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

Article abstract of DOI:10.1021/jm101015z

We investigated a series of sulfonamide boronic acids that resulted from the merging of two unrelated AmpC β-lactamase inhibitor series. The new boronic acids differed in the replacement of the canonical carboxamide, found in all penicillin and cephalosporin antibiotics, with a sulfonamide. Surprisingly, these sulfonamides had a highly distinct structure-activity relationship from the previously explored carboxamides, high ligand efficiencies (up to 0.91), and K_i values down to 25 nM and up to 23 times better for smaller analogues. Conversely, K_i values were 10-20 times worse for larger molecules than in the carboxamide congener series. X-ray crystal structures (1.6-1.8 A?) of AmpC with three of the new sulfonamides suggest that this altered structure-activity relationship results from the different geometry and polarity of the sulfonamide versus the carboxamide. The most potent inhibitor reversed β-lactamase-mediated resistance to third generation cephalosporins, lowering their minimum inhibitory concentrations up to 32-fold in cell culture.

Full text of DOI:10.1021/jm101015z

7852 J. Med. Chem. 2010, 53, 7852–7863
DOI: 10.1021/jm101015z
Design, Synthesis, Crystal Structures, and Antimicrobial Activity of Sulfonamide Boronic Acids
as β-Lactamase Inhibitors†
Oliv Eidam,‡,^ Chiara Romagnoli,‡,§,^ Emilia Caselli,§ Kerim Babaoglu,‡,# Denise Teotico Pohlhaus,‡,¥ Joel Karpiak,‡
Richard Bonnet, Brian K. Shoichet,*,‡ and Fabio Prati*,§
‡Department of Pharmaceutical Chemistry, Byers Hall, University of California San Francisco, 1700 4th Street, San Francisco, California 94158,
United States, §Dipartimento di Chimica, Universitaꢀ degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy, and
Laboratoire de Bacteꢁriologie-Mycologie-Parasitologie 58, Centre de Biologie, Centre Hospitalier Universitaire, Boulevard Montalembert,
63003, Clermont-Ferrand Cedex 1, France. ^Both authors contributed equally to this work. #Current address: Gilead, Foster City, California,
United States. ¥Current address: GlaxoSmithKline, Raleigh, North Carolina, United States.
Received August 4, 2010
We investigated a series of sulfonamide boronic acids that resulted from the merging of two unrelated
AmpC β-lactamase inhibitor series. The new boronic acids differed in the replacement of the canonical
carboxamide, found in all penicillin and cephalosporin antibiotics, with a sulfonamide. Surprisingly,
these sulfonamides had a highly distinct structure-activity relationship from the previously explored
carboxamides, high ligand efficiencies (up to 0.91), and Ki values down to 25 nM and up to 23 times
better for smaller analogues. Conversely, Ki values were 10-20 times worse for larger molecules than
˚
in the carboxamide congener series. X-ray crystal structures (1.6-1.8 A) of AmpC with three of the
new sulfonamides suggest that this altered structure-activity relationship results from the different
geometry and polarity of the sulfonamide versus the carboxamide. The most potent inhibitor reversed
β-lactamase-mediated resistance to third generation cephalosporins, lowering their minimum inhibitory
concentrations up to 32-fold in cell culture.
Introduction
Boronic acids are transition-state analogues that lack the
β-lactam recognition motif and are distinct enough chemically
to evade pre-evolved resistance mechanisms.17-19 They form
dative, rapidly reversible covalent bonds with the active site
serine, forming a tetrahedral adduct that mimics the high-
energy intermediate of β-lactams along the β-lactamase reac-
tion coordinate (Figure 2).20
We found previously that acylglycyl boronic acids bearing
side chains characteristic of penicillins and cephalosporins (R1
group in Figure 2A) have Ki values as low as 20 nM against
AmpC.19 Using a structure-based drug design approach, we
designed m-carboxyphenylboronic acids having a negatively
charged group in a position corresponding to the C4 of cepha-
losporins, such as cephalothin (Figure 1).21,22 Compound 18c
has a Ki value of 1 nM, improving potency up to 300-fold
compared to the corresponding acylglycylboronic acid 10c
(Table 1).
In the crystal structure of the AmpC/18c complex, the oxy-
gen atom of the carboxamide forms a hydrogen bond with
Asn152, a conserved residue in the AmpC active site, while the
carboxamide nitrogen hydrogen-bonds with the backbone
carbonyl of Ala318, a residue that contributes to the enzyme’s
oxyanion hole (Figure 3A). In an unrelated series of non-
covalent β-lactamase inhibitors, discovered by molecular
docking, a sulfonamide group is similarly positioned and
makes the same interactions (Figure 3B).23
It therefore seemed interesting to replace the ubiquitous
carboxamide, present in the R1 side chain of both β-lactams
and their boronic acid mimics, by a sulfonamide (Scheme 1).
Whereas sulfonamide replacements for carboxamides date
The expression of β-lactamases is the most common cause of
resistance to β-lactam antibiotics, such as penicillins and cepha-
losporins.1-3 β-Lactamases catalyze the hydrolysis of the critical
β-lactam ring in β-lactam antibiotics, thereby inactivating them.4
To overcome this problem, β-lactamase inhibitors, such as
clavulanic acid or sulbactam, are coadministered with the
primary β-lactam (Figure 1).5,6 However, the active core of these
inhibitors remains a β-lactam ring, enabling the rapid develop-
ment of resistance.7 For example, AmpC, a class C β-lacta-
mase expressed by many nosocomial pathogens, is not
inhibited by clavulanic acid or sulbactam at clinically relevant
concentrations, leading tosubstantialproblems inthe clinic.8,9
The broad activity of class C β-lactamases has motivated
a search for novel inhibitors. New departures based on the
β-lactam core have shown substantial, typically mechanism-
based activity in vitro and in vivo as inhibitors.10-13 There is
also a motivation to discover inhibitors structurally unrelated
to β-lactams, which might evade pre-evolved bacterial resis-
tance mechanisms.14-16 Lacking the β-lactam core, these inhib-
itors should not be hydrolyzed by mutant enzymes that arise
in response to new β-lactams, should not be recognized by
β-lactam signaling receptors, and might be unaffected by
porin channel mutations that decrease permeability.2,17
†The coordinates and structure factors have been deposited in the
Protein Data Bank with the accession codes 3O86, 3O87, and 3O88 for
AmpC in complex with 4, 9, and 17, respectively.
*To whom correspondence should be addressed. For B.K.S.: phone, 415-
514-4126; fax, 415-514-4260; e-mail, shoichet@cgl.ucsf.edu. For F.P.: phone,
+39-059-2055056; fax, +39-059-373543; e-mail, fabio.prati@unimore.it.
r
pubs.acs.org/jmc
Published on Web 10/14/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7853
Figure 1. Penicillin and cephalosporin antibiotics and two clinically used β-lactamase inhibitors.
Table 1. Ki Values (μM) of Sulfonamide and Carboxamide Boronic
Acidsa
Figure 2. Boronic acids as transition-state analogues: (A) hydro-
lytic attack on the β-lactam ring of a cephalosporin and formation
of the high-energy intermediate; (B) binding of β-lactamase to a
boronic acid and formation of a transition-state analogue.
back to the seminal synthetic work of Sheehan in the
1950s,24 these derivatives were never used clinically and,
as far as we know, have never been explored among
β-lactamase inhibitors previous to this year. Very recent
work by Tan et al. at Merck revealed two arylsulfonamide
boronic acids with activity in the low micromolar range
a
(IC50 between 0.57 and 5.6 μM) against five different
β-lactamases.25
We synthesized 10 sulfonamide boronic acids and tested
their inhibition of AmpC β-lactamase. Among them are seven
different R1 groups including the penicillin G and nafcillin
side chains. We found that the simplest of these new sulfon-
amides were unexpectedly the most potent (Ki values in the
25 nM range), over 23 times more than their carboxamide
analogues, and that the SAR of this series differed completely
from what was observed in the carboxamide series. To under-
stand these differences, we determined X-ray structures of
three inhibitors in complex with AmpC. Finally, we investi-
gated the efficacy of the most potent inhibitors to reverse
antibiotic resistance in bacterial cell cultures.
a (/) Compounds 3c-11c have been published in ref 19 and 18c in
ref 21. Compound 17c has been synthesized and tested in this study. (#) Not
applicable: compound not obtained. (‡) Compounds 17c and 18c have a
thiophene in the R1 side chain and are therefore not exact analogues of 17
and 18, respectively.
Results
Design. The geometry and electronic properties of a sul-
fonamide are quite different from those of 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 amino-
methaneboronate 2 (Scheme 2). The key intermediate 2 was
obtained by treating the chloromethylboronic acid pinacol
ester 1 with lithium bis(trimethylsilyl)amide.19 In situ depro-
tection of 2 with equimolar methanol, followed by conden-
sation with suitable sulfonyl chlorides, afforded the sulfon-
amidomethaneboronic acids 3-9 and 11.
a Abbreviations: LE, ligand efficiency (=(ΔGbinding (kcal/mol))/
(number of non-hydrogen atoms)); PDB, Protein Data Bank; IC50, half-
maximal inhibitory concentration; SAR, structure-activity relation-
ship; MIC, minimum inhibitory concentration; CLSI, Clinical and
Laboratory Standards Institute; CAZ, ceftazidime; FOX, cefoxitin;
THF, tetrahydrofuran; TLC, thin layer chromatography.
Compound 10 (Table 1), bearing the cephalothin side
chain, could not be obtained because of instability of the

7854 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Eidam et al.
suitable sulfonyl chloride. In fact, several attempts to obtain
the (thiophene-2-yl)methanesulfonylchloride led to the cor-
responding 2-chloromethylthiophene instead, as reported
for other electron rich phenylmethanesulfonyl chlorides.26
According to recent literature reports, related sulfonyl-
aminomethaneboronate compounds27,28 can also be prepared
by alkylation of suitable N-tert-butylsulfonamides with pina-
col 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 protodeboronation during sulfo-
nylation reaction occurred. The loss of the boronic moiety
may occur because of stabilization of the intermediate carb-
anion 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 chloromethyl-
lithium.30 Subsequent Matteson stereoselective homologa-
tion with dichloromethyllithium,29,31-33 followed by sub-
stitution with bis(trimethylsilyl)amide and sulfonylation
afforded compounds 15a,b. The use of (þ)-pinanediol as
chiral auxiliary allowed the formation of the final R config-
uration, which mimics the stereochemistry of carbon atom
C6/7 of penicillin/cephalosporin. The conversion of pinane-
diol esters 15a,b to free boronic acids 16 and 17 was achieved
through transesterification with phenylboronic acid in a bi-
phasic acetonitrile/water system.34
Enzymology and Binding Affinities. Except for those bind-
ing 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 was detected for any of the
compounds.
To investigate the effect of the carboxamide replacement
with a sulfonamide, we first measured the potency of methane-
sulfonamide boronic acid 3 (Table 1). Compound 3 inhibits
AmpC with a Ki of 789 nM, a 23-fold improvement of
potency compared to the carboxamide analogue 3c (Ki =
18.5 μM). The high affinity of this relatively small molecule
prompted us to study larger derivatives. Compound 4, bear-
ing the penicillin G side chain has a Ki of 70 nM, a 10-fold
improvement over the initial sulfonamide 3 and an 8-fold
improvement compared to its exact carboxamide analogue
4c (Ki = 570 nM) (Table 1).19 Because compound 5, isomer
of compound 4, has a Ki of 210 nM, we decided to focus on
benzylic derivatives and synthesized compounds 6-9, for
which required sulfonylchlorides were commercially avail-
able or the final products easily accessible. Although most of
those derivatives have a lower potency than 4, compound 9
Figure 3. Inspiration for sulfonamide boronic acids. (A) The
carboxamide of boronic acid 18c hydrogen-bonds with Asn152 and
Ala318 of AmpC β-lactamase (PDB code 1MXO). Carbon atoms
of inhibitor are colored green, oxygens red, nitrogens blue, sulfur
yellow, boron atom amaranth pink. (B) The sulfonamide of a
noncovalent β-lactamase inhibitor hydrogen-bonds with Asn152,
Ala318, Ser64, and Lys67 (PDB code 1L2S). Carbon atoms of inhib-
itor are colored cyan, oxygens red, nitrogens blue, sulfur yellow,
chloride green.
Scheme 1. Conversion of Carboxamide Boronic Acids into
Sulfonamide Boronic Acids
Scheme 2. General Scheme of the Synthesis of Sulfonamidomethaneboronic Acids
Scheme 3. Asymmetric Synthesis of 1-Sulfonamido-2-phenylethaneboronic Acids 16 and 17

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7855
Table 2. Data Collection and Refinement Statistics
AmpC/4
AmpC/9
AmpC/17
PDB code
3O86
3O87
3O88
Data Collection
space group
cell dimension
C2
C2
C2
˚
a, b, c (A)
118.34, 76.88, 97.76
90.00, 116.11, 90.00
30-1.60 (1.69-1.60)a
4.5 (44.0)a
118.13, 76.19, 97.38
90.00, 116.16, 90.00
40-1.78 (1.88-1.78)a
5.8 (45.5)a
118.03, 77.39, 97.41
90.00, 115.90, 90.00
30-1.64 (1.72 - 1.64)a
4.4 (44.8)a
R, β, γ (deg)
˚
resolution (A)
Rmerge (%)
completeness (%)
I/σ(I)
99.0 (98.6)a
97.5 (95.7)a
98.5 (99.5)a
16.4 (3.1)a
3.85 (3.81)a
12.6 (2.7)a
2.95 (2.95)a
15.3 (2.6)a
3.05 (3.05)a
redundancy
Refinement
˚
resolution (A)
29.58-1.60
102 732
16.7/19.3
38.43-1.78
72 662
18.8/23.2
29.54-1.64
95 179
18.1/21.0
no. reflections
R
work/Rfree (%)
no. atoms
proteinb
ligandb
water
5540
30
5448
36
5595
50
819
519
744
2
˚
B-factor (A )
proteinb
ligandb
water
rms deviation
20.97
26.41
33.62
22.31
26.32
31.03
24.59
32.02
35.15
˚
bond length (A)
bond angle (deg)
0.010
1.323
0.011
1.355
0.011
1.374
a Values in parentheses represent values for the highest resolution shells. b Calculated for both molecules in asymmetric unit.
has a Ki of 25 nM, a 3-fold improvement versus compound 4.
We tried to synthesize compound 10, the exact analogue
of the carboxamide 10c, but found that the required
(thiophene-2-yl)methanesulfonylchloride was unstable. Un-
expectedly, compound 11, bearing the nafcillin side chain,
inhibits AmpC with a Ki of 670 nM, 20-fold weaker than the
carboxamide analogue 11c (Ki = 33 nM). This was another
indicator that the SAR changes substantially in the sulfon-
amide series.
In the carboxamide series, addition of a m-carboxybenzyl
group to 10c had resulted in a 7-fold improvement of potency
for compound 17c. We decided to investigate the same modi-
fication for the sulfonamide 4 and synthesized the chiral
compound 17. This latter has a Ki of 430 nM; thus, rather
than a 7-fold improvement, adding a m-carboxybenzyl to
compound 4 led to a 6-fold decrease in inhibition. Com-
pound 16, only with a benzyl as R2 group, has an even worse
Ki 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
cocrystal 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 un-
ambiguously identified in both chains in the initial Fo - Fc
difference density maps (Figure 4). With the exception of the
complex with compound 4, the conformations of the inhib-
itors were very similar in both monomers.
key hydrogen bond interactions in the active site closely
resembled those typically observed in β-lactamase struc-
tures with transition-state analogues 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 back-
bone 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 dis-
tance to the conserved water molecule Wat402.
The crystal structure of compound 4 (Ki = 70 nM) in
˚
complex with AmpC was determined at 1.60 A (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 hydro-
˚
gen-bonds to Asn152 (nitrogen-oxygen distance: 2.6 A).
The other sulfonamide oxygen atom hydrogen-bonds with a
water molecule bridging to Gln120. The benzyl group makes
a T-shaped π-π interaction (90ꢀ 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 3-fold increase affinity of compound 9
(Ki = 25 nM) compared to compound 4, we determined
˚
the crystal structure of the AmpC/9 complex to 1.78 A
resolution (Figure 5B). The conformation of 9 is similar in
both monomers and resembles the conformation of 4 ob-
served in chain A except that the plane of its phenyl ring is
slightly offset 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
For all three inhibitors, electron density connected the
Oγ of the catalytic Ser64 to the boron atoms of the inhib-
itors. The boron geometry was tetrahedral, as expected, and

7856 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Eidam et al.
Figure 4. Stereoviews of initial Fo - Fc electron densities (green, 2.5σ contour level) of sulfonamide boronic acids and final 2Fo- Fc densities
(blue, 1σ contour level) of AmpC and conserved water molecules: (A) compound 4 in chain A; (B) compound 9; (C) compound 17. Inhibitor
carbons are in green, enzyme carbons in gold, oxygens in red, nitrogens in blue, sulfurs in yellow, borons in amaranth pink.
nitrogen of Ser212. The other carboxylate oxygen atom
hydrogen-bonds with an ordered water molecule.
but in a different conformation than observed in the AmpC/4
and AmpC/9 complex structures.
To understand the reduced activity of 17 (Ki = 430 nM)
against AmpC relative to compounds 4 and 9, we determined
˚
its crystal structure in complex with AmpC at 1.64 A resolu-
Antimicrobial Activity. The antiresistance activity of inhib-
itors 4 and 9 was investigated against clinical bacteria exhib-
iting high level of resistance against third-generation cepha-
losporins, such as ceftazidime, via expression of class A or
class C β-lactamases. When the compounds were admin-
istrated alone in a disk diffusion assay, they had little or no
detectable activity on bacteria growth, as expected. How-
ever, in combination with ceftazidime the compounds pro-
duced 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 inhi-
bition 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
tion (Figure 5C). In this complex, the boron-carbon bond is
rotated by 90ꢀ compared to the conformation adopted by
compound 4. This is necessary to accommodate the addi-
tional m-carboxybenzyl R2 group of 17. Consequently, the
sulfonamide is reoriented in the active site, placing the ben-
zylsulfonamide in a totally different location relative to com-
pounds 4 and 9 (Figure 5E). One sulfonamide oxygen of
compound 17 makes stretched hydrogen bonds with Asn152
˚
and Lys67 (3.2 and 3.1 A, respectively), and the nitrogen
atom of the sulfonamide hydrogen-bonds with the backbone
˚
amide of Ala318 (3.1 A), an interaction missed in the other
two structures. Conversely, the second oxygen atom now
˚
abuts the backbone carbonyl of Ala318 (2.8 A) 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

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7857
Figure 5. Polar interactions of sulfonamide boronic acids with AmpC and comparison of conformations. Enzyme residues are depicted
in atom colors (gray carbons, red oxygens, blue nitrogens). Hydrogen bonds are shown as red dashes, water molecules as red spheres.
(A) Compound 4 (green carbon atoms) in chain A. (B) Compound 9 (cyan carbon atoms). (C) Compound 17 (magenta carbon atoms).
(D) Superposition of compound 9 on compound 4. (E) Superposition of compound 17 on compound 4. (F) Superposition of a model of 17
(cyan carbon atoms) on its actual crystal structure (magenta carbon atoms). See Discussion.
(CLSI) standards.40 The compounds had no measurable
antibiotic activity when used alone. In combination with
β-lactams, the compounds decreased MIC values by 8-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 sulfon-
amide boronic acids may inhibit both class C β-lactamases
and class A enzymes, consistent with studies revealing activ-
ity against this class of enzymes in the low micromolar range
described in this study (Table 4 and below) and results from
Tan and colleagues.25 More detailed studies will be necessary
before inhibition of the class A β-lactamases can be shown to
be broad or robust.
A traditional β-lactam resistance mechanism is the up-
regulation 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 potenti-
ate the action of the β-lactam ceftazidime. We compared
the potentiation effect of compounds with that of cefoxitin
(Figure 6B and Figure 6C). Agar plates were inoculated with
K. pneumoniae and E. cloacae strains in which AmpC expres-
sion 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 the cefoxitin disk. This well-known antag-
onist 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 up-regulation
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 R-chymotrypsin, the cysteine protease cru-
zain, and the class A β-lactamase CTX-M-9, which is struc-
turally different from AmpC (Table 4). Compounds 4 and 9
showed no measurable inhibition at 100 μM against cruzain
and R-chymotrypsin. Interestingly, the compounds are ac-
tive against CTX-M-9 with IC50 values in the low micro-
molar range. The Ki values corresponding to the IC50 values
are 2.529 and 0.552 μM, respectively, indicating 36-fold
and 22-fold selectivity toward AmpC versus CTX-M-9
β-lactamase.
Discussion
Three key observations emerge from this study. First, the
new sulfonamide boronicacidsderived fromthe conversionof
the canonical R1 carboxamide retain substantial inhibition
activity against β-lactamases. Second, they rescue antibiotic
resistance when used in combination with third generation
antibiotics in bacterial cell cultures. Third, this superficially
modest substitution changes the geometry of the inhibitors
enough to scramble the SAR observed in the analogue
carboxamides.
A favorable changein the SARwas observed for the smaller
compounds of this study. The most substantial change in
activity was observed for compound 3, which has a Ki of 789
nM against AmpC. Its exact carboxamide analogue 3c is 23-
fold less potent (18.5 μM). The high affinity of 3 corresponds

7858 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Eidam et al.
to a ligand efficiency of 0.91, placing it among the most
efficient of enzyme inhibitors. Given the simplicity of this
inhibitor, it seems clear that its improved affinity can be laid
at the door of the advantages of the sulfonamide versus the
carboxamide group in the AmpC site; this in turn may reflect
the improved hydrogen bonds in this site owing to differences
in polarity and geometry. This advantage was maintained
during the SAR development among the smaller sulfonamide
analogues, with potency improving 11-fold in compound 4
(Ki =70 nM) and by another 3-fold in compound 9 (Ki =
25 nM). The X-ray complexes of these inhibitors underscore
the favorable hydrogenbonds made between themand canon-
ical recognition residues.
Conversely, in larger sulfonamides the SAR is inverted with
affinities generally getting worse and relative to the carbox-
amides getting much worse. An example for the inversed SAR
is the low affinity of the sulfonamide with the nafcillin side
chain. Compound 11 is a 670 nM inhibitor compared to the
carboxamide analogue 11c with a Ki of 33 nM. This result
suggests that adding a bulky side chain to the sulfonamide
weakens, if not completely disrupts, the hydrogen bonds with
residues Asn152 and Gln120 observed in the crystal struc-
tures of compounds 4 and 9. Favorable stacking and van der
Waalsinteraction of theelaborated arylside chain mayalso be
affected negatively.
We observed another change in the SAR when we tested
compounds 16 and 17, having a benzyl and m-carboxybenzyl
as R2 group, respectively. In the carboxamide boronic acids,
adding am-carboxybenzylimproved affinity8-foldfrom com-
pound 10c to 17c. In our sulfonamide series, the same modi-
fication (compound 4 to 17) leads to a 6-fold drop in potency.
In the structure of the AmpC/17 complex, the sulfonamide
hydrogen bonds have been completely disrupted and rear-
ranged versus those made by 4 and 9 (Figure 5E). To under-
stand the flip of the sulfonamide, we modeled compound 17
in the conformation observed for compound 4 (Figure 5F).
The protein-inhibitor interactions in this model looked quite
reasonable, but the ligand internal interactions were strained,
with the aliphatic carbon in the m-carboxybenzyl group very
˚
close to one of the sulfonamide oxygens (3.0 A). We reasoned
that internal 1,5 repulsion between those two atoms could
force the reorientation of the sulfonamide. We therefore
calculated internal energies for both the model and the crys-
tal structure of compound 17 using the semiempirical QM
method AMSOL.41 The internal energy of the ligand alone in
the crystal structure conformation (-90.89 kcal/mol) was
substantially lower than the internal energy of the modeled
ligand (-55.08 kcal/mol), consistent with the internal strain
hypothesis.
The unexpected SAR of these inhibitors suggests that
we cannot simply adopt lessons learned from earlier series.
Perhaps the most encouraging observation to emerge is that
sulfonamide boronic acids with small R1 side chains are very
active against AmpC and can substantially reverse antibiotic
resistance in bacterial cell culture. In addition, their activity
against other β-lactamases classes, as shown here and by
Tan et al.,25 supports further studies on this new class of
β-lactamase inhibitors.
Figure 6. Inhibition of bacterial growth and potentiation effect.
(A) Activity of compounds 4 (CP4) and 9 (CP9) in combination with
ceftazidime (CAZ) and alone against a Klebsiella pneumoniae strain
producing AmpC β-lactamase. Solvent (SOL) was DMSO/water
1:1. (B) Potentiation of β-lactamase inhibition in an E. cloacae strain
in which ampC gene expression is inducible by β-lactam antibiotics,
such as cefoxitin (FOX). (C) Potentiation of β-lactamase inhibition
in a Klebsiella pneumoniae isolate producing the inducible class C
enzyme DHA-1.
Table 3. Minimum Inhibitory Concentrations of Third-Generation Cephalosporins Alone and in Association with Compound 4 or 9 (Ratio 1:1) for
Clinical Bacteria Exhibiting a High Level of Resistance
ceftazidime
(μg/mL)
ceftazidime and
4 (μg/mL)
ceftazidime and
9 (μg/mL)
cefotaxime
(μg/mL)
cefotaxime and
4 (μg/mL)
cefotaxime and
9 (μg/mL)
E. colia
64
64
64
32
32
2
4
4
4
4
8
2
8
4
4
8
4
1
8
64
1
4
2
8
E. cloacaea
C. freundiia
P. aeruginosaa
K. pneumoniaeb
E. colic
16
2
4
>128
8
256
16
4
32
2
16
8
a Bacteria overproducing chromosomally mediated class C β-lactamase. b K. pneumoniae producing the plasmid-mediated class C β-lactamase
DHA-1. c E. coli producing plasmid-mediated class A β-lactamase CTX-M-14.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7859
Table 4. Selectivity of 4 and 9 for AmpC versus other Amide Hydrolases
4.40 (1H, br, SO2NH), 4.27 (2H, s, PhCH2), 7.39 (5H, s, HArom).
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 (Mþ, 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.
enzyme
AmpC
CTX-M-9
IC50 (μM) for 4
IC50 (μM) for 9
0.38
6.87
>500a
>500a
0.13
1.50
>500a
>500a
R-chymotrypsin
cruzain
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 (Mþ-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)methaneboro-
nate (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 (Mþ - 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 dis-
solved in HCl (0.1 M solution in MeOH, 3.6 mL, 0.36 mmol).
After 45 min the mixture was concentrated under reduced pres-
sure and the residue repeatedly washed with diethyl ether, afford-
ing 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, HArom), 7.46 (1H, t, J = 7.60,
HArom), 9.60-10.60 (3H, br, NH3þ). 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 (Mþ, 5%), 156 (8), 107 (64), 106
(100), 104 (15), 77 (11), 59 (11), 41 (8). Anal. Calcd for C14H24-
BClN2O4S: 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.
a No inhibition was observed at 100 μM. The IC50 values assume that
inhibition was no greater than 20% at this concentration.
Methods
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 stan-
dard 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 precooled (-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 Buchi 510 apparatus.
Optical rotations were recorded at þ20 ꢀ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 stan-
dard (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;
results from elemental analyses for the compounds were within
(0.3% of the theoretical values. Data from MS fragmentation
and elemental analyses of free boronic acids were not obtainable
because of the formation of dehydration products. Neverthe-
less, these boronic acids could be converted into analytically
pure pinacol/pinanediol esters by exposure to an equimolar
amount of pinacol/pinanediol in anhydrous THF.
General Procedure for the Synthesis of Sulfonylaminomethan-
eboronates (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 being
stirred for 10 min at -10 ꢀC, the cooling bath was removed.
The reaction mixture was 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 evapo-
rated in vacuo and the residue purified by chromatography
(dichloromethane/diethyl ether 8:2 to methanol), affording the
expected sulfonamides.
Pinacol [4-(Methoxycarbonyl)phenylmethanesufonylamino]-
methaneboronate (8). According to the general procedure de-
scribed above, the compound was recovered as a white solid
(84% yield). Mp 104-107 ꢀ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). 13
C
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 (Mþ - 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
Pinacol (Methanesulfonylamino)methaneboronate (3). Accord-
ing 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 (Mþ -15, 7%), 171 (29), 156 (24), 143 (21), 136 (39), 119
(14), 104 (100), 103 (44), 83 (37), 74 (6). Anal. Calcd for C8H18-
BNO4S: 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),

7860 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Eidam et al.
(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 expo-
sure of 9 to an equimolar amount of pinacol in anhydrous THF
afforded the corresponding pinacol boronate in quantitative
yield and satisfactory elemental analysis results. 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-Ethoxynaphthalene-1-sulfonylamino)methaneboronic Acid
(11). According to the general procedure described above,
reaction of 2 with 2-ethoxynaphthalene-1-sulfonyl chloride, ob-
tained 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, HArom), 7.29
(1H, s, HArom), 7.70-7.91 (3H, m, HArom), 8.32 (1H, s, HArom).
13C 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 quantita-
tive 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.
(þ)-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
solution 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, (þ)-pinanediol (1.12 g, 6.61 mmol) was added and the
solution stirred 1 h at room temperature. The mixture was par-
titioned 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 petro-
leum/ethyl ether 95:5), affording 12b as a yellowish solid (2.10 g,
89% yield). Mp 70-71 ꢀC. [R]D þ7.3 (c 1.1, CHCl3). 1H NMR
(400 MHz, CDCl3): δ 0.94 (3H, s, pinanyl CH3), 1.24 (1H, d, J =
10.9, pinanyl Hendo), 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 (Mþ,
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.
(þ)-Pinanediol Phenylmethaneboronate (13a). n-Butyllithium
(2.5 M solution in hexane, 4.66 mL, 11.64 mmol) was added
dropwise to a stirred solution of (þ)-pinanediol benzeneboro-
nate 12a21 (2.48 g, 9.70 mmol) and bromochloromethane (0.95
mL, 14.55 mmol) in THF (25 mL) at -80 ꢀC under argon atmo-
sphere. 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
(2ꢀ40 mL). The organic phases were dried, filtered, and con-
centrated, 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).
[R]D þ14.2 (c 2.0, CHCl3). 1H NMR (200 MHz, CDCl3): δ 0.87
(3H, s, pinanyl CH3), 1.10 (1H, d, J = 10.8, pinanyl Hendo), 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, HAr). 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 (Mþ þ 1,
2%), 270 (Mþ, 81%), 269 (Mþ - 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.
(þ)-Pinanediol [3-(tert-Butoxycarbonyl)phenyl]methaneboro-
nate (13b). Following the procedure described for the synthesis
of 13a, compound 13b was recovered from 12b as a yellow oil
1
(88% yield). [R]D þ8.7 (c 1.4, CHCl3). H NMR (400 MHz,
CDCl3): δ 0.87 (3H, s, pinanyl CH3), 1.11 (1H, d, J = 11.0,
pinanyl Hendo), 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
(Mþ, 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.
(þ)-Pinanediol (1R)-1-(N-Bis(trimethylsilyl)amino)-2-phenyl-
ethaneboronate (14a). n-Butyllithium (2.5 M solution 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 was 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 af-
forded (þ)-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 solution in tetrahydro-
furan, 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 con-
centrated 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 petro-
leum/ethyl ether/triethylamine 98:2:5) recovering 14a as a pale
1
yellow oil (837 mg, 51% yield). [R]D -14.8 (c 2.3, CHCl3). H
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 Hendo), 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, HArom). 13
C
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.
(þ)-Pinanediol (1R)-2-(3(tert-Butoxycarbonyl)phenyl)-1-(N-
bis(trimethylsilyl)amino)ethaneboronate (14b). Following the
procedure described for the synthesis of 14a, compound 14b
was recovered as a colorless oil (28% yield). [R]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,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7861
pinanyl Hendo), 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.
(þ)-pinanediol in anhydrous THF afforded compound 15a in
quantitative yield and satisfactory elemental analysis results.
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 solution in dichloro-
methane, 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 (þ)-pinanediol (1R)-2-[3-carboxyphenyl]-
1-(phenylmethanesulfonylamino)ethaneboronate (79 mg,
100% yield) as a cream colored solid. Mp 155-158 ꢀC. [R]D
þ25.9 (c 0.6, CHCl3). 1H NMR (400 MHz, CDCl3): δ 0.87 (3H,
s, pinanyl CH3), 1.06 (1H, d, J=10.8, pinanyl Hendo), 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, HArom), 7.97-8.01 (2H, m, HArom), 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.
(þ)-Pinanediol (1R)-1-(Phenylmethanesulfonylamino)-2-phe-
nylethaneboronate (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 tem-
perature and stirred overnight. The solution was partitioned
between diethyl ether (30 mL) and water (10 mL). The aqueous
phase was 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 chro-
matography (dichloromethane/diethyl ether 6:4), affording 15a
as a thick yellowish oil (343 mg, 40% yield). [R]D þ9.7 (c 1.6,
1
CHCl3). H NMR (400 MHz, CDCl3): δ 0.85 (3H, s, pinanyl
CH3), 1.10 (1H, d, J = 10.8, pinanyl Hendo), 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, HArom). 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-(phenyl-
methanesulfonylamino)ethaneboronate (15b). Following the pro-
cedure described for 15a, compound 15b was recovered as a
yellowish oil (35% yield). [R]D þ18.7 (c 0.7, CHCl3). 1H NMR
(400 MHz, CDCl3): δ 0.82 (3H, s, pinanyl CH3), 1.00 (1H, d, J =
10.9, pinanyl Hendo), 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, HArom), 7.83-7.85 (2H, m,
HArom). 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) were sequentially added HCl (1 M aqueous solution,
350 μL, 0.35 mmol), phenylboronic acid (62 mg, 0.51 mmol),
and n-hexane (3 mL). The resulting biphasic solution was
vigorously stirred. After 30 min the n-hexane layer, containing
the pinanediol phenylboronate, was removed and fresh n-hex-
ane (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 afford-
ing 16 as a yellowish solid (159 mg, 92% yield). Mp 107 ꢀC (dec).
[R]D þ78.0 (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, HArom). 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 results were
not obtainable, but exposure of 16 to an equimolar amount of
This compound was then treated according to the procedure
described for 16 to recover 17 as a yellowish solid (54 mg, 92%
1
yield). Mp 118-122 ꢀC (dec). [R]D þ136.0 (c 0.5, MeOH). H
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 (þ)-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 dis-
solved in 100% DMSO at 100 mM. More diluted stocks were
prepared as necessary. AmpC activity was determined by mon-
itoring the change of absorbance at 405 nm due to hydrolyzed
substrate CENTA (Km = 15 μM) by an HP5453 UV-vis spec-
trophotometer. The enzyme was expressed and purified as
described,45 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 nonspecific inhibition via compound aggre-
gation.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 s of
each reaction was 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.).
Ki values were determined by the use of the Cheng-Prusoff
equation.49
The selectivity of compounds 4 and 9 was tested by determin-
ing their activity against CTX-M-9 β-lactamase, R-chymotryp-
sin (bovine pancreatic), and cruzain. R-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 (Km = 35 μM) as substrate.
Reactions were initiated by the addition of 0.3 nM enzyme and
monitored at 405 nm.

7862 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Eidam et al.
R-Chymotrypsin assays were performed in 50 mM Tris buff-
er, 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-amino-
methylcoumarin (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 were initiated by the addition of 2.5 μM Z-FR-AMC
(Km = 2 μM) and monitored for 5 min, determining activity
based on initial rates.
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 con-
tained 30 μg of antibiotic.
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-carboxy-
benzyl 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 re-
sulted in a model with the phenylmethanesulfonamide confor-
mation resembling the conformation observed for compounds 4
and 9, in which the sulfonamide oxygen atoms pointed in the
direction of the m-carboxybenzyl group.
Acknowledgment. This work was supported by NIH Grant
GM63815 (B.K.S. and F.P.), SNF Fellowship PBZHP3-
123277 (O.E.), and Fondazione Cassa di Risparmio di Modena
(F.P., C.R., and E.C.). We thank Drs. Magdalena Korczynska
and Kristin Ziebart for reading this manuscript, Dr. Claudia
Ori for analytical support, and the Centro Interdiparti-
mentale Grandi Strumenti of Modena for NMR and MS
spectra.
References
Crystal Growth and Structure Determination. Cocrystals of
AmpC in complex with compound 4, 9, and 17 were grown by
vapor diffusion in hanging drop vapor diffusion experiments equil-
ibrated 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. The concentrations of the compounds
were 625 μM, and the DMSO concentration was 1.25%. Crys-
tals 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 s 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 inter-
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Muller-Hinton medium. An adequate final concentration was
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5%. The inhibitors were tested for synergy with the third-
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β-lactamases. One Escherichia coli strain showed an AmpC-
overproduced phenotype, and the second one produced the
plasmid-mediated class A β-lactamase CTX-M-14. Citrobacter
freundii, Enterobacter cloacae, and Pseudomas aeruginosa strains
showed AmpC-derepressed phenotype. Klebsiella pneumoniae
isolate produced the inducible plasmid-metiated class C enzyme
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