Mercaptophosphonate compounds as broad-spectrum inhibitors of the metallo-β-lactamases

Article abstract of DOI:10.1021/jm100213c

Although commercialized inhibitors of active site serine β-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-β-lactamases (MBLs) have yet been discovered. In this paper, we investigated t

Full text of DOI:10.1021/jm100213c

4862 J. Med. Chem. 2010, 53, 4862–4876
DOI: 10.1021/jm100213c
Mercaptophosphonate Compounds as Broad-Spectrum Inhibitors of the Metallo-β-lactamases†
^
^
#
‡,
Patricia Lassaux,‡, Matthieu Hamel, Mihaela Gulea, Heinrich Delbruck, Paola Sandra Mercuri, Louise Horsfall,‡,
€
Dominique Dehareng, Michael Kupper, Jean-Marie Frere, Kurt Hoffmann, Moreno Galleni, and Carine Bebrone*,‡,
§
#
#
‡,
€
ꢀ
‡Laboratory of Biological Macromolecules, §Applied Quantum Chemistry and Molecular Modelling Unit, and Centre for Protein Engineering,
University of Lieꢀge, Alleꢁe du 6 Aou^t B6, Sart-Tilman, 4000 Lieꢀge, Belgium, ^Laboratoire de Chimie Moleꢁculaire et Thio-Organique, UMR CNRS
6507, INC3M, FR 3038, ENSICAEN, Universiteꢁ de Caen, 6, Boulevard du Mareꢁchal Juin, 14 050 CAEN, France, and #Institute of Molecular
Biotechnology, RWTH-Aachen University, c/o Fraunhofer IME, Forckenbeckstrasse 6, 52074 Aachen, Germany
Received November 4, 2009
Although commercialized inhibitors of active site serine β-lactamases are currently used in coadminis-
tration with antibiotic therapy, no clinically useful inhibitors of metallo-β-lactamases (MBLs) have yet
been discovered. In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives
against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the
exception of 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13 and 21, all the
mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low
inhibition constants (Ki<15 μM). Interestingly, compound 18 turned out to be a potent broad spec-
trum MBL inhibitor. The crystallographic structures of the CphA-10a and CphA-18 complexes
indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn2þ ion,
respectively. Molecular modeling studies of the interactions between compounds 10a and 18 and the
VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending
on the enzyme and the inhibitor, consistent with the crystallographic structures.
Introduction
MBL inhibitor.5,7 The remaining challenge is thus to find a
specific inhibitor of MBLs that is active on all three subclasses.
Among the already described inhibitors, thiols and mercapto-
acetic acid thiol esters were reported to be broad spectrum MBL
inhibitors.8-11 Indeed, thiomandelic acid was an efficient in-
hibitor (Ki < 0.80 μM) of all the MBLs tested, with the excep-
tion of the subclass B2 CphA enzyme (Ki=244 μM).8 The most
potent broad spectrum MBL inhibitors contain both sulfanyl
(thiolate) and carboxylate functional groups capable of metal
ligation. The sulfanyl group was necessary for the inhibition of
the enzyme, while the carboxylate group was not critical. A
study based on the structure-activity relationship of inhibitors
has emphasized the importance in the proximity of the sulfanyl
group to the carboxylate group in enabling successful inhibi-
tion.8 Therefore, because of the presence of two chelating
functions (sulfanyl and phosphonato), the mercaptophos-
phonic acids (phosphorus analogues of mercaptocarboxylic
acids) are potential candidates for MBL inhibitors. Since the
work of Schwarzenbach in 1949, phosphonates and phos-
phonic acids were known as effective chelating agents.12 Two
reviews dealing with P,S-difunctionalized compounds showed
their various applications (in biomolecular chemistry, catalysis,
and hybrid materials chemistry), mainly arising from their
metal-chelating properties.13 Moreover, phosphonate monoe-
sters are known to be inhibitors of active serine class A and
C β-lactamases.14,15 In 2005, inhibition of class D β-lactamases
by phosphonates was also demonstrated.16 However, to the
best of our knowledge, only two publications described the
use of mercaptophosphonic acids as metallophosphatase in-
hibitors.17,18 Thus, a study of this class of compounds as
promising inhibitors of MBLs seems to be required.
To withstand β-lactam antibiotics, bacteria are able to pro-
duce β-lactamases. Among these enzymes, the metallo-β-lacta-
mase (MBLa) group represents an emerging problem because of
its capacity to hydrolyze almost all β-lactam antibiotics, includ-
ing last generation cephalosporins and carbapenems, and its
capacity to propagate among nosocomial bacterial strains.
MBLs constitute the class B of the β-lactamase family, whereas
classes A, C, and D are composed of different serine active site
enzymes.1 On the basis of the known sequences, three different
lineages of MBLs, identified as subclasses B1, B2, and B3, can be
characterized.2,3 Also, functional and mechanistic factors clearly
distinguish the B1, B2, and B3 enzymes from each other.4,5
In parallel to the search for new non-β-lactamase-susceptible
antibiotics, a second strategy to counter β-lactam resistance is
to coadminister the β-lactam antibiotic with a β-lactamase
inhibitor or inactivator. Commercialized inactivators are cla-
vulanic acid, sulbactam, and tazobactam. Unfortunately, they
are only active against serine β-lactamases and do not inactivate
MBLs.6 Several classes of MBLs inhibitors have already been
described; however, none of these classes constitute the “ideal”
†PDB codes: 3IOF and 3IOG.
*To whom correspondence should be addressed. Phone: þ3243663315.
Fax: þ3243663364. E-mail: carine.bebrone@ulg.ac.be.
a Abbreviations: IC50, concentration of inhibitor leading to 50% inhibi-
tion; Ki, inhibition constant; MBL(s), metallo-β-lactamase(s); MM, mole-
cular mechanics; MMP, mercaptomethylphosphonate; PAA, phosphoracetic
acid; PDB, Protein Data Bank; rmsd, root mean square deviation; SAR,
structure-activity relationship; THF, tetrahydrofuran; TLC, thin-layer
chromatography; TLS, translation/libration/screw; TMS, tetramethylsilane;
Zn1, the Zn2þ ion in the so-called “histidine” binding site; Zn2, the Zn2þ ion
in the so-called “cysteine” binding site.
r
pubs.acs.org/jmc
Published on Web 06/08/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4863
Figure 1. Mercaptophosphonates synthesized as potential MBL inhibitors.
Scheme 1
In the paper, we investigate the inhibitory effect of 14 mer-
captophosphonate derivatives (Figure 1: 1a,b; 10-12a,b; 13;
18-22) belonging to three structural families (R-mercapto-
phosphonates, 2-sulfanyl benzenephosphonates, and 2-(sulf-
anylmethyl)phenylphosphonates) against a representative of
the three subclasses of MBLs (VIM-4 (B1),19 CphA (B2),20
and L1 (B3)21). VIM-4 belongs to the subclass B1 acquired
enzymes such as SPMs,22 SIMs,23 GIMs,24 and IMPs.25 The
VIM-type enzymes are clinically relevant, and they have
caused important outbreaks.26-31 Because they are well char-
acterized enzymes, CphA and L1 or FEZ-132 enzymes were
used as models for their subclasses.
Crystallographic studies were conducted on the enzyme
(CphA, VIM-4, FEZ-1)-inhibitor (10a and 18) complexes.
Both complexes were solved for CphA. Unfortunately, no
complex with the two other MBLs was obtained. Conse-
quently, molecular modeling of the complexes allowed differ-
ent binding modes of the inhibitor in complex with examples
from the MBL subclasses to be proposed.
derivative of 1b was synthesized, the fluorinated compound
13). However, the procedure used for 12b enabled us to prepare
other R-mercaptophosphonates (i.e., aliphatic 11 and aromatic
18-22 derivatives). Derivatives bearing halogen atoms on the
aromatic ring have been particularly targeted (18, 20-22), as it
is now well established that halogen bond interactions could
play an important role in molecular recognition processes.33
Synthesis of Inhibitors 10a and 18. Compound 10a was
synthesized in three steps,34 starting from commercially avail-
able diisopropyl phosphite (Scheme 1). Thiophosphate ammo-
nium salt 8 was obtained quantitatively by reacting diisopropyl
phosphite with elemental sulfur (S8) in the presence of triethyl-
amine. Then, S-alkylation of 1 with 2-iodobenzyl chloride in
refluxing toluene led to thiophosphate 9 in 94% yield. A [1,4]-
sigmatropic rearrangement was used to transform thiopho-
sphate 9 into the 2-(sulfanylmethyl)phenylphosphonate 10a.
The iodide-lithium exchange by reacting 9 with tert-butyl-
lithium led to carbanion i, which rearranged into sulfanyl ii
via the migration of the phosphoryl group from the sulfur atom
on the carbon aromatic ring. At the end of the reaction, the
acidic hydrolysis of the sulfanyl led to the desired compound
10a. The overall yield of the three steps was 66%.
Results
The selected mercaptophosphonates (including phospho-
nic esters and their corresponding acids) belong to three struc-
tural families (R-mercaptophosphonates, 2-sulfanyl benzene-
phosphonates, and 2-(sulfanylmethyl)phenylphosphonates),
depending on the relative positions of the two phosphorus
and sulfur functions in their structures. On the basis of the
promising results obtained in several studies with thiomandelic
acid, its phosphonic analogue 12b was first chosen as a repre-
sentative structure of the R-mercaptophosphonate family. Then
compounds 1b and 10b, belonging to 2-sulfanyl benzenephos-
phonates and 2-(sulfanylmethyl)phenylphosphonates, respec-
tively, have been selected for the study. For a structure-activity
relationship (SAR) study, structural modifications on these
three types of mercaptophosphonic acids have been envisaged.
The structures of compounds 1 and 10 are not easy to modify by
the synthetic procedure described in this paper (only one
Compound 18 was synthesized in five steps, as shown in
Scheme 2. First, R-hydroxyphosphonate 14 was prepared by
condensation of 2,4-dichlorobenzaldehyde with diisopropyl
phosphite. Alcohol 14 was then transformed in three steps
into the corresponding thiol 17 using a sequence described
previously by some of us:35 transformation of 14 into its p-
nitrobenzenesulfonate derivative 15, nucleophilic substitu-
tion using potassium thiocyanate leading to thiocyanato-
methylphosphonate 17, and reduction of the thiocyanate
function with sodium borohydride to afford, after acidic
hydrolysis, the expected R-sulfanylphosphonate 17. Com-
pounds 14, 15, and 16 have been used without purification in
the next step (their purity was >95% by NMR spectro-
scopy). Only R-sulfanylphosphonate 17 was purified by flash
chromatography before its transformation into phosphonic

4864 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Lassaux et al.
Scheme 2
acid 18 by treatment with Me3SiBr followed by metha-
nolysis.36 The overall yield of the five steps was 31%.
position of the substituent on the aromatic ring in the
inhibition. Interestingly, the inhibition of CphA is reduced
when a fluorine atom is added at position C5 of the aromatic
ring (Table 2, 1a vs 13). In contrast, the inhibition of VIM-4
and L1 remains similar to that of the original compound with
Ki values of 2 and 4 μM, respectively. A comparison of 12b to
its substituted derivatives highlighted the most interesting
results obtained with the dichlorinated derivative 18. The
presence of the two chlorine atoms on the aromatic ring (in
C2 and C4) has enhanced the inhibition for all three enzymes
with a clear improvement for the L1 enzyme. Compound 19
inhibits the VIM-4 enzyme similarly to 18 (with a Ki of 2 and
1 μM, respectively); however, for the CphA and L1 enzymes,
its propensity of inhibition is decreased (Table 2). As com-
pound 19 contains an OCH3 function at C2 position, the
difference in inhibition could be due either to the loss of the
chlorine group at C4 or to the different nature of the group at
C2. Compounds 20, 21, and 22 are functionalized at the same
position (C4), and that results in a lower inhibition of VIM-4
compared to 18; thus, the substituent on C4 does not seem
important, whereas position C2 seems relevant for the VIM-4
inhibition. Mercaptophosphonic acids 19, 20, 21, and 22 are
not as efficient as 18 in the inhibition of MBLs subclasses.
They exhibit specific profiles of inhibition: 19 is directed
toward VIM-4,; 20 and 21 are directed toward L1; 22 is
directed toward CphA.
Synthesis of the 12 Other Inhibitors. The o-mercaptoben-
zenephosphonate 1a, its phosphonic acid derivative 1b, and
the fluorinated derivative 13, as well as the R-mercaptopho-
sphonates 11a and 12a, were synthesized as described
previously.35,37,38 The phosphonic acids 10b, 11b, and 12b
were obtained from their phosphonic esters 10a, 11a, and 12a
by the same procedure used to transform ester 17 into its acid
18. The R-mercaptophosphonic acids 19, 20, 21, and 22 were
prepared from diisopropyl phosphite and the corresponding
aromatic aldehyde, in five steps, by the same method used for
the synthesis of compound 18.
Screening of the Inhibitory Activities of Mercaptophospho-
nates on MBLs. The screening for inhibition against the
subclass B1 VIM-4, the subclass B2 CphA, and the subclass
B3 L1 enzymes was investigated with the first set of four
mercaptophosphonates, each of them in both ester and acid
forms (a and b, respectively): the o-mercaptobenzenephos-
phonate 1, the o-phosphonylated benzylic thiol 10, and two
R-mercaptophosphonates, one aliphatic 11 and one aro-
matic 12 (Figure 1). The determined inhibition constants
(Ki) are shown in Table 2. When Ki values were higher than
20 μM, the compounds were not considered as good inhibi-
tors and accurate Ki values were not determined. The o-
mercapto(phenylphosphonic) acid 1b is already known as a
tight-binding inhibitor of the bovine intestinal alkaline
phosphatase (Ki = 0.21 μM) and of the Escherichia coli
alkaline phosphatase (IC50 < 10 μM).17 In this study, while
this compound is an efficient competitive inhibitor of the
three subclasses of MBLs with Ki values ranging from 2 to
12 μM, its ester counterpart (1a) had no inhibitory effect on
the tested enzymes. Compound 10b seems to behave as a
strong chelator, since the residual activities of VIM-4 and L1
are considerably increased upon the addition of an excess of
ZnCl2 (50 μM) in the buffer. The inactivation observed in the
absence of added Zn2þ could be explained by the chelation of
the Zn2þ ion by 10b. With the exception of the inhibition of
CphA, 11a shows no significant inhibitory activity against
any of the other MBLs tested. Its acid counterpart (11b)
inhibits CphA as well as VIM-4 with Ki values of ∼10 μM.
The other three compounds 10a, 12a and 12b behaved as
good inhibitors for the three MBLs with Ki values ranging
from 0.25 to 32 μM. 10a also inhibited the N220G-N116H
extended-spectrum mutant of CphA (the Ki value was 7 μM).
We hoped that compounds 13 and 18-22 bearing various
substituents on the aromatic ring would provide better
inhibitory effect than their unsubstituted counterparts 1b
and 12b, respectively. The study of these compounds also
allowed us to examine the importance of the nature or the
In order to determine the interaction mediating the posi-
tioning of the inhibitors in the active site of each enzyme,
crystallographic and docking studies were performed with
10a and 18, the two inhibitors active against the three
subclasses.
Crystallographic Studies. CphA (B2). To elucidate the
molecular basis of the inhibition of CphA, the crystal
structure of the two inhibitor (10a and 18)/CphA mutant
N220G complexes were solved. The N220G mutant was used
instead of the wild-type CphA enzyme because this mutant
crystallizes much more easily and rapidly than the wild-type
enzyme.39,40 The mutation of this noncatalytically essential
Asn residue in Gly does not modify the kinetic parameters of
the enzyme41 or the three-dimensional structure.39,40 The Ki
values obtained for the 10a and 18 compounds and the
N220G CphA enzyme were 6 and 3 μM, respectively. There-
fore, crystals of N220G CphA were soaked in the presence of
the inhibitors. The crystal structures were solved by mole-
cular replacement using the wild-type structure of CphA
(PDB code 1X8G) as model. After refinement, almost all
residues lay in the allowed or favored region in the Rama-
chandran plot. Stereochemical parameters were calculated
by PROCHECK and WHAT_CHECK and were in the
range expected for structures with similar resolutions. The

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4865
Table 1. X-ray Data Collection and Structure Refinement for CphA-
10a and CphA-18
of the inhibitor molecule could be established by calculating
an anomalous map. As in the native structure, the Ala195
residue is in a disallowed region of the Ramachandran plot.
Ala195 (ψ=105ꢀ, φ=156ꢀ) is located on a loop between two
strands, which belongs to the active site and comprises the
functionally important His196 residue.
CphA-10a
CphA-18
Data Collection Statistics
˚
wavelength [A]
˚
cell parameter [A]
1.5418
C2221
1.5418
C2221
The protein structure of CphA in the complex is very close
to that of the native protein (PDB code 1X8G), with a root-
˚
a, b, c [A]
42.65, 100.66, 1116.99
90, 90, 90
1
42.73, 100.88, 117.56
90, 90, 90
1
R, β, γ [deg]
molecules/
asymmetric unit
˚
mean-square deviation between the CR atoms of 0.307 A.
Superimposition of the two structures shows that the binding
of the inhibitor leads to a movement of the Gly232-Asn233
loop located at the active site entrance so that it encloses the
active site (Figure 2b).
˚
resolution [A]
Rsym
19.59-1.44
0.041/0.112
43889/3054
99.72/96.30
11.6/6.6
19.65-1.41
0.042/0.084
45999/3175
98.33/92.92
9.8/6.3
no. unique reflexes
completeness [%]
multiplicity
I/σ(I)
Rwork
Rfree
The structure of the CphA-10a complex shows that the 10a
inhibitor binds to the catalytic Zn2þ and interacts with the
enzyme active site with both electrostatic and hydrophobic con-
tacts. As previously proposed for thiomandelic acid in the
subclass B1 BcII enzyme,8,42 the inhibitor 10a binds the Zn2þ
ion of CphA via its sulfur (Figure 2b). Typically in CphA struc-
tures, the Zn2þ ion is coordinated to the three MBL conserved
Cys221, Asp120, and His263 residues and an ion from the buffer
solution (e.g., a carbonate ion in 1X8G). Here, the sulfur of the
inhibitor completes the tetrahedral coordination sphere of the
Zn2þ ion. The distance between the Zn2þ ion and the sulfur of
40.7/16.3
0.1225/0.123
0.1408/0.153
37.3/16.6
0.1434/0.146
0.1545/0.176
Root Mean Square Deviation from Ideal
˚
bonds [A]
0.011
1.565
1919
1
0.011
1.510
1889
1
angles [deg]
proteins atoms
Zn2þ
water
290
285
˚
the inhibitor is 2.31 A. Moreover, two water molecules are at
˚
distances of 3.20 (W333) and 3.11 A (W435) from the sulfur
atom. Both water molecules are involved in a large hydrogen
Table 2. Competitive Inhibition Constant Ki (μM) of the Different
Mercaptophosphonate Compounds for One Member of Each MBL
Subclassa
˚
bond network within the protein. W333 is situated 2.85 A away
˚
from the conserved Lys224 residue and is also in contact (2.99 A)
with the backbone nitrogen of Asn233. W435 has additional
VIM-4 (B1)
L1 (B3)
no Zn2þ
CphA (B2)
no Zn2þ
˚
contacts to Asn233 ND2 (2.79 A), to His196 NE2 (2.93 A), and
˚
compd
Zn2þ
no Zn2þ
Zn2þ
˚
also to O21 of the phosphonato group (3.08 A) (Figure 2b).
His196 NE2 and His263 NE2 are also at distances of 3.50 and
1a
1b
>250
>250
2
>250
7
>250
12
14
>250
9
4
12
˚
3.42 A from the sulfur atom of the inhibitor.
There is an interaction between the O1 of the phosphonato
˚
group and CE2 of the Phe156 residue (2.89 A). The methyl
10a
10b
11a
11b
12a
12b
13
3
2
3
>400
>20
1
0.5
2
>50
>40
>40
16
2
>20
11
3
>40
>80
20
groups of the inhibitor molecule are involved in several
interactions with the protein, notably C91 with Val67 CG2
6
8
15
0.25
11
>40
5
˚
(3.19 A), C91 with Leu161 CD1 (3.38 A), C101 with Leu161
˚
3
2.5
2.5
1
32
>100
40
˚
CD1 (3.68 A), C101 with Thr157 O (3.30 A), C92 with Ile153
˚
13
4
4
0.40
18
19
0.40
9
˚
CD1 (2.92 A), C92 with Thr157 OG1 (3.47 A), C102 with
˚
4
13.5
2
15
13
24
1
4
˚
˚
Asp120 OD1 (3.24 A), and C102 with Thr119 OG1 (3.32 A).
A hydrophobic contact is observed between Cβ from Asn233
and the aromatic ring of compound 10a. The Cβ atom is
20
21
5.5
6.7
16
1.7
0.7
8.4
3.8
10
11
22
18
>20
˚
3.8 A from the center point of the ring structure (Figure 2c).
a SD values are below 10%. ND: not determined. Zn2þ: the added
Zn2þ concentration is 50 μM. No Zn2þ: no added Zn2þ; the Zn2þ
concentration is below 0.4 μM.
Structure of the CphA-18 Complex. To investigate the
structural basis of the inhibition of CphA by the [(2,4-dichlo-
rophenyl)(sulfanyl)(methyl)]phosphonic acid (18), crystals of
crystallographic and model statistics for both structures are
summarized in Table 1.
the complex of CphA with the inhibitor were produced. The
˚
structure was refined in the range of 19.65-1.41 A to Rwork
=
Structure of the CphA-10a Complex. To investigate the
structural basis of the inhibition of CphA by the diisopropyl
[2-(sulfanylmethyl)phenyl]phosphonate (10a), crystals of the
inhibitor associated with the CphA enzyme were produced.
0.1435 and Rfree = 0.1548. The CphA-18 complex structure
contains all 227 protein residues. The first two N-terminal
residues are refined in two orientations. The last C-terminal
residue is half occupied only. Moreover, some side chains show
high mobility and are disordered or not fully occupied. One
Zn2þ ion, 5 sulfate ions, 1 molecule of 18 (Figure 3a), 2 glycerol,
and 285 water molecules (Table 1) are found in the structure.
The orientation of the 18 molecule could be verified by calcula-
tion of the anomalous map. Some unexplained density remains
in the active site; however, this supplementary electron density
could not be attributed to water molecules or to an additional
inhibitor molecule. The protein structure of CphA in the
complex is very close to that of the native protein (1X8G),
with a root-mean-square deviation between the CR atoms of
˚
The structure was refined in a resolution range of 19.6 to 1.44 A.
The refinement yielded an Rwork of 0.1228 and Rfree of 0.1415.
The refined CphA-10a complex structure comprises all 227
protein residues. The N-terminus, C-terminus, and some sur-
face oriented residues exhibit high mobility (expressed by high
B-values). Furthermore, 1 Zn2þ ion, 10 sulfate ions, 1 molecule
of 10a, 3 glycerol molecules, and 290 water molecules (Table 1)
were identified in the electron density. In the active site,
the electron density corresponds to the shape of the inhibitor
(Figure 2a). The positions of the sulfur and phosphorus atoms

4866 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Lassaux et al.
Figure 2. Crystallographic structure of the CphA-10a complex. (a) 2Fo - Fc map at 1σ level. The electron density in the active site of CphA
corresponds to the shape of molecule 10a. (b) Inhibitor 10a interacts with the Zn2þ ion via its sulfanyl group. Water molecules (W) and Zn2þ ion are
represented as red and gray spheres, respectively. The “closed” position of the Gly232-Asn233 loop in the complex is represented in green to emphasize
the modification compared to the native structure (in cyan). (c) Interactions between CphA and the phosphonato and methyl groups of 10a depicted
using LIGPLOT.66 The hydrophobic interaction between theAsn233residueandthearomaticringof10a is also represented. In the schematic drawing,
hydrogen bonds are shown as dashed green lines. Ligand and protein hydrophobic contacts are represented as curved red combs.
˚
0.267 A. The difference with the 10a complex structure is
˚
0.171 A. Once more, the binding of the inhibitor leads to the
chlorine atom is also in contact with the CE1 carbon of the
˚
His263 residue (3.35 A), while the second chlorine atom Cl2
˚
binds a water molecule (W284, 3.57 A).
“closed” position of the Gly232-Asn233 loop located at the
active site entrance. Surprisingly, in contrast to the CphA-
10a complex, the Zn2þ ion is bound by the phosphonato
Modeling Studies. Crystals of the VIM-4 (B1) and FEZ-1
(B3) enzymes were obtained as described previously43,44 and
soaked with 10a and 18. Unfortunately, no electron density
corresponding to the inhibitors was observed in the active sites
of the solved structures. Therefore, modeling studies were
performed with VIM-4 (B1), CphA (B2), and the monomeric
FEZ-1 enzyme. This study is much easier than with L1 (B3), as
the latter enzyme is a homotetramer.45 In the following, Zn1
refers to the Zn2þ ion in the so-called “histidine” binding site
where it is coordinated by three conserved histidine residues
(His116-His118-His196) while Zn2 corresponds to that in the
“cysteine” binding site (Asp120-Cys221-His263 in the B1 en-
zyme and Asp120-His121-His263 in the B3 enzyme). In CphA,
the sole Zn2þ ion binds in the “cysteine” site (Asp120-Cys221-
His263).
˚
˚
oxygen atoms O11 (1.98 A) and O12 (3.38 A) of the inhibitor 18
and not by the sulfur (Figure 3b) (it is worth noting that in 10a
the phosphorus function is a phosphonic ester while in 18 it is a
phosphonic acid). Furthermore, O11 is in contact with water
˚
molecule 419 at a distance of 2.57 A. The O12 of the phospho-
nato group also interacts with the NE2 of the His263 residue
˚
(3.38 A). The same oxygen is close to the NZ of the Lys224
˚
residue (2.72 A), and the third oxygen O13 of the phosphonato
group lies in the vicinity of the NE2 of the His196 residue (2.76
˚
A). The water molecule 473 (W473) stabilizes the side chain of
˚
Asn233 through a contact to ND2 (2.77 A). Moreover, W473 is
˚
in contact with the O13 of the phosphonato group (2.88 A) and
˚
with His196 NE2 (3.19 A). The sulfur of the inhibitor is in
˚
contact with a water molecule (W347, 3.06 A) and the backbone
˚
nitrogen of the Asn233 residue (3.45 A). This latter interaction
stabilizes the “closed” form of the Gly232-Asn233 loop. The Cl1
VIM-4-18. Manual docking of compound 18 in the active
site of VIM-4 was the starting point of this study, followed by
three steps of energy minimization. Dynamic simulation and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4867
Figure 3. Crystallographic structure of the CphA-18 complex. (a) 2Fo - Fc map at 1σ level. The electron density in the active site of CphA
reflects the shape of molecule 18. (b) Inhibitor 18 interacts with the Zn2þ ion via two oxygen atoms of the phosphonato group. Water molecules
(W) and Zn2þ ion are represented as red and gray spheres, respectively.
Table 3. Characterization of the Potential Binding Modes between VIM-4 and 18 and 10aa
18
conformation
10a
conformation
parameter
energy (kcal/mol)
interaction energy
1
2
3
4
5
1
2
-144.4
21.53
-141.85
22.75
-144.47
26.04
-123.65
21.33
-81.93
2.08
-210.61
22.45
-95.2
5.84
distortion energy of 18
ꢁ
˚
distance of interaction (A)
Zn1
His116
His118
His196
18-S
2.23
2.12
2.10
2.52
2.16
2.13
2.14
2.09
2.10
2.10
2.16
2.22
2.16
2.13
2.10
2.45
(6.24)
2.21
2.19
2.48
1.86
(4.47)
2.16
2.27
2.36
10a-S
10a-O1
Asp120
18-O11
18-O13
Cys221
2.09
2.05
1.94
1.89
2.25
4.20
2.00
2.01
2.16
2.13
1.92
2.23
3.73
1.96
1.98
2.18
(3.76)
1.89
2.21
4.16
1.97
2.02
2.17
2.15
2.15
Zn1-Zn2
Zn2
5.28
1.91
3.93
1.92
2.27
2.10
2.10
5.74
1.97
1.98
2.13
2.09
4.35
1.91
Asp120-O1
Asp120-O2
Cys221
2.11
2.09
2.12
2.21
2.15
His263
18-O13
His116
a Distances in parentheses are too long for the residue to be a Zn2þ ligand. The interaction energy is the difference between the energy of the complex
and the energies of its constituents in the complex geometry. The distortion energy is the sum of the distortion energies of the constituents, i.e., the
difference between the energy of the constituents in the complex geometry and the energy of their optimized geometries.
energy minimization were successively employed to obtain
optimal conformations of the complexes. Thus, eight differ-
ent conformations representing four different binding modes
were obtained (Table 3). Among them, only one (conformation
5) was mediated by the sulfanyl group of compound 18, with the
smallest interacting energy (-82 kcal/mol). The other three,
with a fifth conformation (conformation 4) obtained after mini-
mization of the VIM-4-18 complex built from the crystallo-
graphic complex CphA-18, were characterized by interactions
between Zn2þ ions and the phosphonato group. Conformation
1 exhibited a coordination between one oxygen (O11) of the
phosphonato group with Zn1 and a second interaction between
a second phosphonate oxygen (O13) and Zn2. Conformation 2
was characterized by a Zn1-O13 bond, whereas conformation
4 had a Zn2-O13 interaction. Finally, conformation 3 had two
of its oxygens (O11 and O13) coordinating Zn1. Apart from
conformation 4 presenting a small interacting value, the other
three conformations (1, 2, 3) were characterized by a strong inte-
raction energy value of -144 kcal/mol with a similar deforma-
tion of compound 18, corresponding to the likely model of
binding. This possible binding mode between VIM-4 and 18
would occur via a binding of the inhibitor phosphonato group
with Zn1 or both Zn2þ ionsinthe active site (Figure 4a). Interes-
tingly, all our models were highly variable. Indeed, as high-
lighted in Table 3, the positions of the Zn2þ ions, particularly
Zn2, and the distance between them were strongly perturbed
because of some rearrangement of the overall fold of the protein.
This mobility is emphasized by the change in the coordination
spheres of both Zn2þ ions, and in conformations 1, 2, and 4,
Cys221 was now interacting with both of them via a rotation of
its position. Furthermore for conformations 1 and 2, Asp120
was binding to Zn2 via two oxygen atoms and not one as usually
described. Thus, out of the three conformations, conformation 3
seems the most probable mode of binding, as the coordination
sphere of the Zn2þ ions is not perturbed (Figure 4a). This model
of interaction is coherent with the crystallographic model of
CphA-18.
VIM-4-10a. As for inhibitor 18, several complex models
were built by manual docking or by using the conformation
of compound 10a from the crystallographic CphA-10a
complex. After minimization conducted as previously, two
conformations were selected corresponding to two different
binding modes both mediated by the inhibitor sulfanyl group
interacting with Zn1 (Table 3). Conformation 1 presented

4868 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Lassaux et al.
Figure 4. Molecular modeling of VIM-4: stereoviews of all conformations. (a) Conformations with the best interactions representing the
possible mode of binding of compound 18 to VIM-4. The inhibitor interacts with Zn1 via two oxygen atoms (O11 and O13) of its phosphonato
group. (b) Conformation representing the possible binding mode of compound 10a to VIM-4. The inhibitor interacts with Zn1 via one oxygen
atom (O1) of its phosphonato group and via its sulfanyl group.
two interactions between 10a and VIM-4, one S-Zn1
and one O11-Zn1; this conformation presented a stronger
interaction than conformation 2 characterized by the sole
sulfanyl-Zn1 coordination (Figure 4b). As for the crystallo-
graphic complex CphA-10a, an interaction via the sulfanyl
group was preferred to an interaction occurring via the
phosphonato group of 10a, and this binding mode seems
to be the most probable.
sulfanyl group to the Zn2þ ion. In the second, it is one oxygen
atom (O11) of the phosphonato group that coordinates the
Zn2þ ion. Finally, in the third conformation, two oxygen
atoms (O11 and O12) of the phosphonato group are binding
to the metal. This last interaction is the one resulting in the
best interaction energy (Figure 5c). This interaction is similar
to the observed crystallography results; however, the rmsd
˚
value of 0.791 A emphasizes that if the interaction remains
CphA-18. The optimization of the crystallographic com-
plex CphA-18 and other manually built complexes was
performed as previously. Three different binding modes were
considered (Table 4). Interestingly, in the first model corre-
sponding to the optimization of the crystallographic com-
plex, the binding of 18 with the protein evolved from two
interactions between the Zn2þ ion and two oxygen atoms
(O11 and O12) of the phosphonate to one interaction via its
the same, the overall fold of the protein is significantly
different. This result is consistent with an interaction taking
place between the phosphonato group and the Zn2þ ion
rather than the sulfanyl group.
CphA-10a. The optimization was performed on various
manually docked complexes and on the crystallographic
CphA-10a complex. Different models were obtained, repre-
senting one major binding mode, since all of them occurred

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4869
Table 4. Characterization of the Potential Binding Modes between CphA and 18 and 10aa
18
conformation
10a
conformation
parameter
energy (kcal/mol)
interaction energy
1
2
3
structure
1
2
3
structure
-87.3
3.56
-52.67
1.84
-91.3
17.66
-114.69
6.9
-144.45
14.67
-62.35
10.54
distortion energy of 10a
˚
distance of interaction (A)
Zn
Asp120-O1
Asp120-O2
Cys221
His263
1.97
1.93
1.98
1.95
2.00
2.02
2.19
2.20
2.31
2.12
2.01
2.17
(6.04)
1.89
1.96
2.14
2.14
2.34
2.18
(4.20)
2.24
2.2.1
2.32
2.08
2.15
2.14
2.28
2.34
2.11
2.30
18-S
18-O11
18-O12
10a-S
10a-O1
His118
1.96
2.11
2.03
1.99
3.38
1.88
2.20
a Conformations 1 (compound 18) and 3 (compound 10a) are the results of the minimization of the X-ray structure of both complexes. Interaction
distances of the crystallographic structure of both complexes are represented in this table. Distances in parentheses are too long for the residue to be a
Zn2þ ligand. The interaction energy is the difference between the energy of the complex and the energies of its constituents in the complex geometry. The
distortion energy is the sum of the distortion energies of the constituents, i.e., the difference between the energy of the constituents in the complex
geometry and the energy of their optimized geometries.
via the sulfanyl counterpart of compound 10a with the
exception of conformation 2 that binds the Zn2þ ion via the
oxygen (O1) of the phosphoryl (PdO) group (Table 4). This
latter conformation exhibits a much stabilized complex, as its
energy of interaction is -144 kcal/mol with a meaningful
deformation of the compound (Figure 5b). The root mean
square deviation of model 3 to the original structure has been
calculated (0.915 A) and has revealed an important change to
the overall fold of the protein. This deviation is even higher in
˚
the case of conformation 2 with a value of 1.728 A making
evident the important changes to the overall structure. In this
case, the minimization process yielded many results consistent
with the crystallography results (Figure 5a); however, the
preferred conformation, which is significantly different to the
initial protein, occurs with a different binding mode when
compared to the crystallography results.
group highlights the complex interactions of this compound.
Once again, all conformations had Zn2þ coordination dif-
ferent from the usual ones as His116 or His121 and/or
His263 was too far from the Zn2þ ions to interact.
Discussion
To date, in contrast to active serine β-lactamases, no
clinically useful inhibitor of MBLs is available despite the
huge number of tested molecules. The development of a broad
spectrum MBL inhibitor is challenging because of the struc-
tural diversity and differences in metal utilization by these
enzymes.5 Many already described MBL inhibitors as “po-
tent” inhibitors active only against one subclass of MBLs or
even only against one specific enzyme,7,46-49 rather than as
“good” inhibitors active against the three subclasses. How-
˚
ꢁ
ever, recently, Lienard et al. reported the most potent broad
FEZ-1-18. Many conformations were obtained for this
complex; however, all the interactions were characterized by
O12-Zn1 and O11-Zn2 binding (Figure 6a), with an energy
of -144 kcal/mol as the best interaction energy (Table 5).
The coordination spheres of both Zn2þ ions were not con-
served in those conformations, as His116 and His121 were
now too far from the Zn2þ ions to coordinate them. This
distortion of the protein was possibly due to the chosen
charge of 1.5 for the Zn2þ. As a matter of fact, the net charge
of the Zn2þ ions was determined at the quantum chemistry
level for a complex involving the inhibitor and two Zn2þ
ions. However, in the case of the FEZ-1-18 complex, the
most favorable interaction occurs via the phosphonato
group, as described for VIM-4 and CphA.
FEZ-1-10a. Four conformations were obtained at the
end of FEZ-1-10a optimization, with two different binding
modes (Table 5). Both of them were characterized by the
binding of the sulfanyl group to the protein, with an inter-
action with Zn1 for the first conformation and the second
one occurring between its sulfanyl and Zn2 and the oxygen
(O1) of the phosphonato group and Zn1. This latter con-
formation, conformation 2 (Figure 6b), exhibited a strong
interaction (-243 kcal/mol) that represented the strongest
interaction of all obtained models. Therefore, conformation
2 is the most probable mode of interaction between FEZ-1
and 10a. The interaction between the sulfanyl group and the
Zn2þ cation is consistent with the crystallographic data;
however, the additional interaction via the phosphonato
spectrum inhibitor ever described that exhibits Ki values lower
than 1 μM for all tested MBLs (BcII, IMP-1, CphA, L1, and
FEZ-1).11 This compound, aswellasthe thiomandelicacid8 or
D-captopril,10 combines in its structure a thiol, a carboxylate,
and an aromatic ring. Thiol, phosphonic acid, and carboxy-
late groups have an affinity for metal ions. Hence, their
phosphorus analogues, i.e., mercaptophosphonic acids and
their ester counterparts, were synthesized and investigated as
broad spectrum MBL inhibitors. Compound 12b is the phos-
phonate analogue of the well described thiomandelic acid.8
Among the 14 tested compounds, 1b, 10a, 12a, and 12b
behaved as good inhibitors for the three subclasses of enzymes
with Ki values ranging from 0.25 to 32 μM. The strategy used
for enhancing the inhibitory potency of phosphonic acids
against metallophosphatases has been to substitute one car-
bon with a halogen;17,18 the same strategy has also been
employed in this study. Here, compound 18, which differs
from compound 12b by the presence of two chlorine atoms on
the phenyl ring, showed an increased inhibition for all the
three representative enzymes with a clear improvement for the
subclass B3 L1 enzyme. The Ki values were 1, 5, and 0.4 μM
for the B1, B2, and B3 enzymes, respectively. Thus, the Ki
values are in the same range as those observed for Lienard’s
compound11 with the exception of the value obtained for the
subclass B2 enzyme which is slightly higher for 18. However,
18 is much better than the thiomandelic acid as a subclass B2
inhibitor8 and in contrast to the latter thus presents a broad
inhibitory spectrum.

4870 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Lassaux et al.
Figure 5. Molecular modeling of CphA: stereoviews of all conformations. (a) CphA-10a: The inhibitor interacts with the Zn2þ ion via its
sulfanyl group. This conformation (3) is similar to the crystallography data but is not that exhibiting the best interaction energy.
(b) CphA-10a: The inhibitor interacts with the Zn2þ ion via one oxygen atom (O1) of the phosphonato group. This conformation represents
the best computed interaction between 10a and CphA. (c) CphA-18: The inhibitor interacts with the Zn2þ ion via two oxygen atoms (O11 and
O12) of the phosphonato group. This conformation is in agreement with the crystallographic data.
To explore the enzyme-inhibitor interactions, compounds
10a and 18 were chosen to obtain the crystallographic structures
of the complexes. We succeeded in obtaining the structures of
the CphA-10a and CphA-18 complexes. Both inhibitor en-
zyme complexes are stabilized by a network of hydrophobic
contacts and hydrogen bonds. Interestingly, these complexes

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4871
Figure 6. Molecular modeling of FEZ-1: stereoviews of all conformations. (a) Conformation representing the possible mode of binding of
compound 18 to FEZ-1. The inhibitor interacts with Zn1 and Zn2 via two oxygen atoms (O11 and O12) of the phosphonato group.
(b) Conformation representing the possible mode of binding of compound 10a to FEZ-1. The inhibitor interacts with Zn1 via one oxygen atom
(O1) of the phosphonato group and with Zn2 via the sulfanyl group.
point to two types of interactions with the enzyme depending of
the complexed inhibitor. Indeed, in the case of 10a, an interaction
of the sulfur atom with the active site Zn2þ ion is observed, while
18 interacts with the Zn2þ ion via its phosphonato group. A simi-
lar situation is observed in the CphA-D-captopril complex
(PDB code 2QDS) where the main interaction between D-capto-
pril and the metal occurs via the inhibitor carboxylate group and
not via the sulfanyl group of D-captopril.11 However, a compar-
ison of the CphA-10a and CphA-18 complex structures with
that previously reported for the carbapenem substrate40 or for
other inhibitors (PDB codes 2QDS and 2GKL)11,50 reveals a
general pattern composed of three important contacts: (i) con-
tact to the Zn2þ ion, (ii) contact to the conserved Lys224 residue
(directly or via a water molecule), and (iii) contact to Asn233 that
stabilizes the Gly232-Asn233 loop closure. In this respect, it is
not surprising that the simplest compound (11a) has a low Ki
constant (2 μM) for CphA.
have also revealed the existence of different binding modes.18
Those compounds differ only by the substitution of the carboxy-
late group of PAA by a sulfanyl group in MMP. The inhibitor
PAA bridges the two Zn2þ ions of the enzyme via two of the
three oxygen atoms of its phosphonato group, while the struc-
ture presenting the interaction between the alkaline phosphatase
and MMP shows that the sulfanyl is a ligand of the first zinc ion
with the phosphonato group directed out of the active site. The
sulfanyl group then seems to be preferred as a ligand to the
phosphonato group. However, the phosphonate is able to form
three very strong salt bridges with both Zn2þ ions. MMP is a
much more efficient inhibitor than PAA, suggesting that mer-
captophosphonates would be more potent than phosphonates.
As we did not succeed in obtaining any MBL-inhibitor com-
plex for subclasses B1 and B3, modeling studies were performed
on the VIM-4 and FEZ-1 enzymes. Those studies revealed that
the most probable binding mode of compound 18 occurs via its
phosphonato group as obtained in the CphA-18 crystallo-
graphic complex. For 10a, the agreement is not so good. In the
crystallographic data, the sulfanyl is interacting with the Zn2þ
ion while the “best” model obtained for the CphA enzyme with
molecular modeling favors an interaction between the phospho-
nato group and the Zn2þ ion. However, the structure observed
by X-rays ranks among the three best ones obtained by model-
ing. Futhermore, the inhibitor-protein interaction in the
A short contact between the O1 of the phosphonato group
˚
and CE2 of the Phe156 residue (2.89 A) is observed for 10a.
This kind of hydrogen bond can be observed when an aromatic
or aliphatic carbon atom is polarized by an adjacent covalently
bonded heteroatom, acidifying the CH hydrogen atoms.51
Previously, the crystal structures of the Escherichia coli
alkaline phosphatase in interaction with a phosphoracetic acid
(PAA) or a mercaptomethylphosphonate (MMP) inhibitor

4872 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Lassaux et al.
Table 5. Characterization of the Potential Binding Modes between
FEZ-1 and 18 and 10aa
Column chromatography was performed using Merck silica gel
Si 60 (40-63 μm). The quality of the solvents used was RS. THF
was purified with a PURESOLV apparatus developed by
Innovative Technology Inc. NMR spectra were recorded with
a Bruker DPX 250 MHz or a Bruker DRX 400 MHz spectro-
meter. The peak patterns are indicated as follows: s, singlet; d,
doublet; t, triplet; sept, septuplet; m, multiplet. Spectra were
recorded with TMS as internal standard or with 85% phospho-
ric acid solution as external reference. The coupling constants
(J) are reported in hertz (Hz). Mass spectra were obtained on a
GC/MS Saturn 2000 spectrometer and HRMS data on a Waters
QTOF microspectrometer. IR spectra were recorded with a
Perkin-Elmer 16 PC FT-IR instrument. The purity of all tested
inhibitors was assessed by elemental analysis or by HPLC
analysis and found to be g95% (details are given in Supporting
Information). A Waters PDA-HPLC instrument with a Waters
XTerra MS-C18 (150 mm ꢀ 4.6 mm, 3.5 μm particle size)
reversed-phase column was used for analytical HPLC analyses.
The eluent was water/acetonitrile: 10/90, flow rate 1 mL/min.
Elemental analysis was performed on a ThermoFinnigan NA
2500 apparatus. Melting points were measured with a Gallen-
kamp apparatus and are uncorrected.
10a
conformation
18
conformation
parameter
1
1
2
energy (kcal/mol)
interacting energy
distortion energy
of compound 10a
-115.45
7.52
-242.54 -161.96
20.44
18.20
˚
distance of interaction (A)
Zn1
His116
His118
His196
18-S
(5.14)
2.07
2.15
(4.24)
2.20
2.12
(4.56)
2.16
2.17
2.27
10a-S
18-O12
2.82
4.52
1.94
10a-O1 1.87
3.18
1.93
Zn1-Zn2
Zn2
6.49
1.93
Asp120-O1
Asp120-O2
His121
2.01
(4.06)
(5.09)
(3.96)
2.17
1.90
(4.31)
(3.94)
10a-S 2.29
His263
18-O11
Inhibitors 1a, 1b,37 11a, and 12a35 have been synthesized and
described previously. Synthesis and analytical data for inhibi-
tors 13, 10b, 11b, 12b, 19-22 and additional spectroscopic data
for compounds 8, 9, 10a, 14-18 are given in Supporting
Information.
Asp120-main
chain
2.06
His118-main
chain
His116
2.05
2.17
Synthesis of Inhibitor 10a. Triethylammonium Diisopropyl
Phosphorothioate (8). To a round-bottom flask equipped with
a condenser diisopropyl phosphite (10.2 g, 1 equiv, 61.4 mmol) and
sulfur (1.9 g, 1 equiv, 61.4 mmol) were added. Then triethylamine
(15 mL) was added dropwise. The reaction is exothermic. After 30
min, when the mixture reached room temperature, the excess
triethylamine was evaporated. The ammonium salt 8 was obtained
as a brown viscous liquid and was used directly in the next step.
a Distances in parentheses are too long for the residue to be a Zn2þ
ligand. The interaction energy is the difference between the energy of the
complex and the energies of its constituents in the complex geometry.
The distortion energy is the sum of the distortion energies of the
constituents, i.e., the difference between the energy of the constituents
in the complex geometry and the energy of their optimized geometries.
1
Yield: quantitative. 31P NMR (101.2 MHz, CDCl3): δ 56.9. H
crystallographic complex could be influenced by the crystal
packing. For VIM-4 and FEZ-1, the most probable model of
binding was characterized by two interactions between the
enzyme and the inhibitor, S-Zn1 and O-Zn2 for VIM-4
and S-Zn2 and O-Zn1 for FEZ-1. The electrostatic surface
potentials of VIM-4 and CphA indicate strongly cationic active
site pockets which could select an interaction with the phos-
phonato group. In contrast FEZ-1 exhibits an anionic active
site pocket that should have an impact on the interaction. The
two methyl groups on phosphonate 10a increase the size of this
part of the molecule. The interaction of this inhibitor via its
phosphonate seems to be impaired by its bulk, which could
explain why the sulfanyl group is the main interacting group in
this case and why the minimization process selects interactions
occurring via both functions even if the protein is highly
destabilized. On this basis, the sulfanyl interaction between
10a and the MBLs seems to be the real interaction.
NMR (250 MHz, CDCl3): δ 1.27 (d, 12H, 3JHH = 6.3 Hz); 1.35 (t,
9H, 3JHH = 7.3 Hz); 3.13 (q, 6H, 3JHH=7.3 Hz); 4.60 (dsept, 2H,
3JHH=6.1 Hz, 3JHP=10.7 Hz); 12.25 (s, 1H). 13CNMR(62.9MHz,
CDCl3): δ 8.8; 23.9 (d, 3JCP=4.7 Hz); 24.1 (d, 3JCP=4.9 Hz); 45.6;
69.3 (d, 2JCP = 6.0 Hz).
Diisopropyl (2-Iodobenzyl)phosphorothioate (9). To a 250 mL
round-bottom flask equipped with a condenser were added ammo-
nium phosphorothioate 8 (9.9 g, 1 equiv, 33.1 mmol) and 2-iodo-
benzyl chloride (10.0 g, 1.2 equiv, 39.7 mmol) in toluene (100 mL).
The mixture was heated for 14 h, then allowed to reach room tem-
perature. The organic layer was washed with a 5% solution of sodi-
um thiosulfate, separated, dried over MgSO4, filtered, and evapo-
rated. The brown oil was purified by flash chromatography (eluent:
pentane/AcOEt 60/40) to afford compound 9 as a colorless liquid.
Yield: 94%. 31P NMR (101 MHz, CDCl3): δ 24.8. 1H NMR (250
MHz, CDCl3): δ 1.26 (d, 6H, 3JHH=6.2 Hz); 1.29 (d, 6H, 3JHH
=
6.2 Hz); 4.16 (d, 2H, 3JHP=13.8 Hz); 4.69 (dsept, 2H, 3JHH=6.2
Hz, 3JHP = 9.1 Hz); 6.94 (dt, 1H, 3JHH = 7.7 Hz, 4JHH = 1.7 Hz);
In conclusion, the investigation of mercaptophosphonate
derivatives as MBL inhibitor has allowed us to find broad
spectrum inhibitors active on representative members of all
the three MBL subclasses. These compounds presentKi values
in the same range as those of the compound previously
7.29 (dt, 1H, 3JHH =7.5 Hz, 4JHH =1.7 Hz);7.51(dd, 1H, 3JHH
=
7.6 Hz, 3JHH=1.6 Hz); 7.82 (dd, 1H, 3JHH = 7.9 Hz, 4JHH = 1.1
Hz). 13C NMR (62.9 MHz, CDCl3): δ 23.9 (d, 3JCP=5.7 Hz); 24.1
(d, 3JCP =3.8 Hz);40.9(d, 2JCP =3.8 Hz);73.1(d, 2JCP =6.3 Hz);
100.6; 128.9; 129.7; 131.1; 140.0; 140.7 (d, 3JCP = 5.0 Hz).
ꢁ
described as a broad-spectrum MBL inhibitor by Lienard
Diisopropyl 2-(Sulfanylmethyl)phenylphosphonate (10a). In a
round-bottom flask, under nitrogen, a solution of 2-iodobenzyl
phosphorothioate 9 (508 mg, 1 equiv, 1.2 mmol) in 5 mL of THF
was added dropwise to a solution of t-BuLi (1.4 mL, 2 equiv, 1.7 M
in hexane, 2.4 mmol) in THF (20 mL) at -78 ꢀC. After the mixture
was stirred for 2 h at -78 ꢀC, the reaction was quenched at -10 ꢀC
with 10 mL of HCl (2 N). The aqueous layer was extracted with
Et2O (2 ꢀ 15 mL). Then the organic layers were separated, dried on
MgSO4, filtered, and evaporated. The obtained product was puri-
fied by chromatography on silica gel (eluent: pentane/AcOEt 50/50)
to give compound 10 as a yellow oil (210 mg, 61%). 31P NMR
et al.11 Moreover, on the basis of structural and modeling
data, these phosphonate compounds could be improved
further.
Experimental Section
Synthesis. General. Reaction progress was monitored by
thin-layer chromatography (TLC) using Merck silica gel 60 alu-
minum sheets (F254). The plates were visualized either by UV
light (254 nm) or by a solution of potassium permanganate.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4873
1
(101.2 MHz, CDCl3): δ 17.6. H NMR (250 MHz, CDCl3): δ
1.26 (d, 3H, 3JHH = 6.2 Hz, CH3); 1.27 (d, 3H, 3JHH = 6.2 Hz);
1.38 (d, 3H, 3JHH = 6.2 Hz); 1.39 (d, 3H, 3JHH = 6.2 Hz); 2.21 (t,
1H, 3JHH=8.4 Hz); 4.05 (d, 2H, 3JHH=8.4 Hz); 4.75 (dsept, 2H,
3JHH=6.2 Hz, 3JHP=9.6 Hz); 7.27-7.32 (m, 1H); 7.45-7.49 (m,
2H); 7.89 (ddd, 1H, 3JHP=14.3 Hz, 3JHH = 6.9 Hz, 4JHH = 0.7
Hz). 13C NMR (62.9 MHz, CDCl3): δ 24.2 (d, 3JCP=5.0 Hz);
J=2.0 Hz, 1H). 13C NMR (100.62 MHz, CDCl3): δ 23.2 (d, 3JCP
3.6 Hz); 23.8 (d, 3JCP = 3.6 Hz); 24.0 (d, 3JCP = 3.6 Hz,); 24.1 (d,
3JCP = 3.6 Hz); 42.7 (d, 1JCP = 150.6 Hz); 73.5 (d, 2JCP = 7.1 Hz);
73.8(d, 2JCP = 7.3 Hz); 109.9 (d, 2JCP = 9.5 Hz), 128.1 (d, JCP=2.0
Hz); 129.6 (d, JCP = 1.2 Hz); 129.7; 131.5 (d, JCP = 3.7 Hz); 135.0
(d, JCP = 9.8 Hz); 135.1 (d, JCP = 2.0 Hz).
=
Diisopropyl r-Sulfanyl(2,4-dichlorobenzyl)phosphonate (17).
A solution of thiocyanatophosphonate 16 (382 mg, 1 mmol, 1
equiv) in EtOH 95% (10 mL) was added dropwise to a stirred
solution of NaBH4 (190 mg, 5 mmol, 5 equiv) in EtOH 95% (10
mL) at room temperature. The mixture was stirred for 2 h (the
reaction was monitored by TLC), then poured into a cooled solu-
tion of 2 N HCl (10 mL) at 0 ꢀC and extracted with AcOEt (3 ꢀ 20
mL). The combined organic phases were dried (MgSO4) and con-
centrated under reduced pressure. The crude product was purified
by flash chromatography (pentane/AcOEt 1/1, Rf = 0.5) to give 17
as a pale-yellow oil, which was stored under argon in the freezer to
avoid oxidation to disulfide. Yield: 64%. 31P NMR (CDCl3, 161.97
MHz): δ 20.2. 1H NMR (400 MHz, CDCl3): δ 0.94 (d, 3JHH = 6.4
Hz, 3H); 1.18 (d, 3JHH = 6.4 Hz, 3H); 1.24 (d, 3JHH = 6.4 Hz, 3H);
1.31 (d, 3JHH = 6.4 Hz, 3H); 2.53 (dd, 3JHH = 9.2, 3JHP = 11.6,
1H); 4.47 (m, 2H); 4.76 (m, 1H); 7.20 (dd, J = 2.0 Hz, 3JHH = 8.4
24.4 (d, 3JCP = 3.8 Hz); 27.3 (d, 3JCP = 3.1 Hz); 71.3 (d, 2JCP
=
6.3 Hz); 126.8 (d, 3JCP=14.4 Hz); 127.6 (d, 1JCP = 185.6 Hz);
130.8 (d, JCP=14.5 Hz); 133.0 (d, JCP = 2.5 Hz); 133.8 (d,
3
4
2JCP=9.4 Hz); 145.5 (d, 2JCP=9.4 Hz).
Synthesis of Inhibitor 18. Diisopropyl r-Hydroxy(2,4-dichloro-
benzyl)phosphonate (14). A mixture of diisopropyl phosphite
(1.66 g, 10 mmol, 1 equiv), 2,4-dichlorobenzaldehyde (1.75 g
10 mmol, 1 equiv), and triethylamine (7 mL, 50 mmol, 5 equiv) was
heated at reflux for 3 h. After addition of HCl 4 N (10 mL), the
resulting mixture was extracted with CH2Cl2 (2 ꢀ 20 mL). The
organic layers were dried on MgSO4 and concentrated under
reduced pressure to afford hydroxyphosphonate 14 as a white
powder, which was used directly in the next step. Yield: 90%.
Mp=92 ꢀC. 31P NMR (CDCl3, 101.25 MHz): δ 18.9. 1H NMR
(400 MHz, CDCl3): δ 1.10 (d, 3JHH=6.1 Hz, 3H); 1.18 (d, 3JHH
=
6.1 Hz, 3H); 1.20 (d, 3JHH=6.1 Hz, 3H); 1.21 (d, 3JHH=6.1 Hz, 3H);
4.55 (m, 1H); 4.65 (m, 1H, CHOP); 5.33 (d, 2JHP = 12.0 Hz, 1H);
7.22 (dd, 3JHH =8.4Hz, J= 2.0 Hz, 1H); 7.31 (dd, J=1.2Hz, J=
2.0 Hz, 1H); 7.59 (dd, 3JHH=8.4 Hz, J = 2.4 Hz, 1H). 13C NMR
(100.62 MHz, CDCl3):δ23.4(d, 3JCP =3.2Hz);23.8(d, 3JCP =3.8
Hz, 1H); 7.31 (dd, J = 1.2 Hz, J = 2.0 Hz, 1H); 7.67 (dd, 3JHH
=
8.4 Hz, J = 2.0 Hz, 1H). 13C NMR (100.62 MHz, CDCl3): δ 23.2
(d, 3JCP = 3.1 Hz); 23.7 (d, 3JCP = 3.7 Hz); 24.1 (d, 3JCP = 3.1 Hz);
24.2 (d, 3JCP = 3.7 Hz); 33.8 (d, 1JCP = 151.0 Hz); 72.4 (d, 2JCP
=
7.3 Hz); 72.6 (d, 2JCP = 7.4 Hz); 127.6 (d, JCP = 2.3 Hz); 129.0 (d,
JCP=1.2 Hz); 131.1 (d, JCP = 3.9 Hz); 133.7 (d, JCP = 3.1 Hz);
134.1 (d, JCP = 9.9 Hz); 134.4 (d, JCP = 1.4 Hz).
Hz); 23.9 (d, 3JCP=3.8 Hz); 24.2 (d, 3JCP=3.8 Hz); 66.8 (d, 1JCP
=
162.8 Hz); 72.0 (d, JCP=7.7 Hz); 72.5 (d, 2JCP =7.5 Hz); 127.1
(d, JCP=3.1 Hz); 128.8 (d, JCP=2.2 Hz); 130.3 (d, JCP = 4.1 Hz);
133.7 (d, JCP = 8.1 Hz); 134.14 (d, JCP = 3.9 Hz); 134.16.
2
r-Sulfanyl(2,4-dichlorobenzyl)phosphonic Acid (18). To a stir-
red solution of 1-sulfanylphosphonate 17 (165 mg, 1 equiv, 0.46
mmol) in CH2Cl2 (2 mL) was added dropwise Me3SiBr (0.5 mL,
3.3 mmol, 7 equiv). The mixture was stirred for 2 days at room
temperature. The volatile products were evaporated. The resi-
due was dissolved in a mixture of CH2Cl2/MeOH (3/1) and
stirred for 3 h. Then the solvents evaporated to give the mer-
captophosphonic acid 18 as a hygroscopic white paste (89 mg,
72%). 31P NMR (161.97 MHz, D2O/MeOD 80/20): δ 20.0. 1H
NMR (400.13 MHz, D2O/MeOD 80/20): δ 4.54 (d, 2JHP =19.6,
1H); 7.23 (dd, 3JHH = 8.8 Hz, J = 1.6 Hz, 1H); 7.38 (s, 1H, H);
7.67 (dd, 3JHH = 8.8 Hz, J = 1.6 Hz, 1H). 13C NMR (100.62
MHz, D2O/MeOD 80/20): δ 34.4 (d, 1JCP = 140.8 Hz); 127.6 (d,
Diisopropyl r-(p-Nitrobenzenesulfonyloxy)(2,4-dichloroben-
zyl)phosphonate (15). p-Nitrobenzenesulfonyl chloride (1.13 g,
5.1 mmol, 1.2 equiv), NEt3 (1.5 mL), and 4-(N,N-dimethylami-
no)pyridine (DMAP) (10 mg/mmol 14, 43 mg) were added to a
solution of hydroxyphosphonate 14 (1.46 g, 4.3 mmol, 1 equiv) in
dry CH2Cl2 (20 mL) under nitrogen at 0 ꢀC. The reaction mixture
was stirred at room temperature for 7 h. After the addition of a
mixture of water (10 mL) and 12 N HCl (15 mL), the organic phase
was separated and the aqueous phase was extracted twice with
CH2Cl2 (10 mL). The combined organic phases were washed with a
saturated aqueous solution of NaHCO3, dried over MgSO4, and
concentrated under reduced pressure. The crude product 15 was
obtained as a white solid and was used directly in the next step.
Yield: 95%. Mp=109 ꢀC. 31P NMR (CDCl3, 161.97 MHz): δ 11.1.
1H NMR (400 MHz, CDCl3):δ0.98 (d, 3JHH=6.4 Hz, 3H); 1.18 (d,
J
133.2 (d, JCP = 2.9 Hz); 133.6 (d, JCP = 8.6 Hz); 135.2 (d, JCP
CP = 2.2 Hz); 128.8 (d, JCP = 1.3 Hz); 130.7 (d, JCP = 3.8 Hz);
=
2.5 Hz).
Chemicals. Buffers and bovine serum albumin (BSA) were
obtained from Sigma-Aldrich (Steinheim, Germany). Dimethyl
sulfoxide (DMSO) and ZnCl2 were purchased from Merck
3JHH = 6.4 Hz, 3H); 1.21 (d, 3JHH=6.4 Hz, 3H); 1.24 (d, 3JHH
=
6.4 Hz, 3H); 4.49 (m, 1H); 4.74 (m, 1H); 6.07 (d, 2JHP = 16.0 Hz,
1H); 7.02 (dd, 3JHH = 8.8 Hz, J = 2.0 Hz, 1H); 7.22 (dd, J = 1.2
Hz, J=2.0 Hz, 1H); 7.37 (dd, 3JHH=8.4 Hz, J=2.4 Hz, 1H); 7.94
(AA BB system, J = 8.9, 4H). 13C NMR (100.62 MHz, CDCl3): δ
(Darmstadt, Germany). Imipenem (Δε300=-9000 M-1 cm-1
)
was from Merck Sharpe and Dohme Research Laboratories
(Rahway, NJ). Nitrocefin (Δε482 = -15 000 M-1 cm-1) was
from Oxoid LTD (Basingstoke, U.K.).
3
3
3
23.3 (d, JCP = 3.2 Hz); 23.7 (d, JCP=3.8 Hz); 24.0 (d, JCP
=
3.8 Hz); 24.1 (d, 3JCP = 3.8 Hz); 73.2 (d, 2 JCP = 7.1 Hz); 73.6 (d,
2JCP=7.1 Hz); 73.8 (d, 1JCP = 175.6 Hz); 124.0; 127.5; 128.3; 128.5;
129.3; 131.3 (d, JCP = 3.5 Hz); 134.3 (d, J CP=8.4 Hz); 136.2 (d,
Enzyme Production and Purification. The CphA, CphA
N220G, CphA N116H-N220G,41 VIM-4,43 L1,52 and FEZ-132
MBLs were produced and purified as described previously.
Determination of the Inhibition Constant. Solutions of 18 and
1b (100 mM) were prepared in DMSO before dilution with a
convenient buffer containing 20 μg/mL BSA and 50 μM ZnCl2
when indicated. The buffers were 10 mM HEPES, pH 7.2, for
VIM-4, 15 mM cacodylate, pH 6.5, for CphA, and 20 mM
HEPES, pH 7.0, for L1. The inhibitors 1a, 10a, 10b, 11a, 11b,
12a, 12b, 13, 19, 20, 21, and 22 were prepared as 100 mM stock
solutions directly into the proper buffer. The VIM-4 subclass B1
enzyme and the L1 subclass B3 enzyme were tested without and
with additional Zn2þ (50 μM final concentration) in the buffer in
order to detect a potential chelating effect of the inhibitor. The
study of the subclass B2 enzyme CphA was done without addition
of Zn2þ tothe buffer, since this subclass only requires one Zn2þ for
its activity and is inhibited by the addition of Zn2þ. VIM-4, CphA,
and L1 enzymes were used at fixed concentrations between
J
CP = 3.1 Hz); 141.9; 150.6.
Diisopropyl r-Thiocyanato(2,4-dichlorobenzyl)phosphonate
(16). A mixture of phosphonate 15 (1.58 g, 3.0 mmol, 1 equiv),
KSCN (1.16 g, 12 mmol, 4 equiv), and 18-crown-6 (50 mg/mmol
of 15, 150 mg) in dry acetonitrile (30 mL) was refluxed for 40 h
under nitrogen. Then acetonitrile was evaporated, and after addi-
tion of water (20 mL), the mixture was extracted with AcOEt (3 ꢀ
20 mL). The combined organic layers were dried with MgSO4 and
concentrated under reduced pressure. The crude product 16 was
obtained as a yellow oil and was used directly in the next step. Yield:
79%. 31P NMR (CDCl3, 161.97 MHz): δ 14.5. H NMR (400
1
MHz, CDCl3): δ 0.99 (d, 3JHH = 6.0 Hz, 3H); 1.18 (d, 3JHH = 6.1
Hz, 3H); 1.24 (d, 3JHH=6.1 Hz, 6H); 4.53 (m, 1H); 4.78 (m, 1H);
5.00 (d, 2JHP = 19.6 Hz, 1H); 7.28 (dd, 3JHH = 8.8 Hz, J = 2.0 Hz,
1H); 7.40 (dd, J=1.2 Hz, J=2.0 Hz, 1H); 7.75 (dd, 3JHH=8.8 Hz,

4874 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Lassaux et al.
0.2 and 0.8 nM. The enzyme and inhibitor (100 μM) were
preincubated for 30 min at 37 ꢀC before the substrate was added.
Hydrolysis of imipenem (CphA and L1) or nitrocefin (VIM-4)
was monitored by following the variation in absorbance at 300
or 482 nm, respectively, using a Uvikon 860 spectrophotometer.
The activity was tested using an initial rate of three samples
without inhibitor. The initial rate conditions were used to
study the inhibition by using the Hanes linearization of the
Henri-Michaelis equation. The competitive inhibition con-
stant, Ki, was calculated using the following equation:53
(the phosphonato and the sulfanyl groups) were possible.
However, it has been demonstrated for previous MBL inhibitor
possessing a sulfanyl (thiol) group that the inhibition mainly
occurs via an interaction between Zn2þ ions and the sulfanyl
function.61 For the modeling studies, we have thus privileged
this interaction. In consequence, two orientations of the inhi-
bitor in the active site cavity were chosen as starting positions:
the sulfanyl in interaction with the Zn2þ ion in the “histidine”
site (Zn1) and the sulfanyl in interaction with the Zn2þ ion in the
“cysteine” site (Zn2). We have used charged phosphonato and
sulfanyl groups because preliminary studies had confirmed that
a deprotonated function was necessary to avoid repulsion by the
Zn2þ ion. Two other models of VIM-4 in complex with 10a and
18 were built using the positions of each compound in the
CphA-10a and CphA-18 crystallographic complexes. The
Vmax
S
υ ¼
S þ Kmð1 þ I=KiÞ
For compounds 1b and 18, it was checked that the rate in
absence of inhibitor remained the same upon addition of 1%
DMSO.
˚
different complexes were soaked in a 5 A layer of water mole-
Crystallography. Crystallization of the CphA-10a and
CphA-18 Complexes and Data Collection. Crystallization of
the N220G CphA was performed as described previously.39
Before data collection, 1 μL of 1.5 mM 10a or 18 was added
to the drops containing the crystals and soaked. After several
days, the crystals were soaked with 1 μL of inhibitor solution
again. For data collection, crystals were transferred to a cryo-
protectant solution containing reservoir solution supplemented
with 30% (v/v) glycerol. The mounted crystals were flash-frozen
in a liquid nitrogen stream. Nearly complete X-ray data sets
were collected using an in-house Bruker FR591 rotating anode
X-ray generator and a Mar345dtb detector. Diffraction data
were processed with XDS54 and scaled with SCALA from the
CCP4 suite55 (Table 1).
Structure Determination and Refinement. Initial phases for the
CphA-10a and CphA-18 complex structures were generated
by molecular replacement, using the structure of the wild-type
CphA as a starting model (Protein Data Bank accession code
1X8G). Molecular replacement was performed with Phaser.56
The obtained molecular replacement models were rebuilt with
ARP/wARP automatically.57 On inspection, the first electron
density map clearly showed the presence of additional density in
the active site, the form of which looked like an inhibitor
molecule. After most of the protein main chain and side chain
atoms and most of the water molecules were built and refined,
compounds 10a and 18 were modeled into the map. Conforma-
tional torsion angle restraints and charge assignments for the
10a and 18 molecules were obtained using CCP4i Libcheck.
The structures were refined in a cyclic process including
manual inspection of the electron density with Coot58 and
refinement with Refmac.59 The refinement to convergence was
done with isotropic B-values and using translation/libration/
screw (TLS) parameter. The positions of the Zn2þ ions and the
positions of the sulfur and phosphorus atoms were verified by
calculating anomalous maps. Alternative conformations were
modeled for a number of side chains, and occupancies were
adjusted to yield similar B-values for the disordered conforma-
tions. Refinement statistics are shown in Table 1.
cules. The geometry was first fully optimized at the molecular
mechanics (MM) level62 with a maximum force convergence
-1
threshold of 0.02 kcal mol-1
A
and then submitted to a
˚
dynamics simulation at 300 K during 100 ps. The chosen force
field was Amber,63,64 the relative dielectric constant was set to 1,
and the programs used were InsightII and Discover running on a
Pentium 4 3.4 GHz under Unix Debian. The Zn2þ ions and the
inhibitors are not recognized as such by the Amber force field.
Thus, each atom must be defined manually as an atom-type
recognized by Amber, such as for instance CA for aromatic
carbons or CU for the Zn2þ atoms which were dealt with as ions
whose net charge had to be defined. The net charges of the
inhibitor and the Zn2þ ions were determined at the quantum
chemistry level, at the RHF/6-31þG** level with the Gaussian
03 program.65 No heating or equilibration step was used in the
dynamics simulation, but the 3D structure submitted to the final
geometry optimization was chosen as the average conformation
built on the end (50 ps) of the simulation dynamics.
In order to validate the achieved VIM-4-18 or VIM-4-10a
conformations, the CphA-10a and CphA-18 complexes were
optimized and a study was performed using the initial orienta-
tions for the inhibitor previously used for VIM-4. Energy
minimization and dynamic simulation were conducted as de-
scribed above.
Finally, FEZ-1-10a and FEZ-1-18 complex models were
built using the structure of FEZ-1 in interaction with D-captotril
(1JT1), which also presented a wide active site ideal for inhibitor
docking. The molecular models of FEZ-1 were based on the
docking on VIM-4 and on the structural conformation of the
inhibitors in CphA. Energy minimization and dynamic simula-
tion were performed as described above.
Acknowledgment. C.B. is a postdoctoral researcher of the
FRS/FNRS (Belgium). P.L. is a fellow of the “Fonds pour la
ꢀ
Formation a la Recherche dans l’Industrie et l’Agriculture”
(FRIA; Brussels, Belgium). We thank Prof. Paulette Charlier,
ꢁ ꢁ
Dr. Frederic Kerff, and Raphael Hermann (CIP, University
of Liege, Belgium) for their help with the FEZ-1 crystals. The
€
Protein Structure Accession Number. Coordinates and struc-
ture factors for CphA-10a and CphA-18 complexes have been
deposited in the Protein Data Bank with the respective accession
codes 3IOF and 3IOG.
ꢀ
ꢀ
work in Liege was supported by the Interuniversity Attraction
Poles (IAP P6/19), Belgian Federal Government, and Grants
2.4511.06, 2.4561.07, 2.4548.10 from the FRS/FNRS (Brussels,
Belgium) and Lot. Nat. 9.4538.03. The work in Aachen
was supported by the European Regional Development
€
Fund (ERDF) and the European Union (“Die Europaische
Kommission investiert in Ihre Zukunft”).
Molecular Modeling. Our methodology was chosen to try to
highlight differences between distinct binding modes involving
Zn2þ-sulfanyl or Zn2þ-phosphonate interactions. The VIM-
4-10a and VIM-4-18 complex models were based on the three-
dimensional structure of VIM-4 enzyme43 (2WHG). This 3D
structure has the advantage of possessing a citrate anion in the
active site of the protein. The active site is then in an ideal “open
conformation” to fit a compound. The starting position of the
complexes between the enzyme and the inhibitor was built by
empirically docking the substrate in the cavity with the pro-
gram InsightII.60 Given the chemical nature of the inhibitor,
interactions between the Zn2þ ions and two chemical groups
Supporting Information Available: Purities of inhibitors 1a,
1b, 10a, 10b, 11a, 11b, 12a, 12b, 13, 18-22 from HPLC or
elemental analysis; additional spectroscopic data for com-
pounds 8, 9, 10a, 14-18; synthesis and analytical details for
inhibitors 10b, 11b, 12b, 13, 19-22. This material is available
free of charge via the Internet at http://pubs.acs.org.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4875
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