Novel naphthalene-N-sulfonyl-D-glutamic acid derivatives as inhibitors of MurD, a key peptidoglycan biosynthesis enzyme

Article abstract of DOI:10.1021/jm800762u

Mur ligases have essential roles in the biosynthesis of peptidoglycan, and they represent attractive targets for the design of novel antibacterials. MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase) is the second enzyme in the series of Mur ligases, and it catalyzes the addition of D-glutamic acid (D-Glu) to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA). Because of the high binding affinity of D-Glu toward MurD, we synthesized and biochemically evaluated a series of N-substituted D-Glu derivatives as potential inhibitors of MurD from E. coli, which allowed us to explore the structure-activity relationships. The substituted naphthalene-N-sulfonyl-D-Glu inhibitors, which were synthesized as potential transitionstate analogues, displayed IC₅₀ values ranging from 80 to 600 μM. In addition, the high-resolution crystal structures of MurD in complex with four novel inhibitors revealed details of the binding mode of the inhibitors within the active site of MurD. Structure-activity relationships and cocrystal structures constitute an excellent starting point for further development of novel MurD inhibitors of this structural class.

Full text of DOI:10.1021/jm800762u

7486
J. Med. Chem. 2008, 51, 7486–7494
Novel Naphthalene-N-sulfonyl-D-glutamic Acid Derivatives as Inhibitors of MurD, a Key
Peptidoglycan Biosynthesis Enzyme‡,§
|
#
|
#
Jan Humljan,#,† Miha Kotnik,#,† Carlos Contreras-Martel, Didier Blanot, Urosˇ Urleb, Andre´a Dessen, Tom Solmajer, and
ˇ
Stanislav Gobec*,¶
Drug DiscoVery, Lek Pharmaceuticals d.d., VeroVsˇkoVa 57, 1526 Ljubljana, SloVenia, Laboratoire des Prote´ines Membranaires, Institut de
Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, UJF, UMR5075, 41 Rue Jules Horowitz, F-38027 Grenoble, France, EnVeloppes
Bacte´riennes et Antibiotiques, IBBMC, UMR 8619 CNRS, UniV Paris-Sud, 91405 Orsay, France, and Faculty of Pharmacy, UniVersity of
Ljubljana, AsˇkercˇeVa 7, 1000 Ljubljana, SloVenia
ReceiVed June 21, 2008
Mur ligases have essential roles in the biosynthesis of peptidoglycan, and they represent attractive targets
for the design of novel antibacterials. MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase) is the
second enzyme in the series of Mur ligases, and it catalyzes the addition of D-glutamic acid (D-Glu) to the
cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA). Because of the high binding affinity
of D-Glu toward MurD, we synthesized and biochemically evaluated a series of N-substituted D-Glu derivatives
as potential inhibitors of MurD from E. coli, which allowed us to explore the structure-activity relationships.
The substituted naphthalene-N-sulfonyl-D-Glu inhibitors, which were synthesized as potential transition-
state analogues, displayed IC50 values ranging from 80 to 600 µM. In addition, the high-resolution crystal
structures of MurD in complex with four novel inhibitors revealed details of the binding mode of the inhibitors
within the active site of MurD. Structure-activity relationships and cocrystal structures constitute an excellent
starting point for further development of novel MurD inhibitors of this structural class.
the resulting acyl phosphate is attacked by the incoming amino
acid (D-Glu) to form a high-energy tetrahedral intermediate,
which subsequently collapses to the amide product and inorganic
phosphate. All Mur ligases (MurC-MurF) act via similar
mechanisms that have been confirmed by X-ray diffraction
analysis,7 by isotope transfer8 and rapid quench9 experiments,
and by the chemical trapping method.10
The crystal structures of the Mur ligase family show that they
have the same three-domain topology, with the N-terminal and
central domains binding UDP precursor and ATP, respectively,
while the incoming amino acid or dipeptide binds to the
C-terminal part.11 Interestingly, sequence alignments of Mur
ligase orthologues and paralogues show relatively low overall
homologies, with the exception of residues comprising the active
sites.12-15 Furthermore, the open and closed X-ray structures
of free and complexed MurD7,16,17 and MurF18,19 show that the
C-terminal domain undergoes substantial conformational changes
upon substrate or inhibitor binding.20
The high stereospecificity of MurD for D-Glu, its ubiquitous
nature among the bacteria, and its absence in mammals all make
MurD a promising target for antibacterial therapy.6 To date,
several phosphinates of the general formula 1 (Figure 1) have
been reported to be tetrahedral transition-state analogue inhibi-
tors of MurD.21-24 Macrocyclic inhibitors25 and peptides26
obtained from phage display screening have also been developed
as MurD inhibitors. Furthermore, pulvinones27 and pyrazole
derivatives28 have been reported as potential inhibitors of
MurA-MurD and MurB-MurD, respectively. However, for all
of these molecules, neither the MurD inhibition mechanisms
nor the crystal structures of their complexes have been reported.
The search for new compounds with the potential to inhibit
MurD led us to synthesize and biochemically evaluate N-
substituted D-Glu derivatives. We postulated that they represent
a promising starting point for the development of MurD
inhibitors not only because of the highly stereospecific affinity
of MurD for D-Glu and the conserved D-Glu-binding site across
1. Introduction
Infectious diseases are one of the leading causes of death
worldwide.1 The emergence and dissemination of resistant
bacterial strains have created an urgent need for the development
of novel antibacterial agents.2 The peptidoglycan, an essential
cell-wall polymer that is unique to prokaryotic cells, is an
important target for antibiotic research.3 While the majority of
approved drugs that target peptidoglycan biosynthesis act on
the extracellular steps,4 there has been increased interest in
exploiting the early intracellular steps that are catalyzed by a
seriesofMurenzymes(MurA-MurFa).5,6Murligases(MurC-MurF)
catalyze a series of reactions that start from UDP-N-acetylmu-
ramic acid (UDP-MurNAc) to yield Park’s nucleotide (UDP-
MurNAc-pentapeptide) by sequentially adding L-Ala (MurC),
D-Glu (MurD), meso-diaminopimelic acid or L-Lys (MurE), and
the D-Ala-D-Ala dipeptide (MurF).3 Recently, we have focused
our attention on MurD, an ATP-dependent, amide-forming
enzyme that acts through the catalytic mechanism illustrated in
Figure 1. Initially, the carboxylic acid is phosphorylated, and
‡
Dedicated to Professor Slavko Pecˇar on the occasion of his 60th
birthday.
§
PDB accession codes: 2UUO, 2UUP, 2VTD, and 2VTE.
* To whom correspondence should be addressed. Phone: +386-1-476-
9500. Fax: +386-1-425-8031. E-mail: gobecs@ffa.uni-lj.si.
#
Lek Pharmaceuticals d.d.
J.H. and M.K. contributed equally to this work.
Institut de Biologie Structurale.
†
|
Univ Paris-Sud.
University of Ljubljana.
¶
a Abbreviations: UDP, uridine 5′-diphosphate; UMA, UDP-N-acetylmu-
ramoyl-L-alanine; UMAG, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate;
MurA, UDP-N-acetylglucosamine enolpyruvyl transferase; MurB, UDP-
N-acetylpyruvylglucosamine reductase; MurC, UDP-N-acetylmuramate:L-
alanine ligase; MurD, UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase;
MurE, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate
ligase; MurF, UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diami-
nopimelate:D-alanyl-D-alanine ligase; GABA, γ-aminobutyric acid; PCC,
pyridinium chlorochromate; DPPA, diphenylphosphoryl azide.
10.1021/jm800762u CCC: $40.75
2008 American Chemical Society
Published on Web 11/13/2008

N-Sulfonyl-D-Glu DeriVatiVes as Inhibitors of MurD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7487
Figure 1. Reaction catalyzed by MurD and design of phosphinate and sulfonamide transition-state analogue inhibitors.
Table 1. Residual Activities of MurD in the Presence of 1 mM Inhibitorsb
a Published previously.37 b Results represent the mean values of two independent experiments. Standard deviations were within (10% of the mean values.
different bacterial species but also because of the 4-fold higher
binding efficiency index29 of D-Glu when compared to other
substrates (UMA and ATP) of MurD. Additionally, it has been
suggested recently that D-Glu represents an essential fragment
of a potent inhibitor.24,30,31
2. Chemistry
Our investigations started with the synthesis of new carboxa-
mides 5a-e (Table 1) that were prepared by the coupling of
C-protected D-Glu with proper carboxylic acids 3a-e using
DPPA, and final deprotection of 4a-c and 4d,e by alkaline
hydrolysis or catalytic hydrogenation, to provide carboxamides
5a-c and 5d,e, respectively (Scheme 1). Carboxylic acid 3b
was prepared from the appropriate starting materials according
to published procedures.34 Sulfonamide compounds 8a,b and
11a-d (Table 1) were synthesized from sulfonyl chlorides 7a,b
and 10a-d, respectively, and free D-Glu in the presence of
aqueous 2 M NaOH (Scheme 1). Sulfonyl chlorides 7a35 and
10a36 were prepared from 2-chloro-1-(chloromethyl)-4-fluo-
robenzene (6a) and N-phenylacetamide (9a) according to
literature procedures (Scheme 1).
Among several crystal structures of Mur ligases11,32 deposited
in the Protein Data Bank, only two crystal structures of MurF
in complex with inhibitors have been reported.19 Recently, we
published the first crystal structures of two isomers of an
N-sulfonyl-Glu inhibitor in complex with MurD from Escheri-
chia coli.33 In the present study, we describe the synthesis and
structure-activity relationships (SARs) of a series of N-
substituted derivatives of D-glutamic acid, leading to naphthalene-
N-sulfonyl-D-Glu derivatives of the general formula 2 (Figure
1), which most probably act as transition-state analogue inhibi-
tors of MurD. In addition, we present four new crystal structures
of small molecule inhibitors in complex with MurD.
With an IC50 of 810 µM, compound 11d (Table 1) was
selected for further optimization (see below). We proceeded with

7488 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Humljan et al.
Scheme 1. Synthesis of Carboxamide and Sulfonamide
alkylated with MeI to give compound 30, which was converted
by alkaline hydrolysis into the target compound 31 (Scheme
Derivativesa
2).
3. Results and Discussion
Target compounds 5a-e, 8a,b, 11a-d, 16c, 17a-o, 18-20,
26, 29, 31, and 38-40 (Tables 1–5) were tested for their
inhibitory activities on MurD from E. coli. The results are
presented as residual activities (RAs) of the enzyme in the
presence of 1 mM of each compound, and as IC50 values for
active compounds. The first series of carboxamides 5a-e and
sulfonamides 8a,b and 11a,b proved to be practically inactive
(Table 1). However, when the bulky biphenyl and naphthalene
substituents were introduced, compounds 11c and 11d were
obtained with IC50 values of 1720 and 810 µM, respectively
(Table 1). It is interesting to compare the inhibitory activities
of sulfonamides 11d and 12 (Table 1). The latter compound,
which contains a sulfonoalanine moiety between the naphthalene
and D-Glu residues, was inactive,37 in contrast to compound
11d, in which the same naphthalene moiety is attached directly
to D-Glu with a sulfonamide bond (Table 1). At this point,
naphthalenesulfonamide 11d was selected as the starting point
for further development to obtain new transition-state analogue
inhibitors of MurD. It is known that sulfonamides possess a
geometry similar to that of the tetrahedral intermediate formed
during the peptide bond cleavage and formation.38 Further
gradual improvements in inhibitor 11d were achieved by
substituting position 6 of the naphthalene ring with alkyloxy
(compounds 17a-f) or cyclopentyloxy (17i) substituents, giving
compounds with IC50 values of between 170 and 590 µM (Table
2). Here, with the extension of the alkyl side chain length from
methyl to pentyl, the inhibitory activity increased more than
3-fold (IC50 values for compound 17a and 17d: 590 and 170
µM, respectively). However, compounds 17g,h bearing an acidic
substituent on the naphthalene ring were less active (the IC50
value of 17g was 630 µM, while 17h was inactive). Inhibitory
activity was further improved by introducing arylalkyloxy
substituents (compounds 17j-o), where the 3-phenylpropyl
a Reagents and conditions: (a) HCl ·D-Glu(OR)2, DPPA, Et3N, DMF,
room temp; (b) for compounds 5a-c, 1 M NaOH, dioxane, room temp;
for compounds 5e,d, H2, Pd/C, glacial acetic acid, room temp; (c) (i)
Na2S2O3, MeOH/H2O, reflux; (ii) Cl2, glacial acetic acid, 0 °C to room
temp; (d) D-Glu, 2 M NaOH, pH 10-11, 70 °C; (e) ClSO3H, 5-65 °C.
the synthesis of analogues of 11d bearing substitutions on the
naphthalene ring. Here, sodium hydroxynaphthalenesulfonic
acids (13) were first alkylated with the corresponding alkyl
halides to give alkyloxynaphthalenesulfonic acids 14a-o and
32-34, followed by chlorination using thionyl chloride to
provide the desired sulfonyl chlorides 15a-o. Treatment with
C-protected D-Glu then provided sulfonamides 16a-o and
35-37, which were converted by alkaline hydrolysis, catalytic
hydrogenation, or acidolysis into the target sulfonamide inhibi-
tors 17a-o and 38-40, respectively (Scheme 2, Table 2). The
same procedure was also used for the synthesis of the Gly
derivative 19 (Table 3), using C-protected Gly instead of
C-protected D-Glu, while the GABA and L-Glu derivatives 18
and 20 (Table 3), respectively, were synthesized from sulfonyl
chloride 15c and the appropriate free amino acid, using aqueous
2 M NaOH in acetone. For the production of compounds 38-40
(Table 5), a general synthetic approach for the synthesis of
17a-o was used (Scheme 2). However, in the first step, Na/
EtOH was replaced with powdered NaOH for the preparation
of compounds 32-34. Furthermore, in the synthesis of sul-
fonamides 16m-o and 37, the isolation of the corresponding
sulfonyl chloride was omitted (Scheme 2) (see Experimental
Section).
(17k, IC50 ) 132 µM) and 4-cyanobenzyl (17l-o, IC50
≈
80-120 µM) derivatives were seen to be the most potent
inhibitors of MurD.
To further investigate the SARs, we prepared analogues of
representative naphthylsulfonamide 17c, incorporating changes
that would help the elucidation of the structural features required
for MurD inhibition (Tables 3-5). To determine the importance
of the D-Glu R- and γ-carboxylate groups, we prepared
compounds 18 and 19 as analogues of 17c but lacking the
R-carboxylate group and the glutamic acid side chain, respec-
tively (Table 3). The importance of the γ-carboxylate group
for the inhibition of MurD was recently demonstrated in the
phosphinodipeptide series, where truncated analogues were seen
to be devoid of inhibitory activity.24 Our truncated sulfonamide
analogues 18 and 19 did not inhibit MurD either, confirming
that the presence of both R- and γ-carboxylate groups of the
glutamate residue in this type of inhibitor is essential for good
inhibition. In the complex of MurD with its product UMAG
(MurD·UMAG, pdb entry 4UAG),7 the R-carboxylate of D-Glu
is hydrogen-bonded to Thr321 and forms a charge-based
interaction with Nꢀ of Lys348 and the γ-carboxylate is
positioned through hydrogen bonds with Ser415 and Phe422.
The absence of inhibition by analogues 18 and 19 is consistent
with the loss of binding energy of the two hydrogen bonds
provided by the R-carboxylate and γ-carboxylate group, re-
spectively. Furthermore, the dimethyl ester derivative 16c proved
To determine the importance of the sulfonamide group,
analogues of compound 17c with carboxamide (26), a reduced
amide bond (29) at the position of the sulfonamide bond, and
the N-methyl derivative (31) were prepared (Table 4). Thus,
6-hydroxy-2-naphthoic acid (21) was first protected as the
methyl ester to generate ester 22, followed by O-alkylation with
butyl bromide to give the corresponding ether 23 and alkaline
hydrolysis for the carboxylic acid 24. Subsequent DPPA-
mediated coupling of carboxylic acid 24 with C-protected D-Glu
gave 25, followed by deprotection providing the desired
carboxamide 26 (Scheme 3). Alternatively, treatment of inter-
mediate 23 with LiAlH4 gave alcohol 27, which was converted
to aldehyde 28 by oxidation with pyridinium chlorochromate
(PCC). Treatment with D-Glu under reductive amination condi-
tions gave the desired reduced amide 29 (Scheme 4). To probe
the role of the sulfonamide proton of 17c, the N-methyl
derivative 31 was synthesized. Here, intermediate 16c was first

N-Sulfonyl-D-Glu DeriVatiVes as Inhibitors of MurD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7489
Scheme 2. Synthesis of Substituted Naphthalenesulfonamide Derivativesa
a Reagents and conditions: (a) for compounds 14a-o (i) Na/EtOH, room temp, (ii) R1-X, DMSO, room temp; for compounds 32-34, R1-Br, NaOH,
DMSO, 60 °C; (b) SOCl2, DMF, 0 °C to room temp; (c) HCl ·D-Glu(OR)2, Et3N, CH2Cl2, room temp; (d) for compounds 17a–e, 17g–n and 38–40 1 M
NaOH, dioxane, room temp; for compound 17f, H2, Pd/C, MeOH, room temp; for compound 17o, CF3COOH, CH2Cl2, room temp; (e) MeI, K2CO3, DMF;
(f) 1 M NaOH, dioxane, room temp.
Table 2. MurD Inhibitory Activity of Naphthalene-N-sulfonyl-D-Glu Derivativesb
a Published previously.33 b Results represent the mean values of two independent experiments. Standard deviations were within (10% of the mean values.
to be inactive, further confirming the importance of the free
carboxylate groups of D-Glu. On the other hand, we were
surprised by the small difference in potency between the
enantiomeric compounds 17c (D-Glu derivative) and 20 (L-Glu
derivative) (Table 3), which is somewhat contradictory to the
high stereospecificity of MurD for the D-Glu substrate.39 It was
unexpected that a compound containing the L-Glu moiety would
act as an inhibitor of this enzyme. Steady-state kinetics and
X-ray diffraction studies unambiguously demonstrated that the
L-Glu moiety of compound 20 binds into the D-Glu-binding site
of MurD in a manner similar to that of D-Glu in UMAG or in
compound 17c.33 Small differences in binding modes of the
naphthalenesulfonamido group contributed to 3-fold better
inhibition by compound 17c.
ment of the sulfonamide in 17c with carboxamide (26) led to
diminished activity against MurD, demonstrating that the
introduction of the planar carboxamide is not tolerated and that
the tetrahedral geometry on the nitrogen substituent is preferred
for inhibitory activity. To examine the importance of H-bond
donor/acceptor properties of the sulfonamide group, the analogue
with a reduced amide group 29 was synthesized. Compound
29 with a tetrahedral carbon atom and basic secondary amine
group proved to be inactive (Table 4), indicating the importance
of the hydrogen bond between the sulfonamide group oxygens
and the enzyme. The acidic sulfonamide moiety is thus favored
over the basic reduced amide. In further investigations, inhibitor
17c was N-methylated to obtain compound 31. The latter
compound was devoid of inhibitory activity (Table 4), clearly
demonstrating that proton donor properties on the sulfonamide
nitrogen are also crucial for good inhibition.
In the next step, we wanted to investigate the importance of
the intact sulfonamide group. As illustrated in Table 4, replace-

7490 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Table 3. Influence of Glutamate Moiety on Inhibitory Activityb
Humljan et al.
the binding mode of the inhibitors, we also solved the high-
resolution crystal structures of MurD in complex with four novel
inhibitors (17d, 17l, 17n, 17o).
3.1. Crystal Structures of MurD in Complex with
Compounds 17d, 17l, 17n, and 17o. The crystal structures of
the MurD-inhibitor complexes were solved at a high resolution
to determine the binding mode of the inhibitors within the active
site of MurD.
In the structures of MurD complexed to 17d and 17l (Figures
2 and 3, respectively) the D-Glu moiety of both compounds
occupies the same site as seen for related sulfonamide inhibitors
(compounds 17c and 20)33 and the product UMAG.7 The
R-carboxyl group of these inhibitors forms a charge based
interaction with Nꢀ of Lys348 and is additionally hydrogen-
bonded with a conserved water molecule, W107 and W415 in
the complexes with 17d and 17l, respectively. Within the active
site, 17l forms further hydrogen bonds to Oγ of Thr321 and the
carboxyl group of Asp182. The carboxyl group of the D-Glu
side chain is held in place by hydrogen bonds with Ser415 and
Phe422. One of the carboxylic oxygen atoms forms hydrogen
bonds with both the backbone nitrogen and the Oγ of Ser415,
whereas the other oxygen atom interacts with the backbone
nitrogen of Phe422 and, in the case of the MurD-17l complex,
also with water molecule W413. The latter is further hydrogen-
bonded to water molecule W412, which has a direct interaction
with the nitrogen of the inhibitor, forming an extended ring-
type system. The sulfonic group of both inhibitors also
contributes to recognition within the active site by interacting
with water molecule W185 (e.g., in complex with compound
17l), which bridges between the oxygen atom of the sulfonic
group and Oγ of Ser159, as well as Oγ of Asn138. The
naphthalene ring of the inhibitors is positioned in the cleft
between all three domains, making hydrophobic interactions
with Leu416, Phe161, and CR of Gly73. The functional groups
of both compounds at position 6 of the naphthalene ring extend
into the uracil-binding pocket. While the pentoxy group of
compound 17d probably makes hydrophobic interactions, the
cyanobenzyloxy moiety of 17l forms an additional hydrogen
bond with the backbone nitrogen of Thr36 and makes π-π
interactions through its phenyl ring with the salt bridge
Asp35-Arg37.
a Published previously.33 b Results represent the mean values of two
independent experiments. Standard deviations were within (10% of the
mean values.
Table 4. Activities of Derivatives Obtained by Replacement or
Methylation of the Sulfonamide Bondb
X
R
IC50 (µM)
17c
26
29
-SO2-
-CO-
-CH2-
-SO2-
H
H
H
280a
>1000
>1000
>1000
31
-CH3
a Published previously.33 b Results represent the mean values of two
independent experiments. Standard deviations were within (10% of the
mean values.
Table 5. Influence of Substitution Patterns on Inhibitory Activityb
Both close analogues of 17l, 17n, and 17o bind to MurD in
a similar manner to 17I. The additional fluorine atom at the
ortho position of the p-cyanobenzyloxy group of 17n faces the
solvent area, similar to one of the carbonyl oxygen atoms of
the uracil part of the product UMAG.7 Not surprisingly, the
naphthalene group of 17o, the 2,7-substituted analogue of 17l,
occupies a slightly different position compared to its 2,6-
substituted counterpart. Nevertheless, the cyanobenzyloxy group
extends to the uracil-binding pocket in a similar manner, forming
a hydrogen bond with the backbone nitrogen atom of Thr36.
substitution
IC50 (µM)
17c
38
39
2,6
2,7
1,5
1,4
280a
180
>1000
>1000
40
a Published previously.33 b Results represent the mean values of two
independent experiments. Standard deviations were within (10% of the
mean values.
Having identified that both the sulfonamido and D-Glu
moieties are necessary for good inhibition, we next focused our
attention on understanding the effects of naphthalene substitution
patterns (Table 5). Here, 2,6- and 2,7-regioisomers (compounds
17c and 38, respectively) were preferred over 1,5- and 1,4-
regioisomers (compounds 39 and 40, respectively). In addition,
compound 17l and its 2,7-regioisomer 17o were equipotent
inhibitors of MurD (Table 2).
Finally, a common feature of all of the active compounds is
N-(2-naphthylsulfonyl)-D-glutamic acid, which is substituted on
position 6 or 7 by a lipophilic substituent. Because of the highly
conserved active site of MurD among different bacteria,15 it is
expected that the identified inhibitors will also inhibit other
bacterial MurD enzymes. To obtain additional information about
Experimentally determined positions and conformations of
compounds 17d, 17l, 17n, and 17o in the MurD active site offer
a solid foundation for optimization of inhibitors of this structural
class. As suggested in Figure 4, the design of derivatives of
inhibitor 17l with improved interaction patterns within the active
site of MurD could proceed in three directions. First, with the
appropriate substitution of the NR atom of 17l we could gain
additional interactions with Asn138 or with amino acid residues
constituting the ATP-binding site (e.g., Lys115 or/and Lys319).
The functional group added to NR atom should be carefully
selected, since the newly formed interactions between the
introduced moiety and the protein have to energetically over-
come a loss of a hydrogen bond that the sulfonamide hydrogen

N-Sulfonyl-D-Glu DeriVatiVes as Inhibitors of MurD
Scheme 3. Synthesis of Carboxamide Analogue 26a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7491
a Reagents and conditions: (a) SOCl2, MeOH, reflux; (b) BuBr, K2CO3, acetone, reflux; (c) 1 M NaOH, dioxane, room temp; (d) HCl ·D-Glu(OMe)2,
DPPA, Et3N, DMF, room temp.
Scheme 4. Synthesis of Reduced Amide Analogue 29a
ammonium formate, pH 4.7. The compounds were detected and
quantified with an LB 506 C-1 HPLC radioactivity monitor
(Berthold France, Thoiry, France) using Quickszint Flow 2 scin-
tillator (Zinsser Analytic, Maidenhead, U.K.) at 0.6 mL/min. The
residual activities for each of the inhibitor concentrations were
calculated with respect to a similar assay without inhibitor. The
values are expressed as the mean of two independent experiments,
and standard deviations were within (10% of the mean values.
The IC50 values were calculated from the fitted regression equation
using the logit-log plot.
a Reagents and conditions: (a) LiAlH4, THF, 0 °C to room temp; (b)
PCC, Na+CH3COO-, 4 Å molecular sieves, argon, CH2Cl2; (c) D-Glu,
MeOH, NaOH, NaBH4, 0 °C to room temp.
Control experiments were performed in the presence of Tween-
20 (0.003%) to exclude nonspecific binding of inhibitors.41 No
significant differences were seen when compared with measure-
ments without Tween-20.
forms with a water molecule (W412, Figure 3b). Second, by
substitution or bioisosteric replacement of the naphthalene ring,
the strengthening of hydrophobic interactions with Leu416,
Phe161, and CR of Gly73 is expected (Figure 4b). Finally, the
additional substitution or bioisosteric replacement of the p-
cyanophenyl moiety of compound 17l should result in the
formation of additional hydrogen bonds with Ser71 and Thr36,
both being part of the “uracil-binding pocket”.
5.2. Crystallization, Preparation of the Inhibitor Complexes,
and Data Collection. Crystals were obtained from the 6×His-tagged
enzyme as described previously33 and were subsequently used for
soaking with inhibitors by transfer to fresh reservoir solutions
containing 2 mM inhibitor and 5% DMSO. After 5 h of soaking,
the crystals were flash cooled in liquid nitrogen using Paratone oil
as a cryoprotectant. All of the crystals tested belonged to space
group P41 and had one molecule per asymmetric unit. Data sets
were collected at the European Synchrotron Radiation Facility
(ESRF, Grenoble, France) BM30A, ID14-EH1, and ID29 beamlines.
5.3. Materials and Methods. Chemistry. Chemicals from
Sigma-Aldrich, Acros Organics, and TCI-Europe were used without
further purification. Solvents were used without purification or
drying, unless otherwise stated. Analytical TLC was performed on
Merck silica gel (60F254) plates (0.25 mm). Compounds were
visualized with ultraviolet light. Column chromatography was
carried out on silica gel 60 (particle size 240-400 mesh). Melting
points were determined on a Reichert hot stage microscope and
are uncorrected. 1H NMR spectra were recorded on a Bruker
AVANCE DPX300 spectrometer in CDCl3 or DMSO-d6 solution,
with TMS as the internal standard. IR spectra were obtained on a
Perkin-Elmer 1600 FT-IR spectrometer. Optical rotation was
measured on a Perkin-Elmer 1241 MC polarimeter. Microanalyses
were performed on a Perkin-Elmer C, H, N analyzer 240 C. Mass
spectra were obtained using a VG-Analytical Autospec Q mass
spectrometer. LC-MS samples were analyzed with an ACQUITY
UPLC separations module interfaced to a Waters Quattro Micro
API mass spectrometer.
4. Conclusions
We have synthesized a series of naphthalene-N-sulfonyl-D-
Glu derivatives as MurD inhibitors that show IC50 values from
80 to 600 µM. The SARs for these types of inhibitors were
determined. Additionally, we solved four new crystal structures
of MurD complexed with inhibitors to determine the binding
mode of inhibitors within the enzyme active site. We have
described the possible ways of optimizing the structures of
reported inhibitors. Combinations of advantageous substitutions
or/and bioisosteric replacements should considerably improve
the interactions of these derivatives within the MurD active site
and should have a beneficial impact on the inhibitory activities
of these compounds against MurD. These compounds represent
promising starting points for the development of MurD inhibitors
with antibacterial activity.
5. Experimental Section
5.1. Inhibition Assay. The compounds were tested for their
ability to inhibit the addition of D-[14C]Glu to UMA in a mixture
(final volume, 50 µL) containing 0.1 M Tris-HCl, pH 8.6, 5 mM
MgCl2, 25 µM UMA, 25 µM D-[14C]Glu (50 000 cpm), 5% (v/v)
DMSO, purified MurD from E. coli40 (diluted with 20 mM
potassium phosphate, pH 7.0, 1 mM DTT, 1 mg/mL BSA), and
the test compound (compounds were soluble at all of the concentra-
tions used in the enzyme assay mixture, containing 5% DMSO).
The mixture was incubated for 30 min at 37 °C and the reaction
stopped by adding 10 µL of glacial acetic acid. The mixture was
lyophilized and taken up in HPLC elution buffer. The radioactive
substrate and product were separated by reverse-phase HPLC with
a Nucleosil 5C18 column (150 mm × 4.6 mm) as stationary phase
and isocratic elution at a flow rate of 0.6 mL/min with 50 mM
5.4. Experimental Procedures. Example for the Synthesis
of Naphthalene-N-sulfonyl-D-Glu Analogues 17a-o. These com-
pounds were prepared according to the five-step sequence outlined
below.
Step 1. Anhydrous ethanol (80 mL) was put in a dry flask fitted
with a reflux condenser and calcium chloride guard tube. Sodium
(1.9 g, 82.6 mmol) was added portionwise to it to form sodium
ethanolate. After the solution was cooled to room temperature,
6-hydroxy-2-naphthalenesulfonic acid sodium salt (13) (10.0 g, 40.6
mmol) was added and the reaction mixture was stirred for 2 h.
The mixture was then filtered and the solid residue washed with
MeOH to provide sodium 6-oxidonaphthalene-2-sulfonate (quan-
tative), which was used in the next step without further purification.

7492 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Humljan et al.
Figure 2. (a) Details of the ligand-binding site in the MurD complex with compound 17d. The Fo - Fc residual map is contoured at 3.0σ (green).
(b) Binding mode of compound 17d in the active site of E. coli MurD.
Figure 3. (a) Details of the ligand-binding site in the MurD complex with compound 17l. The Fo - Fc residual map is contoured at 3.0σ (green).
(b) Binding mode of compound 17l in the active site of E. coli MurD.
Figure 4. Possible directions of optimization of compound 17l. The protein surface is colored in accordance with the protein-ligand distance (the
legend in Å) (a) and by the calculated values of the lipophilicity potential (b).
Step 2. Sodium 6-(4-Cyanobenzyloxy)naphthalene-2-sulfonate
(14l). To a solution of sodium 6-oxidonaphthalene-2-sulfonate (2.97
g, 11.10 mmol) generated in step 1 in DMSO (20 mL), 4-(bro-
momethyl)benzonitrile (4.35 g, 22.20 mmol) was added. The
reaction mixture was stirred at room temperature overnight and then
poured into ice-cold acetone (200 mL) with continuous stirring.
The precipitated product was filtered off and washed with ice-cold
acetone to provide compound 14l, which was used without further
purification. White solid (4.01 g, 98%); 1H NMR (300 MHz,
DMSO-d6) δ 5.35 (s, 2H, CH2), 7.28 (dd, J ) 9.0, 2.5 Hz, 1H,
naph-H), 7.42 (d, J ) 2.5 Hz, 1H, naph-H), 7.64-7.78 (m, 4H,
naph-H and Ar-H), 7.83-7.95 (m, 3H, naph-H and Ar-H), 8.10
(s, 1H, naph-H); MS m/z 384 (M + Na)+.
Step 3. 6-(4-Cyanobenzyloxy)naphthalene-2-sulfonyl Chloride
(15l). To an ice-cooled suspension of the above sodium sulfonic
acid 14l (775 mg, 2.15 mmol) in DMF (10 mL), thionyl chloride
was slowly added (0.32 mL, 4.40 mmol). The resulting mixture
was stirred at room temperature for an additional 3 h. The
reaction was then quenched by pouring onto crushed ice with
continuous stirring, and the resulting mixture was filtered to
provide the title compound 15l, which was used in the next step
without further purification.
Step 4. (R)-Dimethyl 2-(6-(4-cyanobenzyloxy)naphthalene-2-
sulfonamido)pentanedioate (16l). Sulfonyl chloride 15l (720 mg,
2.01 mmol) was dissolved in CH2Cl2 (10 mL), filtered to remove
undissolved material if any, and added to a mixture of HCl · D-
Glu(OMe)2 (550 mg, 2.60 mmol) and Et3N (0.6 mL, 4.30 mmol)
in CH2Cl2 (10 mL). The resulting mixture was stirred overnight,
quenched with ice-cold 2 M HCl (10 mL), and extracted with
CH2Cl2 (3 × 20 mL). The combined organic layers were washed
with brine (30 mL), dried over Na2SO4, filtered, and evaporated
under reduced pressure. The resulting residue was purified by
column chromatography to provide 16l. White solid (224 mg,
21%, two steps); mp 163-165 °C; [R]2D3 -19.1 (c 0.287,
1
CH2Cl2); H NMR (300 MHz, DMSO-d6) δ 1.60-1.78 (m, 1H,
CHCH2), 1.79-1.95 (m, 1H, CHCH2), 2.16-2.36 (m, 2H,
CH2COO), 3.30 (s, 3H, CH3, overlaps with water), 3.40 (s, 3H,
CH3), 3.78-3.97 (m, 1H, CH), 5.40 (s, 2H, OCH2), 7.41 (dd, J

N-Sulfonyl-D-Glu DeriVatiVes as Inhibitors of MurD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7493
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MurC and MurD synthetases of peptidoglycan biosynthesis: borohy-
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) 9.0, 2.4 Hz, 1H, naph-H), 7.55 (d, J ) 2.4 Hz, 1H, naph-H),
7.66-7.77 (m, 3H, 1 × naph-H and 2 × Ar-H), 7.86-7.93 (m,
2H, Ar-H), 7.97 (d, J ) 8.8 Hz, 1H, naph-H), 8.10 (d, J ) 9.0
Hz, 1H, naph-H), 8.30 (d, J ) 1.6 Hz, 1H, naph-H), 8.37 (d,
1H, J ) 7.5 Hz, NH); MS m/z 497 (M + H)+. Anal.
(C25H24N2O7S) C, H, N.
ˇ
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Step 5. N-(6-(4-Cyanobenzyloxy)naphthalene-2-sulfonamido)-
D-glutamic Acid (17l). To a stirred solution of protected sulfona-
mide 16l (124 mg, 0.25 mmol) in dioxane (2 mL), 1 M NaOH
(2 mL) was added, and the reaction mixture was stirred for 3 h.
The reaction mixture was diluted with H2O (15 mL) and washed
with EtOAc (2 × 15 mL). The aqueous phase was acidified to
pH 1-2 using 2 M HCl and extracted with EtOAc (3 × 15 mL).
The combined organic layers were washed with brine (1 × 20
mL), dried over Na2SO4, filtered, and evaporated under reduced
pressure to provide the target compound 17l. White solid (105
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(19) Longenecker, K. L.; Stamper, G. F.; Hajduk, P. J; Fry, E. H.; Jakob,
C. G.; Harlan, J. E.; Edalji, R; Bartley, D. M.; Walter, K. A.; Solomon,
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1
mg, 90%); mp 180-185 °C; [R]2D3 -16.0 (c 0.225, MeOH); H
NMR (300 MHz, DMSO-d6) δ 1.58-1.75 (m, 1H, CHCH2),
1.76-1.94 (m, 1H, CHCH2), 2.21 (t, J ) 7.4 Hz, 2H, CH2COO),
3.77-3.90 (m, 1H, CH), 5.40 (s, 1H, OCH2), 7.40 (dd, J ) 9.0,
2.4 Hz, 1H, naph-H), 7.54 (d, J ) 2.4 Hz, 1H, naph-H),
7.57-7.79 (m, 3H, 1 × naph-H and 2 × Ar-H), 7.86-7.99 (m,
3H, 1 × naph-H and 2 × Ar-H), 8.08 (d, J ) 9.0 Hz, 1H, naph-
H), 8.31 (d, J ) 1.6 Hz, 1H, naph-H); MS m/z 469 (M + H)+.
Anal. (C23H20N2O7S) C, H, N.
Acknowledgment. This work was supported by the
European Union FP6 Integrated Project EUR-INTAFAR
(Project No. LSHM-CT-2004-512138) under the thematic
priority Life Sciences, Genomics and Biotechnology for
Health and by the Ministry of Education, Science and Sport
of the Republic of Slovenia, the Centre National de la
Recherche Scientifique, Lek Pharmaceuticals d.d., and the
Institut Franc¸ais Charles Nodier. The authors thank Dr. Chris
Berrie for critical reading of the manuscript.
Supporting Information Available: Description of crystal
structure solution and model refinement, full synthetic experi-
mental section, and microanalysis and HRMS data. This material
is available free of charge via the Internet at http://pubs.acs.org.
(20) Perdih, A.; Kotnik, M.; Hodoscek, M.; Solmajer, T. Targeted molecular
dynamics simulation studies of binding and conformational changes
in E. coli MurD. Proteins 2007, 68, 243–254.
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inhibitors of the D-glutamic acid-adding enzyme of peptidoglycan
biosynthesis. J. Org. Chem. 1996, 61, 1756–1760.
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