D-Proline-based peptidomimetic inhibitors of anthrax lethal factor

Article abstract of DOI:10.1016/j.ejmech.2012.08.028

In this work we reported the generation of d-proline-derived hydroxamic acids as inhibitors of anthrax lethal factor (LF), taking advantage of a pyrrolidine ring as the central scaffold and a hydroxamate group as the Zn ²⁺ chelating agent. The

Full text of DOI:10.1016/j.ejmech.2012.08.028

European Journal of Medicinal Chemistry 56 (2012) 96e107
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European Journal of Medicinal Chemistry
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Original article
D-Proline-based peptidomimetic inhibitors of anthrax lethal factor
,
b
a
Chiara Calugi a, Andrea Trabocchi a , Claudia Lalli , Antonio Guarna
*
a Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Florence, Italy
b Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work we reported the generation of D-proline-derived hydroxamic acids as inhibitors of anthrax
Received 13 June 2012
Received in revised form
17 August 2012
Accepted 20 August 2012
Available online 28 August 2012
lethal factor (LF), taking advantage of a pyrrolidine ring as the central scaffold and a hydroxamate group
as the Zn2þ chelating agent. The introduction of two hydrophobic groups addressing the S10 subsite and
a long substrate-binding groove was conceived by overlapping the bioactive conformations of two re-
ported LF inhibitors. Micromolar affinity of compound 38 suggested cis-3-substituted-1-sulfonamido-D-
proline hydroxamic acids as a promising class of peptidomimetic inhibitors for developing novel LF
inhibitors.
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
Amino acid
Small molecule
Molecular docking
Enzyme
Drug design
1. Introduction
formation of oedema toxin and of lethal toxin, respectively [10]. PA
is activated proteolitically on the host cell surface by the furin-like
Anthrax is a disease caused by Bacillus anthracis a Gram positive
bacterium usually found in the soil [1,2]. It is an epizootic disease to
which most mammals are considered susceptible. This disease can
be transmitted to humans through contact with infected animals,
and no cases of human-to-human transmission have been reported,
so far. Due to the risk of using B. anthracis spores as a weapon of bio-
terrorism against human populations, there has been a growing
interest towards the development of a treatment for anthrax during
the last decade. The clinical effect of this infectious disease depends
on its entry route to the body. While entry of B. anthracis spores in
the human body through skin causing cutaneous anthrax is easily
curable with antibiotics, inhalation of anthrax spores is usually
fatal. After inhalation, the spores survive and sprout within
macrophages, allowing them to reach lymph nodes, and ultimately
to start bacteria proliferation in the circulation system [3e5]. The
anthrax toxin [6] consists of three secreted proteins, the protective
antigen (PA) the lethal factor (LF) and the oedema factor (EF), which
are crucial for killing host macrophages and suppressing the
immune system’s activity [7e9]. The protective antigen PA is
necessary for lethal and oedema factors to enter the cell, and binary
combinations of PA with the two proteins LF and EF lead to the
proprotein convertase, then the bioactive PA fragment hepta-
merizes to form a membrane pore, and binds to both LF and EF. This
complex is then endocytosed, and lethal and oedema factor are
released in the cytosol by a denaturationerenaturation process,
where they exert their toxic action. EF is a calmodulin-activated
adenylate cyclase which causes an increase of cAMP concentra-
tion in the cells [11], and LF, is a zinc-dependent metalloproteinase,
that has been recognized as the primary and most important
virulence factor of B. anthracis. The anthrax lethal factor cleaves
several members of the mitogen-activated protein kinase kinase
(MAPKK) family near their N-terminus [12e15]. This proteolytic
activity prevents phosphorylation of MAPKs, and consequently
leads to disruption of many signaling pathways and ultimately to
cell-death. Given the crucial role that anthrax LF plays in the
infectious process, this enzyme is considered a potential thera-
peutic target of anthrax. Indeed, a possible therapeutic approach
envisages the combination of antibiotics with LF inhibitors to clear
the active infection while blocking the toxin already present in the
cells. There are a number of successful LF inhibitors reported to date
[16e21], and important structureeactivity studies for under-
standing binding mode features of chemically diverse inhibitors
have been published during the last decade [22,23]. Also, recent
reports on potent hydroxamic acid-based LF inhibitors [24,25], and
new SAR studies have been reported [26]. Herein, we describe
a rational drug design approach that allowed for the discovery of
* Corresponding author. Tel.: þ39 055 4573507; fax: þ39 055 4573531.
E-mail address: andrea.trabocchi@unifi.it (A. Trabocchi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.028

C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
97
new D-proline-derived peptidomimetic LF inhibitors as a new entry
to LF inhibitors, characterized by a pyrrolidine ring as the central
scaffold and a hydroxamate group as the Zn2þ chelating agent.
2. Results and discussion
2.1. Structure alignment and rational design
Preliminary experiments validated the applicability of arylsul-
fonyl-D-pyrrolidine hydroxamic acids as LF inhibitors in the micro-
molar range, and both 4-chloro and 3,4-dichlorobenzenesulfonyl
groups were selected as substituents at the nitrogen atom. Never-
theless, unsatisfactory logP values for this set of compounds
precluded further consideration as inhibitors for intracellular assays
(unpublished data). Thus, we reasoned to develop a new set of D-
proline-based inhibitors possessing two hydrophobic groups, in
order to allow for better pharmacokinetic properties of the inhibitor,
and possibly improving their specific binding activity. Accordingly,
weconsidered the design of second generation inhibitors taking into
account the binding mode of two diverse inhibitors known in the
literature to enable the design of new peptidomimetics possessing
Fig. 2. Superimposition of two reference inhibitors (top) as obtained from the struc-
ture alignment of two LF coordinates from X-ray data (PDB codes: 1YQY and 1ZXV),
and the resulting designed D-proline-based class of compounds (bottom). ZBG is Zn-
Binding Group; HG1 is Hydrophobic Group 1; HG2 is Hydrophobic Group 2.
a pharmacophoric model, which was the starting point for the
design of new LF inhibitors (Fig. 2). The binding interaction model
obtained by such superimposition is characterized by a central
template, a Zn2þ chelating agent as represented by the hydrox-
amate moiety, and by the presence of two hydrophobic groups
(HG1 and HG2) addressing two different cavities explored by the
reference inhibitors herein taken into account. Taking advantage of
D-proline as the core scaffold. Two well-known anthrax lethal factor
inhibitors were chosen as model structures, namely the inhibitor
L915 developed by Merck and BI-MFM3 by Cengent laboratories, in
order to generate a binding interaction model, which provided key
structural features required to design novel anthrax LF inhibitors
(Fig. 1) [27,28].
BI-MFM3 is a phenylfuran-2-yl-methylene-rhodanine acetic
acid derivative. In this case, crystallographic data displayed the
Zn2þ metal ion interacting at the rhodanine ring via the sulfur
atoms of 2-thioxothiazolidine-4-one moiety. Moreover, the phenyl
ring group established interactions with hydrophobic side chains of
LF, which belong to a binding pocket different from that addressed
by the 4-fluoro-3-methylphenyl substituent of L915. L915 is
a competitive hydroxamate LF inhibitor, for which the crystal
structure in complex with LF was solved. The oxygen atoms of the
hydroxamate moiety chelates the Zn2þ ion in a bidentate and
planar conformation, and the substituted phenyl ring is located in
a deep hydrophobic pocket adjacent to the catalytic center. The
tetrahydropyranyl moiety, which is located in a large cavity
between two different protein domains, is not crucial for the
ligandeprotein interaction. These crystallographic data highlighted
that the binding affinity of L915 to LF is driven by the interactions
between the hydroxamic acid and the metal ion, and between the
4-fluoro-3-methylphenyl substituent and the S10 pocket. The
coordinates of these two complexes were superimposed using
Swiss PDB viewer, and then protein superimposition was discarded
allowing for retaining the alignment of the inhibitors. From
the superimposition of the two inhibitors we obtained
this analysis, we considered the pyrrolidine ring of D-proline as the
central scaffold, and 4-chloro- or 3,4-dichlorophenylsulfonyl
moieties at the nitrogen atom, similarly to L915 inhibitor, as the
hydrophobic groups for addressing the deep hydrophobic S10
subsite (Fig. 2).
Accordingly, we synthesized an array of D-proline hydroxamic
acid peptidomimetics containing an additional substituent in order
to study the introduction of hydrophobic (HG) groups, as well as
the preferred stereochemical arrangement. Initially, we explored
the effect of changing substituents and stereochemistry starting
from 4-hydroxy-D-proline scaffold.
2.2. Synthesis
Taking into account cis-4-hydroxy-D-proline as the chiral
template, the key intermediates (2R,4R) and (2R,4S)-methyl
1-((3,4-dichlorophenyl)sulfonyl)-4-hydroxypyrrolidine-2-
carboxylate were achieved in good yield via the route as shown in
Scheme 1. After esterification of cis-4-hydroxy- -proline (3) with
D
Scheme 1. Reagent and conditions: (a) SOCl2, MeOH, r.t., 16 h; (b) 3,4 dichloroben-
zene-1-sulfonyl chloride, Et3N, DMAP, dry CH2Cl2, N2, r.t., 16 h; (c) ClCH2CO2H, PPh3,
DIAD, dry toluene; (d) NaHCO3, MeOH.
Fig. 1. Merck L915 LF inhibitor (top) and Cengent BI-MFM3 LF inhibitor (bottom).

98
C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
thionyl chloride in methanol to give 4, and subsequent sulfonamide
formation, an inversion of configuration through a Mitsunobu
reaction was performed on 5 to obtain the corresponding (2R,4S)-
isomer 6.
Sulfonamides 5 and 6 were used to prepare the final methyl ester
derivatives 7e14 through the synthetic pathway as shown in
Scheme 2. Benzylation of compound
5
using benzyltri-
chloroacetimidate afforded the ether 7 in nearly quantitative yield.
The corresponding isomer 8 was obtained in the same way starting
from compound 6. Mesylation and subsequent nucleophilic substi-
tution on sulfonamide 5 with NaN3 produced azide 9 with inverted
configuration at the C-4 stereocenter. This compound was combined
with ethynylbenzene through click-chemistry to prepare the cor-
responding methyl ester 11 characterized by a 4-phenyltriazole 11.
Compound 9 was also subjected to catalytic hydrogenation using
10% Pd/C to give amine 13. Similarly, trans-4-hydroxy-proline
derivative 6 gave access to cis-4-azido-derivative 10, and conse-
quently the corresponding click-adduct 12 and cis-4-aminoproline
14. Compounds 13e14 proved to be a good starting point to intro-
duce amides at position 4 of the pyrrolidine scaffold (Scheme 3).
With exception of compound 17 obtained from 14, 4-aminoproline
precursors 13e14 were applied for generating a pool 4-amido
derivatives as represented by 15e19, in order to have chemical and
stereochemical diversity for the second hydrophobic group (HG2),
as shown in Fig. 2. The methyl ester of compounds 7e8, 11e12 and
15e19 was then directly converted to corresponding hydroxamic
acids using NH2OK/NH2OH in MeOH, as shown in Scheme 4, to
produce the corresponding compounds 20e28 (Table 1) [29].
In order to modulate the position of HG2 and to study its effect
on LF inhibition, we also considered position 3 of the pyrrolidine
Scheme 3. Reagents and conditions: (a) 2,4-dimethoxybenzoyl chloride, Et3N, dry
CH2Cl2, r.t., 16 h; (b) (Z)-3-(furan-2-yl)acryloyl chloride, Et3N, dry CH2Cl2, r.t., 16 h; (c)
thiophene-2-carbonyl chloride, Et3N, dry CH2Cl2, r.t., 16 h.
H2N
N3
O
scaffold to install the second hydrophobic group, while retaining
the preferred cis-configuration (see inhibition data). A relative
straightforward route to achieve the desired cis-3-hydroxyproline
scaffold involved the stereoselective reduction of 3-ketoproline
(Scheme 5) [30,31]. N-Boc-protected diester 30 was prepared in
two steps starting from ethyl glycinate 29 and ethyl acrylate. A
Dieckmann cyclization was then conducted to yield racemic
O
N
S
N
S
e
O
O
O
O
O
O
13: (4S) from 9, 97%
14: (4R) from 10, 96%
9: (4S) from 5, 75%
10: (4R) from 6, 77%
Cl
Cl
Cl
Cl
Cl
Cl
b
-ketoester 31. Finally, baker’s yeast reduction of the ketone affor-
N
N
d
b,c
ded the optically active -hydroxyester 32, which was used to
obtain final hydroxamic acids 39e41 (Scheme 6).
b
N
HO
O
O
N
S
N
S
O
O
O
O
O
O
Cl
5: (4R)
6: (4S)
11: (4S) from 9, 73%
12: (4R) from 10, 56%
Cl
Cl
a
O
O
N
S
O
O
O
7: (4R) from 5, 99%
8: (4S) from 6, 99%
Cl
Scheme 2. Representative synthesis of sulfonamides 7e14 starting from intermediates
5 and 6. Reagent and conditions: (a) benzyltrichloroacetimidate, CF3SO3H, 3Å MS, Et2O,
r.t., 3 h; (b) MsCl, Et3N, DCM, r.t., 2 h; (c) NaN3, DMF, 90 ꢀC, 16 h; (d) ethynylbenzene,
Cu(OAc)2, sodium ascorbate, H2O/t-BuOH, r.t., 16 h; (e) 10% Pd/C, H2, MeOH, r.t., 16 h.
Scheme 4. General procedure for the synthesis of hydroxamic acids. Reagents and
conditions: (a) NH2OH$HCl, KOH 0.87M in MeOH, r.t., 48e72 h.

C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
99
Table 1
Array of 4-hydroxy-proline derivatives assayed towards anthrax lethal factor.
Compound
R
C-4 absolute configuration
% Inhibitiona
20
21
22
R
S
S
50
16
e
Scheme 6. Reagents and conditions: (a) benzyl bromide, NaH, dry THF, TBAI, N2, r.t.,
16 h for 33, or 1-iodohexane, NaH, dry DMF, r.t., 16 h for 34; (b) 1:1 TFA/DCM, r.t., 2e
3 h; (c) 3,4 dichlorobenzene-1-sulfonyl chloride, Et3N, DMAP, dry CH2Cl2, N2, r.t., 16 h
for 35, or 4 chlorobenzene-1-sulfonyl chloride, Et3N, DMAP, dry CH2Cl2, N2, r.t., 16 h for
23
24
R
S
8
36 and 37; (d) NH2OH$HCl, KOH, MeOH, m
W, 80 ꢀC, 1h or NH2OH$HCl, KOH, MeOH, r.t.,
48 h.
11
2.3. Enzyme inhibition assay
The ability of this set of peptidomimetics to inhibit anthrax
lethal factor activity was evaluated through a spectrophotometric
assay using the anthrax lethal factor protease substrate II. Cleavage
was measured by monitoring the increase in absorbance at 405 nm
corresponding to the release of p-nitroaniline. Initially, inhibition
studies were carried out at a fixed concentration of potential
25
R
65
26
27
S
46
27
inhibitors (10 mM), and the percentage of inhibition was calculated
(Table 1). The evaluation of the 4-substituted hydroxamic acid-
based peptidomimetics as LF inhibitors resulted in the identifica-
tion of compounds 20 and 25, exhibiting inhibition potency equal
R
or above 50% at 10 mM concentration. The first round of biological
28
S
22
screening on this set of molecules revealed a preference for the cis-
configuration (4R configuration) in giving better inhibition toward
the target enzyme. This tendency suggested a role of the absolute
configuration at the C-4 stereocenter for inhibition of LF.
a
The inhibitory potency was determined using the initial velocity value, which
was compared with that in absence of potential inhibitors. Values are reported as
the mean of three experiments.
IC50 values for compounds 20 and 25 were calculated by dosee
response measurements using inhibitors in a range of concentra-
tions between 0.5 and 10 mM (Table 2). The two compounds showed
The hydroxylic group at position 3 of the pyrrolidine ring was
treated with NaH and benzyl bromide or 1-iodohexane, affording
the corresponding ethers 34 and 35, respectively. After Boc
removal, the sulfonamide functionality was introduced as for 5, and
the final hydroxamic acids were prepared by a procedure similar to
that followed for 4-hydroxyproline derivatives 20e28.
moderate inhibitory potency, possessing IC50 values in low
mM
Table 2
IC50 values of 3-hydroxyproline derivatives.
Compound
R1
R2
IC50 (mM)
38
39
Cl
H
1.4
1.7
Scheme 5. Reagents and conditions: (a) ethyl acrylate, Et3N, EtOH, r.t., 48e72 h; (b)
Boc2O, 5% NaOH, H2O, r.t., 16 h; (c) t-BuOK, toluene dry, N2, 0 ꢀC, 1.5 h; (d) Baker’s yeast,
H2O, 30 ꢀC, 48 h.
40
H
>10

100
C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
range. Compound 20 displayed low inhibition potency toward LF
activity (IC50 ¼ 9.5 M), whereas 25 showed better inhibition
M), suggesting the beneficial effect of
catalytic Zn2þ binding site. In particular, the phenylsulfonyl moiety
was located in the S10 cavity, whereas the benzyl group was
oriented in the long substrate-binding groove. These results are
particularly attractive because, as shown elsewhere [22,23], this
long recognition groove and the deep S10 pocket are the main
determinants for tight-binding inhibition.
m
capability (IC50 ¼ 2.1
m
including an aromatic amido group at the 4-cis position.
In order to study the effect of the position of HG2 towards
inhibition, cis-3-substituted compounds were assayed for LF inhi-
bition, and IC50 values obtained from the screening are shown in
Table 2. Compounds 38e40 displayed a clear increase in the inhi-
bition potency as compared to the 4-hydroxy-proline analogue 20.
The substituents in that position of the scaffold may fit in the
hydrophobic substrate-binding groove better than 4-hydroxy-
proline derivatives, by establishing favorable interactions with
enzymatic hydrophobic side chains. This result highlighted this
substituent arrangement to match our initial hypothesis of binding
interaction model better than 4-hydroxyproline derivatives. On the
other hand, compound 40 showed a complete loss of potency
toward LF, indicating a detrimental effect of the linear alkyl
substituent in the inhibition mechanism. The lack of efficacy could
be caused by flexibility of this substituent, which can assume an
unfavorable conformation in LF binding groove or by the absence of
the aromatic ring which may have a specific role in the interaction
with the enzyme.
2.5. Structureeactivity relationship
Taking advantage of the structural informations obtained from
the superimposition of the bioactive conformations of two refer-
ence inhibitors, we developed an array of peptidomimetics pos-
sessing the key hydroxamate group as the Zn2þ chelating agent and
a pyrrolidine ring as the central scaffold. The introduction of two
hydrophobic groups addressing the S10 subsite and
a long
substrate-binding groove was explored in the proline scaffold in
order to access an improved lethal factor inhibitor. As regarding to
the S10 subsite, both 4-chloro and 3,4-dichlorobenzenesulfonyl
groups were selected as substituents at the nitrogen atom. The
application of both benzyl or benzoyl residues at the nitrogen atom
of the scaffold proved to impair the inhibitory activity (data not
shown), thus suggesting a key role of the sulfonamide in addressing
the catalytic site. This hypothesis was corroborated by molecular
docking calculation, as a hydrogen-bond between an oxygen atom
of this group and Gly656 amide proton was observed (Fig. 3).
As regarding to the second hydrophobic group (HG2), an array of
the position on the scaffold and the stereochemistry was taken into
account to introduce hydrophobic substituents with some chemical
diversity between them. Benzyloxy derivative 20 and 2,4-
dimethoxybenzamido compound 25, both possessing the (R)
configuration, showed best inhibition profile for 4-substituted
proline derivatives (Table 1). Highly constrained aromatic groups
for HG2, as represented by phenyltriazolyl derivatives 22 and 23
proved to impair the inhibition profile in either stereochemical
orientation, suggesting a stringent topological requirement to
address the corresponding region of the catalytic site identified as
the long substrate-binding groove. Finally, differences in the type of
amido substituent at this position were found to be not significant,
2.4. Molecular modeling
Docking studies were carried out with active compound 38 and
the crystal structure of anthrax LF (PDB code: 1YQY) to give more
insight into the possible binding mode of this
D-proline-based
compound to the target enzyme. GOLD software was used to assess
the interactions established between the peptidomimetic and the
LF active site. Docking results of compound 38 showed a main
cluster of conformations characterized by the typical binding mode
of hydroxamate-based LF inhibitors (Fig. 3). Specifically, in agree-
ment with the crystallographic structure of LF-inhibitor L915 ob-
tained by Merck, compound 38 showed the oxygen atoms of the
hydroxamic acid moiety chelating the catalytic zinc ion.
The aromatic substituents addressed both the hydrophobic
substrate-binding groove and the S10 subsite adjacent to the
Fig. 3. Best-scoring docked conformations resulting from the docking calculation of compound 38.

C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
101
as observed by similar IC50 values of thienyl derivatives as
compared to 2,4-dimethoxybenzamido compounds (Table 1).
The improved inhibition of compounds 38 and 39, bearing
a benzyloxy group at position cis-3, with respect to the corre-
sponding isomer 20, possessing the same group at position 4 with
similar stereochemistry, suggested that an HG2 at position 3 of the
scaffold may fit in the hydrophobic substrate-binding groove better
than 4-substituted-proline derivatives. Moreover, the complete loss
of potency toward LF observed for compound 40 suggest a specific
role of aromatics as HG2 in the interaction with the enzyme, as
compared to inhibitory activity of compounds 38 and 39.
Thus, all these informations suggest that the proline scaffold
may be a feasible peptidomimetic scaffold to achieve a lethal factor
inhibitor with improved performance. The HG1 consisting of
a substituted benzenesulfonyl group may be further explored
taking into account more interactive moieties such as polar groups
like guanidine-isosteres or hydroxyls. As regarding to HG2, the
position 3 was established as being the most promising with a cis
geometry with respect to HG1. In this view, an array of hetero-
aromatic group possessing more interactive moieties for the
interaction within the long substrate-binding groove, and an
overall hindrance similar to the benzyloxy-derivative 38 may give
access to an improved inhibitor.
3.71 (s, 3H), 3.06e2.87 (m, 2H), 2.69e2.59 (br, 2H), 2.32e2.19
(m, 1H), 1.98e1.93 (m, 1H) ppm. Anal. C6H11NO3 (C, H, N).
4.1.2. (2R,4R)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-
hydroxypyrrolidine-2-carboxylate (5)
To a solution of 4 (1.1 g, 7.63 mmol), Et3N (2.1 mL, 15.3 mmol)
and DMAP (186 mg, 1.53 mmol) in anhydrous CH2Cl2 (76 mL), 2,4-
dichlorobenzenesulfonyl chloride (1.3 mL, 8.39 mmol) was added at
0
ꢀC. The mixture was allowed to warm to room temperature, and
was left overnight stirring under a nitrogen atmosphere. Succes-
sively, the mixture was washed with aqueous 5% NaHCO3, 1 N HCl,
and brine. The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Compound 5 was isolated by
flash chromatography (pet. ether/EtOAc 1:1) thus giving a white
23
solid (1.9 g, 70%). M.p. 104.2e104.9 ꢀC. [
a
]
¼ þ60.7 (c ¼ 1.0,
D
CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
7.88 (d, J ¼ 1.9 Hz, 1H), 7.63
(dd, J ¼ 1.9, 8.8 Hz, 1H), 7.55 (d, J ¼ 8.8 Hz, 1H), 4.36-4.33 (m, 2H),
3.68 (s, 3H), 3.41e3.31 (m, 3H), 2.21e2.06 (m, 2H) ppm. 13C NMR
(50 MHz, CDCl3):
d 173.5 (s), 137.7 (s, 2C), 133.6 (s), 131.1 (d), 129.2
(d), 126.5 (d), 70.7 (d), 59.4 (d), 56.7 (t), 53.0 (q), 38.8 (t) ppm. ESI-
MS m/z (ESþ) 353.91 [(M þ H)þ, 100], 375.87 [(M þ Na)þ, 3.9]. Anal.
C12H13Cl2NO5S (C, H, N).
4.1.3. (2R,4S)-methyl 1-((3,4-dichlorophenyl)sulfonyl)-4-
3. Conclusion
hydroxypyrrolidine-2-carboxylate (6)
To a solution of 5 (800 mg, 2.26 mmol), PPh3 (653 mg, 2.49 mmol)
and ClCH2CO2H (235 mg, 2.49 mmol) in anhydrous toluene (12 mL),
DIAD (0.5 mL, 2.49 mmol) was added dropwise at 0 ꢀC. The mixture
was allowed to reach room temperature, and then it was irradiated
under microwave conditions for 20 min at 90 ꢀC. Successively, the
mixture was concentrated under reduced pressure. The crude
product was dissolved in MeOH (7.5 mL) and NaHCO3 (40 mg,
0.45 mmol) was added. The mixture was irradiated under micro-
wave conditions for 15 min at 90 ꢀC. The mixture was concentrated
under reduced pressure, then the crude dissolved in EtOAc and
washed with aqueous 5% NaHCO3 and brine. The organic phase was
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Compound 6 was isolated by flash chromatography (pet.
In conclusion, we reported the design, synthesis and enzyme
inhibition assay of an array of
D-proline-derived peptidomimetic
hydroxamic acids possessing an aromatic sulfonyl group at the
nitrogen atom and another hydrophobic group at position 3 or 4 of
the pyrrolidine ring. The structureeactivity analysis of this class of
compounds showed the importance of the cis-configuration of the
hydrophobic substituent with respect to the hydroxamic acid, and
improved activity when the HG2 was found at position 3 of the
pyrrolidine ring. Docking calculations confirmed the HG2 at posi-
tion 3 to adapt in the long substrate-binding groove, thus enabling
future hit-to-lead processes on this class of peptidomimetic LF
inhibitors taking into account this molecular geometry.
ether/EtOAc 3:1), thus giving a colorless oil (720 mg, 90% over two
23
4. Experimental section
steps). [
d
a]
¼ þ92.3 (c ¼ 0.95, CH2Cl2). 1H NMR (400 MHz, CDCl3):
D
7.96 (d, J ¼ 1.9 Hz,1H), 7.70 (dd, J ¼ 1.9, 8.8 Hz,1H), 7.58 (d, J ¼ 8.8 Hz,
4.1. General
1H), 4.45e4.39 (m, 2H), 3.73 (s, 3H), 3.55 (dd, J ¼ 3.9, 11.7 Hz, 1H),
3.43 (d, J ¼ 11.7 Hz, 1H), 2.29e2.23 (m, 2H), 2.11-2.05 (m, 1H) ppm.
Chromatographic separations were performed on silica gel
(Kieselgel 60, Merck) using flash-column techniques or automatic
Biotage Isolera system; Rf values refer to TLC carried out on 25-mm
silica gel plates (Merck F254) with the same eluant as indicated for
column cromatography. 1H NMR spectra were recorded with Varian
Gemini and Inova NMR spectrometers operating at 200, 300 and
400 MHz for the proton and 50 and 100 MHz for the carbon,
respectively. ESI mass spectra were carried out on an ion-trap
double quadrupole mass spectrometer using electrospray (ESþ)
ionization techniques, and a normalized collision energy within the
range of 21e28 eV for MSMS experiments. All the compounds were
found to be >95% pure by HPLC analysis. Analyses indicated by the
symbols of the elements or functions were within ꢁ0.4% of the
theoretical values.
13C NMR (50 MHz, CDCl3):
d 171.9 (s), 137.5 (s), 137.3 (s), 133.2 (s),
130.6 (d),129.2 (d),126.4 (d), 69.6 (d), 59.3 (d), 56.3 (t), 52.4 (q), 39.2
(t) ppm. ESI-MS m/z (ESþ) 353.82 [(M þ H)þ, 45.7], and 375.93
[(M þ Na)þ, 24.02]. Anal. C12H13Cl2NO5S (C, H, N).
4.1.4. (2R,4R)-methyl 4-(benzyloxy)-1-((3,4-dichlorophenyl)
sulfonyl)pyrrolidine-2-carboxylate (7)
To
trichloroacetimidate (105
anhydrous Et2O (2.8 mL) was added 30
a
solution of
5
(100 mg, 0.28 mmol), benzyl 2,2,2-
L, 0.56 mmol) and 3Å MS (200 mg) in
of tri-
m
mL
fluoromethansulfonic acid (0.34 mmol) at 0 ꢀC. The mixture was
allowed to reach room temperature, then it was left stirring under
a nitrogen atmosphere for 3 h. Successively, the mixture was
washed with aqueous 5% NaHCO3 and brine. The organic phase was
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Compound 7 was isolated by flash chromatography (pet.
ether/EtOAc 2:1) to give a white solid (125 mg, 99%). 1H NMR
4.1.1. (2R,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate (4)
cs-4-Hydroxy- -proline 3 (1 g, 7.63 mmol) was added to
D
a solution of SOCl2 (0.61 mL, 8.39 mmol) in MeOH (38 mL). The
mixture was stirred overnight. Successively, the mixture was
concentrated under reduced pressure and filtered over Amberlyst
A-21 resin, thus giving compound 4 as a colorless oil (1.1 g, 99%). 1H
(400 MHz, CDCl3):
d
7.95 (d, J ¼ 1.9 Hz, 1H), 7.68 (dd, J ¼ 1.9, 8.8 Hz,
1H), 7.51 (d, J ¼ 8.8 Hz, 1H), 7.25e7.13 (m, 5H), 4.55 (dd, J ¼ 2.4,
9.2 Hz, 1H), 4.34 (q, J ¼ 11.7 Hz, 2H), 4.09e4.05 (m, 1H), 3.55 (s, 3H),
3.53-3.50 (m, 1H), 3.30 (dd, J ¼ 2.4, 10.7 Hz, 1H), 2.43 (d, J ¼ 10.7 Hz,
NMR (200 MHz, CDCl3):
d
4.38e4.30 (br, 1H), 3.79e3.72 (m, 1H),
1H), 2.15e2.08 (m, 1H) ppm. 13C NMR (50 MHz, CDCl3):
d 171.4 (s),

102
C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
138.5 (s), 136.8 (s), 136.4 (s), 133.5 (s), 130.6 (d), 129.2 (d), 128.1 (d,
2C), 127.6 (d), 127.1 (d, 2C), 126.3 (d), 76.7 (t), 70.6 (d), 59.3 (d), 52.9
(t), 52.2 (q), 35.6 (t) ppm. ESI-MS m/z (ESþ) 443.15 [Mþ, 21.3],
466.09 [(M þ Na)þ, 100]. Anal. C19H19Cl2NO5S (C, H, N).
was diluted with EtOAc, and washed with aqueous NaHCO3 and
brine. The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Compound 11 was isolated
by flash chromatography (pet. ether/EtOAc 1:1) thus giving a white
23
solid (91 mg, 73%). M.p. 160.5e161.4 ꢀC. [
a]
¼ þ42.6 (c ¼ 0.75,
D
4.1.5. (2R,4S)-methyl 4-(benzyloxy)-1-(3,4-dichlorophenylsulfonyl)
pyrrolidine-2-carboxylate (8)
Prepared as reported for 7 starting from 6 (100 mg, 0.28 mmol).
Compound 8 was isolated by flash chromatography (pet. ether/
CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
7.95 (d, J ¼ 1.9 Hz, 1H), 7.76e
7.73 (m, 3H), 7.59 (dd, J ¼ 1.9, 8.8 Hz, 1H), 7.52 (d, J ¼ 8.8 Hz, 1H),
7.43e7.39 (m, 2H), 7.36e7.32 (m, 1H), 5.25e5.22 (m, 1H), 4.69 (t,
J ¼ 7.8 Hz, 1H), 4.06 (dd, J ¼ 5.8, 10.7 Hz, 1H), 3.88 (dd, J ¼ 3.9,
10.7 Hz, 1H), 3.79 (s, 3H), 3.04e2.97 (m, 1H), 2.65e2.58 (m, 1H)
EtOAc 2:1) thus giving
a colourless oil (125 mg, 99%).
23
[
a]
¼ þ84.65 (c ¼ 0.85, CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
7.95
ppm. 13C NMR (50 MHz, CDCl3):
d 171.5 (s), 147.9 (s), 137.9 (s), 137.1
D
(d, J ¼ 1.9 Hz, 1H), 7.66 (dd, J ¼ 1.9, 8.8 Hz, 1H), 7.45 (d, J ¼ 8.8 Hz,
1H), 7.33e7.25 (m, 3H), 7.06 (d, J ¼ 7.8 Hz, 2H), 4.32 (t, J ¼ 7.8 Hz,
1H), 4.28 (s, 2H), 4.12e4.11 (br, 1H), 3.77 (s, 3H), 3.59 (d, J ¼ 2.4 Hz,
2H), 2.41e2.36 (m, 1H), 2.12-2.06 (m, 1H) ppm. 13C NMR (50 MHz,
(s), 133.4 (s), 130.9 (d), 129.4 (d), 128.7 (d), 128.5 (d, 2C), 128.1 (d),
125.9 (d), 125.3 (d, 2C), 117.8 (d), 59.3 (d), 58.1 (d), 53.5 (t), 52.6 (q),
36.1 (t) ppm. ESI-MS m/z (ESþ) 480.99 [(Mþ1)þ, 31.37], 502.98
[(M þ Na)þ, 100]. Anal. C20H18Cl2N4O4S (C, H, N).
CDCl3):
d 172.0 (s), 137.5 (s, 2C), 136.8 (s), 133.4 (s), 130.8 (d), 129.4
(d), 128.4 (d, 2C), 127.8 (d), 127.1 (d, 2C), 126.6 (d), 76.6 (t), 70.8 (d),
59.9 (d), 55.7 (t), 52.7 (q), 37.0 (t) ppm. ESI-MS m/z (ESþ) 443.15
[Mþ, 18.1], 466.09 [(M þ Na)þ, 100]. Anal. C19H19Cl2NO5S (C, H, N).
4.1.9. (2R,4R)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-(4-phenyl-
1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxylate (12)
Prepared as reported for 11 starting from 10 (100 mg,
0.26 mmol). Compound 12 was isolated by flash chromatography
4.1.6. (2R,4S)-methyl 4-azido-1-(3,4-dichlorophenylsulfonyl)
pyrrolidine-2-carboxylate (9)
(pet. ether/EtOAc 1:1) thus giving a white solid (70 mg, 56%). M.p.
23
177.3e178.0 ꢀC. [
a
]
¼ þ2.0 (c ¼ 0.45, CH2Cl2). 1H NMR (400 MHz,
D
To a solution of 5 (250 mg, 0.70 mmol), Et3N (118
in anhydrous CH2Cl2 (4.2 mL), methanesulfonyl chloride (55
m
L, 0.84 mmol)
L,
CDCl3):
d
7.99 (d, J ¼ 1.9 Hz, 1H), 7.87 (s, 1H), 7.73e7.70 (m, 3H), 7.57
m
(d, J ¼ 8.2 Hz, 1H), 7.37e7.33 (m, 2H), 7.29e7.25 (m, 1H), 5.22e5.15
(m, 1H), 4.56 (dd, J ¼ 5.3, 9.3 Hz, 1H), 3.97 (dd, J ¼ 7.0, 10.8 Hz, 1H),
3.71 (dd, J ¼ 5.3, 10.8 Hz, 1H), 3.65 (s, 3H), 2.96e2.88 (m, 1H), 2.64e
0.84 mmol) was added at 0 ꢀC. The mixture was allowed to reach
room temperature, and was left stirring under a nitrogen atmo-
sphere for 2 h. Successively, the mixture was washed with NaHCO3
and brine, then the organic phase was dried over anhydrous Na2SO4
and concentrated under reduced pressure. The crude was dissolved
in DMF (1.7 mL) and NaN3 (92 mg, 1.40 mmol) was added. The
mixture was heated at 90 ꢀC overnight. Successively, the mixture
was diluted with EtOAc, and washed with H2O and brine. The
organic phase was dried over anhydrous Na2SO4, and concentrated
under reduced pressure. Compound 9 was isolated by flash chro-
2.58 (m, 1H) ppm. 13C NMR (50 MHz, CDCl3):
d 170.7 (s), 138.3 (s),
137.6 (s, 2C), 133.8 (s), 131.2 (d), 130.0 (s), 129.5 (d), 128.8 (d, 2C),
128.3 (d, 2C), 126.6 (d, 2C), 125.6 (d, 2C), 118.3 (d, 2C), 59.1 (d), 58.1
(d), 53.0 (t þ q, 2C), 36.6 (t) ppm. ESI-MS m/z (ESþ) 481.09 [(Mþ1)þ,
27.73], 502.94 [(M þ Na)þ, 100]. Anal. C20H18Cl2N4O4S (C, H, N).
4.1.10. (2R,4S)-methyl 4-amino-1-(3,4-dichlorophenylsulfonyl)
pyrrolidine-2-carboxylate (13)
matography (pet. ether/EtOAc1:1) to give awhite solid (200 mg, 75%
Compound 9 (400 mg, 1.06 mmol) was dissolved in MeOH
(5 mL), and 10% Pd/C (53 mg) was added. The resulting mixture was
stirred overnight at room temperature under a hydrogen atmo-
sphere. The suspension was then filtered over Celite, and MeOH
was removed under reduced pressure. The mixture was filtered
over Amberlyst A-21 resin, thus giving compound 13 as a colorless
oil, which was used directly for subsequent steps (363 mg, 97%). 1H
23
over two steps). M.p. 119.7e120.6 ꢀC. [
a
]
¼ þ53.6 (c ¼ 0.95,
D
CH2Cl2).1H NMR (400 MHz, CDCl3):
d
7.95 (d, J ¼ 1.9 Hz,1H), 7.69 (dd,
J ¼ 1.9, 8.8 Hz,1H), 7.60 (d, J ¼ 8.8 Hz,1H), 4.34 (t, J ¼7.8Hz,1H), 4.23e
4.19 (m, 1H), 3.74 (s, 3H), 3.64 (dd, J ¼ 4.8, 11.2 Hz, 1H), 3.46 (ddd,
J ¼ 1.4, 2.9, 11.2 Hz, 1H), 2.29e2.16 (m, 2H) ppm. 13C NMR (50 MHz,
CDCl3):
d 171.1 (s), 137.3 (s), 137.1 (s), 133.3 (s), 130.7 (d), 129.0 (d),
126.2 (d), 59.1 (d), 59.0 (d), 52.9 (t), 52.5 (q), 36.1 (t) ppm. ESI-MS m/z
NMR (200 MHz, CDCl3):
d
7.88 (d, J ¼ 6.9 Hz, 1H), 7.63e7.48 (m, 2H),
(ESþ) 401.04 [(M þ Na)þ, 100]. Anal. C12H12Cl2N4O4S (C, H, N).
4.40 (dd, J ¼ 5.4, 8.4 Hz, 1H), 3.70 (s, 3H), 3.66e3.60 (m, 2H), 3.03e
2.99 (m, 1H), 2.20e2.08 (m, 1H), 1.94e1.80 (m, 1H) ppm. 13C NMR
4.1.7. (2R,4R)-methyl 4-azido-1-(3,4-dichlorophenylsulfonyl)
pyrrolidine-2-carboxylate (10)
Prepared as reported for 9 starting from 6 (250 mg, 0.70 mmol).
Compound 10 was isolated by flash chromatography (pet. ether/
(50 MHz, CDCl3): d 171.6 (s), 137.2 (s), 137.2 (s), 133.4 (s), 130.6 (d),
129.2 (d), 126.3 (d), 58.3 (d), 51.9 (q), 51.2 (t), 45.4 (d), 33.5 (t) ppm.
Anal. C12H14Cl2N2O4S (C, H, N).
EtOAc 1:1), thus giving a white solid (203 mg, 77% over two steps).
4.1.11. (2R,4R)-methyl 4-amino-1-(3,4-dichlorophenylsulfonyl)
pyrrolidine-2-carboxylate (14)
Prepared as reported for 13 starting from 10 (400 mg,
1.06 mmol). After filtration of the mixture over Amberlyst A-21
resin, compound 14 was obtained as a colorless oil, and directly
used for subsequent steps (358 mg, 96%). 1H NMR (200 MHz,
23
M.p. 69.8e70.9 ꢀC. [
a
]
¼ þ29.5 (c ¼ 1.0, CH2Cl2). 1H NMR
D
(400 MHz, CDCl3):
d
8.02 (d, J ¼ 1.9 Hz, 1H), 7.77 (dd, J ¼ 1.9, 8.8 Hz,
1H), 7.60 (d, J ¼ 8.8 Hz, 1H), 4.62e4.59 (m, 1H), 4.21e4.18 (m, 1H),
3.72 (s, 3H), 3.68 (dd, J ¼ 5.8, 10.7 Hz, 1H), 3.26 (dd, J ¼ 3.4, 10.7 Hz,
1H), 2.46-2.93 (m, 1H), 2.31-2.26 (m, 1H) ppm. 13C NMR (50 MHz,
CDCl3):
d
170.6 (s), 138.2 (s), 137.5 (s), 133.2 (s), 130.7 (d), 129.2 (d),
CDCl3):
d
7.91 (d, J ¼ 6.9 Hz, 1H), 7.61e7.49 (m, 2H), 4.54 (dd, J ¼ 4.7,
126.3 (d), 59.0 (d), 58.9 (d), 52.4 (t), 52.2 (q), 35.7 (t) ppm. ESI-MS
8.7 Hz, 1H), 4.21 (t, J ¼ 5.8 Hz, 1H) 3.89 (dd, J ¼ 5.8, 10.9 Hz,1H), 3.71
(s, 3H), 3.48 (dd, J ¼ 4.7, 10.9 Hz, 1H), 2.69e2.54 (m, 1H), 2.44e2.32
m/z (ESþ) 401.04 [(M þ Na)þ, 100]. Anal. C12H12Cl2N4O4S (C, H, N).
(m, 1H) ppm. 13C NMR (50 MHz, CDCl3):
d 171.9 (s), 137.6 (s), 137.0
4.1.8. (2R,4S)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-(4-phenyl-
1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxylate (11)
(s), 133.2 (s), 130.5 (d), 129.1 (d), 126.4 (d), 57.7 (d), 52.3 (q), 50.9 (t),
45.1 (d), 33.2 (t) ppm. Anal. C12H14Cl2N2O4S (C, H, N).
To a solution of 9 (100 mg, 0.26 mmol) and ethynylbenzene
(27 mg, 0.26 mmol) in H2O/tBuOH (0.7 mL/0.7 mL) a solution of
0.9 M sodium ascorbate (0.29 mL, 0.26 mmol) and a solution of
0.3 M Cu(OAc)2 (0.87 mL, 0.26 mmol) were added. The mixture was
stirred overnight at room temperature. Successively, the mixture
4.1.12. (2R,4S)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-(2,4-
dimethoxybenzamido)pyrrolidine-2-carboxylate (15)
To a solution of 13 (50 mg, 0.14 mmol) and Et3N (39
0.28 mmol) in anhydrous CH2Cl2 (0.7 mL) 2,4-dimethoxybenzoyl
mL,

C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
103
chloride (28 mg, 0.14 mmol) was added at 0 ꢀC. The mixture was
allowed to warm to room temperature, and it was left overnight
stirring under a nitrogen atmosphere. Successively, the mixture
was washed with NaHCO3, 1 N HCl and brine. The organic phase
was dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Compound 15 was isolated by flash chromatography
chromatography (pet. ether/EtOAc 1:2) thus giving a brown solid
22
(49 mg, 37%). M.p. 69.4e70.2 ꢀC. [
NMR (400 MHz, CDCl3):
a
]
¼ þ45.2 (c ¼ 0.3, CH2Cl2). 1H
D
d
7.82 (d, J ¼ 6.8 Hz, 1H), 7.48e7.41 (m, 2H),
7.35e7.34 (br, 1H), 7.20 (d, J ¼ 15.1 Hz, 1H), 6.45 (d, J ¼ 3.4 Hz, 1H),
6.35 (dd, J ¼ 1.9, 3.4 Hz, 1H), 6.27 (d, J ¼ 6.3 Hz, 1H), 6.00 (d,
J ¼ 15.1 Hz, 1H), 4.49 (t, J ¼ 7.8 Hz, 2H), 3.60 (s, 3H), 3.58e3.57 (m,
1H), 3.43 (dd, J ¼ 2.9, 10.7 Hz, 1H), 2.25-2.19 (m, 2H) ppm. 13C NMR
(pet. ether/EtOAc 1:2) thus giving a white solid (44 mg, 60%). M.p.
22
75.5e76.5 ꢀC. [
a
]
¼ þ69.23 (c ¼ 0.3, CH2Cl2). 1H NMR (400 MHz,
(50 MHz, CDCl3): d 171.7 (s), 165.6 (s), 150.9 (s, 2C), 144.0 (d), 137.9
D
CDCl3):
d
8.07 (d, J ¼ 8.7 Hz, 1H), 7.97 (d, J ¼ 6.3 Hz, 1H), 7.89 (d,
(s), 133.1 (d), 129.1 (d), 128.0 (d), 127.4 (d þ s, 2C), 117.7 (d), 114.0 (d),
112.1 (d), 59.3 (d), 53.5 (t), 52.6 (q), 49.5 (d), 36.8 (t) ppm. Anal.
C19H18Cl2N2O6S (C, H, N).
J ¼ 6.8 Hz, 1H), 7.47-7.43 (m, 2H), 6.55 (dd, J ¼ 1.9, 8.7 Hz, 1H), 6.44
(d, J ¼ 1.9 Hz, 1H), 4.64e4.62 (br, 1H), 4.58 (t, J ¼ 7.8 Hz, 1H), 3.91 (s,
3H), 3.82 (s, 3H), 3.65 (s, 3H), 3.59-5.58 (m, 2H), 2.44e2.38 (m, 1H),
2.29e2.22 (m, 1H) ppm. 13C NMR (50 MHz, CDCl3):
d
171.9 (s), 164.8
4.1.16. (2R,4R)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-
(s), 163.5 (s), 158.8 (s, 2C), 138.7 (s), 133.6 (d), 133.7 (d), 128.8 (s þ d,
2C), 127.4 (d), 113.5 (s), 105.2 (d), 98.4 (d), 58.9 (d), 56.0 (q), 55.6 (q),
53.5 (d), 52.6 (t), 49.7 (q), 37.6 (t) ppm. Anal. C21H22Cl2N2O7S (C,
H, N).
(thiophene-2-carboxamido)pyrrolidine-2-carboxylate (19)
Prepared as reported for 18 starting from 14 (100 mg,
0.28 mmol). Compound 19 was isolated by flash chromatography
(pet. ether/EtOAc 1:2) thus giving a white solid (54 mg, 42%). M.p.
22
56.8e58.2 ꢀC. [
a]
¼ þ51.9 (c ¼ 0.3, CH2Cl2). 1H NMR (400 MHz,
D
4.1.13. (2R,4S)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-
(thiophene-2-carboxamido)pyrrolidine-2-carboxylate (18)
CDCl3) major rotamer:
d
7.79 (d, J ¼ 6.8 Hz, 1H), 7.68 (d, J ¼ 9.2 Hz,
1H), 7.54 (d, J ¼ 7.8 Hz, 1H), 7.47 (t, J ¼ 8.2 Hz, 2H), 7.41 (dd, J ¼ 0.9,
4.8 Hz, 1H), 7.01 (t, J ¼ 4.8 Hz, 1H), 4.71e4.69 (m, 1H), 4.20 (dd,
J ¼ 1.9, 10.2 Hz, 1H), 3.73 (s, 3H), 3.51 (d, J ¼ 9.7 Hz, 1H), 3.30 (dd,
J ¼ 4.8, 9.7 Hz, 1H), 2.27e2.20 (m, 1H), 1.95 (dd, J ¼ 1.9, 12.1 Hz, 1H)
To a solution of 13 (100 mg, 0.28 mmol) and Et3N (79
mL,
0.57 mmol) in anhydrous CH2Cl2 (1.4 mL) thiophene-2-carbonyl
chloride (41 mg, 0.28 mmol) was added at 0 ꢀC. The mixture was
allowed to reach room temperature, and it was left overnight stir-
ring under a nitrogen atmosphere. Successively, the mixture was
washed with aqueous NaHCO3, 1 N HCl and brine. The organic
phase was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. Compound 18 was isolated by flash chromatog-
ppm. 13C NMR (50 MHz, CDCl3) major rotamer:
d 174.0 (s), 160.9 (s),
138.6 (s), 136.4 (s), 133.3 (d), 130.4 (d), 129.2 (d), 129.0 (s), 128.2 (s),
128.1 (d),127.7 (d), 127.5 (d), 58.9 (d), 55.3 (t), 53.3 (q), 48.9 (d), 36.6
(t) ppm. Anal. C17H16Cl2N2O5S2 (C, H, N).
raphy (pet. ether/EtOAc 1:2) thus giving a yellow oil (32 mg, 25%).
4.1.17. Ethyl 3-((tert-butoxycarbonyl)(2-ethoxy-2-oxoethyl)amino)
propanoate (30)
22
[
a]
¼ þ59.5 (c ¼ 0.25, CH2Cl2). 1H NMR (400 MHz, CDCl3) major
D
rotamer:
d
7.85 (d, J ¼ 6.8 Hz, 2H), 7.46e7.39 (m, 4H), 7.37e7.34 (m,
To a suspension of ethyl glycinate hydrochloride 29 (7.0 g,
50.1 mmol) in 95% ethanol (115 mL) was added ethyl acrylate
(6.0 mL, 55.1 mmol) and Et3N (7.0 mL, 50.1 mmol). The reaction
mixture was allowed to stir at room temperature for 48 h. The
reaction was concentrated and water was added. The aqueous
phase was extracted with EtOAc. The organic layer was dried over
anhydrous Na2SO4 and evaporated. The crude product, isolated as
a yellow oil, was dissolved in CHCl3 (80 mL), and Boc2O (15.3 g,
70.1 mmol) was added at 0 ꢀC, followed by 5% aqueous NaOH
(80 mL). The reaction was stirred at 0 ꢀC for 1 h, and then at room
temperature for 72 h. The mixture was extracted with CHCl3 and
the organic layer was washed with brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The crude
product was purified by automated flash chromatography (pet.
ether/EtOAc 4:1, Rf 0.56), to afford 30 as a yellow oil (11.78 g, 77%
yield over two step). 1H NMR (CDCl3, 200 MHz) major rotamer:
1H), 6.98e6.93 (m, 1H), 6.58 (d, J ¼ 6.3 Hz, 1H), 4.56 (t, J ¼ 7.8 Hz,
2H), 3.61 (s, 3H), 3.54e3.50 (m, 2H), 2.38e2.31 (m, 1H), 2.25e2.18
(m, 1H) ppm. 13C NMR (50 MHz, CDCl3) major rotamer:
d 171.6
(s), 161.6 (s), 138.1 (s), 138.0 (s), 133.1 (d), 130.5 (d), 129.0 (d), 128.7
(s), 128.3 (d), 127.6 (d, 2C), 126.6 (s), 58.8 (d), 53.3 (t), 52.7 (q), 50.0
(d), 37.1 (t) ppm. Anal. C17H16Cl2N2O5S2 (C, H, N).
4.1.14. (2R,4R)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-(2,4-
dimethoxybenzamido)pyrrolidine-2-carboxylate (16)
Prepared as reported for 15 starting from 14 (75 mg, 0.21 mmol).
Compound 16 was isolated by flash chromatography (pet. ether/
EtOAc 1:2) thus giving a white solid (44 mg, 40%). M.p. 68.3e
22
69.2 ꢀC. [
CDCl3):
a]
¼ þ16.2 (c ¼ 0.2, CH2Cl2). 1H NMR (400 MHz,
D
d
8.46 (d, J ¼ 8.3 Hz, 1H), 8.07 (d, J ¼ 8.7 Hz, 1H), 7.87 (d,
J ¼ 7.3 Hz, 1H), 7.58 (d, J ¼ 7.3 Hz, 1H), 7.52 (t, J ¼ 7.3 Hz, 1H), 6.53
(dd, J ¼ 2.4, 8.7 Hz, 1H), 6.44 (d, J ¼ 2.4 Hz, 1H), 4.79e4.75 (br, 1H),
4.29 (dd, J ¼ 3.4, 10.2 Hz, 1H), 3.93 (s, 3H), 3.80 (s, 3H), 3.71 (s, 3H),
3.51 (dd, J ¼ 5.8, 10.2 Hz, 1H), 3.44 (dd, J ¼ 2.4, 10.2 Hz, 1H), 2.39e
2.31 (m, 1H), 2.06e2.01 (m, 1H) ppm. 13C NMR (50 MHz, CDCl3):
d
4.16 (q, J ¼ 7.1 Hz, 2H), 4.11 (qd, J ¼ 3.6, 7.1 Hz, 2H), 4.00 and 3.94
(s, 2H), 3.55 and 3.51 (t, J ¼ 6.6 Hz, 2H), 2.62 and 2.59 (t, J ¼ 6.6 Hz,
2H), 1.46 and 1.40 (s, 9H), 1.25 (m, 6H). Anal. C14H25NO6 (C, H, N).
d
172.6 (s), 164.5 (s), 163.4 (s), 159.0 (s, 2C), 137.0 (s), 133.7 (d), 133.0
4.1.18. (R/S)-1-tert-Butyl 2-ethyl 3-oxopyrrolidine-1,2-
dicarboxylate (31)
To a suspension of potassium tert-butoxide (791 mg, 7.05 mmol)
in anhydrous toluene (19 mL) at ꢂ10 ꢀC a solution of 30 (1.43 g,
4.7 mmol) in toluene was added dropwise. The reaction was stirred
at 0 ꢀC for 90 min and then it was quenched with acetic acid
(d), 129.1 (d), 128.8 (s), 127.5 (d), 113.6 (s), 105.1 (d), 98.3 (d), 59.2
(d), 55.8 (q), 55.6 (q), 55.0 (d), 52.6 (t), 48.4 (q), 36.9 (t) ppm. Anal.
C21H22Cl2N2O7S (C, H, N).
4.1.15. (2R,4S)-methyl 1-(3,4-dichlorophenylsulfonyl)-4-((E)-3-
(furan-2-yl)acrylamido)pyrrolidine-2-carboxylate (17)
(500 mL), followed by the addition of a cold solution of NaH2
-
To a solution of 14 (100 mg, 0.28 mmol) and Et3N (79
m
L,
PO4$H2O (2.5 g) in 25 mL of water. The layers were separated, and
the aqueous phase was extracted with CHCl3. The combined
organic layers were dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude product was purified by flash
chromatography (silica gel, pet. ether/EtOAc 5:1, Rf 0.44) obtaining
the title compound 31 as a yellow oil (497 mg, 41% yield). 1H NMR
0.57 mmol) in anhydrous CH2Cl2 (1.4 mL) (E)-3-(furan-2-yl)acryloyl
chloride (44 mg, 0.28 mmol) was added at 0 ꢀC. The mixture was
allowed to reach room temperature, and it was left overnight stir-
ring under a nitrogen atmosphere. Successively, the mixture was
washed with aqueous NaHCO3, 1 N HCl and brine. The organic
phase was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. Compound 17 was isolated by flash
(CDCl3, 200 MHz) major rotamer:
d 4.55 and 4.47 (s, 1H), 4.27e4.22
(m, 2H), 3.91-3.84 (m) and 3.82 (t, J ¼ 8.2 Hz) 2H, 2.69 (t, J ¼ 8.0 Hz,

104
C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
24
2H), 1.49 and 1.43 (s, 9H), 1.30 (t, J ¼ 7.1 Hz, 3H). Anal. C12H19NO5
compound 34 as a colorless oil (130 mg, 66% yield). [
a
]
ꢂ13.0
D
(C, H, N).
(c ¼ 1.14, CHCl3); 1H NMR (CDCl3, 300 MHz) major rotamer:
d 4.40
(d, J ¼ 7.1 Hz, 1H), 4.21e4.15 (m, 2H), 3.69e3.58 (m, 1H), 3.56e3.29
(m, 2H þ 2H), 2.07e2.0 (m, 2H), 1.53e1.47 (m, 2H), 1.40 (s, 9H),
1.30e1.23 (m, 9H), 0.89e0.85 (m, 3H) ppm. 13C NMR (50 MHz,
4.1.19. (2R,3S)-1-tert-Butyl 2-ethyl 3-hydroxypyrrolidine-1,2-
dicarboxylate (32)
Immobilized baker yeast preparation: separate solutions of
dried baker’s yeast (4.0 g) and sodium alginate (971 mg), each in
40 mL of water were prepared by slow addition of reagent into
water. When both reagents were completely dissolved, the two
solutions were combined and added dropwise by means of an
addition funnel to a solution of 10% calcium chloride (137 mL).
Upon impact with calcium chloride solution, the drops formed
gelatinous beads, containing the baker’s yeast. The beads were
washed three times with 130 mL of water and used for the reaction.
The beads were placed in a 500 mL round flask and 31 mL of water
were added. Sucrose (5.8 g) was added, the mixture was stirred at
room temperature for 30 min, and then 31 (385 mg, 1.5 mmol) was
added. The reaction was stirred at 30 ꢀC for 48 h. The reaction
mixture was then filtered, and yeast beads were washed twice with
Et2O (40 mL). The aqueous layer was centrifuged, and then
extracted with Et2O. The combined organic extracts were dried
over anhydrous Na2SO4 and evaporated. The crude product was
purified by automated flash chromatography (pet. ether/EtOAc 1:1,
CDCl3) major rotamer: d 169.9 (s), 153.7 (s), 79.7 (s), 79.6 (d), 78.8
(s), 70.6 (t), 62.0 (d), 60.5 (t), 43.3 (t), 31.5 (t), 29.5 (t), 29.0 (t), 28.2
(q), 25.6 (t), 22.4 (t), 14.2 (q), 13.8 (q) ppm. Anal. C18H33NO5 (C, H,
N).
4.1.22. (2R,3S)-ethyl 3-(benzyloxy)-1-((3,4-dichlorophenyl)
sulfonyl)pyrrolidine-2-carboxylate (35)
To a stirred solution of 33 (352 mg, 1.01 mmol) in CH2Cl2 (10 mL)
was added TFA (10 mL). The reaction mixture was stirred at room
temperature for 3 h. After evaporation of the solvent and filtration
over Amberlyst A-21 resin, crude product, isolated as a yellow oil,
was used in the next step without further purification. To
a suspension of crude in dry CH2Cl2 (13.6 mL) Et3N (281
2.02 mmol) and DMAP (24 mg, 0.2 mmol) were added under N2
atmosphere. The mixture was cooled to
ꢀC and 3,4-
mL,
0
dichlorobenzylsulfonyl chloride (272 mg, 1.11 mmol) was added
in portions. The mixture was allowed to reach room temperature
and stirred overnight. Then it was washed with a saturated NaHCO3
solution, 5% HCl and brine. The organic layer was dried over
anhydrous Na2SO4 and evaporated in vacuo. The crude product was
Rf 0.35), affording desired compound 32 as a yellow oil (185 mg,
24
47% yield). [
a
]
¼ þ29.0 (c ¼ 1.0, CHCl3). 1H NMR (CDCl3,
D
300 MHz) major rotamer:
d
4.6e4.58 (m, 1H), 4.31 (d, J ¼ 6.9 Hz,
purified by automated flash chromatography (pet. ether/EtOAc 4:1,
24
1H), 4.24e4.17 (m, 2H), 3.68e3.56 (m, 1H), 3.52e3.43 (m, 1H), 2.62
Rf 0.51) to obtain 306 mg of 35 as a yellow oil (67% yield). [a]
D
(br, 1H), 2.14e1.96 (m, 2H), 1.40 (s, 9H), 1.31e1.25 (m, 3H) ppm. 13C
ꢂ11.8 (c ¼ 1.0, CHCl3). 1H NMR (CDCl3, 300 MHz):
d 7.95 (d,
NMR (50 MHz, CDCl3) major rotamer:
d
170.5 (s), 154.0 (s), 80.0 (s),
J ¼ 2.2 Hz, 1H), 7.71 (dd, J ¼ 2.2, 8.5 Hz, 1H), 7.58 (d, J ¼ 8.5 Hz, 1H),
7.36e7.24 (m, 5H), 4.65e4.60 (m, 3H), 4.31e4.14 (m, 3H), 3.53e3.45
(m, 2H), 2.18e2.04 (m, 2H), 1.25e1.17 (m, 3H) ppm. 13C NMR
72.0 (d), 63.9 (d), 60.8 (t), 43.7 (t), 31.9 (t), 28.1 (q), 14.2 (q) ppm.
ESI-MS m/z (ESþ) 259.65 [(Mþ1)þ,18.34], 281.12 [(M þ Na)þ, 24.52],
540.84 [(2M þ Na)þ, 100]. Anal. C12H21NO5 (C, H, N).
(50 MHz, CDCl3):
d 168.4 (s), 138.4 (s), 137.2 (s), 136.8 (s), 133.1 (s),
130.7 (d), 128.9 (d), 128.1 (d), 127.6 (d), 127.2 (d) 126.2 (d), 78.3 (d),
72.0 (t), 62.6 (d), 61.2 (t), 45.3 (t), 29.8 (t), 13.8 (q) ppm. Anal.
C20H21Cl2NO5S (C, H, N).
4.1.20. (2R,3S)-1-tert-Butyl 2-ethyl 3-(benzyloxy)pyrrolidine-1,2-
dicarboxylate (33)
To a stirred solution of 32 (317 mg,1.22 mmol) in dry THF, benzyl
bromide (173
m
L, 1.46 mmol) and TBAI (137 mg, 0.37 mmol) were
4.1.23. (2R,3S)-ethyl 3-(benzyloxy)-1-((4-chlorophenyl)sulfonyl)
pyrrolidine-2-carboxylate (36)
Prepared as reported for 35 starting from 33 (352 mg,
1.01 mmol) and 4-chlorobenzylsulfonyl chloride (325 mg,
1.54 mmol). The crude product was purified by automated flash
added under N2 atmosphere. The mixture was cooled to 0 ꢀC, and
60% NaH (58 mg, 1.46 mmol) was added. The reaction mixture was
stirred at room temperature overnight. The mixture was poured
into ice water and extracted with EtOAc. The combined organic
layers were washed with brine, dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude product was purified by auto-
chromatography (pet. ether/EtOAc 4:1, Rf 0.49) to give compound
24
36 as a yellow oil (157 mg, 26% yield). [
a
]
D ꢂ18.0 (c ¼ 1.0, CHCl3);
mated flash chromatography (pet. ether/EtOAc 2:1, Rf 0.28)
1H NMR (CDCl3, 300 MHz):
d
7.82 (dd, J ¼ 1.9 Hz, J ¼ 6.6 Hz, 2H), 7.48
24
affording 33 as colorless oil (41% yield). [
a
]
ꢂ41.3 (c ¼ 0.45,
(dd, J ¼ 1.9, 6.6 Hz, 2H), 7.33e7.24 (m, 5H), 4.63e4.53 (m, 2H), 4.24
(q, J ¼ 7.1 Hz, 2H), 4.20e4.03 (m, 2H), 3.58e3.39 (m, 2H), 2.21e2.01
(m, 2H), 1.18 (t, J ¼ 7.1 Hz, 3H) ppm. 13C NMR (50 MHz, CDCl3):
D
CHCl3). 1H NMR (CDCl3, 300 MHz) major rotamer:
d 7.37e7.26 (m,
5H), 4.71e4.46 (m, 3H), 4.18 (q, J ¼ 7.1 Hz, 2H), 3.77e3.59 (m, 2H),
3.40e3.32 (m, 1H), 2.20e2.03 (m, 2H), 1.42 (s, 9H), 1.24 (t, J ¼ 7.1 Hz,
d
168.8 (s), 139.2 (s), 137.2 (s), 137.1 (s), 129.2 (d), 128.8 (d), 128.4 (d),
3H) ppm. 13C NMR (50 MHz, CDCl3) major rotamer:
d
169.7 (s),153.4
127.8 (d), 127.4(d), 78.5 (d), 72.3 (t), 62.7 (d), 61.3 (t), 45.4 (t), 30.0
(t), 14.0 (q) ppm. Anal. C20H22ClNO5S (C, H, N).
(s), 137.2 (s), 127.9 (d), 127.3 (d), 127.0 (d), 79.7 (d), 78.4 (s), 71.7 (t),
61.7 (d), 60.5 (t), 43.1 (t), 28.8 (t), 28.0 (q), 14.0 (q) ppm. ESI-MS m/z
(ESþ) 349.93 [(Mþ1)þ, 28.24], 372.09 [(M þ Na)þ, 100]. Anal.
C19H27NO5 (C, H, N).
4.1.24. (2R,3S)-ethyl 1-((4-chlorophenyl)sulfonyl)-3-(hexyloxy)
pyrrolidine-2-carboxylate (37)
Prepared as reported for 35 starting from compound 34
(130 mg, 0.4 mmol) and 4-chlorobenzylsulfonyl chloride (93 mg,
0.44 mmol). The crude product was purified by flash cromatog-
4.1.21. (2R,3S)-1-tert-Butyl 2-ethyl 3-(hexyloxy)pyrrolidine-1,2-
dicarboxylate (34)
Compound 32 (148 mg, 0.6 mmol) was dissolved in DMF
raphy (silica gel, pet. ether/EtOAc 4:1, Rf 0.52) to obtain 37 as
28
(1.8 mL) and 1-iodohexane (177
mL, 1.2 mmol) was added. The
a yellow oil (102 mg, 60% yield). [
NMR (CDCl3, 300 MHz):
a
]
ꢂ30.2 (c ¼ 0.7, CHCl3). 1H
D
mixture was cooled to 0 ꢀC, and 60% NaH (72 mg, 1.8 mmol) was
added. The reaction mixture was allowed to reach room tempera-
ture, and it was stirred overnight. The mixture was then diluted
with H2O and extracted with EtOAc. The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, filtered and
evaporated. A yellow oil was obtained and purified by automated
flash chromatography (EP/Et2O 3:1, Rf 0.28), giving the title
d
7.81 (dd, J ¼ 1.9, 6.6 Hz, 2H), 7.48 (dd,
J ¼ 1.9, 6.6 Hz, 2H), 4.55 (d, J ¼ 6.9 Hz, 1H), 4.20e4.0 (m, 3H), 3.55e
3.37 (m, 4H), 2.1e1.99 (m, 2H), 1.50e1.45 (m, 2H), 1.30e1.20 (m,
9H), 0.88e0.84 (m, 3H) ppm. 13C NMR (50 MHz, CDCl3):
d 168.8 (s),
139.1 (s),137.3 (s), 129.2 (d), 128.8 (d), 79.4 (d), 70.9 (t), 62.8 (d), 61.1
(t), 45.4 (t), 31.5 (t), 30.0 (t), 29.5 (t), 25.5 (t9, 22.4 (t), 14.0 (q), 13.9
(q) ppm. Anal. C19H28ClNO5S (C, H, N).

C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
105
4.2. General procedure for the synthesis of hydroxamic acids [29]
ESI-MSMS m/z (ESþ) 447.36 [(M
C19H17Cl2N5O4S (C, H, N).
þ
H-Cl)þ, 100]. Anal.
Preparation of NH2OK/NH2OH solution: NH2OH$HCl (8 eq) was
solubilized in MeOH (0.4 mL/mmol) by heating to reflux. Most, but
not all of the salt dissolved. The solution was cooled to <40 ꢀC, and
a solution of KOH (12 eq) in MeOH (0.2 mL) was added in one
portion. The resulting suspension was cooled to room temperature
before use and was used without prior removal of precipitated
material. A solution of ester (1 eq) in NH2OK/NH2OH solution
previously prepared was stirred at room temperature 3 days. The
reaction mixture was taken up in 1N HCl, extracted with EtOAc,
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by column chromatography (EtOAc/MeOH
4:1) to afford hydroxamic acids.
4.2.5. (2R,4S)- 1-(3,4-dichlorophenylsulfonyl)-4-(2,4-
dimethoxybenzamido)-N-hydroxypyrrolidine-2-carboxamide (24)
22
White solid, 49% yield. M.p. 120.4e122.7 ꢀC. [
a
]
¼ þ12.9
D
(c ¼ 0.65, CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
8.07 (d, J ¼ 8.7 Hz,
1H), 7.97 (d, J ¼ 6.3 Hz, 1H), 7.89 (d, J ¼ 6.8 Hz, 1H), 7.47e7.43 (m,
2H), 6.55 (dd, J ¼ 1.9, 8.7 Hz, 1H), 6.44 (d, J ¼ 1.9 Hz, 1H), 4.64e4.62
(br, 1H), 4.58 (t, J ¼ 7.8 Hz, 1H), 3.91 (s, 3H), 3.82 (s, 3H), 3.65 (s, 3H),
3.59-5.58 (m, 2H), 2.44e2.38 (m, 1H), 2.29e2.22 (m, 1H) ppm. 13C
NMR (50 MHz, CDCl3): d 178.3 (s), 164.5 (s), 162.9 (s), 158.5 (s), 136.6
(s), 136.5 (s), 133.0 (d), 131.6 (d), 128.3 (d), 127.0 (d), 113.1 (s), 113.2
(s), 104.6 (d), 97.5 (d), 62.4 (d), 55.4 (q), 55.1 (q), 54.1 (d), 49.4 (t),
36.4 (t) ppm. ESI-MS m/z (ESþ) 518.83 [(Mþ1)þ, 51.29], 535.84
[(M þ Na)þ, 100]. Anal. C20H21Cl2N3O7S (C, H, N).
4.2.1. (2R,4R)- 4-(Benzyloxy)-1-(3,4-dichlorophenylsulfonyl)-N-
hydroxypyrrolidine-2-carboxamide (20)
23
White solid, 38% yield. M.p. 77.4e79.5 ꢀC. [
a
]
¼ þ29.6 (c ¼ 1,
4.2.6. (2R,4R)- 1-(3,4-dichlorophenylsulfonyl)-4-(2,4-
D
CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
9.44 (br, 1H), 7.88 (s, 1H), 7.62
dimethoxybenzamido)-N-hydroxypyrrolidine-2-carboxamide (25)
22
(d, J ¼ 8.4 Hz, 1H), 7.52 (d, J ¼ 8.4 Hz, 1H), 7.22e7.17 (m, 5H), 7.10e
7.06 (br, 1H), 4.43 (d, J ¼ 11.4 Hz, 1H), 4.26 (d, J ¼ 11.4 Hz, 1H), 4.23
(d, J ¼ 9.3 Hz, 1H), 3.91 (br, 1H), 3.62 (d, J ¼ 10.8 Hz, 1H), 3.07 (dd,
J ¼ 4.0, 10.8 Hz, 1H), 2.53 (d, J ¼ 10.8 Hz, 1H), 1.65e1.58 (m, 1H)
White solid, 23% yield. M.p. 115.9e118.2 ꢀC. [
a]
¼ þ9.0
D
(c ¼ 0.45, CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
8.07 (d, J ¼ 8.7 Hz,
1H), 7.97 (d, J ¼ 6.3 Hz, 1H), 7.89 (d, J ¼ 6.8 Hz, 1H), 7.47-7.43 (m,
2H), 6.55 (dd, J ¼ 1.9, 8.7 Hz, 1H), 6.44 (d, J ¼ 1.9 Hz, 1H), 4.64e4.62
(br, 1H), 4.58 (t, J ¼ 7.8 Hz, 1H), 3.91 (s, 3H), 3.82 (s, 3H), 3.65 (s, 3H),
3.59e5.58 (m, 2H), 2.44e2.38 (m, 1H), 2.29e2.22 (m, 1H) ppm. 13C
ppm. 13C NMR (50 MHz, CDCl3):
d 168.5 (s), 138.6 (s), 136.7 (s),
134.9 (s), 134.2 (s), 131.5 (d), 129.5 (d), 128.4 (s, 2C), 127.9 (d), 127.8
(d, 2C), 126.8 (d), 76.2 (t), 70.9 (d), 60.2 (d), 55.1 (t), 34.7 (t) ppm.
ESI-MS m/z (ESþ) 466.92 [(M þ Na)þ, 100]. Anal. C18H18Cl2N2O5S
(C, H, N).
NMR (50 MHz, CDCl3): d 168.4 (s), 165.0 (s), 163.6 (s), 159.1 (s), 135.1
(s), 133.7 (s), 133.6 (d, 2C),129.5 (d), 127.7 (d), 113.5 (s, 2C), 105.3 (d),
98.4 (d), 59.6 (d), 56.2 (q), 55.9 (q), 55.5 (d), 49.0 (t), 34.4 (t) ppm.
ESI-MS m/z (ESþ) 518.89 [(Mþ1)þ, 45.34], 535.79 [(M þ Na)þ, 100].
Anal. C20H21Cl2N3O7S (C, H, N).
4.2.2. (2R,4S)- 4-(Benzyloxy)-1-(3,4-dichlorophenylsulfonyl)-N-
hydroxypyrrolidine-2-carboxamide (21)
23
White solid, 23% yield. M.p. 59.8e61.9 ꢀC. [
a
]
¼ þ82.5 (c ¼ 1,
4.2.7. (2R,4S)- 1-(3,4-dichlorophenylsulfonyl)-N-hydroxy-4-
(thiophene-2-carboxamido)pyrrolidine-2-carboxamide (26)
D
CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
8.09 (s, 1H), 7.80 (d, J ¼ 8.3 Hz,
22
1H), 7.53 (d, J ¼ 8.4 Hz, 1H), 7.46-7.42 (m, 4H), 7.18e7.14 (m, 2H),
4.42 (t, J ¼ 7.8 Hz, 1H), 4.33 (q, J ¼ 11.7 Hz, 2H), 4.20 (br, 1H), 3.80-
3.61 (m, 2H), 2.58-2.53 (m, 1H), 2.39-2.36 (m, 1H) ppm. 13C NMR
Yellow solid, 60% yield. M.p. 134.8e137.2 ꢀC. [
a
]
¼ þ29.3
D
(c ¼ 0.6, CH2Cl2). 1H NMR (400 MHz, CDCl3) major rotamer:
d 7.79
(d, J ¼ 6.8 Hz,1H), 7.68 (d, J ¼ 9.2 Hz,1H), 7.54 (d, J ¼ 7.8 Hz,1H), 7.47
(t, J ¼ 8.2 Hz, 2H), 7.41 (dd, J ¼ 0.9, 4.8 Hz,1H), 7.01 (t, J ¼ 4.8 Hz,1H),
4.71-4.69 (m, 1H), 4.20 (dd, J ¼ 1.9, 10.2 Hz, 1H), 3.73 (s, 3H), 3.51 (d,
J ¼ 9.7 Hz, 1H), 3.30 (dd, J ¼ 4.8, 9.7 Hz, 1H), 2.27e2.20 (m, 1H), 1.95
(dd, J ¼ 1.9, 12.1 Hz, 1H) ppm. 13C NMR (50 MHz, CDCl3) major
(50 MHz, CDCl3):
d 169.1 (s), 138.1 (s), 136.7 (s), 135.8 (s), 133.5 (s),
131.0 (d), 129.7 (d), 128.3 (s, 2C), 59.8 (d), 55.0 (t), 36.8 (t) ppm. ESI-
MS m/z (ESþ) 444.93 [(M þ H)þ, 24.83], 466.94 [(M þ Na)þ, 100].
Anal. C18H18Cl2N2O5S (C, H, N).
rotamer: d 179.2 (s), 162.0 (s), 138.1 (s), 137.1 (s), 136.0 (d), 132.4 (s),
4.2.3. (2R,4S)-1-(3,4-dichlorophenylsulfonyl)-N-hydroxy-4-(4-
phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxamide (22)
White solid, 49% yield. M.p. 186.8e188.8 ꢀC. 1H NMR (400 MHz,
132.2 (d), 129.8 (s), 128.6 (d, 2C), 127.0 (d, 2C), 62.1 (d), 54.0 (t), 40.0
(dH), 35.2 (t) ppm. ESI-MS m/z (ESþ) 464.09 [(Mþ1)þ, 61.54], 486.13
[(M þ Na)þ, 100]. Anal. C16H15Cl2N3O5S2 (C, H, N).
DMSO):
d
9.13 (br, 1H), 8.41 (s, 1H), 7.87 (d, J ¼ 1.9 Hz, 1H), 7.69 (d,
J ¼ 7.0 Hz, 2H), 7.58 (dd, J ¼ 1.9, 8.4 Hz, 1H), 7.51 (d, J ¼ 8.4 Hz, 1H),
7.41 (t, J ¼ 7.6 Hz, 2H), 7.30 (t, J ¼ 7.6 Hz, 1H), 5.22 (br, 1H), 4.32 (t,
J ¼ 7.6 Hz, 1H), 4.00 (dd, J ¼ 4.00, 11.4 Hz, 1H), 3.87 (d, J ¼ 11.4 Hz,
1H), 2.93-2.90 (m, 1H), 2.52-2.50 (m, 1H) ppm. 13C NMR (50 MHz,
4.2.8. (2R,4R)- 1-(3,4-dichlorophenylsulfonyl)-N-hydroxy-4-
(thiophene-2-carboxamido)pyrrolidine-2-carboxamide (27)
22
Yellow solid, 30% yield. M.p. 136.3e138.2 ꢀC. [
a
]
¼ þ17.6
D
(c ¼ 0.35, CH2Cl2). 1H NMR (400 MHz, CDCl3) major rotamer:
d 7.79
DMSO):
d
167.3 (s), 146.7 (s), 137.4 (s), 136.7 (s), 132.5 (s), 131.5 (d),
(d, J ¼ 6.8 Hz,1H), 7.68 (d, J ¼ 9.2 Hz,1H), 7.54 (d, J ¼ 7.8 Hz,1H), 7.47
(t, J ¼ 8.2 Hz, 2H), 7.41 (dd, J ¼ 0.9, 4.8 Hz,1H), 7.01 (t, J ¼ 4.8 Hz,1H),
4.71e4.69 (m,1H), 4.20 (dd, J ¼ 1.9,10.2 Hz,1H), 3.73 (s, 3H), 3.51 (d,
J ¼ 9.7 Hz, 1H), 3.30 (dd, J ¼ 4.8, 9.7 Hz, 1H), 2.27e2.20 (m, 1H), 1.95
(dd, J ¼ 1.9, 12.1 Hz, 1H) ppm. 13C NMR (50 MHz, CDCl3) major
130.6 (s), 129.0 (d, 2C), 128.9 (d), 128.1 (d), 127.2 (d), 125.3 (d, 2C),
120.6 (d), 58.9 (d), 58.8 (d), 55.6 (t), 35.5 (t) ppm. ESI-MS m/z (ESþ)
482.87 [(M þ H)þ, 100]. Anal. C19H17Cl2N5O4S (C, H, N).
4.2.4. (2R,4R)-1-(3,4-dichlorophenylsulfonyl)-N-hydroxy-4-(4-
rotamer: d 168.6 (s), 161.4 (s), 138.1 (s), 135.7 (s), 133.1 (d), 130.1 (d),
phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxamide (23)
128.9 (d, 2C), 128.2 (s), 127.3 (s), 127.1 (d), 127.5 (d), 57.9 (d), 55.3 (t),
48.5 (d), 34.7 (t) ppm. ESI-MS m/z (ESþ) 464.06 [(Mþ1)þ, 67.23],
486.11 [(M þ Na)þ, 100]. Anal. C16H15Cl2N3O5S2 (C, H, N).
22
White solid, 53% yield. M.p. 75.9e77.8 ꢀC. [
a
]
¼ ꢂ15.4
D
(c ¼ 0.5, EtOAc). 1H NMR (400 MHz, DMSO):
d 8.78 (s, 1H), 7.81
(s, 1H), 7.55 (d, J ¼ 7.8 Hz, 2H), 7.66 (d, J ¼ 8.7 Hz, 1H), 7.38 (t,
J ¼ 7.3 Hz, 2H), 7.28 (d, J ¼ 6.8 Hz, 1H), 7.13 (t, J ¼ 8.7 Hz, 1H), 5.00 (t,
J ¼ 6.3 Hz, 1H), 4.14 (t, J ¼ 6.3 Hz, 1H), 3.87 (t, J ¼ 10.7 Hz, 1H), 3.60
(dd, J ¼ 6.3, 10.7 Hz,1H), 2.82e2.75 (m, 1H), 2.37e2.31 (m, 1H) ppm.
4.2.9. (2R,4S)-1-(3,4-dichlorophenylsulfonyl)-4-((E)-3-(furan-2-yl)
acrylamido)-N-hydroxypyrrolidine-2-carboxamide (28)
22
Brown solid, 33% yield. M.p. 181.3e183.9 ꢀC. [
a]
¼ ꢂ108.8
D
13C NMR (50 MHz, DMSO):
d
173.8 (s), 159.9 (s), 146.7 (s), 130.9 (s),
(c ¼ 0.5, CH2Cl2). 1H NMR (400 MHz, CDCl3):
d
7.82 (d, J ¼ 6.8 Hz,
129.4 (d), 129.1 (d, 2C), 128.3 (d), 128.1 (d), 126.5 (d), 125.3 (d, 2C),
120.8 (s), 120.7 (s), 117.4 (d), 61.3 (d), 57.9 (d), 53.3 (t), 36.6 (t) ppm.
1H), 7.48e7.41 (m, 2H), 7.35e7.34 (br, 1H), 7.20 (d, J ¼ 15.1 Hz, 1H),
6.45 (d, J ¼ 3.4 Hz, 1H), 6.35 (dd, J ¼ 1.9, 3.4 Hz, 1H), 6.27 (d,

106
C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
J ¼ 6.3 Hz, 1H), 6.00 (d, J ¼ 15.1 Hz, 1H), 4.49 (t, J ¼ 7.8 Hz, 2H), 3.60
(s, 3H), 3.58e3.57 (m, 1H), 3.43 (dd, J ¼ 2.9, 10.7 Hz, 1H), 2.25e2.19
66.1(d), 48.0 (t), 31.5 (t), 30.0 (t), 29.3 (t), 25.4 (t), 22.5 (t), 14.0 (q)
ppm. ESI-MSMS m/z (ESþ) 404.42 (Mþ, 10.99), 175.05 (100). Anal.
C17H25ClN2O5S (C, H, N).
(m, 2H) ppm. 13C NMR (50 MHz, CDCl3):
d 168.7 (s), 165.9 (s), 150.9
(s, 2C), 143.6 (d), 136.3 (s), 132.7 (d), 129.0 (d), 128.2 (d), 127.2 (s þ d,
2C), 118.3 (d), 113.0 (d), 111.7 (d), 62.3 (d), 54.2 (t), 49.6 (d), 35.2
(t) ppm. ESI-MS m/z (ESþ) 474.06 [(Mþ1)þ, 100]. Anal.
C18H17Cl2N3O6S (C, H, N).
4.3. Enzyme inhibition assay
The inhibition of LF activity by final compounds was measured
by a spectrophotometric assay. It was conducted using anthrax
lethal factor protease substrate II (AceGlyeTyreßAlaeArgeArge
ArgeArgeArgeArgeArgeArgeValeLeueArgepNA) as peptide
substrate. Peptide bond cleavage was measured monitoring the
release of p-nitroaniline as an increase in absorbance at 405 nm
wavelength. Each reaction contained the colorimetric substrate
4.2.10. (2R,3S)-3-(benzyloxy)-1-((3,4-dichlorophenyl)sulfonyl)-N-
hydroxypyrrolidine-2-carboxamide (38)
Compound 35 (100 mg, 0.22 mmol) was dissolved in anhydrous
MeOH in an oven-dried, sealed microwave vial under N2 atmo-
sphere, and a preformed solution of hydroxylamine hydrochloride
(46 mg, 0.66 mmol) and KOH (74 mg, 1.32 mmol) in MeOH was
added. The vessel was heated for 1 h at 80 ꢀC in a microwave
reactor. The mixture was acidified with 1 M HCl to pH 3. The
resulting solution was evaporated, and then diluted with H2O.
Aqueous solution was extracted with EtOAc, and the organic layer
was dried over anhydrous Na2SO4 and concentrated in vacuo. The
crude product was purified by flash chromatography (silica gel, et.
(10 mM), the inhibitors at increasing concentrations, and anthrax
lethal factor (10 nM) in 250 mM Na2HPO4, 150 mM NaCl, pH ¼ 7.4 at
25 ꢀC. After LF addition to the reaction mixture, decrease of
absorbance at 405 nm was followed for 10 min. The IC50 values
were obtained by doseeresponse measurements using the inhib-
itor in a range of concentrations: 500 nM, 1
mM, 2.5 mM, 5 mM,
10 M. All the experiments were repeated twice. The IC50 values
m
pet. ether/EtOAc 1:1, Rf 0.4), affording 38 as colorless oil (13 mg, 14%
were determined by fitting binding inhibition data by non-linear
regression using GraphPad Prism 4.0 Software Package (GraphPad
Prism, San Diego, CA).
24
yield). [
d
a
]
ꢂ86.0 (c ¼ 0.65, CHCl3). 1H NMR (CDCl3, 300 MHz):
D
9.58 (br, 1H), 7.89 (d, J ¼ 1.9 Hz, 1H), 7.57 (dd, J ¼ 1.9, 8.2 Hz, 1H),
7.30 (d, J ¼ 8.2 Hz, 1H), 7.21e7.19 (m, 3H), 6.87e6.84 (m, 2H), 4.26e
4.20 (m, 2H), 3.64 (m, 1H), 3.26e3.17 (m, 1H), 1.98e1.94 (m, 2H),
0.88e0.79 (m, 2H) ppm. 13C NMR (50 MHz, CDCl3):
d 166.0 (s), 138.1
4.4. Tridimensional structure alignment
(s), 136.3 (s), 134.4 (s), 133.5 (s), 130.7 (d), 129.5 (d), 128.0 (d), 127.5
(d), 126.6 (d), 80.7 (d), 70.3 (t), 66.1 (d), 47.7 (t), 29.9 (t) ppm. ESI-
MSMS m/z (ESþ) 445.44 [(Mþ1)þ, 20.37], 385.38 [(M-CONHOH)þ,
100]. Anal. C18H18Cl2N2O5S (C, H, N).
The Swiss PDB viewer (SPDBV) program (4.0.1 version, Swiss
Institute of Bioinformatics) was used to superimpose three
dimensional (3D) structures of anthrax lethal factor in complex
with L915 (PDB code: 1YQY), and LF in complex with BI-MFM3 (PDB
code: 1ZXV) [32,33]. The two PDB structures were superimposed
using the “magic fit” option. Then, using the “improve fit” option
implemented in SPDBV, iterations were performed until conver-
gence of RMS and maintaining the number of matching residues as
high as possible. Structural alignment was generated after protein
superimposition. The residues of the superimposed protein that
were spatially close to residues of the static one were aligned.
Appropriate gaps were inserted in the sequences to indicate a lack
of structural correspondence. The quality of the superimposition
was evaluated by calculating the RMS at each residue.
4.2.11. (2R,3S)-3-(benzyloxy)-1-((4-chlorophenyl)sulfonyl)-N-
hydroxypyrrolidine-2-carboxamide (39)
To a solution of 36 (122 mg, 0.29 mmol) in MeOH (1 mL) was
added dropwise a preformed solution of NH2OK (0.87 mmol) in
MeOH (522 mL). The mixture was stirred at room temperature for
48 h. The reaction mixture was then acidified with 1 M HCl to pH
3 and extracted with EtOAc. The organic layers were dried over
anhydrous Na2SO4, filtered and evaporated. The crude product
was purified by automated flash chromatography (pet. ether/
EtOAc 1:2), to obtain compound 39 as white solid (35 mg, 29%
28
yield). [
d
a
]
ꢂ80.8 (c ¼ 1.5, CHCl3). 1H NMR (CDCl3, 300 MHz):
D
7.76 (d, J ¼ 8.5 Hz, 2H), 7.33 (d, J ¼ 7.8 Hz, 2H), 7.27 (m, 3H),
4.5. Docking calculation
6.90 (m, 2H), 4.25 (m, 2H), 4.24 (s, 2H), 3.69 (m, 1H), 3.25 (m,
1H), 1.99 (m, 2H). 13C NMR (50 MHz, CDCl3):
d
179.9 (s), 140.2 (s),
Docking analysis was performed using the GOLD (Genetic
Optimization for Ligand Docking) software package 5.1 version
(CCDC, Cambridge, UK) [34]. The 3D structure of the ligand was
generated using SPARTAN version 5.147 running on an SGI IRIX
6.5 workstation, and the AM1 semiempirical method [35] was
used to optimize the global minimum conformer. The coordi-
nates of LF enzyme were retrieved from the Protein Data Bank
(PDB code:1YQY). The ligand-protein complex was unmerged to
achieve free enzyme structure, water molecules were removed
and hydrogen atoms were added to the enzyme. The binding site
137.8 (s), 134.4 (s), 129.6 (d, 2C), 128.5 (d), 127.9 (d), 127.3 (d),
81.4 (d), 70.7 (t), 66.5 (d), 48.2 (t), 30.4 (t) ppm. ESI-MSMS m/z
(ESþ) 433.00 [(M
C18H19ClN2O5S (C, H, N).
þ
Na)þ, 20.80], 244.00 (100). Anal.
4.2.12. (2R,3S)-1-((4-chlorophenyl)sulfonyl)-3-(hexyloxy)-N-
hydroxypyrrolidine-2-carboxamide (40)
To a solution of 37 (102 mg, 0.24 mmol) in MeOH (1 mL) was
added dropwise a preformed solution of NH2OK (0.72 mmol) in
ꢀ
MeOH (600
m
L). The resulting mixture was stirred at room
was defined as all residues of the protein within 10 A from the
temperature for 48 h. Then, it was acidified with 1 M HCl (pH 3)
and extracted with EtOAc. The organic layers were dried over
anhydrous Na2SO4, filtered and concentrated under reduced
pressure, to give orange oil. The crude product was purified by
flash cromatography (silica gel, pet. ether/EtOAc 1:2, Rf 0.42),
giving compound 40 as a yellow oil (12 mg, 12% yield). 1H NMR
g
-carbon of His686. Goldscore was chosen as the fitness func-
tion, and standard default settings were used in all the calcu-
lations. The zinc metal ion was set up in tetrahedral
a
coordination. For each of the 50 independent genetic algorithm
runs, a maximum of 10e6 genetic operations were carried out,
using the default operator weights and a population size of 100
ꢀ
(CDCl3, 300 MHz):
d
7.75 (d, J ¼ 8.5 Hz, 2H), 7.43 (d, J ¼ 8.5 Hz, 2H),
chromosomes. Results differing by less than 1.5 A in ligand-all
4.04 (s, 1H), 3.60 (1H), 3.18 (m, 1H), 3.02 (m, 2H), 1.86 (m, 2H),
atom RMSD, were clustered together. The analysis of binding
mode of the docked conformations were carried out using
PyMol software v.0.99 [36].
1.22e0.95 (m, 8H), 0.80 (t, J ¼ 6.9 Hz, 3H). 13C NMR (50 MHz,
CDCl3):
d 166.6 (s), 140.2 (s), 129.5 (d), 129.4 (d), 81.3 (d), 68.9 (t),

C. Calugi et al. / European Journal of Medicinal Chemistry 56 (2012) 96e107
107
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Acknowledgments
Financial support received from Fondazione Roma, University of
Florence, CINMPIS is gratefully acknowledged. Dr. Fiorella Tonello is
acknowledged for carrying out preliminary inhibition assays.
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