D. Tondi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3416–3419
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Table 1
Phenyl boronic acid used for structural comparative analysis of AmpC b-lactamase binary complexes
1
2
3
4
5
O
O2N
COOH
COOH
OH
HO
N
H
B
S
S
O
B
O
OH
HO
B
S
B
HO
OH
O
HO
OH
B
HO OH
B
HO
OH
Ki (l
M)
Ki (l
M)
Ki (lM)
Ki (
lM)
Ki (lM)
0.083
1.7
2.9
4.2
0.20
Five X-ray crystallographic complexes were selected for
comparison and analysis: AmpC_compound 1 (pdb code GA9),
AmpC_3-nitrophenyl boronic acid, compound 2 (1KDS), AmpC_
4-carboxyphenyl boronic acid, compound 3 (1KDW), AmpC_4-
(2-carboxy-vinyl)-phenyl boronic acid, compound 4 (1KE0) and
AmpC_biphenyl-4,4’-diboronic acid, compound 5 binary com-
plexes (1KE3) (Table 1).10
of 1 while the 4-carboxy group is hydrogen bonded to Gln120
and to an ordered water molecule. Moreover, a polar interaction
is acquired with Asn152 (Fig. 2). The other two complexes, one
bearing a 4-boronic acid-phenyl group in the para position (5,
IKE3) and the other with a propenoic acid (4, IKE0) group in the
comparable site, show interactions with Gln120 accompanied by
large conformational changes (Fig. 2).
When all five complexes were superimposed for comparison,
conserved binding sites, already described as critical for the
enzyme’s molecular recognition, were clearly visible: the amide
site, a hydroxyl site, a hydrophobic site, an aryl group site, a
carboxylate site, and several conserved water sites (Fig. 2).10 The
unique functionalities that characterize each of the inhibitors make
relatively few interactions with the protein. The nitro group of 2
does not make any interactions with AmpC. The carboxylate group
The comparative analysis of the five selected crystal structures
identified the para-phenyl position as the most convenient site to
introduce appropriate chemical groups to modulate the PK proper-
ties of compound 1. Our analysis highlighted Gln120 as having
high conformational freedom which would allow the binding site
to accommodate bulkier derivatives (Fig. 2). That said, a hydro-
philic group introduced at the para position could interact with
Gln120 and modulate the PK properties of the final designed mol-
ecule. We chose to derivatize lead 1 with a para-carboxylic group
that could fit the site and has the optimal properties to improve
PK properties; therefore a small set of compounds (10–14) was de-
signed and synthesized as 4-carboxy-derivatives of 1. We consid-
ered that moving from lead 1 to the corresponding carboxy
derivative 11 would considerably decrease the lipophilicity
(Log D = +2.32 for 1; Log D = +0.026 for 11 at pH 7.4).16
of compound 3 is 3.3 Å from Ne2 of Gln120 while that in com-
pound 2 is only 2.9 Å away due to the different conformation of
Gln120. The carboxylate group in compound 4 interacts with a sin-
gle ordered water molecule. In contrast, the structure of AmpC/
compound 5 shows specific polar interactions between the inhibi-
tor and the enzyme. One hydroxyl of the terminal boronic acid
group interacts with both atom Ne2 of Gln120 and atom Od1 of
Asp123 (2.5 and 2.9 Å, respectively). In the analyzed complex,
the para-phenyl position is oriented towards the entrance of the
AmpC binding site, in proximity to Gln120 (distances ranging from
3.05 and 5.35 Å). In particular, 1KDW, AmpC/3 binary complex
shows that the phenyl ring is oriented exactly as the phenyl ring
Compounds 11–14 were synthesized using 3-amino-4-carboxy-
phenyl boronic acid (10) prepared in house as a starting scaffold as
described in Scheme 1 (Table 2).18
When compared to their non-carboxylate analogs (6–9), the
affinities of the new derivatives 10–14 for AmpC increased
between 2.6–4.5 fold, with the exception of 12 which performed
worse with respect to its analog. For all new compounds, the pre-
dicted Log D improved because of the presence of the carboxylate
group.
The X-ray crystallographic structure of 11 in complex with
AmpC b-lactamase, determined to 2.1 Å resolution, was obtained.
The coordinates and the structure factors have been deposited with
the RCSB Protein Data Bank (PDB) database under accession code
3BM6.18,19 X-ray analysis confirmed our design hypothesis and
the predicted binding geometry (Fig. 3). Overall, the complex with
11 closely resembled the AmpC/1 complex, with a few differences.
CH3
COOH
COOH
COOH
H
N
NO2
NO2
NH2
R
S
O2
b
a
c
B
B
B
B
HO
OH
HO
OH
HO
OH
HO
OH
Figure 2. Superimposition of the five complexes used in the design of compound 1
follows up. The flexibility of Gln120 and then plasticity of AmpC in allocating
functional groups different for chemistry and size is highlighted. Picture was
generated using Pymol.17
10
11-14
Scheme 1. Reagents and conditions: (a) KMnO4, NaOH (0.26 M), 50 °C; (b) EtOH,
MeOH, Pd/C 10%, H2 (2 atm), rt; (c) NaHCO3 (0.5 M), acetone, RSO2Cl, rt, NaOH (2 N).