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

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 Genovese¹Sandra Lazzari¹Ettore Venturi¹Luca Costantino¹

●

Jesus Blazquez^2,3Claudia Ibacache-Quiroga^2,3Maria Paola Costi¹

●

1

Donatella Tondi

Received: 18 May 2016 / Accepted: 13 February 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 K_iof 1 µM, a

ligand efﬁciency of 0.38 kcal mol⁻¹and 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

●

Keywords Structure-based de novo design Thiophene

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derivatives Non covalent β-lactamases inhibitor

●

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 F₂₅₄plates (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, CD₃OCD₃at 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 (K_i= 1 µM); its physical properties

R

HOOC

OH

H

N

NH

O

R

H

S

H

NH

S

O

NH

S

O

N

R¹

OH

COOH

O

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 NaHCO₃saturated solution

(20 mL) and the solution was extracted three times with

ethyl acetate. The collected organic layers were washed with

NaHCO₃saturated 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, OCH₃53.3, 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 H₂SO₄were

added. The mixture was heated under reﬂux overnight. The

reaction was neutralized with NaHCO₃s.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, OCH₃53.4, 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), NaHCO₃s.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, OCH₂60.8,

OCH₃53.7, C-13 45.2, C-15 18.4, CH₃14.2. 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 CH₂Cl₂and washed with NH₄Cl s.s. (2 × 30

mL), brine (2 × 30 mL), NaHCO₃s.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,

OCH₃53.4, OCH₃52.8, 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, OCH₂61.6, OCH₃53.2, C-7 44.8,

CH₃14.0. 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,

OCH₃53.4, OCH₃52.3, 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, OCH₃53.2, OCH₃52.4, 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, OCH₃53.7, OCH₃52.8.

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, OCH₂60.9, OCH₃53.2, C-7 39.1, C-8 34.3,

CH₃14.1. 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, ½αꢀ_D¼ 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, ½αꢀ_D¼ ꢁ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 K_mof

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 K_ivalues

were obtained from IC₅₀plots 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

Na₂SO₄anhydrous 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 C₁₄H₁₃N₂O₄S₂337.0317, 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 Na₂SO₄anhydrous 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

C₁₃H₁₂N₅O₄S₂366.0331, 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 O_γand 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 O_γof Ser64 and the N_ζof Lys67;

the other hydrogen binds to the N_δ2of 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 C_δ1of

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 N_εof 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 K_i^avalues of lead 1

and its derivatives vs. AmpC

#

1

Structure

K_i(µM)

#

Structure

K_i(µM)

1.05

11f

1.8

NH

O

NH

O

COOH

O

S

O

S

COOH

HO

COOH

S

11a

11b

11c

11d

11e

1.7

3.0

19.8

1.0

11g

11h

11i

27

NH

O

NH

S

COOH

O

S

O

COOH

S

HOOC

COOH

S

148

212

44

NH

O

NH

O

S

COOH

O

COOH

S

NH

O

NH

O

S

COOH

S

11l

NH

O

NH

S

COOH

O

S

O

NH₂

COOH

S

HOOC

COOH

S

11m

1.6

F

NH

O

S

N

NH

O

N

S

COOH

N

H

COOH

S

COOH

S

a

Inhibitory constant K_iwas calculated from experimentally determined IC₅₀using the Cheng-Prusoff

equation: K_i= IC₅₀/(1 + S/K_m)

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

62.5

125

>500

250

>500

31.25

250

62.5

15.63

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

125

250

125

62,5

250

125

31,25

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

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