Design, synthesis and biological evaluation of non-covalent AmpC β-lactamases inhibitors

Article abstract of DOI:10.1007/s00044-017-1809-x

Abstract: Bacterial resistance represents a worldwide emergency threatening the efficacy of all available antibiotics. Among the several resistance mechanisms developed by bacteria, β-lactamase enzymes?(BLs), which are able to inactivate most β-lactam core antibiotics, represent a key target to block, thus prolonging antibiotics half-life. Several approaches aimed at inhibiting β-lactamases have been so far undertaken, mainly involving β-lactam-like or covalent inhibitors. Applying a structure-based de novo design approach, we recently discovered a novel, non-covalent and competitive inhibitor of AmpC β-lactamase:?lead 1. It has a K_i of 1 μM, a ligand efficiency of 0.38 kcal mol^?1 and lead-like physical properties. Moreover, it reverts resistance to ceftazidime in bacterial pathogens expressing AmpC and does not up-regulate β-lactamases expression in cell culture. Its features make it a good candidate for chemical optimization: starting from lead 1 crystallographic complex with AmpC, 11 analogs were designed to complement additional AmpC sites, then synthesized and tested against clinically resistant pathogens. While the new inhibitors maintain similar in vitro activity as the starting lead, some of them, in biological assays, extert a higher potency showing improved synergic activity with ceftazidime in resistant clinically isolated strains. Graphical Abstract: [InlineMediaObject not available: see fulltext.].

Full text of DOI:10.1007/s00044-017-1809-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1809-x
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of non-covalent AmpC
β-lactamases inhibitors
Filippo Genovese1 Sandra Lazzari1 Ettore Venturi1 Luca Costantino1
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Jesus Blazquez2,3 Claudia Ibacache-Quiroga2,3 Maria Paola Costi1
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1
Donatella Tondi
Received: 18 May 2016 / Accepted: 13 February 2017
© Springer Science+Business Media New York 2017
Abstract Bacterial resistance represents a worldwide emer-
gency threatening the efficacy of all available antibiotics.
Among the several resistance mechanisms developed by
bacteria, β-lactamase enzymes (BLs), which are able to
inactivate most β-lactam core antibiotics, represent a key
target to block, thus prolonging antibiotics half-life. Several
approaches aimed at inhibiting β-lactamases have been so far
undertaken, mainly involving β-lactam-like or covalent inhi-
bitors. Applying a structure-based de novo design approach,
we recently discovered a novel, non-covalent and competitive
inhibitor of AmpC β-lactamase: lead 1. It has a Ki of 1 µM, a
ligand efficiency of 0.38 kcal mol−1 and lead-like physical
properties. Moreover, it reverts resistance to ceftazidime in
bacterial pathogens expressing AmpC and does not up-
regulate β-lactamases expression in cell culture. Its features
make it a good candidate for chemical optimization: starting
from lead 1 crystallographic complex with AmpC, 11 analogs
were designed to complement additional AmpC sites, then
synthesized and tested against clinically resistant pathogens.
While the new inhibitors maintain similar in vitro activity as
the starting lead, some of them, in biological assays, extert a
higher potency showing improved synergic activity with
ceftazidime in resistant clinically isolated strains.
Graphical Abstract
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Keywords Structure-based de novo design Thiophene
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derivatives Non covalent β-lactamases inhibitor
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Synthesis Minimum inhibitory concentration (MIC)
Introduction
Microbial resistance to antibiotics is a serious threat to
public health, menacing available antibiotics armamentar-
ium (Rice and Bonomo 2000); (Cosgrove and Carmeli
2003). The most widespread resistance mechanism to β-
lactam antibiotics, such as penicillins and cephalosporins,
remains the expression of β-lactamase enzymes (BLs), able
to cleave the lactam bond present in the β-lactam ring of
these drugs, inactivating them (Tondi et al. 2016); (Farina
et al. 2014); (Bush and Jacoby 2010). β-lactamases threa-
tening last resort antibiotics represents a worldwide emer-
gency (WHO global report 2014).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1809-x) contains supplementary material,
which is available to authorized users.
* Donatella Tondi
tondid@unimore.it
1
Department of Life Sciences, University of Modena and Reggio
Emilia, Via Campi 103, Modena 41125, Italy
2
Among β-lactamases, AmpC BLs are clinically relevant
cephalosporinases, chromosomally encoded and mediating
Enterobacteriaceae resistance to cephalothin, cefazolin,
cefoxitin, most penicillins, and β-lactam-β-lactamases
Instituto de Biomedicina de Sevilla, Calle Antonio Maura
Montaner, Seville 41013, Spain
3
National Centre for Biotechnology-CSIC, Campus of
Cantoblanco, Madrid 28049, Spain

Med Chem Res
inhibitor combinations (Jacoby 2009); (Drawz and Bonomo
2010). In many bacteria, AmpC enzymes are inducible and
can be expressed at high levels due to mutation, thus,
conferring resistance to broad-spectrum last generation
cephalosporins. Resistance due to plasmid-mediated AmpC
enzymes is particularly dangerous in terms of cross-species
spreading, already documented in bacteria such as Escher-
ichia coli, Klebsiella pneumoniae, and Salmonella spp.,
Citrobacter freundii, Enterobacter aerogenes and Proteus
mirabilis, and is often associated with multidrug resistance
isoforms (Kenneth and Thomson 2010). Among AmpC-
producing bacteria, resistance to last resort antibiotics like
carbapenems is arising in some organisms by mutations that
reduce influx (outer membrane porin loss) or enhance efflux
(efflux pump activation).
(molecular weight, hydrophobicity and hydrogen bond
profile), its ligand efficiency of 0.38 kcal mol‒1, its
mechanism of action and the relatively potent activity in
whole cells pose it as a good candidate for chemical deri-
vatization (Hopkins et al. 2004). In our continuing effort to
identify new β-lactamases inhibitors, we report here the
structure-based design, synthetic optimization and biologi-
cal evaluation of a novel series of thiophene carboxylic
derivatives of 1. Our goal was to improve potency and
biological activity toward clinically resistant strains taking
advantage of additional binding site in AmpC BL.
Materials and methods
There is a strong need for AmpC β-lactamase inhibitors
that are able to effectively restore the activity of β-lactams
in a scenario where the number of effective antibiotics is
reduced on a daily basis.
Synthesis
All intermediates and final products were characterized
through monodimensional H-NMR, whereas all final car-
1
Two main strategies have been developed for counter-
acting β-lactamases-mediated resistance: developing anti-
biotics not susceptible of inactivation by BLs or designing
inhibitors to be administered with antibiotics that reversibly
or irreversibly bind the BLs. Among new BLs inhibitors,
only few resemble new chemical entities and/or possess
novel mechanism of action, like the boronic acid deriva-
tives, missing the classical β-lactam ring and extensively
investigated as covalent reversible inhibitors of serine BLs
(Fig. 1), acting as transition state analogs (Tondi et al.
2001); (Venturelli et al. 2007); (Tondi et al. 2010); (Eidam
et al. 2010, 2012); (Tondi et al. 2014); (Hecker et al. 2015).
Recently, we reported the structure-based optimization of
lead 1, a first non-covalent inhibitor of class C β-lactamase
AmpC (Fig. 1) (Tondi et al. 2005). This compound is
chemically dissimilar to penicillins and cephalosporins, and
binds to the enzymes non-covalently and reversibly, in
contrast to known β-lactamases inhibitors (Fig. 1).
boxylic acids were characterized through monodimensional
1H-NMR and 13C-NMR. Elemental analyses were per-
formed on a Perkin–Elmer Elemental Analyzer 240C. In a
few cases, mass spectroscopy high-resolution analysis was
performed to confirm elemental formula. The structures of
all compounds were consistent with their analytical and
spectroscopic data.
All commercial chemicals and solvents were reagent
grade, and were used without further purification unless
otherwise specified. Reaction progress was monitored by
TLC on pre-coated silica gel 60 F254 plates (Merck) and
visualization was accomplished with UV light (254 nm).
Merck silica gel (Kieselgel 60) was used for flash chroma-
1
tography (230–400 mesh) when required. H and 13C NMR
spectra were recorded on a Bruker DPX200 spectrometer or a
Bruker FT-NMR AVANCE 400. Chemical shifts are repor-
ted as δ values (ppm) referenced to residual solvent (CHCl3
at δ 7.26 ppm, dimethyl sulfoxide (DMSO) at δ 2.50 ppm,
MeOD at δ 3.31 ppm, CD3OCD3 at δ 2.05 ppm). When peak
multiplicities are given, the following abbreviations are used:
Lead 1 proved to be a druggable low micromolar inhi-
bitor of AmpC (Ki = 1 µM); its physical properties
R
HOOC
OH
H
N
NH
O
R
H
S
H
NH
S
S
O
NH
S
O
O
N
N
R1
OH
COOH
O
O
O
COOH
COOH
S
COOH
1
2
3
4
Fig. 1 Novel lead compound 1, discussed here, with respect to classical β-lactam substrates (2, penicillins, 3, cephalosporins) and classical
β-lactam inhibitor (4, clavulanic acid)

Med Chem Res
s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br,
broadened signal. Two-dimensional NMR techniques (het-
eronuclear single quantum coherence and heteronuclear
multiple bond correlation) were used to drive the assignment
CH2Cl2 (25 mL). The mixture was cooled to 0 °C and
(Boc)2O (10 mmol, 2.18 g, dissolved in 12 mL CH2Cl2)
was added in dropwise with stirring. Then the mixture was
warmed to r.t. and stirred for 40 h. The reaction was
quenched with an NH4Cl-saturated solution (40 mL) and
extracted with CH2Cl2 (2 × 60 mL). The collected organic
layers were washed with NH4Cl-saturated solution (1 × 60
mL), then dried with anhydrous Na2SO4. After concentra-
tion under reduced pressure, a brown oil was obtained. The
product was purified by flash chromatography (n-hexane/
diethyl ether 1:1). Scheme S1. Yield: 26%. 1H-NMR
(CDCl3, 400 MHz): δ = 8.24 (s, H-4, 1H), 8.18 (d, J = 8.2
Hz, H-6, 1H), 7.76 (d, J = 7.5 Hz, H-8, 1H), 7.54 (t, J =
8.0 Hz, H-7, 1H), 5.81 (br, NH, 1H), 5.44 (s, H-2, 1H),
3.75 (s, OCH3, 3H), 1.43 (s, Boc, 9H). Anal. calcd. for
C14H18N2O6: C, 54.19; H, 5.85; N, 9.03. Found: C, 54.19;
H, 5.85; N, 9.03.
1
of signals in H and 13C spectra (refer to Supplementary
TableS1 for atom numbering). Optical rotations were mea-
sured at 25 °C on a Perkin-Elmer 241 polarimeter and are
expressed in deg cm2/g. Synthesis schemes are provided as
supplemental information.
Amino(3-nitrophenyl)acetic acid (1) (Sassatelli et al. 2006)
Amino(phenyl)acetic acid (33 mmol, 5 g) was cooled at 0 °
C and added to a mixture of concentrated H2SO4 (98% w/w,
5 mL) and fuming HNO3 (90% w/w, 5 mL). The mixture
was stirred for 1 h at 0 °C then 30 min at r.t. After this, the
reaction was hydrolyzed with cold water (60 mL). The pH
was adjusted by addition of 10 M NH4OH. The resulting
mixture was stirred at 4 °C for 10 h, 3-nitrophenylglycine
was isolated by filtration. After washing with cold water,
the compound was isolated as a yellow powder. Scheme S1.
Yield: 64%. 1H-NMR (DMSO, 200 MHz): δ = 8.27 (s, H-4,
1H), 8.10 (d, J = 8.1 Hz, H-6, 1H), 7.83 (d, J = 7.9 Hz, H-8,
1H), 7.64 (t, J = 7.9 Hz, H-7, 1H), 4.51 (s, H-2, 1H). Anal.
calcd. for C8H8N2O4: C, 48.98; H, 4.11; N, 14.28. Found:
C, 48.92; H, 4.19; N, 14.31.
Methyl (3-aminophenyl)[(tert-butoxycarbonyl)amino]
acetate (4)
A mixture of methyl [(tert-butoxycarbonyl)amino](3-
nitrophenyl)acetate (3) (2.6 mmol, 0.76 g), MeOH (90 mL)
and 5% Pd/C (80 mg) was hydrogenated at r.t. under 40 psi
for 24 h. After filtration over Celite® and washing with
MeOH, the filtrate was evaporated to give the product as a
light yellow solid. Scheme S1. Yield: 83%. 1H-NMR
(CDCl3, 200 MHz) δ = 7.03 (t, J = 8.0 Hz, H-7, 1H), 6.64
(d, J = 8.1 Hz, H-8, 1H), 6.59 (s, H-4, 1H), 6.56 (d, J = 8.0
Hz, H-6, 1H), 5.70 (br, NH, 1H), 5.12 (s, H-2, 1H), 3.62 (s,
OCH3, 3H), 1.37 (s, Boc, 9H). Anal. calcd. for
C14H20N2O4: C, 59.99; H, 7.19; N, 9.99. Found: C, 59.93;
H, 7.11; N, 10.07.
Methyl amino(3-nitrophenyl)acetate (2) (Li and Zhao 2006)
SOCl2 (0.9 mL) was added dropwise to a suspension of
amino(3-nitrophenyl)acetic acid (1) (5 mmol, 1 g) in dry
MeOH (4 mL). After the addition was completed, the solu-
tion was heated under reflux overnight. The solution was
cooled to r.t., then diethyl ether was added until a white solid
was formed. The solid was filtered out and dried in vacuum.
The solid was solubilized in a NaHCO3 saturated solution
(20 mL) and the solution was extracted three times with
ethyl acetate. The collected organic layers were washed with
NaHCO3 saturated solution (2 × 10 mL) and brine (2 × 10
mL), then dried with anhydrous Na2SO4. After concentra-
tion under reduced pressure, methyl 3-nitrophenylglycinate
was obtained as a light yellow solid. Scheme S1. Yield:
Methyl (3-amino-2,6-difluorophenyl)acetate (5)
7.5 mmol (1.54 g) of 2,6-difluorophenylacetic acid were
added over 20 min to 15 mL of cooled (−10 °C) HNO3.
After 15 min at −10 °C, the reaction was poured into water/
ice and the mixture was extracted with diethyl ether (4 × 10
mL). The organic layer was washed with brine (3 × 10 mL)
and dried with anhydrous Na2SO4. The product obtained
after concentration under reduced pressure was solubilized
in EtOH (150 mL) and 5% Pd/C (150 mg) was added. The
compound was hydrogenated at r.t. under 30 psi of H2
overnight. After filtration over Celite®, the filtrate was
evaporated. The obtained compound was stirred for 10 min
with EtOH/HCl conc (37% w/w, 10% V/V), after eva-
poration of the solvent, the glue was treated with ethyl
acetate and the product was isolated by filtration. SOCl2
(0.2 mL) was added dropwise to a suspension of the
obtained solid in dry MeOH (4 mL). After the addition was
completed, the solution was heated under reflux overnight.
1
70%. H-NMR (CDCl3, 200 MHz): δ = 8.29 (s, H-4, 1H),
8.14 (d, J = 8.1 Hz, H-6, 1H), 7.76 (d, J = 7.8 Hz, H-8, 1H),
7.52 (t, J = 8.0 Hz, H-7, 1H), 4.76 (s, H-2, 1H), 3.72 (s,
OCH3, 3H). Anal. calcd. for C9H10N2O4: C, 51.43; H, 4.8;
N, 13.33. Found: C, 51.39; H, 4.84; N, 13.36.
Methyl [(tert-butoxycarbonyl)amino](3-nitrophenyl)acetate
(3)
Et3N (21 mmol, 3 mL) was added to a suspension of methyl
amino(3-nitrophenyl)acetate (2) (10 mmol, 2.1 g) in

Med Chem Res
The solvent was removed at low pressure, the compound
was solubilized with NaHCO3 s.s. (until gas was devel-
oped) and extracted with diethyl ether (4 × 5 mL). The
collected organic layers were washed with water (3 × 5
mL), then dried with anhydrous Na2SO4. After concentra-
tion under reduced pressure, methyl (3-amino-2,6-difluor-
ophenyl)acetate was obtained. Scheme S2. Yield: 28%.
1H-NMR (CDCl3, 200 MHz): δ = 6.86–6.71 m, (H-5 and
H-6, 2H), 3.73 (s, OCH3, 3H), 3.71 (t, J = 1.3 Hz, H-7, 2H).
A correlation between 1H and 19F was observed. Anal.
calcd. for C9H9F2NO2: C, 53.73; H, 4.51; N, 6.96. Found:
C, 53.68; H, 4.55; N, 6.89.
evaporated to give the product. Yield: 40%. Scheme S3. 1H-
NMR (CDCl3, 200 MHz): δ = 7.13 (t, J = 7.8 Hz, H-5, 1H),
6.77 and 6.69 (two d, J = 7.8 Hz, H-6 and H-4, 1H each),
6.75 (s, H-2, 1H), 4.26 (br, NH2, 2H), 4.12 (q, J = 7.1 Hz,
OCH2, 2H), 3.62 (q, J = 7.2 Hz, H-7, 1H), 1.46 (d, J = 7.2
Hz, H-9, 3H), 1.21 (t, J = 7.1 Hz, CH3, 3H). Anal. calcd. for
C11H15NO2: C, 68.37; H, 7.82; N, 7.25. Found: C, 68.37;
H, 7.82; N, 7.25.
[3-({[2-(methoxycarbonyl)thiophen-3-yl]sulfonyl}amino)
phenyl]acetic acid (9a)
3-aminophenylacetic acid (0.55 mmol, 0.083 g) was dis-
solved in 4 mL of dry pyridine, and methyl 3-
chlorosulfonylthiophene-2-carboxylate (0.83 mmol, 0.2 g)
was slowly added to the solution under stirring. The reac-
tion was stirred for 24 h at 50 °C, under nitrogen. The
resulting solution was treated with water and ice; the pro-
duct was isolated through filtration. Scheme S4. Yield:
2-(3-nitrophenyl)propionitrile (6)
30 mmol (4.86 g) of 2-(3-nitrophenyl)acetonitrile, 50 mmol
(6.91 g) of potassium carbonate and 500 mmol (45 g) of
dimethylcarbonate were reacted 3.5 h at 180 °C and 10 bar
of pressure. Activated carbon was added to the reaction and
filtered off on Celite®. The solvent was evaporated under
low pressure, the obtained compound was treated with
cyclohexane (3 × 5 mL) and the product was isolated by
1
80%. H-NMR (CDCl3, 200 MHz): δ = 8.08 (s, 1H, NH),
7.42 (d, J = 5.2 Hz, 1H, H-5), 7.38 (d, J = 5.2 Hz, 1H, H-4),
7.19 (t, J = 7.4 Hz, 1H, H-11), 7.10 (s, 1H, H-8), 7.05 (d, J
= 7.0 Hz, 1H, H-10), 7.02 (d, J = 7.6 Hz, 1H, H-12), 3.98
(s, 3H, OCH3), 3.57 (s, 2H, H-13); 13C-NMR (CDCl3, 50
MHz): δ = C-14 176.2, C-6 160.9, C-3 143.1, C-7 136.1, C-
9 134.0, C-2 132.2, C-5 131.5, C-4 130.2, C-11 129.4, C-12
126.6, C-8 122.7, C-10 120.8, OCH3 53.3, C-13 40.5. Anal.
calcd. for C15H15NO6S2: C, 48.77; H, 4.09; N, 3.79. Found:
C, 48.82; H, 4.01; N, 3.82.
1
filtration. Scheme S3. Yield: 23%. H-NMR (CDCl3, 200
MHz): δ = 8.23 (s, H-2, 1H), 8.21 (d, J = 7.8 Hz, H-6, 1H),
7.75 (d, J = 8.0 Hz, H-4, 1H), 7.61 (dd, J = 7.8, 8.0 Hz, H-
5, 1H), 4.04 (q, J = 7.3 Hz, H-7, 1H), 1.72 (d, J = 7.3 Hz,
H-9, 3H). Anal. calcd. for C9H8N2O2: C, 61.36; H, 4.58; N,
15.9. Found: C, 61.3; H, 4.54; N, 15.98.
Ethyl 2-(3-nitrophenyl)propionate (7)
[4-({[2-(methoxycarbonyl)thiophen-3-yl]sulfonyl}amino)
2 mmol (0.31 g) of 2-(3-nitrophenyl)propionitrile were dis-
solved in 5 mL of ethanol and 0.3 mL of H2SO4 were
added. The mixture was heated under reflux overnight. The
reaction was neutralized with NaHCO3 s.s., the aqueous
solution was extracted with CH2Cl2 (4 × 5 mL). The
organic layer was dried with anhydrous Na2SO4, after
concentration under reduced pressure, the product was
purified through flash chromatography (cyclohexane/diethyl
ether 97:3). Scheme S3. Yield: 92%. 1H-NMR (CDCl3, 200
MHz): δ = 8.18 (s, H-2, 1H), 8.12 (d, J = 8.1 Hz, H-6, 1H),
7.65 (d, J = 7.7 Hz, H-4, 1H), 7.50 (dd, J = 8.1, 7.7 Hz, H-
5, 1H), 4.15 (q, J = 7.1 Hz, OCH2, 2H), 3.82 (q, J = 7.2 Hz,
H-7, 1H), 1.56 (d, J = 7.2 Hz, H-9, 3H), 1.22 (t, J = 7.1 Hz,
CH3, 3H). Anal. calcd. for C11H13NO4: C, 59.19; H, 5.87;
N, 6.27. Found: C, 59.15; H, 5.87; N, 6.3.
phenyl]acetic acid (9b)
The compound 9b was synthesized in a similar manner to
9a using 4-aminophenylacetic acid. Scheme S4. Yield:
1
89%. H-NMR (CDCl3, 400 MHz): δ = 7.50 and 7.49 (d,
J = 5.3 Hz, H-4 and H-5, 1H each), 7.20 (d, J = 8.5 Hz,
H-8, 2H), 7.14 (d, J = 8.5 Hz, H-9, 2H), 4.04 (s, OCH3,
3H), 3.61 (s, H-11, 2H); 13C-NMR (CDCl3, 100 MHz): δ
= C-12 176.1, C-6 160.7, C-3 143.3, C-7 136.7, C-2
132.9, C-5 131.5, C-4 130.4, C-8 130.2, C-10 123.7, C-9
122.1, OCH3 53.4, C-11 39.7. Anal. calcd. for
C15H15NO6S2: C, 48.77; H, 4.09; N, 3.79. Found: C,
48.75; H, 4.07; N, 3.77.
3-[3-({[2-(methoxycarbonyl)thiophen-3-yl]sulfonyl}amino)
phenyl]propanoic acid (9c)
Ethyl 2-(3-aminophenyl)-propionate (8)
The compound 9c was synthesized in a similar manner to
9a using 3-(3-aminophenyl)propionic acid as synthetic
scaffold. Scheme S4. Yield: 71%. H-NMR (DMSO, 200
MHz): δ = 8.54 (br, NH, 1H), 7.64 (d, J = 5.3 Hz, H-5, 1H),
7.39 (d, J = 5.2 Hz, H-4, 1H), 7.18 (t, J = 7.8 Hz, H-11,
A mixture of ethyl 2-(3-nitrophenyl)propionate (7) (3.7
mmol, 0.7 g), ethanol (90 mL) and 5% Pd/C (100 mg) was
hydrogenated at r.t. under 30 psi overnight. After filtration
over Celite® and washing with ethanol, the filtrate was
1

Med Chem Res
1H), 7.03 (s, H-8, 1H), 6.91 (d, H-10 and H-12, J = 7.9 Hz,
2H), 3.87 (s, OCH3, 3H), 2.72 (t, J = 7.7 Hz, H-13, 2H),
2.43 (t, J = 7.7 Hz, H-14, 2H). Anal. calcd. for
C16H17NO6S2: C, 48.77; H, 4.09; N, 3.79. Found: C, 50.12;
H, 4.47; N, 3.65.
Methyl 3-{[3-(1-ethoxy-1-oxopropan-2-yl)phenyl]
sulfamoyl}thiophene-2-carboxylate (9f)
The compound 9f was synthesized in a similar manner
to 9d using ethyl 2-(3-aminophenyl)-propionate (1.25
mmol, 0.24 g). After 20-h stirring at r.t., the solvent was
evaporated under low pressure. The reaction was
diluted with 50 mL of ethyl acetate and washed with 2N
HCl (2 × 30 mL), brine (2 × 30 mL), NaHCO3 s.s.
(2 × 30 mL), then dried with anhydrous Na2SO4. After
concentration under reduced pressure, the product was
obtained as a white solid. Scheme S5. Yield: 48%. 1H-NMR
(CDCl3, 400 MHz): δ = 8.22 (s, NH,1H), 7.45 (d, J = 5.2
Hz, H-5, 1H), 7.43 (d, J = 5.2 Hz, H-4, 1H), 7.17 (t, J = 7.8
Hz, H-11, 1H), 7.05 (s, H-8, 1H), 7.06-7.01 (m, H-10 and
H-12, 2H), 4.07 (q, J = 7.1 Hz, OCH2, 2H), 3.83 (s,
OCH3,3H), 3.61 (q, J = 7.2 Hz, H-13, 1H), 1.40 (d, J = 7.2
Hz, H-15, 3H), 1.18 (t, J = 7.1 Hz, CH3,3H); 13C-NMR
(CDCl3, 100 MHz): δ = C-14 173.9, C-6 161.1, C-3 143.9,
C-9 141.9, C-7 136.5, C-2 132.3, C-5 131.7, C-4 130.2, C-
11 129.3, C-10 124.8, C-8 121.0, C-12 120.5, OCH2 60.8,
OCH3 53.7, C-13 45.2, C-15 18.4, CH3 14.2. Anal. calcd.
for C17H19NO6S2: C, 51.37; H, 4.82; N, 3.52. Found: C,
51.44; H, 4.78; N, 3.5.
Methyl3-{[3-(1-{[(tert-butoxy)carbonyl]amino}-2-methoxy-
2-oxoethyl)phenyl]sulfamoyl} thiophene-2-carboxylate (9d)
To a suspension of methyl (3-aminophenyl)[(tert-butox-
ycarbonyl)amino]acetate (4) (1.8 mmol, 0.5 g) in dry
CH2Cl2 (7.5 mL) was added methyl 3-(chlorosulfonyl)
thiophene-2-carboxylate (2.1 mmol, 0.5 g, dissolved in 7.5
mL of dry CH2Cl2). Under nitrogen atmosphere, 500 μL of
pyridine were added dropwise with stirring. The mixture
was kept to r.t. and stirred for 21 h. The reaction was diluted
with 50 mL of CH2Cl2 and washed with NH4Cl s.s. (2 × 30
mL), brine (2 × 30 mL), NaHCO3 s.s. (2 × 30 mL), brine
(2 × 30 mL), then dried with anhydrous Na2SO4. After
concentration under reduced pressure (the residual pyridine
was removed through evaporation at low pressure after
addition of a small amount of H2O (azeotrope H2O/pyridine
1:1)), the product was obtained as a brown solid. Scheme
S5. Yield: 83%. 1H-NMR (CDCl3, 400 MHz,): δ = 8.29 (br,
NH-SO2, 1H), 7.44 (br, H-5 and H-4, 2H), 7.21 (t, J = 7.8
Hz, H-11, 1H), 7.12 (d, J = 8.0 Hz, H-12, 1H), 7.10 (s, H-8,
1H), 7.09 (d, J = 7.4 Hz, H-10, 1H), 5.50 (br, NH-Boc, 1H),
5.22 (s, H-13, 1H), 4.01 (s, OCH3, 3H), 3.68 (s, OCH3, 3H),
1.43 (s, Boc, 9H); 13C-NMR (CDCl3, 100 MHz): δ = C-14
171.1, C-6 161.3, CO-Boc 154.7, C-3 143.9, C-7 138.3, C-
9 136.2, C-5 131.6, C-2 131.1, C-4 130.4, C-11 129.8, C-10
124.2, C-12 121.7, C-8 120.3, C-Boc 80.3, C-13 57.2,
OCH3 53.4, OCH3 52.8, CH3-Boc 28.3. Anal. Calcd. for
C15H16N2O6S2: C, 46.86; H, 4.2; N, 7.29. Found: C, 46.82;
H, 4.14; N, 7.32.
Methyl 3-[(2-ethoxy-2-oxoethyl)sulfamoyl]thiophene-2-
carboxylate (9g)
The compound 9g was synthesized in a similar manner to
9a using glycine ethyl ester (4 mmol, 0.41 g). The mixture
was kept for stirring at r.t. for 4 h. The solvent was removed
at low pressure. Four milliliter of 2 N HCl was added to the
obtained glue and the solution was extracted with CH2Cl2
(3 × 5 mL), then dried with anhydrous Na2SO4. After
concentration under reduced pressure, the product was
1
obtained as brown oil. Scheme S6. Yield: 44%. H-NMR
Methyl 3-{[2,4-difluoro-3-(2-methoxy-2-oxoethyl)phenyl]
sulfamoyl}thiophene-2-carboxylate (9e)
(CDCl3, 400 MHz): δ = 7.54 (d, J = 5.2 Hz, H-5, 1H), 7.50
(d, J = 5.2 Hz, H-4, 1H), 6.89 (br, 1H NH,), 4.09 (q, J =
7.1 Hz, OCH2,2H), 4.00 (s, OCH3,3H), 3.94 (d, J = 5.7, H-
7, 2H), 1.20 (t, J = 7.1 Hz, CH3,3H); 13C-NMR (CDCl3,
100 MHz): δ = C-8 168.6, C-6 161.0, C-3 144.6, C-2 142.3,
C-5 131.3, C-4 129.4, OCH2 61.6, OCH3 53.2, C-7 44.8,
CH3 14.0. Anal. calcd. for C8H9NO6S2: C, 34.4; H, 3.25; N,
5.02. Found: C, 34.37; H, 3.29; N, 4.99.
The compound 9e was synthesized in
a
similar
manner to 9d using methyl (3-amino-2,6-difluorophenyl)
acetate (5). Scheme S5. Yield: 44%. 1H-NMR (CDCl3, 400
MHz): δ = 8.47 (s br, NH,1H), 7.51 (dt, J = 5.8, 8.9 Hz, H-
12, 1H) 7.46 and 7.42 (2 × d, J = 5.2 Hz, H-4 and H-5, 1H
each), 6.85 (dt, J = 1.6, 8.8 Hz, H-11, 1H), 4.00 (s,
OCH3,3H), 3.66 (s, OCH3,3H), 3.59 (s, H-13, 2H); 13C-
NMR (CDCl3, 100 MHz): δ = C-14 169.5, C-6 161.0,
C-10 158.9, It is possible to observe the correlation 13C-19F
C-8 153.8, C-3 144.0, C-2 131.6, C-4 and C-5 131.0 and
130.1, C-12 125.6, C-7 120.4, C-11 111.2, C-9 110.8,
OCH3 53.4, OCH3 52.3, C-13 27.9. Anal. calcd. for
C15H13F2NO6S2: C, 44.44; H, 3.23; N, 3.46. Found: C,
44.44; H, 3.23; N, 3.46.
Methyl 3-[(1-methoxy-1-oxopropan-2-yl)sulfamoyl]
thiophene-2-carboxylate (9h/9i)
A solution of triethylamine (23.6 mmol, 3.2 mL) in CH2Cl2
(5.5 mL) was added dropwise, under stirring to a mixture of
the correspondent alanine methyl ester hydrochloride
enantiomer (3.2 mmol, 0.45 g) and methyl 3-(chlor-
osulfonyl)thiophene-2-carboxylate (3.2 mmol, 0.77 g). The

Med Chem Res
reaction was stirred at r.t. for 4 h. After removing the sol-
vent under reduced pressure, 1 N HCl (50 mL) was added
and the product was extracted with ethyl acetate (3 × 15
mL). The combined organic layers, washed with NaHCO3
0.3 M (6 mL) and water (2 × 10 mL), were dried with
anhydrous Na2SO4. After concentration under reduced
pressure, the product was obtained as a brown solid.
Scheme S7. For methyl 3-{[(2S)-1-methoxy-1-oxopropan-
2-yl]sulfamoyl}thiophene-2-carboxylate (9h): 81% yield,
for methyl 3-{[(2R)-1-methoxy-1-oxopropan-2-yl]sulfa-
moyl}thiophene-2-carboxylate (9i): 66% yield, 1H-NMR
(CDCl3, 400 MHz): δ = 7.51 and 7.47 (, d, J = 5.2 Hz, H-4
and H-51H each), 6.83 (d, J = 8.5 Hz, NH,1H), 4.10 (m, H-
7, 1H), 3.93 (s, OCH3, 3H), 3.55 (s, OCH3, 3H), 1.38 (d, J
= 7.2 Hz, H-9, 3H); 13C-NMR (CDCl3, 100 MHz): δ = C-8
171.9, C-6 160.8, C-3 144.8, C-2 131.2, C-4 and C-5 130.3
and 130.0, OCH3 53.2, OCH3 52.4, C-7 52.1, C-9 19.7.
Anal. calcd. for C9H11NO6S2: C, 36.85; H, 3.78; N, 4.78.
Found: C, 36.89; H, 3.75; N, 4.71.
133.1, C-5 132.5, C-4 131.0, C-11 130.5, C-10 123.9, C-12
121.2, C-8 119.9, C-13 55.6, OCH3 53.7, OCH3 52.8.
3-{[3-(carboxymethyl)phenyl]sulfamoyl}thiophene-2-
carboxylic acid (11a)
9a (0.40 mmol, 0.14 g) was suspended in 4 mL of 2 N
NaOH, and the mixture was stirred at 80 °C until the
starting material reacted completely; the reaction was fol-
lowed by TLC. Thereafter, the solution was cooled, and 2 N
HCl was added until the solution reached pH 2. A pre-
cipitate, corresponding to 3-(3-carboxymethyl-phenylsulfa-
moyl)-thiophene-2-carboxylic acid was formed and isolated
through filtration. Scheme S4. Yield: 73%. 1H-NMR
(DMSO, 200 MHz): δ = 12.3 (br, COOH, 2H), 9.88 (s,
NH, 1H), 7.84 (d, J = 5.2 Hz, H-5, 1H), 7.38 (d, J = 5.2 Hz,
H-4, 1H), 7.17 (t, J = 7.7 Hz, H-11, 1H), 7.04 (s, H-8, 1H),
6.97 (d, J = 7.9 Hz, H-10, 1H), 6.93 (d, J = 7.5 Hz, H-12,
1H), 3.46 (s, H-13, 2H). Anal. calcd. for C13H11NO6S2: C,
45.74; H, 3.27; N, 4.1. Found: C, 45.78; H, 3.25; N, 4.
Methyl 3-[(3-ethoxy-3-oxopropyl)sulfamoyl]thiophene-2-
carboxylate (9l)
3-{[4-(carboxymethyl)phenyl]sulfamoyl}thiophene-2-
carboxylic acid (11b)
The compound 9l was synthesized in a similar manner to 9h
using β-alanine ethyl ester hydrochloride. Scheme S8.
Yield: 76%. 1H-NMR (CDCl3, 400 MHz): δ = 7.54-7.52
(br, H-4 and H-5, 2H), 6.61 (t, J = 6.4 Hz, NH, 1H), 4.08
(q, J = 7.1 Hz, OCH2, 2H), 3.91 (s, OCH3, 3H), 3.23 (q, J
= 6.3 Hz, H-7, 2H), 2.47 (t, J = 6.3 Hz, H-8, 2H), 1.21 (t, J
= 7.1 Hz, CH3, 3H); 13C-NMR (CDCl3, 100 MHz): δ = C-9
171.4, C-6 160.9, C-3 145.1, C-2 131.3, C-4 and C-5 131.0
and 130.6, OCH2 60.9, OCH3 53.2, C-7 39.1, C-8 34.3,
CH3 14.1. Anal. calcd. for C9H11NO6S2: C, 36.85; H, 3.78;
N, 4.78. Found: C, 36.87; H, 3.8; N, 4.79.
Compound 11b was synthesized in a similar manner to 11a
using 9b. Scheme S4. Yield: 69%. H-NMR (DMSO, 200
1
MHz) δ = 11.9 (br, COOH, 2H), 8.50 (bt, NH, 1H), 7.85 (d,
J = 5.5 Hz, H-5, 1H), 7.53 (d, J = 5.3 Hz, H-4, 1H), 7.13 (d,
J = 8.6 Hz, H-8, 2H), 7.10 (d, J = 8.6 Hz, H-9, 2H), 3.55 (s,
H-11, 2H). Anal. calcd. for C13H11NO6S2: C, 45.74; H,
3.25; N, 4.1. Found: C, 45.7; H, 3.22; N, 3.98.
3-{[3-(2-carboxyethyl)phenyl]sulfamoyl}thiophene-2-
carboxylic acid (11c)
2-amino-{3-[2-(methoxycarbonyl)thiophen-3-sulfonamido]
phenyl}acetic acid (10)
Compound 11c was synthesized in a similar manner to 11a
using 9c. Scheme S4. Yield: 68%. H-NMR (DMSO, 400
1
MHz): δ = 12.1 (br, COOH, 2H), 9.54 (br, NH, 1H), 7.84
(d, J = 5.3 Hz, H-5, 1H), 7.39 (d, J = 5.2 Hz, H-4, 1H), 7.14
(t, J = 7.8 Hz, H-11, 1H), 6.98 (s, H-8, 1H), 6.91 (d, H-10
and H-12, J = 7.9 Hz, 2H), 2.72 (t, J = 7.7 Hz, H-13, 2H),
2.43 (t, J = 7.7 Hz, H-14, 2H); 13C-NMR (DMSO, 100
MHz): δ = C-15 174.0, C-6 161.8, C-9 142.4, C-3 142.2, C-
7 137.8, C-2 135.1, C-5 130.6, C-4 130.1, C-11 128.7, C-10
123.4, C-8 119.6, C-12 117.5, C-14 34.7, C-13 29.9. Anal.
calcd. for C14H13NO6S2: C, 47.32; H, 3.69; N, 3.94. Found:
C, 47.30; H, 3.70; N, 3.92.
Trifluoroacetic acid (2 mmol, 0.15 ml) was added dropwise
with stirring to a cooled (0 °C) solution of 9d (1.5 mmol,
0.56 g) in CH2Cl2 (4 mL) . The mixture was stirred at 0 °C
for 30 min, then it was warmed to r.t. and stirred for 1 h.
After concentration under reduced pressure, an oil was
obtained. The compound was treated with 10 mL of diethyl
ether, the resulting mixture was stirred at r.t. for 1 h. The
product was isolated by filtration as a pink solid. Yield:
99%. 1H-NMR (DMSO, 400 MHz): δ = 10.36 (br, NH, 1H),
8.91 (br, NH3+, 3H), 7.98 (d, J = 5.2 Hz, H-5, 1H), 7.47 (d,
J = 5.2 Hz, H-4, 1H), 7.36 (t, J = 7.9 Hz, H-11, 1H), 7.26 (s,
H-8, 1H), 7.17 (d, J = 7.4 Hz, H-10, 1H), 7.15 (d, J = 7.6
Hz, H-12, 1H), 5.26 (s, H-13, 1H), 3.89 (s, OCH3, 3H), 3.69
(s, OCH3, 3H); 13C-NMR (DMSO, 100 MHz): δ = C-14
169.1, C-6 160.3, C-3 142.7, C-7 138.5, C-9 133.9, C-2
3-({3-[carboxy(carboxyamino)methyl]phenyl}sulfamoyl)
thiophene-2-carboxylic acid (11d)
Compound 11d was synthesized in a similar manner to 11a
using methyl 3-(N-(3-(methylphenylglycinate)sulfamoyl)

Med Chem Res
thiophene-2-carboxylate (9d). After acidification, the reac-
tion was washed with ethyl acetate (3 × 10 mL). The aqu-
eous layer was concentrated under reduced pressure and the
white solid was ground with 20 mL of acetone. The solution
was filtered and after concentration under reduced pressure,
the product was obtained as a brown wax. Scheme S5.
Yield: 40%. 1H-NMR (MeOD, 400 MHz): δ = 7.68 (d, J =
5.2 Hz, H-5, 1H), 7.41 (d, J = 5.2 Hz, H-4, 1H), 7.36 (s, H-
8, 1H), 7.35 (t, J = 8.0 Hz, H-11, 1H), 7.23 (d, J = 7.9 Hz,
H-10, 1H), 7.22 (d, J = 8.0 Hz, H-12, 1H), 5.35 (s, H-13,
1H); 13C-NMR (MeOD, 100 MHz): δ = C-14 170.3, C-6
163.0, C-3 143.5, C-7 139.4, C-2 134.9, C-9 134.8, C-4
132.2, C-5 131.8, C-11 131.3, C-10 125.5, C-12 123.7, C-8
122.0, C-13 56.5. Anal. calcd. for C13H12N2O6S2: C, 43.81;
H, 3.39; N, 7.86. Found: C, 43.84; H, 3.41; N, 7.83.
Scheme S6. Yield: 79%. 1H-NMR (DMSO, 400 MHz): δ =
12.4 (br, COOH,2H), 9.84 (s, NH,1H), 7.89 and 7.41 (d, J
= 5.2 Hz, H-5 and H-4, 1H each), 3.74 (s, H-7, 2H); 13C-
NMR (DMSO, 100 MHz): δ = C-8 170.7, C-6 161.7, C-2
143.7, C-3 134.0, C-5 and C-4 131.4 and 130.3, C-7 44.8.
Anal. calcd. for C7H7NO6S2: C, 31.7; H, 2.66; N, 5.28.
Found: C, 31.89; H, 2.62; N, 5.2.
3-[(1-carboxyethyl)sulfamoyl]thiophene-2-carboxylic acid
(11h/11i)
Compounds 11h and 11i were synthesized in a similar
manner to 11d using 9h and 9i. Scheme S7. For 3-{[(1S)-1-
carboxyethyl]sulfamoyl}thiophene-2-carboxylic
acid
25
(11h): 70% yield, ½αꢀD ¼ þ80 (c = 28 mg/mL, acetone); for
3-{[(1R)-1-carboxyethyl]sulfamoyl}thiophene-2-car-
25
3-{[3-(carboxymethyl)-2,4-difluorophenyl]sulfamoyl}
thiophene-2-carboxylic acid (11e)
boxylic acid (11i): 68% yield, ½αꢀD ¼ ꢁ79 (c = 37 mg/mL,
1
acetone). H-NMR (MeOD, 400 MHz): δ = 7.72 and 7.49
(d, J = 5.2 Hz, H-4 and H-5, 1H each), 4.02 (q, J = 7.2 Hz,
H-7, 1H), 1.36 (d, J = 7.2 Hz, H-9, 3H); 13C-NMR
(MeOD, 100 MHz): δ = C-8 174.5, C-6 162.7, C-3 145.1,
C-2 134.2, C-4 and C-5 31.5 and 131.0, C-7 53.0, C-9
19.8. Anal. calcd. for C8H9NO6S2: C, 34.41; H, 3.25; N,
5.02. Found (h): C, 34.38; H, 3.2; N, 5. Found (i): C, 34.4;
H, 3.18; N, 5.03.
Compound 11e was synthesized in a similar manner to 11a
using 9e. Scheme S5. Yield: 57%. H-NMR (DMSO, 400
1
MHz): δ = 12.6 (br, COOH, 2H), 9.60 (br, NH, 1H), 7.87 (d,
J = 5.2 Hz, H-5, 1H), 7.32 (d, J = 5.2 Hz, H-4, 1H), 7.23
(dt, J = 6.0, 8.9 Hz, H-12, 1H) 7.05 (t, J = 8.9 Hz, H-11,
1H), 3.56 (s, H-13, 2H); 13C-NMR (DMSO, 100 MHz): δ =
C-14 170.3, C-6 161.1, C-10 158.5, C-3 142.2, C-2 134.6,
C-5 131.1, C-4 129.8, C-12 126.1, C-7 120.6, C-9 111.8, C-
11 110.7, C-13 27.8. Anal. calcd. for C13H9F2NO6S2: C,
41.38; H, 2.4; N, 3.71. Found: C, 41.35; H, 2.39; N , 3.7.
3-[(2-carboxyethyl)sulfamoyl]thiophene-2-carboxylic acid
(11l)
Compound 11l was synthesized in a similar manner to 11d
using 9l, the reaction was performed at 60 °C for 20 min.
Scheme S8. Yield: 88%. 1H-NMR (MeOD, 400 MHz): δ =
7.77 and 7.51 (d, J = 5.2 Hz, H-4 and H-5, 1H each), 3.20
(t, J = 6.3 Hz, H-7, 2H), 2.46 (t, J = 6.3 Hz, H-8, 2H); 13C-
NMR (MeOD, 100 MHz δ = C-9 173.8, C-6 161.9, C-3
144.2, C-2 133.0, C-4 and C-5 131.0 and 130.5, C-7 39.2,
C-8 33.9. Anal. calcd. for C8H9NO6S2: C, 34.41; H, 3.25;
N, 5.02. Found: C, 34.45; H, 3.28; N, 5.06.
3-{[3-(1-carboxyethyl)phenyl]sulfamoyl}thiophene-2-
carboxylic acid (11f)
Compound 11f was synthesized in a similar manner to 11a
using 9f. After acidification, the crude reaction mixture was
extracted with CH2Cl2 (3 × 10 mL), then dried with anhy-
drous Na2SO4. After concentration under reduced pressure,
the product was obtained. Scheme S5. Yield: 50%. 1H-NMR
(DMSO, 400 MHz δ = 12.3 (br, COOH, 2H), 9.93 (s, NH,
1H), 7.85 (d, J = 5.2 Hz, H-5, 1H), 7.38 (d, J = 5.2 Hz, H-4,
1H), 7.18 (t, J = 7.6 Hz, H-11, 1H), 7.06 (s, H-8, 1H), 6.97
(d, J = 7.6 Hz, H-12, 1H), 6.95 (d, J = 7.6 Hz, H-10, 1H),
3.56 (q, J = 7.1 Hz, H-13, 1H), 1.25 (d, J = 7.1 Hz, H-15,
3H); 13C-NMR (DMSO, 100 MHz): δ = C-14 175.3, C-6
161.3, C-9 142.6, C-3 142.1, C-7 138.0, C-2 135.1, C-5
130.8, C-4 130.4, C-11 128.8, C-10 123.0, C-8 118.7, C-12
118.2, C-13 44.3, C-15 18.1. Anal. calcd. for C14H13NO6S2:
C, 47.32; H, 3.69; N, 3.94. Found: C, 47.3; H, 3.71; N, 3.9.
3-Aminophenylacetonitrile (10a)
Pd/C (0.48 g) was added to
a
solution of 3-
nitrophenylacetonitrile (1.0 mol, 3.80 g,) in methanol (130
mL) and the suspension was hydrogenated for 2 h at 30 psi.
At the end of the reaction the suspension was filtered over
Celite® and the solvent removed under reduced pressure.
The oil thus obtained was used in the next step without
purification. Scheme S9. Yield 98%. 1H-NMR (CDCl3,
200 MHz): δ = 7.20-7.10 (m, 1H), 6.7-6.6 (m, 3H); 13C-
NMR (CDCl3, 100 MHz): δ = 130.05, 117.84, 114.53,
114.17, 23.50. Anal. calcd. for C8H8N2: C, 72.7; H, 6.1; N,
21.2. Found: C, 72.63; H, 6.08; N, 21.12.
3-[(carboxymethyl)sulfamoyl]thiophene-2-carboxylic acid (11g)
Compound 11g was synthesized in a similar manner to 11d
using 9g. The product was obtained as a yellow solid.

Med Chem Res
Methyl 3-{[3-(cyanomethyl)phenyl]sulfamoyl}thiophene-2-
carboxylate (10b)
measurements were performed using nitrocefin as a sub-
strate (200 μM) in 50 mM Tris buffer, pH 7.0, and mon-
itored in an HP8453 UV–Vis spectrophotometer. The Km of
nitrocefin for AmpC determined in this buffer was 127 μM.
Synthesized inhibitors 11a–11m were dissolved in DMSO
at a concentration of 10 mM; further diluted stocks were
subsequently prepared as necessary. Reactions were initi-
ated by the addition of 1.75 nM enzyme and monitored in
methacrylate cuvettes. No incubation effect on inhibi-
tion was detected for any compound. Inhibition Ki values
were obtained from IC50 plots assuming competitive inhi-
bition, an assumption consistent with both previous inhi-
bition patterns in this series and with experiments
investigating the effect of increasing substrate concentra-
tions (not shown) (Powers et al. 2002).
To a solution of 3-aminophenylacetonitrile (10a) (13.0
mmol, 1.73 g) in dry pyridine (60 mL) at r.t. under stirring
in a nitrogen atmosphere, 3-chlorosulfonylthiophene-2-
carboxylic acid methyl ester (14.5 mmol, 3.50 g) was
added portionwise, then the mixture was heated at 50 °C for
8 h. The solution thus obtained was cooled at r.t. and poured
in ice bath (300 mL); the precipitate obtained was filtered,
washed with water, then dissolved in CH2Cl2 (50 mL),
washed with 1 N HCl; the organic layer was dried with
Na2SO4 anhydrous and evaporated under reduced pressure.
Scheme S9. Yield 66%, m.p. 110–112 °C; 1H-NMR
(DMSO, 400 MHz): δ = 10.40 (1H, s), 8.10 (1H, d, J
5.4), 7.61 (1H, d, J 5.4), 7.40 (1H, t, J 7.8); 7.28 (1H, d, J
8.1); 7.15 (1H, d, J 7.6); 4.10 (2H, s), 4.00 (3H, s); 13C-
NMR (DMSO, 100 MHz): δ = 160.35, 142.70, 138.38,
133.09, 132.86, 132.51, 131,07, 130.23, 124.14, 119.47,
119.39, 119.08, 53.68, 22.77. ESI-HRMS: m/z [MH]+
calculated for C14H13N2O4S2 337.0317, found 337.0311.
Anal. calcd. for C14H12N2O4S2: C, 49.99; H, 3.6; N, 8.33.
Found: C, 49.97; H, 3.55; N, 8.38.
Microbiology
Compounds in the thiophene series were tested for synergy
with the β-lactam ceftazidime (CAZ) against pathogenic
bacteria from clinical isolates (Spain). The bacteria were
resistant to β-lactams because of the expression of class C β-
lactamases. Strains tested were K. pneumoniae 20/013, E.
coli 40/012, Enterobacter cloacae 558349, E. aerogenes
563334, Pseudomonas aeruginosa 12, P. aeruginosa 59, P.
aeruginosa 120, P. aeruginosa 148, P. aeruginosa 199.
(From the collection of the Spanish Network for Research
in Infectious Pathology-REIPI).
Minimum inhibitory concentration (MIC) values were
determined with Mueller–Hinton broth using the micro-
dilution method according to NCCLS guidelines (Clinical
and Laboratory Standards Institute 2010). To test the inhi-
bitory activity, compounds were dissolved in DMSO and
dilutions were performed using growth medium. After
dilution, the final concentration of DMSO was <5% in all
the tests. The inhibitor/CAZ ratio was 1:1. A control
experiment showed that DMSO did not have any effect on
the bacteria growth at the concentration used.
3-{[3-(2H-tetrazol-5-ylmethyl)phenyl]sulfamoyl}thiophene-
2-carboxylic acid (11m)
To a solution of methyl 3-{[3-(cyanomethyl)phenyl]sulfa-
moyl}thiophene-2-carboxylate (10b) (4.48 mmol, 1.50 g) in
toluene (60 mL), sodium azide (13.43 mmol, 0.87 g) and
triethylamine hydrochloride (13.43 mmol, 1.85 g,) were
added under stirring and the mixture was heated at 100 °C
under a nitrogen atmosphere for 12 h. After, the mixture
was cooled at r.t, toluene was decanted and the residue was
acidified with 1 N HCl. The solid thus obtained was
extracted with EtOAc (3 × 15 mL), the organic layer was
dried with Na2SO4 anhydrous and concentrated under
reduced pressure. The product was purified by silica gel
column chromatography (CH2Cl2:CH3OH 9:1, then 100%
CH3OH). Scheme S9. Yield 0.46 g (28%), m.p. 177 °C
1
(dec.); H-NMR (DMSO, 200 MHz): δ = 11.50 (1H, broad
Results and discussion
s), 7.50 (1H, d, J 5.4), 7.10 (2H, m), 7.08-6.90 (3H, m), 4.15
(2H, s); 13C-NMR (DMSO 400 MHz): δ = 162.24, 139.03,
137.32, 129.74, 129.20, 128.79, 125.04, 121.86, 120.09,
29.26. ESI-HRMS m/z [MH]+ calculated for
C13H12N5O4S2 366.0331, found 366.0326. Anal. calcd. for
C13H11N5O4S2: C, 42.73; H, 3.03; N, 19.17. Found: C,
42.76; H, 3; N, 19.15.
In its available crystallographic complex with AmpC, lead 1
highly complements the core of the active site, interacting
with all key residues involved in β-lactam recognition, such
as the catalytic Ser64 and conserved residues such as
Lys67, Asn152, and Tyr221 (Fig. 2) (Tondi et al. 2005).
The thiophene carboxylate binds at the heart of the oxya-
nion hole of β-lactamase, a hot spot for substrate and
inhibitor recognition, orienting the inhibitor in the binding
site. The thiophene carboxylate moiety in 1 interacts via
hydrogen bonds with the Oγ and the main-chain NH of the
hydrolytic Ser64. An additional hydrogen bond is formed
Enzymology
AmpC from E. coli was expressed and purified to homo-
geneity as described before (Powers et al. 1999). Kinetic

Med Chem Res
conserved Tyr221 lies, in the proximity of polar and apolar
residues such as Arg204, Gly320, Thr319, and Val211.
Compounds 11a–11c were designed to investigate the
effects of the elongation of the distal carboxylate function,
while changing its point of derivatization on the phenyl
ring. Inhibition levels indicate that this parameter has no
significant impact on the binding process (Table 1). Rea-
sonably, the elongation increases the distal carboxylate
mobility, without affecting its interaction with water and
Arg204.
While the changes in the carboxylate position on the
distal phenyl ring or its distance from the ring resulted in no
significant effects on the activity, the chemistry of com-
pound 11d, a 2‐amino‐2‐phenylacetic acid derivative,
determined a drop of 20-fold in affinity. The presence in
solution of a zwitterion form seems to affect binding
negatively, probably determining a rearrangement of the
ligand in the binding site to minimize steric hindrance and,
thus, determining the loss of interaction between the car-
boxylate and Arg204 (Table 1). The introduction in com-
pound 11e of two fluorine atoms, hydrogen isosteres with
an electron-withdrawing effect, did not improve lead 1
potency as for compound 11f, where an additional methyl
group meant to interact with the apolar residues of the
binding site was added. Derivative 11f affinity, not dis-
similar from the lead one, confirmed our hypothesis for
compound 11d activity reduction: it is a matter of size and
charge, with bulkier moieties not allowed in the satellite
binding site we aimed to target with this series. Derivative
11h and its enantiomer 11i, in which the phenyl ring was
omitted, were both worse inhibitors of AmpC, stressing the
importance of dipole–quadrupolar interactions engaging
between the ligand phenyl ring and Tyr221. Phenyl ring
deletion induces a loss in activity of 150–200-fold. The
reduced potency we observed when the phenyl ring is
missing is less dramatic in derivative 11l, where the distal
chain is not branched and the spacer between the sulfona-
mide and the distal carboxylate is longer. Therefore, in the
case of 11h and 11i, we can postulate bumping interactions
with the protein and/or loss of the interaction with Arg204.
In fact, deletion of phenyl ring together with ramification of
carboxylate chain (as in derivatives 11h and 11i) sig-
nificantly reduces the binding affinity toward AmpC. This
reduction is mitigated when the ramification is deleted (11g)
or when the distance between sulfonamide and carboxylate
is kept longer (11l). In fact, as the distance between the two
functionalities (derivatives 11l) increases, the affinity for
AmpC improves compared to 11h and 11i (Table 1).
Finally, in compound 11m, a tetrazole derivative, we meant
to investigate the effect of one of the most commonly
adopted carboxylic acid bioisosteres (Ballatore et al. 2013).
In our most active derivatives, the carboxylic acid func-
tional group is always present and its chemistry is important
Fig. 2 Lead 1 in its binding orientation in AmpC BL (PDB code
XGJ1)
with the main-chain nitrogen of Ala318. Hydrogen bond
interactions are also observed with the highly conserved
Wat401. The thiophene ring is within van der Waals dis-
tance from Leu119 and Leu293 residues. A sulfonamide
oxygen interacts with the Oγ of Ser64 and the Nζ of Lys67;
the other hydrogen binds to the Nδ2 of Asn152. The nitro-
gen atom of the sulfonamide interacts with the main-chain
oxygen of Ala318. The distal 4′-carboxylate-2′-hydroxy
benzene ring establishes dipole–quadrupole interactions
with Tyr221 (the distance between 2′-hydroxyl and Cδ1 of
Tyr221 is 3.43 Å). The 4′-carboxylate points toward the
entry of the binding site in an open and solvent accessible
area of the protein. In the first monomer, this carboxylate
hydrogen binds to an ordered water molecule, Wat521
(distance 2.9 Å), which interacts via hydrogen bond with
the Nε of Arg204 (distance, 2.8 Å). In the second monomer,
Arg204 adopts a different conformation and these interac-
tions are lost. Interestingly, here a new interaction is
observed between Gln120 and the sulfonamide of 1, with
the Gln120 in this monomer pointing toward the inhibitor
rather than out of the site, as it does in the first monomer.
Considering that recent results coming from decon-
structing fragment-based studies confirmed the 3-(sulfony-
lamino)-2-carboxylic acid thiophene moiety as the driving
force for lead 1 binding in the active site, we kept this
structural core unchanged while introducing chemical
diversity on the distal site of lead 1, where new chemistry
could be used to infer improved affinity toward AmpC
(Babaoglu and Shoichet 2006).
In drawing and synthesizing eleven derivatives of 1
(compounds 11a–11m, Table 1) our main goal was to
improve the complementarity of the distal phenyl ring of 1,
located in an open region of the active site where the

Med Chem Res
Table 1 Kia values of lead 1
and its derivatives vs. AmpC
#
1
Structure
Ki (µM)
#
Structure
Ki (µM)
1.05
11f
1.8
NH
O
NH
O
COOH
O
S
O
S
COOH
HO
COOH
COOH
S
S
11a
11b
11c
11d
11e
1.7
1.7
3.0
19.8
1.0
11g
11h
11i
27
NH
O
O
NH
S
COOH
O
S
O
COOH
S
HOOC
COOH
S
148
212
44
NH
O
NH
O
O
S
S
COOH
COOH
O
COOH
COOH
S
S
NH
O
NH
O
O
O
S
S
COOH
COOH
COOH
COOH
S
S
11l
NH
O
NH
S
COOH
O
O
S
O
NH2
COOH
S
HOOC
COOH
S
11m
1.6
F
F
NH
O
O
S
N
NH
O
O
N
S
COOH
N
N
H
COOH
S
COOH
S
a
Inhibitory constant Ki was calculated from experimentally determined IC50 using the Cheng-Prusoff
equation: Ki = IC50/(1 + S/Km)
in AmpC molecular recognition. However, the presence of
the carboxylic function can be responsible for significant
drawbacks in passive diffusion across biological membrane.
No effect was detected in the inhibitory activity of the
isolated enzyme: 11m kept the same affinity as 1, thus
supporting our hypothesis. The effect we meant to reach
was confirmed in the whole cells test, where an improved
in vivo affinity was detected.
In general, we confirmed the importance of the carbox-
ylate and its optimal distance off from the phenyl ring: the
key role of the phenyl ring was clear when we removed it,
causing the worst deterioration of affinity (Table 1).
Moreover, we successfully introduced a tetrazole moiety
with a better pharmacokinetic profile without affecting
potency toward AmpC.
To investigate the ability of compounds 11a–11m to
reverse antibiotic resistance, we studied their antimicrobial
activity in bacterial cell cultures. Compounds were tested
for synergy with the third-generation cephalosporin CAZ
against clinically isolated bacteria resistant to third-
generation cephalosporins (Nukaga et al. 2004). The mini-
mum inhibitory concentrations (MICs) of the CAZ/inhibitor
combination were determined. Strains tested were K.
pneumoniae 20/013 E. coli 40/012, E. cloacae 558349, E.
aerogenes 563334, P. aeruginosa 12, P. aeruginosa 59, P.
aeruginosa 120, P. aeruginosa 148, and P. aeruginosa 199,

Med Chem Res
Table 2 MICs of third-generation CAZ alone and in combination with compounds 11a–11m
MICs (μg/mL) of CAZ alone or in combination
Bacterial strains
alone
1
11c
11d
11f
11h
11l
11g
11m
K. pneumoniae 20/013
E. coli 40/012
≥1000
500
250
125
>500
250
500
250
250
62.5
125
>500
250
>500
>500
31.25
250
62.5
15.63
125
125
P. aeruginosa 199
125
31.25
62.5
not tested
31.25
62.5
Table 3 MICs of third-generation CAZ alone and in combination
with lead compound 1 and with compounds 11c
Conclusions
MICs (μg/mL) of CAZ alone or in combination
In this study 11 novel thiophene carboxylate derivatives
were designed, synthesized and evaluated for their ability to
inhibit AmpC. We introduced chemical diversity on the
distal part of a low micromolar inhibitor of AmpC, lead 1.
Although a marked increase of potency in in vitro tests
was not registered for the 11 designed and synthesized new
chemical entities, as none of the introduced chemistry was
able to trigger stronger interactions with the open region of
AmpC they were meant to target, this study yielded a better
comprehension of the key pharmacophoric features and the
catalytic pocket of this relevant enzyme. Our study provided
a better understanding of how additional binding pockets
could be critical in designing inhibitors with improved
potency.
Bacterial strains
Alone
1
11c
E. cloacae 558349
E. aerogenes 563334
P. aeruginosa 12
P. aeruginosa 59
P. aeruginosa 120
P. aeruginosa 148
500
250
250
250
125
250
250
125
125
125
62,5
250
125
31,25
62,5
62,5
62,5
125
from the collection of the Spanish Network for Research in
Infectious Pathology-REIPI.
The MIC of CAZ by itself against these pathogens ran-
ged from 125 μg/mL to >1 mg/mL (Table 2), a value sig-
nificantly higher than the threshold for empirical resistance
levels ( ≥ 2 μg ∕ mL) (Performance standards for anti-
microbial susceptibility testing 2010).
Indeed, considering the opened area of the pocket, lar-
gely accessible to the solvent, we can conclude that the
binding affinity is mainly stabilized by the first part of the
molecule, the sulfamoyl-thiophene carboxylic acid moiety.
The rest of the ligand, or at least the functional groups out
of the phenyl ring, contributes in a less extent to the bind-
ing, suggesting that none of the introduced moieties binds
efficiently enough: the anchor for the binding remains the
thiophene carboxylate core and the sulfonamide. In
designing larger more potent inhibitors, additional second-
ary binding sites need to be considered (Babaoglu and
Shoichet 2006).
Interestingly, improvement in the activity toward whole
bacterial cells for this new series of compounds was
achieved. All compounds were able to lower the MIC of
third generation cephalosporin CAZ by a minimum 2-fold
to a maximum of 8-fold compared to the starting lead 1. In
particular, compound 11c achieved the best results and its
synergistic effect in combination with CAZ was tested
against a larger panel of resistant clinically isolated strains,
showing a significant decrease in MIC values compared to
CAZ alone.
Interestingly, in combination with the new inhibitors, the
MIC values of CAZ improved in all the tested strains, typically
by an 8-fold factor. In some cases, the MIC of the combination
CAZ/inhibitor were lower than CAZ/lead 1 (Table 2).
Analyzing the results for the best combination 11c/CAZ,
MICs improved by at least 8-fold in K. pneumoniae strains,
enough to partially restore detectable susceptibility, and by 8-
fold in E. coli and P. aeruginosa strains. The 11c/CAZ
synergistic effect was higher than the lead 1/CAZ association.
Due to the encouraging results, compound 11c was
evaluated in a larger panel of clinically isolated strains
producing AmpC and including E. cloacae, E. aerogenes
and several strains of P. aeruginosa. Compound 11c was
active in all cases, potentiating CAZ activity from two up to
8-fold and being, in combination with CAZ, the most active
against E. aerogenes (Table 3).
When comparing the synergistic effect of compound 11c
with lead 1, improvement in potency was detected vs. all
tested strains, except P. aeruginosa 120 strain vs. other
clinically resistant strains synergist effect improvement
ranged from 1-fold to 4-fold with respect to the initial lead.
Further medicinal chemistry work is ongoing to obtain a
more significant increase in potency for these derivatives,
taking advantage of other additional recognition sites in
AmpC.

Med Chem Res
Acknowledgements This work was supported by NIH GM63815,
by FAR 2014, University of Modena to DT, by Ministerio de Econ-
omía y Competitividad, Instituto de Salud Carlos III, the Spanish
Network for Research in Infectious Diseases RD12/0015/0029 and
Fondo de Investigación Sanitaria Grant PI13/00063. C.I-Q. was sup-
ported by a grant from “Becas para estudios de doctorado en el
extranjero” from Chile Scholarship Program (72110933), National
Council for Scientific and Technological Research (CONICYT),
Chilean Government. The authors acknowledge the “Fondazione Cassa
di Risparmio di Modena” for funding the HPLC-ESI-QTOF system at
the Centro Interdipartimentale Grandi Strumenti of the University of
Modena and Reggio Emilia. We acknowledge Dr. Pasquale Linciano
for support in NMR compounds characterization.
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Conflict of interest The authors declare that they have no com-
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