28; N, 2.53; S 5.79. Found: C, 64.88; H, 7.02; N, 2.29; S, 5.47.

(1R)-1-(phenylmethanesulfonylamino)-2-phenylethaneboronic acid (16)

To a solution of 15a (245 mg, 0.54 mmol) in CH₃CN (3 mL), HCl (1M aqueous solution, 350 μL, 0.35 mmol), phenylboronic acid (62 mg, 0.51 mmol) and n-hexane (3 mL) were sequentially added and the resulting biphasic solution was vigourously stirred. After 30 min the n-hexane layer, containing the pinanediol phenylboronate, was removed and fresh n-hexane (3 mL) was added. This last procedure was repeated several times until a TLC analysis revealed no remaining pinanediol boronate. The acetonitrile phase was then concentrated affording 16 as a yellowish solid (159 mg, 92% yield). Mp 107 °C (dec). [α]_D +78.0 (c 1.0, MeOH). ¹H-NMR (400 MHz, MeOD): δ 2.83 (2H, d, J 7.1, CHCH₂), 3.05 (1H, t, J 7.1, BCH), 4.20 (2H, s, CH₂SO₂), 7.16–7.44 (10H, m, H_Arom). ¹³C-NMR (100 MHz, MeOD): δ 38.8, 39.3 (CB), 58.6, 126.1, 127.2, 128.0, 128.2, 129.0, 130.1, 130.6, 133.3. EI-MS and elemental analysis were not obtainable, but exposure of 16 to an equimolar amount of (+)-pinanediol in anhydrous THF afforded compound 15a in quantitative yield and satisfactory elemental analysis; Anal. Calcd. for C₂₅H₃₂BNO₄S: C, 66.23; H, 7.11; N, 3.09; S 7.07. Found: C, 66.14; H, 6.91; N, 3.32; S, 6.89.

(1R)-2-(3-carboxyphenyl)-1-(phenylmethanesulfonylamino)ethaneboronic acid (17)

A solution of 15b (90 mg, 0.16 mmol) in dichloromethane (2 mL) was cooled to −10 °C and treated with an excess of trifluoroacetic acid (10% v/v soln in dichloromethane, 2 mL). After 10 min at −10 °C, the mixture was allowed to react at room temperature for 5 h. The solution was concentrated in vacuo and the residue crystallized from diethyl ether affording (+)-pinanediol (1R)-2-[3-carboxyphenyl]-1-(phenylmethanesulfonylamino)ethaneboronate (79 mg, 100% yield) as a cream coloured solid. Mp 155–158 °C. [α]_D +25.9 (c 0.6, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ 0.87 (3H, s, pinanyl CH₃), 1.06 (1H, d, J 10.8, pinanyl H_endo), 1.31 (3H, s, pinanyl CH₃), 1.33 (3H, s, pinanyl CH₃), 1.88–2.38 (5H, m, pinanyl protons), 2.99 (1H, dd, J 13.7, 6.0, CHCH₂), 3.15 (1H, dd, J 13.7, 5.6, CHCH₂), 3.37 (1H, br, BCH), 4.31 (2H, s, CH₂SO₂), 4.40 (1H, d, J 7.7, CHOB), 4.51 (1H, br, SO₂NH), 7.30–7.58 (7H, m, H_Arom), 7.97–8.01 (2H, m, H_Arom), 10.2 (1H, br, COOH). ¹³C-NMR (100 MHz, CDCl₃): δ 24.0, 26.3, 27.0, 28.3, 35.0, 38.2, 38.5, 39.4, 42.4 (CB), 51.1, 59.2, 78.8, 87.3, 128.5, 128.6, 128.7, 127.2, 129.3, 129.4, 130.8, 131.2, 135.6, 139.0, 171.8. Anal. Calcd. for C₂₆H₃₂BNO₆S: C, 62.78; H, 6.48; N, 2.82; S 6.45. Found: C, 62.61; H, 6.72; N, 2.64; S, 6.21.

This compound was then treated according to the procedure described for 16 to recover 17 as a yellowish solid (54 mg, 92% yield). Mp 118–122 °C (dec). [α]_D +136.0 (c 0.5, MeOH). ¹H-NMR (400 MHz, MeOD): δ 2.87 (2H, d, J 7.0, CHCH₂), 3.05 (1H, t, J 7.0, BCH), 4.24 (2H, s, CH₂SO₂), 7.31–7.88 (9H, m, H_Arom). ¹³C-NMR (100 MHz, MeOD): δ 38.4, 42.9 (CB), 58.8, 127.5, 128.0, 128.1, 128.2, 129.6, 130.1, 130.2, 130.6, 133.8, 139.1, 168.4. EI-MS and elemental analysis were not obtainable, but exposure of 11 to an equimolar amount of (+)-pinanediol in anhydrous THF afforded the corresponding pinanediol boronate in quantitative yield and satisfactory elemental analysis; Anal. Calcd. for C₂₆H₃₂BNO₆S: C, 62.78; H, 6.48; N, 2.82; S 6.45. Found: C, 62.56; H, 6.71; N, 2.59; S, 6.28.

Enzymology

Sulfonamidomethaneboronic acids were dissolved in 100% DMSO at a concentration of 100 mM; more diluted stocks were prepared as necessary. AmpC activity was determined by monitoring the change of absorbance at 405 nm due to hydrolyzed substrate CENTA (K_m 15 μM) by an HP5453 UV-vis spectrophotometer. The enzyme was expressed and purified as described⁴⁵ and CENTA was purchased from Tydock Pharma.

Kinetic measurements were run at room temperature in 50 mM sodium-cacodylate pH 6.5 in the presence of 0.01% Triton-X to prevent non-specific inhibition via compound aggregation.^{46, 47} These conditions also ensure the hydrolysis of pinacol esters to free boronic acids.⁴⁸ Reactions were performed in 1 mL cuvettes with 60 μM CENTA and initiated by adding 1.2 nM enzyme. No incubation effect was detected for any compound, consistent with earlier studies.¹⁸ The first 180 sec of each reaction were used to measure initial rates. IC₅₀ values were obtained by fitting binding data to a sigmoidal dose-response equation using GraphPad Prism (GraphPad software, Inc). K_i values were determined by the use of Cheng-Prusoff equation.⁴⁹

The selectivity of compounds 4 and 9 was tested by determining their activity against CTX-M-9 β-lactamase, α-chymotrypsin (bovine pancreatic) and cruzain. α-Chymotrypsin and all necessary assay components were purchased from Sigma-Aldrich. Recombinant cruzain was provided by Dr. Rafaela Ferreira and CTX-M-9 by Dr. Yu Chen.

CTX-M-9 activity was measured under the same conditions as with AmpC, with 60 μM CENTA (K_m 35 μM) as substrate. Reactions were initiated by the addition of 0.3 nM enzyme and monitored at 405 nm.

α-Chymotrypsin assays were performed in 50 mM Tris buffer pH 7.5 with 0.01% Triton-X. Inhibitor and 0.001 mg/mL enzyme were incubated at their final concentration for 10 min before the reaction was initiated by the addition of 200 μM N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and monitored at 410 nm.

Cruzain assays were performed in 100 mM sodium acetate pH 5.5 with 0.01% Triton-X and 5 mM dithiothreitol. Cruzain activity was measured in a 96-well format fluorimetric assay, monitoring the cleavage of the substrate Z-Phe-Arg-aminomethylcoumarin (Z-FR-AMC), in a SpectraMax M5, Molecular Devices spectrofluorimeter. Inhibitor and 0.4 nM enzyme were incubated at their final concentration for 10 min before the reactions was initiated by the addition of 2.5 μM Z-FR-AMC (K_m 2 μM) and monitored for five minutes, determining activity based on initial rates.

Modeling

Compound 17 was modeled in complex with AmpC by superposing 17 on the crystal structure of compound 4 in chain A. To remove clashes of the additional m-carboxybenzyl group with the protein, we minimized 17 in the AmpC active site by an all-atom energy minimization using PLOP.⁵⁰ The oxygen atom of Ser64 was allowed to move while the rest of the enzyme was kept rigid during the minimization. This resulted in a model with the phenylmethanesulfonamide conformation resembling the conformation observed for compounds 4 and 9, in which the sulfonamide oxygen atoms pointed in the direction of the m-carboxybenzyl group.

Crystal growth and structure determination

Co-crystals of AmpC in complex with compound 4, 9 and 17 were grown by vapour diffusion in hanging drop vapor diffusion experiments equilibrated over 1.7 M potassium phosphate buffer (pH 8.7–8.9) using microseeding technique. The initial concentration of the protein was 3.9 mg/ml, and the concentrations of the compounds were 625 μM, the DMSO concentration was 1.25%. Crystals appeared 4–7 days after equilibration at 20 °C. Before data collection, crystals were immersed in a cryoprotectant solution of 25% sucrose, 1.7 M potassium phosphate, pH 8.7, for about 30 sec, and were flash cooled in liquid nitrogen.

Diffraction data were measured at beamline 8.3.1 of the Advance Light Source (ALS, Lawrence Berkeley Lab, CA). Reflections were indexed, integrated and scaled using the XDS package.⁵¹ The space group was C2 with two molecules in the asymmetric unit. The initial phasing model was an apo AmpC model (PDB entry 1KE4), with water molecules and ions removed. The model was positioned initially by rigid body refinement and further refined with L-BFGS minimization, simulated annealing, individual B-factor refinement and water picking using PHENIX.⁵² Coot was used for model building and the PRODRG server was used to generate ligand restraints.^{53, 54} Ligand restraints were modified manually because the PRODRG server does not support boron atoms.

Microbiology

Susceptibility testing was performed and interpreted following the guidelines of CLSI.⁴⁰ The compounds were dissolved in 50% DMSO, and dilutions were performed using Muller–Hinton medium. An adequate final concentration was obtained to determine the minimum inhibitory concentrations (MICs). The concentration of DMSO was maintained below 5%. The inhibitors were tested for synergy with the third-generation β-lactams ceftazidime and cefotaxime against several clinical bacteria. The ratio of β-lactams to inhibitors was 1:1. Each value reported reflects the average of three independent experiments. The bacteria exhibited high level of resistance to β-lactams because of the expression of class C or class A β-lactamases. One Escherichia coli strain showed an AmpC-overproduced phenotype and the second one produced the plasmid-mediated class A β-lactamase CTXM-14. Citrobacter freundii, Enterobacter cloacae and Pseudomas aeruginosa strains showed AmpC-derepressed phenotype. Klebsiella pneumoniae isolate produced the inducible plasmid-metiated class C enzyme DHA-1.

For disk diffusion plate assays, bacterial strains were diluted in sterile water to a turbidity equivalent to 0.5 McFarland turbidity standards. After a subsequent 10-fold dilution, the bacterial suspensions were inoculated on Mueller Hinton agar. The plates were dried for 10 min before applying the disks containing cefotaxime or ceftazidime antibiotics (30 μg) or the inhibitor (30 μg) or both. After overnight incubation at 37 °C, the zones of bacterial-growth inhibition were measured.

For the β-lactamase induction experiments, plates of Mueller Hinton agar were inoculated with two clinical strains (E. cloacae and K. pneumoniae) in which production of AmpC-type enzyme is inducible by β-lactam antibiotics. Inhibitors were added to blank disks, and the final content of inhibitor per disk was 64 μg of compounds and 32 μg cefoxitin. Disks of ceftazidime contained 30 μg of antibiotic.