ATP competitive inhibitors of d-alanine-d-alanine ligase based on protein kinase inhibitor scaffolds

Article abstract of DOI:10.1016/j.bmc.2008.02.046

d-Alanine-d-alanine ligase (DDl) is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. Here, we describe a new approach to identify new inhibitor scaffolds for DDl based on similarity in the ATP

Full text of DOI:10.1016/j.bmc.2008.02.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 17 (2009) 1079–1087
ATP competitive inhibitors of D-alanine–D-alanine ligase based
on protein kinase inhibitor scaffolds
Gemma Triola,a,b Stefan Wetzel,a,b Bernhard Ellinger,a,b Marcus A. Koch,a,b
Katja Hubel,a,b Daniel Rauha,c and Herbert Waldmanna,b,
¨
aMax Planck Institute of Molecular Physiology, Department of Chemical Biology, Otto Hahn Straße 11, 44227 Dortmund, Germany
bUniversity of Dortmund, Department of Chemistry, Otto Hahn Straße 6, 44227 Dortmund, Germany
cChemical Genomics Centre of the Max Planck Society, Otto Hahn Straße 15, 44227 Dortmund, Germany
Received 20 December 2007; revised 12 February 2008; accepted 13 February 2008
Available online 16 February 2008
Abstract—D-Alanine–D-alanine ligase (DDl) is an essential enzyme in bacterial cell wall biosynthesis and an important target for
developing new antibiotics. Here, we describe a new approach to identify new inhibitor scaffolds for DDl based on similarity in
the ATP binding region of different kinases and DDl. After an initial screening of several protein kinase inhibitors, we found that
the Brutons’s tyrosine kinase inhibitor LFM-A13, an analog of the Leflunomide metabolite A771726, inhibits DDl with a Ki of
185 lM. A series of malononitrilamide and salicylamide derivatives of LFM-A13 has been synthesized to confirm the validity of
this scaffold as an inhibitor of DDl.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
mycin-intermediate Staphylococcus aureus (VISA) need
higher levels of vancomycin to be killed and are
thought to be an intermediate stage to the development
of bacteria that are fully resistant.3 There con-
sequently remains a strong need for novel antibiotics,
particularly directed against multiresistant Gram-nega-
tive bacteria.
Bacteria resist hypotonic shock and cell lysis through
the biosynthesis of a peptidoglycan-containing cell wall
which gives the cell shape and structural strength and
insulates the organism from external osmotic pressure.
Most antibiotics exploit the vulnerability of the assem-
bly of this extra cellular polymer. For example, the
cross-linking transpeptidation is a major target for b-
lactam antibiotics (penicillines, cephalosporines). Gly-
copeptides such as vancomycin and teicoplanin target
the peptidoglycan cell wall of Gram-positive bacteria
by selectively binding to the D-alanyl–D-alanine termini
of peptidoglycan precursors and preventing their cross
linking to adjacent strands.1,2 However, bacteria are
continually mutating and resistance has evolved to
every antibiotic ever placed into clinical practice, irre-
spective of their chemical class or the molecular target
of the drug. More strikingly, first signs of alarm appear
with increasing resistance to vancomycin which had
been reserved as a drug of ‘last resort’, used only after
treatment with other antibiotics had failed. Bacteria
with reduced vancomycin susceptibility such as vanco-
1.1. DDl as potential target for new antibiotics
D-Alanine–D-alanine ligase (DDl) (E.C. 6.3.2.4) cata-
lyzes the ATP-dependent assembly of the dipeptide D-
alanyl–D-alanine (D-ala–D-Ala) which is an essential
building block for bacterial cell wall biosynthesis.4
DDl consists of four domains, whose interfaces create
binding sites for D-Ala and ATP, respectively. The for-
mation of D-Ala–D-Ala proceeds via a two-step mecha-
nism. First, a D-Ala unit is phosphorylated by ATP to
produce an intermediate which subsequently will react
with the second D-Ala moiety to form the dipeptide.
For Escherichia coli two isoforms of DDl, DDlA, and
DDlB, have been described and share a sequence
homology of 35%.5 D-Ala–D-Ala is common to both
Gram-negative and Gram-positive organisms and
highly conserved among prokaryotes and has no
eukaryotic counterparts. Therefore, DDl has emerged
as an attractive target to develop novel antibiotics.5
Keywords: D-Ala–D-ala ligase; Kinase inhibitor; Antibiotics.
Corresponding author. Tel.: +49 231 133 2400; fax: +49 231 133
2499; e-mail: herbert.waldmann@mpi-dortmund.mpg.de
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.046

1080
G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
D-cycloserine, a structural analog of D-Ala, is the only
small molecule known to inhibit DDl activity and to re-
sult in an antibiotic effect (Scheme 1). D-Cycloserine
inhibits enzyme activity as a D-Ala competitive inhibi-
tor with a reported Ki of 27 lM and is proposed to
bind to the first D-Ala binding site.5–7 However, its
poor selectivity and toxicity limits its use.6,8 Other
inhibitors of DDl such as phosphonate and phosphi-
nate dipeptides have been described as transition-state
analogs that bind to the enzymes active site and inhibit
enzyme function with IC50 values in the low micromo-
lar range. However, these molecules failed to show sig-
nificant antibacterial activity.9–11 Using a de novo
structure-based molecular design software SPROUT,
a new inhibitor for DDlB was recently designed.12 In
another approach, 3-chloro-2,2-dimethyl-N-4-trifluoro-
methyl)phenyl-propan-amide was found as an inhibitor
of Staphylococcus aureus DDl in a high–throughput
screen. This molecule does not interfere with direct bind-
ing of ATP or D-Ala but binds to a hydrophobic pocket
near the first D-Ala binding site.13 Finally, a small
family of diazenedicarboxamides were developed as
inhibitors of DDlB with activities in the micromolar
range.14
2. Results and discussion
2.1. Kinase inhibitors as starting points for new inhibitors
of DDl
Over the last decades, kinases have evolved as a major
class of target proteins in medicinal chemistry and chem-
ical biology. In order to shut down unwanted kinase
activities, a plethora of small organic molecules has
since been developed to inhibit kinase function by tar-
geting the ATP binding pocket of the kinase do-
main.21–25 Typically, such inhibitors bind to the ATP
binding site of the kinase through the formation of 2–
3 hydrogen bonds and through hydrophobic interac-
tions in and around the region occupied by the adenine
ring of ATP. Although some selective ATP competitive
kinase inhibitors have been developed, the highly con-
served nature of the ATP binding region often results
in inhibitor scaffolds with poor kinase selectivity. This
conserved nature of ATP binding sites among other pro-
tein classes and the wealth of ATP competitive kinase
inhibitors could serve as a starting point for the develop-
ment of inhibitors that target proteins other than
kinases.
2.2. Biochemical screening
1.2. A link between protein and lipid kinases and DDl
Based on the described similarities between the ATP
binding sites of DDl and protein and lipid kinases we
developed a new approach to find new inhibitor scaf-
folds binding to the ATP binding site of DDl. A collec-
tion of 27 different ATP competitive kinase inhibitors
was selected and screened at mM concentrations against
DDl measuring residual activity. ATP competitive ki-
nase inhibitors identified as DDl inhibitors are given in
Table 1 (complete list of compounds tested is provided
as supporting information).
In order to find new inhibitor scaffolds for DDl, we drew
from earlier investigations15–17 and based our search on
the similarity that exists between the ATP binding do-
mains of various kinases and the glutathione synthase
fold (ATP-grasp) to which DDl belongs. Preliminary
studies reported similarities between DDl and protein
kinases, such as casein kinase (CK1), AMPc, cyclin
dependent kinase 2 (CDK2), the Src family tyrosine ki-
nase Hck, the insulin receptor tyrosine kinase (IRK),
phosphorylase kinase (PhK), the extra cellular signal
regulated protein kinase (ERK2), and lipid kinases such
as PI3K.18–20 Structural alignments of the ATP binding
sites of DDl and different classes of kinases highlight a
conserved topology of the co-factor binding site as well
as the same relative orientation of the adenine part of
ATP (Fig. 1). These common features served as starting
points toward our investigations of novel DDl
inhibitors.
Under the conditions tested, DDl was better inhibited
by the EGFR kinase inhibitors Tyrphostin-47 (T-47),
Tyrphostin-51 (T-51) and by the Bruton’s tyrosine ki-
nase inhibitor LFM-A13. Slight inhibition could be de-
tected for the AMPc inhibitor HA-1004, the CDK2
inhibitor Olomoucine, and the Src family tyrosine ki-
nase inhibitor Piceatannol. Interestingly, the known
PI3K inhibitors Wortmannin, and LY294002 were also
found to inhibit DDl.
Detailed inhibition studies were then performed for the
most potent DDl inhibitors identified in the initial
screen. DDl enzyme inhibition was measured at different
inhibitor and ATP concentrations in order to determine
Ki values and the type of inhibition. For Ki determina-
tions, parallel series of experiments were made under
the same conditions in the presence of various inhibitor
concentrations and data were analyzed by using Linewe-
aver–Burk plots (Table 1). Tyrphostin T-47, Olomou-
cine, LY29004, and LFM-A13 were found to be ATP
competitive inhibitors of DDl. However, the Tyrphostin
For our contributions to the development of kinase inhibitors see
Refs. 26–29.
Scheme 1. Structures of reported DDl inhibitors.

G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
1081
Figure 1. Structural alignments of the ATP binding sites of DDl and various kinases. (A) Structural alignment of the ATP binding sites of DDl (PDB
code 2DLN)9 in salmon and the protein kinase Hck (PDB code 1AD5)46 in green. (B) Structural alignment of the ATP binding sites of DDl (PDB
code 2DLN) in salmon and lipid kinase PI3K-c (PDB code 1E8X47) in green. The alignments highlight similar binding modes for the ligands ADP
(DDl), AMP-PNP (Hck), and ATP (PI3K-c). Structural alignments were performed using DaliLite.48 Images were generated using PyMol (http://
www.pymol.org).
Table 1. Kinase inhibitors that showed inhibition of DDl
Compound
Target
IC50 values of kinase inhibition (lM)
Scaffold
DDl residual activity
Ki values for DDl inhibition
1
2
3
4
p56lck, syk
PKA, PKG
PI3K, PKC
PI3K
I
84.63 7.5
85.3 12.86
81.37 4.4
83.94 3.5
II
III
IV
1049
1.4 mM
(ATP competitive)
CK2
PI3K, PKC
BTK
6.949,50
5
6
IV
V
77.00 14.9
41.19 15.5
2.5
185 lM
Jak, Tec, Plk
CDK2/cycB,A,E
ERK1/MAPK
EGFR, PDGFR
IRK
(ATP competitive)
1.8 mM
7
8
9
7
25
VI
79.7 10.3
(ATP competitive)
51.79 19.08
2.4–3.5
640
0.8
VII
VII
290 lM
(ATP competitive)
Mixed inhibition
EGFR
61.92 2.44
Scaffold I (natural product, resverastrol analog, phenolic stilbene), scaffold II (sulfonamide), scaffold III (natural product, stereoidal furanoids),
scaffold IV (flavonoid), scaffold V (leflunomide), scaffold VI (purine), scaffold VII (Tyrphostins).
(Solubility of compounds could be increased by adding BSA (1 mg/ml) or chaps instead of 0.01% of the detergent NP-40). Ki values were determined
for the best inhibitors.

1082
G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
T-51, although a structural homolog of T-47, showed
the profile of a mixed inhibitor for DDl. Interestingly,
mixed inhibition has also been reported for T-51 when
screened against kinase activity.30–32 The Tyrphostins
were originally designed on the basis of the polyphenol
erbstatin,33 a mixed ATP and substrate competitive ki-
nase inhibitor. T-51 and T-47 are reported as potent
inhibitors of EGFR and PDGFR with IC50 values in
the low micromolar range.30–32 The malononitrilamide
LFM-A13 belongs to a series of compounds that were
synthesized based on the active Leflunomide metabolite
A77172634 (Scheme 2).
Leflunomide is an antirheumatic drug (AravaÒ) known
to inhibit dihydroorotate dehydrogenase. However, its
metabolite A771726 (11) has been hypothesized to exert
immunosuppressive activity by the inhibition of several
protein kinases such as PDGFR,35 EGFR36,37, and to
prevent phosphorylation of Jak1, and Jak3 that are nec-
essary for Interleukin-2 receptor signaling.38 Several
analogs of A771726 such as LFM-A12 and LFM-A13
were also reported to inhibit EGFR,35 Bruton’s tyrosine
kinase (BTK),34 Jak2,39 Tec40, and Polo-like kinases
(Plk).41
Scheme 3. Synthesis of Leflunomide analogs. Reagents and condi-
tions: (i) acetylsalicylic acid chloride, NEr3, DCM, o/n; (ii) imidazole,
MeOH, overnight; (iii) cyanoacetic acid, DIC, THF, 24 h, rt; (iv) NaH,
acetyl chloride, THF, 0 °C 30 min, then o/n at rt.
A77172642,43 and stabilized by an intramolecular hydro-
gen bond. Finally, the extended analog of LFM-A13 19,
which is further functionalized with a carboxylate and
the simplified analog 20, that retains the aryl substitu-
tion, however, lacks the nitrile group and the enol and
thus is not able to form the discussed intramolecular
hydrogen bond were synthesized to study the structural
features required for inhibition.
2.3. Synthesis
Based on the findings that the binding of ATP to DDl
can be inhibited by ATP competitive kinase inhibitors
of the malononitrilamide class in the micromolar range,
we developed a focused library based on LFM-A13 to
establish further structure–activity relationships. To ex-
plore substitution patterns at the aniline moiety, com-
pounds 11 and 15–18 were synthesized according to
published procedures.34 Briefly, different substituted ani-
lines were coupled to cyanoacetic acid in the presence of
diisopropylcarbodiimide followed by treatment with
NaH and acylation with acetylchloride (Scheme 3). Sim-
plified salicylic acid derivatives such as 12–14 were syn-
thesized by coupling of anilines to acetylsalicylic acid
and further deprotected to investigate whether the b-
keto nitrile group is needed for DDl inhibitory activity.
Such analogs are thought to mimic a planar conforma-
tion that is considered the active conformation of
The prepared derivatives were evaluated for their ability
to inhibit recombinant DDl. Compounds were screened
in a DDl assay that was carried out in the presence of
250 lM ATP and 500 lM D-Ala. Orthophosphate was
detected with malachite green. Structure–activities are
provided in Table 2.
The inhibitory activity of LFMA13 could be retained
by the salicylic derivative 12 and slightly increased by
15, 18. Compound 18 with CF3-substituents in the 2-
and 5-position showed higher activity compared to
LFM-A13 (bromo-substitution in 2,5-position). Similar
results were obtained for the 2-chloro-6-methyl deriva-
tive 15. However, neither A771726 (CF3 in the 4-posi-
tion) nor the dimethyl- (16) or trimethyl-analog 17
showed any DDl inhibiting activity. Changes in the ali-
phatic region of the molecules were not tolerated and
the extended analogs 19 and the carbamate 20 were
not active.
3. Conclusions
Using structural alignments of the ATP binding sites of
the bacterial ligase DDl and protein and lipid kinases in
complex with ATP analogs, we rationalized that inhibi-
tion of DDl activity may be achieved by ATP competi-
tive kinase inhibitors. We tested a series of commercially
available kinase inhibitors and found LFM-A13 and
Tyrphostine derivatives to inhibit DDl enzyme activity.
Based on the initial screening results we synthesized a
series of malononitrilamide and salicylamide derivatives
Scheme 2. Leflunomide, active metabolites of leflunomide A771726
and analogs.

G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
Table 2. Activity of leflunomide analogs on DDl
1083
Molecule No.
Class
Substitution
DDl Residual activity (%)
Ki (lM)
R1
R2
R3
R4
R5
R0
R00
11
12
B
A
A
A
B
B
B
B
B
B
C
CF3
CH3
114.52 10.7
65.43 10.7
Not soluble
Not soluble
41.19 15.5
46.15 3.14
95 13
Br
Br
Br
13
14
CH3
CH3
Br
CH3
CH3
CH3
CH3
LFM-A13
CH3
CH3
185
215
15
16
17
18
19
20
Cl
CH3
CH3
CH3
CH3
CH3
CF3
Br
CH3
CH3
116.37 7.25
58.61 0.3
88.10 12.45
112.02 8.24
CF3
Br
CH3
CH2COOH
60
COCH3
and were able to confirm the validity of these scaffolds as
inhibitors of DDl. From this investigation we gained a
better understanding of the structural requirements
and limitations necessary for the preparation of ATP
competitive DDl inhibitors. The compounds in this
study may serve as starting points for the development
of bi-substrate inhibitors that incorporate both an
ATP competitive and a substrate competitive moiety.
Bisubstrate inhibitors that block the ATP and D-Ala
binding sites should exhibit enhanced selectivity and po-
tency profiles by preferentially inhibiting DDl over
kinases.
of CDCl3 (d = 7.26 and 77.00 ppm), CD3OD (d = 3.31
and 49.05 ppm), DMSO-d6 (d = 2.50 and 39.43 ppm)
CD3CN (d = 1.94 and 1.24 ppm) and the following
abbreviations are used to indicate signal multiplicities:
s (singlet), d (doublet), t (triplet), q (quartet), quint
(quintet), sext (sextet), sept (septet), br (broad), ap
(apparent). LCMS was performed on a Hewlett–Pack-
ard 1100 series connected to a Finnigan LCQ ESI-spec-
trometer using a gradient of 20% acetonitrile to 100%
acetonitrile in 10 min. Preparative HPLC was per-
formed on a Hewlett–Packard Agilent Series 1100 Sys-
tem equipped with a Nucleodur C18 gravity 5 lm
column from Macherey&Nagel. GCMS (EI) analysis
was performed on a Hewlett–Packard 6890 series gas
chromatograph connected to a Hewlett–Packard 5973
series mass spectrometer; column: H&W 19091s-102
HP-5mS, capillary: 25.0U201mU0.33mmm nominal.
IR spectra were measured on a Bruker Vector 22 spec-
trometer with an A527 diffuse reflectance head from
Spectra Tech.
4. Experimental
4.1. General
Unless otherwise noted, chemicals were obtained from
Aldrich, Acros, and Fluka and were used without fur-
ther purification. The following materials were obtained
from Gerbu Biochemicals: EDTA, IPTG, and ampicil-
lin. The following materials were obtained from Serva:
HEPES, PMSF, and DTT. MgCl2 and KCl were ob-
tained from J.T. Baker and (NH4)2SO4 from Riedel de
Ha¨en. Kinase inhibitors were obtained from Sigma, To-
cris or Biomol.
4.2. Expression and purification
The plasmid W3110/pTB2 containing the E. coli DDlB
gene was a gift of C.T. Walsh (Havard Medical School,
Boston). Expression and purification of E. coli DDlB
was performed according to the modified protocol of
Zawadzke et al.5 All steps were performed at 4 °C unless
otherwise specified. Enzyme activity was monitored by
detection of orthophosphate using malachite green. Pro-
tein concentration was determined by the method of
Bradford.44 The standard column buffer consisted of
50 mM HEPES, 5 mM MgCl2, 1 mM EDTA and
5 mM DTT, pH 7.2. BL21(DE3)RIL cells were trans-
formed with W3110/pTB2 in and grown at 25 °C in
LB medium containing ampicillin to an OD595 of 0.6,
whereupon IPTG was added to a final concentration
All solvents were distilled by standard procedures. All
reactions were performed under argon with freshly dis-
tilled and dried solvents. Analytical chromatography
was performed by using Merck silica gel 60 F254 alumi-
num plates. Flash chromatography was performed by
using Acros silica gel (0.035–0.07 mm). 1H and 13C
NMR spectroscopic data were recorded on a Bruker
DRX 500 or Varian Mercury VX 400 spectrometer at
RT. NMR spectra were calibrated to the solvent signals

1084
G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
of 1 mM for induction. Cells were then grown for 4 h at
25 °C and harvested by centrifugation at 7000 rpm for
10 min. The cell pellet (ca. 8 g) was resuspended in
20 mL of lysis buffer (100 mM HEPES, 5 mM MgCl2,
1 mM EDTA, 5 mM DTT, and 1 mM PMSF, pH 7.2)
and was passed through a microfluidizer (Microfluidics
Corporation, Newton, MA). Cell debris was removed
by centrifugation at 10,000 rpm for 30 min. Powdered
ammonium sulfate was gradually added to the superna-
tant to a concentration of 50% saturation, and the solu-
tion was stirred for 45 min. The solution was cleared by
centrifugation at 10,000 rpm for 30 min, and ammo-
nium sulfate was added to the supernatant to a concen-
tration of 50% saturation. After being stirred for
additional 45 min, the precipitate was removed by cen-
trifugation at 10,000 rpm for 30 min. The precipitate
was resuspended in column buffer and the resuspended
pellet dialysed (Spectrum regenerated Cellulose MWCO
5000 kDa) over night against column buffer. After cen-
trifugation (15 min, 10,000 rpm) the supernatant was
applied to a Source Q30 (XK6/20, GE Healthcare) with
2 mL/min and washed with column buffer (5 mL/min).
Protein was eluted at 4.0 mL/min with a gradient up
to 300 mM KCl in column buffer. The activity eluted
at ꢀ150 mM KCl. The purified protein appeared as a
32-kDa band on SDS–polyacrylamide gel (15 %).
Crude acyl chloride was dissolved in chloroform, and
the solvent was removed under reduced pressure (3
times). The oily residue was redissolved in dichloro-
methane and the resulting solution slowly added at
0 °C to a mixture of substituted anilines (1 equiv)
and triethylamine (3.3 equiv) in dichloromethane. The
resulting mixture was stirred overnight at room tem-
perature, followed by successive washing of the organic
phase with 1 N HCl, 1 N NaOH, and water, drying
with anhydrous magnesium sulfate and evaporation
under reduced pressure. The crude product was puri-
fied by flash chromatography on silica gel (eluent,
cyclohexane/ethyl acetate, 9:1) to afford compounds
12a–14a.
Step (ii): Imidazole (4 equiv) was added to a 0.03 M
solution of 12a–14a (1 equiv) in methanol and was stir-
red at room temperature overnight. The resulting white
precipitate was filtered off and dried under vacuum.
4.4.2. General procedure B. Steps (iii) and (iv): Cyano-
acetic acid (1.6 equiv) and substituted aniline (1 equiv)
were dissolved in THF (25 mL) and stirred at 0 °C.
Diisopropylcarbodiimide (DIC) was added over 5 min.
The reaction was stirred for 24 h at room temperature.
AcOEt was added and the organic phase was sequen-
tially washed with a 1 N HCl solution, with brine, dried
over anhydrous magnesium sulfate, filtered, and the sol-
vent was removed under reduced pressure. Finally, the
crude solid product was recrystallized from ethyl alcohol
or used without further purification. Sodium hydride (2
equiv) was added slowly to a solution of amide in tetra-
hydrofuran (1 equiv; 0.3 M) at 0 °C. After stirring for
30 min at 0 °C, acetyl chloride (1.1 equiv) was added
to the reaction mixture. The reaction was continued
overnight and then quenched by the addition of acetic
acid. The mixture was poured into water containing
hydrochloric acid to precipitate the crude product,
which was collected by filtration and washed with water.
The final pure product was obtained in some cases by
recrystallization.
4.3. D-Alanine–D-alanine ligase activity
The assay was carried out in a 96-well format at a total
reaction volume of 50 lL at 37 °C. The reaction mixture
contained the following components (final concentra-
tion): 100 mM Hepes (pH 7.4), 10 mM KCl, 10 mM
MgCl2, 250 lM ATP, 500 lM D-Ala, (2 mM) DDlB
(0.13 lg), and compound (2 mM). The antibiotic D-
cycloserine, a known substrate competitive inhibitor of
DDl was used as positive control (Ki for DDlB
27 lM). Compounds were pre-incubated with all assay
components except the substrates for 30 min. D-Ala
and ATP were added to start the reaction. After
15 min, DDlB activity was monitored by detection of
orthophosphate using malachite green45 (80 lL of mala-
chite green-molybdate solution were added (2.45 mM
malachite oxalate salt, 16 mM of ammonium molybdate
tetrahydrate in 4 N HCl), followed by 10 lL of 34% so-
dic citrate solution). The plates were read at 620 nm
after additional 20 min. Ki determinations were per-
formed under similar conditions using D-Ala (32 mM),
ATP (600 lM, 300 lM, 200, lM, 100 lM) and inhibitor
(1 mM, 2 mM, 3 mM, 4 mM) with 15 min incubation at
25 °C.
4.5. N-(2,5-Dibromophenyl)-2-acetoxybenzamide (12a)
Following general procedure A (Step 1), compound
12a was obtained in 32% yield. Selected data: 1H
NMR (400 MHz, CDCl3): 2.37 (s, 3 H), 7.15 (dd, 1H,
J 3.6, 8.8 Hz), 7.20 (dd, 1H, J 0.8, 8.8 Hz), 7.38 (dt,
1H, J 1.6, 6.4 Hz), 7.40 (d, 1H, J 8.8 Hz), 7.55 (dt,
1H, J 2, 7.6 Hz), 7.93 (dd, 1H, J 1.6, 7.6 Hz), 8.62
(bs, 1H), 8.78 (d, 1H,
J
2.4 Hz); 13C NMR
(100 MHz, CDCl3): 21.3, 111.5, 122.1, 123.5, 124.5,
126.5, 127.5, 128.2, 130.3, 132.8, 133.2, 136.9, 148.0,
163.3, 168.7; LC-MS (ESI): calcd for C15H11 79Br2NO3
411.92 [M+H]+, found 411.74 [M+H]+, 371.98 [M-acet-
yl+H]+, Rt 10.05 min; IR (KBr): 582, 1028, 1084, 1190,
1656, 1761, 3305.
4.4. Chemistry
All syntheses were performed by following literature
procedures.34,43
4.4.1. General procedure A. Step (i): Acetylsalicylic acid
(1.6 equiv) was added to thionyl chloride (3.2 equiv),
and the suspension was refluxed until complete dissolu-
tion of the acid (1 h). The resulting yellow solution was
then cooled to room temperature, and remaining thio-
nyl chloride was evaporated under reduced pressure.
4.6. N-(2,6-Dimethylphenyl)-2-acetoxybenzamide (13a)
Following general procedure A (Step 1), compound 13a
was obtained in 41% yield. Selected data: 1H NMR
(400 MHz, CDCl3): 2.29 (s, 6H), 2.31 (s, 3H), 7.15 (m,

G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
1085
4H), 7.35 (dt, J 7.58, 0.96 Hz, 1H), 7.46 (bs, 1H), 7.53
4.11. N-(2-Chloro-6-methyl)phenyl)-2-cyano-3-hydroxy-
but-2-enamide (15)
(dt, J 7.84, 1.65 Hz, 1H), 7.86 (dd, J 7.70, 1.66 Hz,
1H). 13C NMR (100 MHz, CDCl3): 18.1, 20.8, 26.7,
123.1, 126.0, 127.2, 128.3, 128.5, 129.5, 132.0, 133.5,
135.4, 148,2, 164.1, 169.3; ESI-MS: calcd for
C17H18NO3 284.13 [M+H]+, found 284.00 [M+H]+,
242.77 [M-acetyl+H]+; IR (KBr): 751, 914, 1191, 1131,
1521, 1649, 1765, 2965, 3223.
Following general procedure B, compound 15 (80 mg,
35%) was obtained. H NMR (400 MHz, DMSO-d6):
1
2.19 (s, 3H), 2.28 (s, 3H), 7.25 (q, J 7.70 Hz, 1H), 7.25
(s, 1H), 7.37 (dd, J 6.29, 2.95 Hz, 1H), 10.16 (bs, 1H);
13C NMR (100 MHz, DMSO-d6): 18.9, 23.3, 43.8,
46.8, 122.8, 125.7, 127.5, 129.0, 129.7, 132.5, 133.5,
177.5, 195.5; 13C NMR (100 MHz, CD3CN) 19.1, 22.8,
23.9, 81.0, 117.2, 128.7, 130.2, 130.7, 130.8, 132.9,
132.2, 170.3, 190.9; LC-MS (ESI): calcd for
C12H11ClN2O2 251.06 [M+H]+, found 251.11 [M+H]+;
Rt 7.11 min; ESI-MS 249.07 [MꢁH]ꢁ.
4.7. N-(2,4,6-Trimethylphenyl)-2-acetoxybenzamide
(14a)
Following general procedure A (Step 1), compound 14a
was obtained in 28% yield. Selected data: 1H NMR
(400 MHz, CDCl3): 2.20 (s, 6H), 2.27 (s, 3H), 2.30 (s,
3H), 6.29 (s, 2H), 7.15 (dd, J 8.13, 1.12 Hz, 1H), 7.30
(dt, J 7.57, 1.15 Hz, 1H), 7.50 (ddd, J 8.12, 7.50,
1.70 Hz, 1H), 7.52 (bs, 1H), 7.78 (dd, J 7.69, 1.69 Hz,
1H). 13C NMR (100 MHz, CDCl3): 18.0, 20.7, 20.9,
123.1, 126.0, 128.4, 128.7, 129.2, 130.8, 131.6, 135.0,
136.8, 148.1, 164.3, 169.2; LC-MS (ESI): calcd for
C18H21NO3298.14 [M+H]+, found 594.68 [2M+H]+,
297.95 [M+H]+, 256.20 [M-acetyl+H]+, Rt 10.94 min;
IR (KBr): 713, 1011, 1198, 1764, 2976, 3278.
4.12. N-(2,6-Dimethyl)phenyl)-2-cyano-3-hydroxybut-2-
enamide (16)
Following General procedure B, product 16 was obtained
(0.31 g; 67%). Selected data: 1H NMR (400 MHz,
CD3CN): 2.22 (s, 6H), 2.32 (s, 3H), 7.15 (m, 3H), 8.03
(s, 1H); 13C NMR (100 MHz, CD3CN): 18.2, 22.2, 80.2,
116.9, 118.2, 128.9, 133.6, 137.8, 169.7, 190.1; ESI-MS:
calcd for C13H13N2O2 229.10 [MꢁH]ꢁ, found 229.13
[MꢁH]ꢁ.
4.8. N-(2,5-Dibromophenyl)-2-hydroxybenzamide (12b)
4.13. N-(2,6-Trimethyl)phenyl)-2-cyano-3-hydroxybut-2-
enamide (17)
Following general procedure A (Step 2), compound 12b
was obtained in 12% yield. Selected data: 1H NMR
(400 MHz, DMSO-d6): 7.01 (t, 1H, J 8 Hz), 7.04 (d, 1H,
J 8 Hz), 7.29 (dd, 1H, J 2.4, 8.4 Hz), 7.45 (dt, 1H, J 1.6,
8.4 Hz), 7.66 (d, 1H, J 8.4 Hz), 8.03 (dd, 1H, J 1.6,
8 Hz), 8.66 (d, 1H, J 2.4 Hz), 10.91 (bs, 1H), 11.90 (bs,
1H); 13C NMR (100 MHz, CDCl3): 112.1, 112.7, 116.9,
117.7, 119.7, 120.7, 121.6, 124.9, 127.9, 130.7, 134.0,
137.9, 143.9, 154.0, 156.4, 164.0; ESI-MS: calcd for
C13H879Br2NO2 367.89 [MꢁH]ꢁ, found 367.93 [MꢁH]ꢁ;
IR (KBr): 682, 1025, 1082, 1229,1399, 1608, 3198.
Following general procedure B, product 17 was obtained
(0.085 g; 20%). Selected data: 1H NMR (400 MHz,
DMSO-d6): 2.11, 2.13 (2 s, 9H), 2.25, 2.28 (2 s, 3H), 6.93
(m, 2H); ESI-MS: calcd for C14H17N2O2 245.13
[M+H]+, found 245.87 [M+H]+, 262.98 [M+H2O]+.
4.14. N-(2,5-Bis(trifluoromethyl)phenyl)-2-cyano-3-
hydroxybut-2-enamide (18)
Following general procedure B, product 18 was ob-
tained (0.19 g; 90%) after recrystallization from toluene.
4.9. N-(2,6-Dimethylphenyl)-2-hydroxybenzamide (13b)
1
Selected data: H NMR (400 MHz, CD3CN): 2.36 (s,
Following general procedure A (Step 2), compound 13b
was obtained in 40% yield. Selected data: 1H NMR
(400 MHz, DMSO-d6): 2.19 (s, 6H), 6.96 (ddd, J 6.22,
3.51, 0.97 Hz, 1H), 7.14, (s, 2H), 7.50–7.43 (m, 1H),
8.05 (dd, J 8.19, 1.63 Hz, 1H), 10.06 (bs, 1H), 12.32
(bs, 1H); 13C NMR (100 MHz, CH3CN): 18.3, 115.1.
118.8, 119.7, 127.9, 128.5, 128.9, 134.6, 135.4, 137.2,
162.6, 170.2; LC-MS (ESI): calcd for C15H16NO2
242.12 [M+H]+, found 242.15 [M+H]+, Rt 3.43 min.
3H), 7.98 (d, J 8.25 Hz, 1H), 7.82 (d, J 8.26 Hz, 1H).
8.14 (s, 1H), 8.33 (bs, 1H); 13C NMR (100 MHz,
CD3CN): 23.0, 55.5, 55.9, 117.1, 118.9, 123.2, 123.4,
125.7, 125.8, 126.0, 126.1, 126.9, 126.9, 129.7, 129.6,
130.0, 130.1, 170.3, 191.0; GC/MS: calcd for
C12H8F6N2O2 338.05, found m/z 338, 296, 229. ESI-
MS: calcd for C12H9F6N2O2 339.06 [M+H]+, found
338.93 [M+H]+, 355.27 [M+NH3]+.
4.15. 4-Cyano-5-(2,5-dibromophenylamino)-3-hydroxy-5-
oxopent-3-enoic acid (19)
4.10. N-(2,4,6-Trimethylphenyl)-2-hydroxybenzamide
(14b)
General procedure B was applied with a slight modifica-
tion: ethylmalonyl chloride (1.1 equiv.) was used instead
of acetyl chloride to afford compound 19 (0.49 g; 60%).
1H NMR (400 MHz, DMSO-d6): 3.98 (s, 2 H), 7.35 (dd,
J 8.57, 2.42 Hz, 1H), 7.61 (d, J 8.58 Hz, 1H), 7.83 (d, J
2.40 Hz, 1H), 9.97 (bs, 1H);13C NMR (100 MHz,
CD3CN): 32.6, 35.0, 45.1, 49.8, 69.4, 119.9, 126.7, 130.8,
131.9, 133.7, 134.5, 134.9, 141.9, 159.8, 166.7, 184.7; IR
(KBr): 1035, 1400, 1531, 1580, 1671, 2190, 2928, 3279;
GC/MS: m/z 405, 318, 251, 237; ESI-MS: 403.27 [MꢁH]ꢁ.
Following general procedure A (Step 2), compound 14b
was obtained in 38% yield. Selected data: 1H NMR
(400 MHz, DMSO-d6): 2.14 (s, 6H), 2.26 (s, 3H), 6.95
(m, 3H), 7.46 (dt, J 7.81, 1,21 Hz, 1H), 8.05 (dd, J
8.12, 1.41 Hz, 1H), 9.98 (bs, 1H), 12.39 (bs, 1H); 13C
NMR (100 MHz, CD3CN): 17.9, 20.9, 118.8, 119.7,
127.8, 129.5, 131.9, 135.3, 136.8, 138.1, 162.5; LC-MS
(ESI): calcd for C16H18NO2 256.13 [M+H]+, found
256.18 [M+H]+, Rt 10.12 min.

1086
G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
13. Liu, S.; Chang, J. S.; Herberg, J. T.; Horng, M.; Tomich,
P. K.; Lin, A. H.; Marotti, K. R. Proc Natl Acad Sci
U.S.A. 2006, 103, 15178.
14. Kovac, A.; Majce, V.; Lenarsic, R.; Bombek, S.; Bostock,
J. M.; Chopra, I.; Polanc, S.; Gobec, S. Bioorg. Med.
Chem. Lett. 2007.
15. Koch, M. A.; Wittenberg, L. O.; Basu, S.; Jeyaraj, D.
A.; Gourzoulidou, E.; Reinecke, K.; Odermatt, A.;
Waldmann, H. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 16721.
16. Charette, B. D.; Macdonald, R. G.; Wetzel, S.; Berkowitz,
D. B.; Waldmann, H. Angew. Chem. Int. Ed. 2006, 45,
7766.
17. Wetzel, S.; Schuffenhauer, A.; Silvio, R.; Ertl, P.; Wald-
mann, H. Chimia 2007, 61, 355.
18. Kobayashi, N.; Go, N. Eur. Biophys. J. 1997, 26, 135.
19. Grishin, N. V. J. Mol. Biol. 1999, 291, 239.
20. Denessiouk, K. A.; Johnson, M. P. Proteins: Structure,
Function and Genetics 2000, 38, 310.
21. Bridges, A. I. Chem. Rev. 2001, 101, 2541.
22. Toledo, L. M.; Lydon, N. B.; Elbaum, D. Curr. Med.
Chem. 1999, 9, 775.
4.16. Methyl 2,5-dibromophenylcarbamate (20)
2,5-Dibromoaniline (0.8 g; 3.2 mmol) was dissolved in
pyridine (5.23 mL; 65 mmol) and stirred at 0 °C. Meth-
ylchloroformiate (0.38 mL; 4.9 mmol) was then slowly
added at 0 °C and the reaction was stirred at room tem-
perature for 4 h. AcOEt (5 mL) and water (5 mL) were
added to the reaction and the organic phase was subse-
quently washed with 1 M CuSO4, 1 M HCl, and brine,
dried over anhydrous MgSO4, filtered, and the solvent
was removed under reduced pressure. Compound 20
(0.72 g; 73%) was isolated by column chromatography
using a mixture of 9:1 cyclohexane/AcOEt as eluent.
1H NMR (400 MHz, CDCl3): 3.81 (s, 3H), 7.06 (dd, J
8.52, 2.34 Hz, 1H), 7.12 (bs, 1H), 7.36 (d, J 8.53 Hz,
1H), 8.38 (d, J 2.13 Hz, 1H); 13C NMR (100 MHz,
CDCl3): 52.7, 109.7, 110.7, 122.1, 122.8, 127.1, 133.2,
136.2, 136.9, 152.1, 153.3; GCMS: calcd for
C8H779Br2NO2 306.88, found m/z 307, 250, 228, 171.
23. Traxler, P. M. Exp. Opin. Ther. Patents 1998, 8, 1599.
24. Lawrence, D. S.; Niu, J. Pharmacol. Ther. 1998, 77, 81.
25. Lindsley, C. W.; Barnett, S. F.; Yaroschak, M.; Bilodeau,
M. T.; Layton, M. E. Curr. Top. Med. Chem. 2007, 7,
1349.
26. Kissau, L.; Stahl, P.; Mazitschek, R.; Giannis, A.;
Waldmann, H. J. Med. Chem. 2003, 46, 2917.
27. Stahl, P.; Kissau, L.; Mazitschek, R.; Giannis, A.;
Waldmann, H. Angew. Chem. Int. Ed. 2002, 41, 1174.
28. Stahl, P.; Kissau, L.; Mazitschek, R.; Huwe, A.; Furet, P.;
Giannis, A.; Waldmann, H. J. Am. Chem. Soc. 2001, 123,
11586.
Acknowledgments
We are grateful to C.T. Walsh for the kind gift of the
plasmid W3110/pTB2 containing the E.coli DDlB gene,
and B. Scho¨lermann for excellent Technical Assistance.
G.T. thanks Generalitat de Catalunya for a postdoc-
toral fellowship. SW is grateful to Novartis for a
Ph.D. scholarship.
29. Rosenbaum, C.; Baumhof, P.; Mazitschek, R.; Muller, O.;
¨
Giannis, A.; Waldmann, H. Angew.Chem. Int. Ed. 2004,
43, 224.
30. Gazit, A.; Yaish, P.; Gilon, C.; Levitzki, A. J. Med. Chem.
1989, 32, 2344.
31. Levitzki, A. Biochem. Pharmacol. 1990, 40, 913.
32. Yaish, P.; Gazit, A.; Gilon, C.; Levitzki, A. Science 1988,
242, 933.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.02.046.
References and notes
33. Posner, I.; Engel, M.; Gazit, A.; Levitzki, A. Mol.
Pharmacol. 1994, 45, 673.
34. Mahajan, S.; Ghosh, S.; Sudbeck, E. A.; Zheng, Y.;
Down, S.; Hupke, M.; Uckun, F. J. Biol. Chem. 1999, 274,
9587.
35. Ghosh, S.; Zheng, Y.; jun, X.; Mahajan, S.; Mao, C.;
Sudbeck, E. A.; Uckun, F. Clin. Cancer Res. 1999, 5,
4264.
36. Xu, X.; Williams, J. W.; Bremer, E. G.; Finnegan, A.;
Chong, A. S. F. J. Biol. Chem. 1995, 270, 12398.
37. Mattar, T.; Kochhar, K.; Barlett, R.; Bremer, E. G.;
Finnegan, A. FEBS Letts. 1993, 334, 161.
38. Elder, R. T.; Xu, X.; Williams, J. W.; Gong, H.; Finnegan,
A.; Chong, A. S. J. Immunol. 1997, 159, 22.
39. Van den Akker, E.; van Dijk, T. B.; Schmidt, U.; Felida,
L.; Beug, H.; Lo¨wenberg, B.; von Lindern, M. Biol. Chem.
2004, 385, 409.
1. Waxman, D. J. Annu. Rev. Biochem. 1983, 52, 825.
2. Gale, E.F.; Cundliffe, E.; Reynolds, P.E.; Richmond,
M.H.; Waring, M.J. In The Molecular Basis of Antibiotic
Action 2nd Ed.; Sons, J.W.A., Ed.: London, 1981, p. 646.
3. Howe, R.; Monk, A.; Wootton, M.; Walsh, T. R.;
Enright, M. C. Emerg. Infect. Dis. 2004, 10, 855.
4. Ellsworth Chem. Biol. 1996, 3, 37.
5. Zawadzke, L. E.; Bugg, T. D. H.; Walsh, C. T. Biochem-
istry 1991, 30, 1673.
6. Neuhaus, F. C.; Lynch, J. L. Biochemistry 1964, 3, 471.
7. Strominger, J. L.; Ito, E.; Threnn, R. H. J. Am. Chem.
Soc. 1960, 82, 998.
8. Stammer, C.H.; McKinney, J.D. 1965, 30, 3436.
9. Fan, C.; Moews, P. C.; Walsh, C. T.; Knox, J. R. Science
1994, 266, 439.
´
40. Fernandes, M. J.; Lachance, G.; Pare, G.; Rollet-Labelle,
10. Vo-Quang, Y.; Carniato, D.; Vo-Quang, L.; Lacoste,
A.; Neuzi, E.; Le Goffic, F. J. Med. Chem. 1986, 29,
579.
11. Parsons, H.; Patchett, A. A.; Bull, H. G.; Schoen, W. R.;
Taub, D.; Davidson, J.; Combs, P. L.; Springer, J. P.;
Gadebusch, H.; Weissberger, B.; Valiant, M. E.; Mellin, T.
N.; Busch, R. D. J. Med. Chem. 1988, 31, 1772.
12. Besong, G. E.; Bostock, J. M.; Stubbings, W.; Chopra, I.;
Roper, D. I.; Lloyd, A. J.; Fishwick, C. W.; Johnson, A. P.
Angew. Chem. Int. Ed. 2005, 6403.
E.; Naccache, P. H. J. Leukoc. Biol. 2005, 78, 524.
41. Uckun, F.; Tibbles, H.; Venkataraman, K.; DuMez, D.;
Erbeck, D. Bioorg. Med. Chem. 2007, 15, 800.
42. Silva, H. T.; MOrris, R. E. Exp. Opin. Invest. Drugs 1997,
6, 51.
43. Bertolini, G.; Aquino, M.; Biffi, M.; d’Atri, G.; di Pierro,
F.; Ferrario, F.; Mascagni, P.; Somenzi, F.; Zaliani, A.;
Leoni, F. J. Med. Chem. 1997, 40, 2011.
44. Bradford, M. M. Anal. Biochem. 1976, 72, 248.

G. Triola et al. / Bioorg. Med. Chem. 17 (2009) 1079–1087
1087
45. Lanzetta, P. A.; Alvarez, P. S.; Reinach, P. S.; Candia, O.
A. Biochemistry 1979, 18, 95.
46. Sicheri, F.; Moarefi, I.; Kuriyn, J. Nature 1997, 385,
602.
47. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.;
Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Mol.
Cell. 2000, 6, 909.
48. Holm, L.; Park, J. Bioinformatics 2000, 16, 566.
49. Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P.
Biochem. J. 2000, 351, 95.
50. Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, J.;
McLauchlan, H.; Klevernic, I.; Arthur, S.; Alessi, D.;
Cohen, P. Biochem. J. 2007, Sep 13, Epub ahead of
print.