Towards Gram-negative antivirulence drugs: New inhibitors of HldE kinase

Article abstract of DOI:10.1016/j.bmc.2008.12.021

Gram-negative bacteria lacking heptoses in their lipopolysaccharide (LPS) display attenuated virulence and increased sensitivity to human serum and to some antibiotics. Thus inhibition of bacterial heptose synthesis represents an attractive target for the development of new antibacterial agents. HldE is a bifunctional enzyme involved in the synthesis of bacterial heptoses. Development of a biochemical assay suitable for high-throughput screening allowed the discovery of inhibitors 1 and 2 of HldE kinase. Study of the structure-activity relationship of this series of inhibitors led to highly potent compounds.

Full text of DOI:10.1016/j.bmc.2008.12.021

Bioorganic & Medicinal Chemistry 17 (2009) 1276–1289
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Towards Gram-negative antivirulence drugs: New inhibitors of HldE kinase
*
Nicolas Desroy , François Moreau, Sophia Briet, Géraldine Le Fralliec, Stephanie Floquet, Lionel Durant,
Vanida Vongsouthi, Vincent Gerusz, Alexis Denis , Sonia Escaich
MUTABILIS SA, 102 Avenue Gaston Roussel, 93230 Romainville, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Gram-negative bacteria lacking heptoses in their lipopolysaccharide (LPS) display attenuated virulence
and increased sensitivity to human serum and to some antibiotics. Thus inhibition of bacterial heptose
synthesis represents an attractive target for the development of new antibacterial agents. HldE is a
bifunctional enzyme involved in the synthesis of bacterial heptoses. Development of a biochemical assay
suitable for high-throughput screening allowed the discovery of inhibitors 1 and 2 of HldE kinase. Study
of the structure–activity relationship of this series of inhibitors led to highly potent compounds.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 7 August 2008
Revised 1 December 2008
Accepted 9 December 2008
Available online 24 December 2008
Keywords:
Antivirulence drugs
Antibacterial
Membrane permeability
HldE
RfaE
Lipopolysaccharide
Escherichia coli
Gram-negative
1. Introduction
pathogens are normally constitutive of the normal flora of healthy
humans. Therefore an interesting approach relies on targeting the
Nowadays, it is estimated that 5–10% of patients are expected to
develop a nosocomial infection during their stay in hospitals in the
US and Europe. Considering their contribution to morbidity and
mortality, these hospital-acquired infections therefore represent
a public health problem with an increasing economic impact.1–4
Bacteria are the most common nosocomial pathogens, and their
resistance to existing therapies is increasing at an alarming rate.5,6
Gram-positive organisms such as MRSA (methicillin resistant
Staphylococcus aureus) account for the majority of nosocomial
infections. However, there has been in the last few years a dra-
matic increase in multi-drug resistant Gram-negative bacteria.
Among those bacteria, extended-spectrum b-lactamase (ESBL) pro-
ducing Enterobacteriaceae (e.g., Escherichia coli, Proteus, Klebsiella,
Enterobacter) and Pseudomonas aeruginosa have become resistant
to most of the currently available antibacterial agents. Although
appropriate antimicrobial selection, surveillance systems, and
effective infection-control procedures intend to limit the occur-
rence and spread of resistant pathogens, new effective therapies
are urgently needed.7,8
factors affecting the pathogenicity of these bacteria. The so-called
antivirulence strategy intends to increase the sensitivity of bacteria
to the innate immunity components such as complement of
human serum but also to classic antibiotics.9,10
Lipopolysaccharide (LPS) is one of the major constituent of the
outer leaflet of the outer membrane of Gram-negative bacteria.
LPS plays a major role in the host/pathogen interaction, helps to
anchor proteins in the outer membrane and provides an efficient
protective barrier against immune effectors.11 Lipopolysaccharide
consists of lipid A, a core oligosaccharide and a repeating polysac-
charide O-antigen. The core oligosaccharide can be further divided
into an inner core made of 3-deoxy-D-manno-oct-2-ulosonic acid
(Kdo) and heptose residues, and an outer core comprising hexoses
derivatives. The requisite LPS structure for cell viability is ReLPS: a
LPS constituted only of lipid A and Kdo residues (deep-rough phe-
notype). Bacteria having the deep rough phenotype or a truncated
LPS exhibit an increased sensitivity to antibiotics like erythromy-
cin, novobiocin or rifampicin due to permeabilization of the outer
membrane.11,12 Furthermore, strains of E. coli, Salmonella typhimu-
rium and Neisseria meningitidis harboring a ReLPS display attenu-
ated virulence.13–16
Many nosocomial infections are caused by pathogens that take
advantage of compromised host defenses, and some of these
Chemical inhibition of heptose synthesis or transfer should lead
to bacteria having a truncated LPS. Such bacteria should display an
increased sensitivity to host defenses and an increased permeabil-
ity to antibiotics. Thus inhibition of heptose synthesis or transfer
* Corresponding author. Tel.: +33 (0)1 57 14 05 38; fax: +33 (0)1 57 14 05 24.
E-mail address: nicolas.desroy@mutabilis.fr (N. Desroy).
Present address: GlaxoSmithKline, Les Ulis, France.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.021

N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
1277
O
P
O
P
O
P
O
O
O
O
O
O
O
O
OH
OH
O
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
OH
O
OH
O
HO
HO
GmhA
HldE
GmhB
O
P
O
O
P
O
OH
O
O
O
O
O
HO
OH
ATP ADP
OH
sedoheptulose-7-phosphate
Pi
D-glycero-D-manno-
heptose-7-phosphate
D-glycero-β-D-manno-
heptose-1,7-bisphosphate
D-glycero-β-D-manno-
heptose-1-phosphate
ATP
PPi
HldE
OH
OH
NH2
NH2
N
HO
HO
HO
ADP Kdo2-Lipid A
HO
HO
HO
OH
O
OH
O
N
N
N
N
N
O
O
WaaC
HldD
O
O
O
O
(L-glycero-β-D-manno-
O
N
O
O
P
O
P
O
O
N
P
O
P
heptosyl)-Kdo2-Lipid A
O
O
O
OH OH
ADP-L-glycero-β-D-manno-heptose
-glycero-b-
OH OH
ADP-D-glycero-β-D-manno-heptose
Scheme 1. Synthesis of ADP-
L
D
-manno-heptose from sedoheptulose-7-phosphate and incorporation into LPS.
represents an attractive target for the development of new Gram-
negative antimicrobial agents with antivirulence and membrane
permeabilizing properties.
Table 1
Conservation of HldE protein among Gram-negative bacteria
We previously disclosed the structure of E. coli WaaC enzyme,17
which catalyzes the addition of the first heptose molecule to a LPS
Kdo residue, and the discovery of inhibitors of this enzyme.18 Here
we report our efforts towards inhibition of HldE (previously named
RfaE), an enzyme involved in the biosynthesis of ADP-heptose.
Bacteria
Identity/similarity (%)
Escherichia coli
Shigella sonnei
100/100
99/99
93/98
93/97
92/97
86/92
69/81
68/82
58/72
50/66
38/58
Enterobacter sp. 638
Salmonella typhimurium LT2
Klebsiella pneumoniae MGH 78578
Yersinia pestis
Haemophilus influenzae
Pasteurella multocida
Pseudomonas aeruginosa
Neisseria meningitidis Z2491
Helicobacter pylori
2. Biochemistry
2.1. Biosynthesis of bacterial heptose
The bacterial synthesis of ADP-L-glycero-b-D-manno-heptose
from sedoheptulose-7-phosphate in E. coli is presented in Scheme
1.19 Sedoheptulose-7-phosphate is first obtained from the reaction
of fructose-6-phosphate with ribose-5-phosphate catalyzed by
transketolase TktA. Ketose-aldose isomerase GmhA transforms
A biochemical assay for HldE-kinase (E. coli) activity was set up
using -glycero- -manno-heptose-7-phosphate (H-7-P) and ATP as
substrates. Readout was either luminescent (ATP depletion) or
fluorescent (ADP formation) depending on possible compound
interferences (Scheme 2).
As for all carbohydrate kinases, the kinase activity requires a
divalent cation as electrophilic activator. Here, Mg2+ and Mn2+ cat-
ions were shown to sustain HldE activity, the latter being approx-
imately 3-fold more efficient than the former at the optimal
concentration of 1 mM (Fig. 1A). As often observed within the car-
bohydrate kinase family,22,23 a monovalent cation is absolutely
required for catalysis: while Na+ did not affect activity, K+ strongly
activated HldE-kinase with a Kd of 12 mM, and an optimal concen-
tration of 25 mM (Fig. 1B). The optimal pH was 7.5. Surprisingly,
DMSO (up to 15%) had a significant activating effect. We used a
maximum of 5% DMSO in the assays for dissolution of compounds.
D
D
sedoheptulose-7-phosphate into
D-glycero-D-manno-heptose-7-
phosphate, which is next phosphorylated at the anomeric hydroxyl
by HldE. At this step only the b anomer is formed. Next, dephos-
phorylation at position 7 is achieved by phosphatase GmhB, and
adenylyl transfer to the phosphate group in position 1 catalyzed
by HldE leads to ADP-
the -stereochemistry of the C6 hydroxyl by the epimerase HldD
(previously named RfaD) affords ADP- -glycero-b- -manno-heptose
D-glycero-b-D-manno-heptose. Inversion of
D
L
D
which is next incorporated into LPS by the heptosyltransferases
WaaC, WaaF and WaaQ if a third heptose molecule is present.
2.2. Biochemical characterization of HldE-kinase
HldE in E. coli is a bifunctional cytoplasmic enzyme comprising
2 functional domains: a carbohydrate kinase and an adenylyltrans-
ferase one.20 We focused in this work on the inhibition of the first
one: the kinase activity. HldE is well conserved among Gram-neg-
ative bacteria as shown in Table 1. However, this enzyme is absent
of genera producing LPS core devoid of heptose like Acinetobacter,
Moraxella and Chlamydia.21 In bacteria like N. meningitidis, the two
catalytic activities found in HldE of E. coli are split on two separate
enzymes HldA (kinase) and HldC (adenylyltransferase). Identity/
similarity of HldE (kinase domain) with human enzymes is poor:
a significant sequence similarity is essentially found with human
ribokinase (40%). Inhibition of HldE-kinase has therefore a low
potential for toxicity by interfering with human enzymes.
HldE
H-7-P
+
ATP
H-1,7-P + ADP
Pyruvate kinase
+ Lactate dehydrogenase
Luciferase
Luminescent
readout
Fluorescent
readout
Scheme 2. Biochemical assays for HldE-kinase. H-7-P =
tose-7-phosphate; H-1,7-P = -glycero-b- -manno-heptose-1,7-bisphosphate.
D-glycero-D-manno-hep-
D
D

1278
N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
4.6
4
A
B
[2] = 300µM
[2] = 100µM
[2] = 33µM
[2] = 11µM
[2] = 0
6
5
4
3
2
1
0
3.4
2.8
2.2
1.6
1
0.6
0.4
Mg2+
Mn2+
0.4
-0.2
0.2
0
5000
10000
1000
10000
Metal (μM)
KCl (μM)
Figure 1. HldE-kinase initial velocity (vi): (A) Effect of the divalent cation added;
0
(B) effect of K+ ions.
0
0.01
0.02
0.03
1/[ATP] (µM-1)
-0.2
Table 2
Steady-state kinetic parameters of HldE-kinase of E. coli
Figure 2. Competition between
presence of 1.8 M of H-7-P substrate: double reciprocal plot (1/initial velocity vs
1/[ATP]) showing intersecting lines on y-axis at 1/Vmax
2 and ATP on HldE-kinase measured in the
a
l
Entry
Compound
KM
(l
M)
.
1
2
3
4
ATP at [H-7-P] = 1.8
ATP at [H-7-P] = 0.2
H-7-P at [ATP] = 50
H-7-P at [ATP] = 0.3
l
l
M
M
147 14
>0.8b
0.60 0.07
0.23 0.03
lM
to 15 KM) (data not shown). Finally, as shown in Figure 2, com-
pound 2 behaved as a competitive reversible inhibitor with respect
to ATP. The Ki of compound 2 determined using the fluorescent
lM
a
Values standard deviations.
No defined value could be determined due to rapid consumption of H-7-P at
b
assay was 38
inhibitor of HldE reported to date is a sulfonic acid derivative with
a slightly higher Ki of 63
M for HldE-kinase of E. coli.20b
3 lM. The only other published small molecule
high ATP concentrations.
l
Taken together, these data showed that compound 2 was not a
promiscuous hit as analyzed by Feng and Shoichet:24 this com-
pound was a specific inhibitor of HldE-kinase, binding reversibly
to its ATP site.
The steady-state kinetic parameters of HldE-kinase of E. coli
were measured at pH 7.5, in the presence of 1 mM MnCl2,
25 mM KCl and 5% DMSO (Table 2). We found
a kcat of
1714 24 minꢀ1 for the enzyme. The KM of ATP for HldE-kinase
was in the high micromolar range (Table 2, entry 1) as observed
for E. coli ribokinase.23 On the contrary, the KM of H-7-P was below
2.4. Selectivity of the HldE-kinase inhibitors
1
l
M (Table 2, entries 3 and 4). These values were extremely low
compared to the KM of -ribose for the same ribokinase (280
D
l
M).23
The selectivity of our inhibitors of HldE-kinase of E. coli
(HldE-K) with respect to the ribokinase (RK) of E. coli, and to
HldA (the equivalent of HldE-kinase for N. meningitidis) was
studied. The goal was to investigate the ability of such chemical
scaffold to recognize the ATP binding pocket of closely related
enzymes: on the one hand HldA, an enzyme catalyzing the same
reaction but with only 66% sequence similarity with respect to
HldE-K, and on the other hand RK, an enzyme catalyzing another
reaction, although belonging to the same carbohydrate kinase
family (38% sequence similarity with respect to HldE-K). Com-
parison with human ribokinase was also envisaged in order to
take into consideration potential interactions. RK, HldA and HldE
share highly conserved amino-acids in the ATP binding site as
depicted from the structure of E. coli RK25 (Fig. 3). Therefore
an ATP competitor like compound 2 could potentially interact
with any of them.
2.3. Identification and characterization of HldE-kinase
inhibitors
The high-throughput screening (HTS) of 40,000 compounds
from Mutabilis internal library was performed on HldE-kinase of
E. coli using the luminescent assay. HTS was performed in the pres-
ence of 3 nM of HldE, 1 mM MnCl2, 25 mM KCl and 5% DMSO at pH
7.5. In this assay, concentrations of ATP and H-7-P were both set at
0.2
centration for each compound to be screened was 50
HTS campaign led to the identification of compounds 1 and 2 as
lM in order to favor detection of substrate competitors. Con-
l
M. This
inhibitors of HldE-kinase with IC50 of 51 11
respectively (Scheme 3).
lM and 69 16 lM,
The mechanism of action of compound 2 was investigated in or-
der to rule out non specific (promiscuous) inhibition. Inhibition
kinetic of 2 was linear. It neither depended on order of addition
nor on enzyme/inhibitor pre-incubation. The inhibition was nei-
A biochemical assay was set up for RK of E coli with D-ribose and
ATP as substrates, and for HldA with H-7-P and ATP as substrates
(see Section 6 for details). The KM of ATP for both enzymes is pre-
sented in Table 3.
ther shifted by Triton-X100 (100
lM to 1 mM) nor by H-7-P (up
The IC50 of compounds 1 and 2 were measured on RK and were
found to be higher than 300
ATP was 3 M, i.e., far below the apparent KM of ATP in the same
conditions (18 M). Their IC50 is therefore a reasonable approxima-
lM. In this assay, the concentration of
l
l
N
N
tion of their affinity for the ATP site of RK.
O
O
On this basis, we concluded that the series related to 1 and 2
had the potential for being selective of HldE with respect to RK
and was therefore of interest for chemical optimization. In order
to study the structure–activity relationship (SAR) of these com-
pounds, the synthesis of analogues was investigated. The synthesis
of derivatives and their activity is described herein.
N
O
O
N
N
OH
OH
N
O
O
1
2
Scheme 3. Structure of HTS hits 1 and 2.

N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
1279
200
|
210
|
220
|
230
|
240
|
250
|
260
|
270
|
280
|
HldE_Ecoli
HldA_Nmeningitidis
RK_Ecoli
GATLLTPNLSEFEAVVGKCKTEEEIVERGMKLIADYELSALLVTRSEQGMSLLQPGKA-PLHMPTQAQEVYDVTGAGDTVIGV
GATLITPNRAELKEVVGSWKNENDLTEKAQNLRRHLDLTAILLTRSEEGMTLFSEGE--PIYQPTRAQEVYDVSGAGDTVIAG
LVDIITPNETEAEKLTGIRVENDEDAAKAAQVLHEKGIRTVLITLGSRG--VWASVNGEGQRVPGFRVQAVDTIAAGDTFNGA
LSDVFCCNESEAEILTGLTVGSAADAGEAALVLLKRGCQVVIITLGAEGCVVLSQTEPEPKHIPTEKVKAVDTTGAGDSFVGA
RK_Human
Consensus
l d itpNesEae ltG
v
eda kaa vl
g
vliTlg eG vls e ep h Pt v avDttgAGDtf ga
290
|
300
|
310
|
320
|
330
|
340
|
350
|
360
|
HldE_Ecoli
HldA_Nmeningitidis
RK_Ecoli
LA--ATLAAGNSLEEACFFANAAAGVVVGKLGTSTVSPIELENAVRGRADTGFGVMTEEELKLAVAAARKRGEKVVMTNG
MG--LGLAAGCTMPEAMYLANTAAGVVVAKLGTAVCSFAELTKALSGQSTM-----------------------------
LI--TALLEEKPLPEAIRFAHAAAAIAVTRKGAQPSVPWREEIDAFLDRQR-----------------------------
LAFYLAYYPNLSLEDMLNRSNFIAAVSVQAAGTQSSYPYKKDLPLTLF--------------------------------
RK_Human
Consensus
$a lal
s$e#a fanaaAa! V
Gtq s p
e
l l
Figure 3. Sequences alignment: HldE of E. coli, HldA of N. meningitidis, RK of E. coli and human are shown. Conserved sequence motifs found in the ATP binding site of E. coli
RK25 are underlined. Alignment done with Multalin.26
Table 3
O
Steady-state kinetic parameters of HldA and RK of E. coli
R1
Cl
O
NH2
OEt
+
Entry
Enzyme
KM of ATPa
(lM)
>0.4b
1
2
HldA at [H-7-P] = 0.2
l
M
M
a
3a, R1 : pyridin-2-yl
3b, R1 : pyridin-3-yl
3c, R1 : pyridin-4-yl
3d, R1 : phenyl
3e, R1 : 2-aminophenyl
3f, R1 : furan-2-yl
RK at [
D
-ribose] = 3
l
18
6
a
Values standard deviations.
No defined value could be determined due to rapid consumption of H-7-P at
b
H
R1
high ATP concentrations.
N
OEt
3a-i
3g, R1 : thiazol-2-yl
3h, R1 : quinolin-2-yl
3i, R1 : benzothiazol-2-yl
b
3. Chemistry
O
The synthesis of analogues of compounds 1 and 2 was first car-
ried out by coupling an oxazole-carboxylic acid to a secondary
amine. In order to avoid side reactions, the acidic function of the
desired amines needed to be masked.
R1
H2N
O
+
OEt
Scheme 4. Reagents and conditions: (a) NEt3, CH3CN, rt to 50 °C, overnight; (b)
AcOH, NaBH3CN, CH2Cl2, 0 °C to rt, overnight.
A set of secondary amines 3a–i was prepared by nucleophilic
substitution or reductive amination as depicted in Scheme 4. The
2-pyridinyl group R1 of 1 and 2 was replaced by regioisomers or
other heterocycles. The carboxylic acid of the final analogues was
protected as an ethyl ester.
The synthesis of oxazole-carboxylic acids 4 and 6 with varying
substituents on the phenyl ring was realized according to known
procedures (Scheme 5).27–29 Reaction of methyl 2-aminoacetoace-
tate hydrochloride with aroyl chlorides gave the corresponding
amides which were cyclized using triphenylphosphine, iodine
and triethylamine. Saponification led to oxazolecarboxylic acids
4. Another route involved the thermal reaction of ethyl 2-chloro-
acetoacetate with aryl amides followed by saponification to afford
oxazolecarboxylic acids 6. Coupling of 4 and 6 to secondary amines
3a–i was performed using EDAC as coupling agent. Saponification
finally afforded analogues 5a–t and 7a–c. Compounds 5b, 5f, 5k
in which R2 is NH2 were obtained by reduction of the correspond-
ing nitro groups of 5a, 5e, 5j, respectively.
The replacement of the central oxazole ring by other hetero-
cycles was also implemented. Compounds 10–15 (Scheme 6)
were prepared from commercially or readily available carboxylic
acids which were reacted with amine 3a followed by
saponification.
A straightforward synthetic pathway to thiazole derivatives 9a–
h was also devised from 2-bromo-4-methyl-1,3-thiazole-5-carbox-
ylic acid (Scheme 7). Since activation of the carboxylic acid with
oxalyl chloride led to partial replacement of the bromine atom
by chlorine, oxalyl bromide was used. Coupling with amines 3a
and 3i followed by one-pot Suzuki coupling and saponification
afforded compounds 9a–h.
OH
O
R2
O
R2
O
N
O
O
O
O
O
d, c
d, c
a, b, c
N
O
N
+
+
OH
OH
Ar
Ar
Cl
O
O
R1
NH2
O
4
6
5a-t
OH
O
R2
O
R2
N
O
O
e, c
O
N
NH2
R1
N
Cl
O
7a-c
R1, R2 : see Tables 4 and 6
Scheme 5. Reagents and conditions: (a) NEt3, CH2Cl2, rt, overnight; (b) PPh3, I2, NEt3, CH2Cl2, rt, overnight; (c) LiOH, THF, H2O, rt, overnight; (d) EDAC, 4-DMAP, amine 3a–3i,
CH2Cl2, rt to 50 °C, overnight; (e) toluene, 120–140 °C, overnight.

1280
N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
Table 4
Influence of R2 on the inhibitory activity
N
N
a
O
O
Entry
Compound
R1
R2
IC50 (lM)
S
N
N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
2
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
None
None
o-NO2
o-NH2
o-CF3
51 11
69 16
141 24
31 11
121 30
44 10
5.8 1.6
153 40
35 11
Ph
Ph
OH
OH
N
10
O
5a
5b
5c
5d
5e
5f
5g
5h
5i
7a
5j
5k
5l
O
11
O
o-Br
N
N
m-NO2
m-NH2
m-CF3
m-Br
m-OMe
m-OMe
p-NO2
p-NH2
p-CF3
p-Br
O
O
O
O
N
N
N
N
Ph
Ph
OH
OH
10
15
17
2
4
4
N
N
Ph
O
O
12
13
15
151 30
N
N
22
142
27
3
9
3
6
Ph
5m
5n
O
p-OMe
75
N
OH
OH
O
a
Values standard deviations are means of three experiments.
O
O
14
Scheme 6. Structures of analogues 10–15.
Table 5
Influence of the central ring on the inhibitory activity
4. Results and discussion
a
Entry
Compound
IC50 (lM)
1
2
3
4
5
6
10
11
12
13
14
15
52 14
42 13
57 15
>300
100 20
>300
The activity on HldE kinase of compounds synthesized was
measured using the biochemical assay described previously.
Results are reported in Tables 4–7.
As seen in Table 4, substituents were introduced on the phenyl
group in different positions. Although it appeared difficult to
deduce from the results a clear trend concerning the required ste-
ric and electronic properties of the best substituent in each posi-
tion, some important observations were made. The amino group
was the best substituent in ortho (5b) and para (5k) positions
(Table 4, entries 4 and 14). In contrast it led to the greatest loss
of activity in meta position (5f, Table 4, entry 8). An opposite
behavior was observed for the nitro substituent: a great loss of
activity was seen in ortho (5a) and para (5j) positions (Table 4,
entries 3 and 13) whereas it displayed the best activity in meta
position (5e, Table 4, entry 7). The trifluoromethyl group in meta
(5g) position (Table 4, entry 9) gave only a slight increase in activ-
ity compared to 1, however a loss of activity was observed in ortho
(5c) and para (5l) positions (Table 4, entries 5 and 15). Introduction
of a bromine in any position gave an increase of activity (5d, 5h,
5m, Table 4, entries 6, 10 and 16), the most significant effect being
observed in meta position. The methoxy substituent in meta (5i)
afforded a gain of activity (Table 4, entry 11), and in contrast a
slight loss of activity in para position (5n, Table 4, entry 17). Sim-
ilarly, as seen by the comparison between 2 and 7a (Table 4, entries
2 and 12), introduction of a methoxy substituent in meta position
also led to an increase of activity with this scaffold.
a
Values standard deviations are means of three experiments.
Table 6
Influence of R1 on the inhibitory activity
a
Entry
Compound
R1
R2
IC50
(l
M)
1
2
3
4
5
6
7
8
5o
5p
5q
7b
5r
5s
5t
Pyridin-3-yl
Pyridin-4-yl
Phenyl
2-Aminophenyl
Furan-2-yl
Thiazol-2-yl
Quinolin-2-yl
Benzothiazol-2-yl
None
None
None
None
None
None
m-OMe
m-OMe
>300
>300
>300
>300
>300
110 10
12
0.27 0.07
4
7c
a
Values standard deviations are means of three experiments.
bromine or a methoxy group gave the most significant gain in
potency: 5.8 M (5e), 10 M (5h) and 15 M (5i), respectively. In
contrast, only a moderate increase of activity could be obtained
with ortho and para substitutions by an amino group, at 31
(5b) and 22 M (5k), respectively.
As seen in Table 5, the central oxazole ring could be replaced by
other heterocycles such as thiazole (10), furane (11) or pyrazole
(12) without affecting the potency (Table 5, entries 1–3). Com-
l
l
l
l
M
Analogues having a heteroaromatic ring (pyridinyl, pyrazinyl,
furanyl) or an ethyl group instead of the phenyl ring were also pre-
l
pared, but they were all inactive (IC50 > 300
lM). Therefore, we
concluded that the phenyl ring was required for activity and that
with respect to the IC50 of 1 at 51 lM, meta substitution by a nitro,
pound 14 having no methyl group displayed an IC50 of 100 lM
R1
R1
O
O
O
R2
S
N
S
S
a
b
N
N
OH
Br
Br
N
OEt
OH
N
O
O
8
R1, R2 : see Table 7
9a-h
Scheme 7. Reagents and conditions: (a) (COBr)2, CH2Cl2, DMF, 0 °C to rt, 2 h, then amine 3a or 3i, DIPEA, 0 °C to rt, overnight; (b) boronic acid or boronate ester, PdCl2dppf,
Cs2CO3, 1,4-dioxane, water, 110 °C, 2 days.

N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
1281
Table 7
OH
N
O
Further R2 modifications in meta and para positions
O
a
S
N
Entry
Compound
R1
R2
IC50
(
l
M)
N
1
2
3
4
5
6
7
8
9
5u
5v
5w
9a
9b
9c
9d
9e
9f
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
m-Morpholinyl
m-NHCOCH3
m-NHCOC3H5
m-COCH3
m-NO2, p-NH2
m-OMe
70 30
28
14
216 75
13
0.42 0.04
0.50 0.09
2
3
S
O
Pyridin-2-yl
2
O
O2N
S O
Benzothiazol-2-yl
Benzothiazol-2-yl
Benzothiazol-2-yl
Benzothiazol-2-yl
Benzothiazol-2-yl
Benzothiazol-2-yl
Benzothiazol-2-yl
m-NO2
m-SO2Me
75
2
m-OMe, p-NH2
0.26 0.06
0.48 0.04
0.33 0.04
0.11 0.02
b
9c
9d
9e
10
11
12
9g
9h
5x
b
m-Pyrazol-3-yl
O
a
Values standard deviations are means of three experiments.
R2 substituents are fused with the phenyl ring, see Scheme 9 for structures
b
N
H2N
H
corresponding to 9g and 9h.
9f
9g
9h
Scheme 9. Meta and meta–para substitutions with R1 = benzothiazol-2-yl.
(Table 5, entry 5).When the substitution pattern was modified or
the central ring replaced by a 6-membered ring as in 13 and 15,
a complete loss of activity was observed (Table 5, entries 4 and 6).
The carboxylic acid on one of the amide side chains of 1 and 2
proved to be essential for activity. Replacement by an amide
(CONH2), alcohol, tetrazole, or acylsulfonamide (CO–NH–SO2Me)
gave inactive analogues. Additionally, lengthening of this side
chain by one methylene led to a fivefold decrease in activity (IC50
amide functionality in meta position (5v, 5w, Table 7, entries 2
and 3) improved the activity. The most notable effect was observed
with the cyclopropylcarboxamide substituted 5w.
Replacement of the central oxazole ring of 7c by a thiazole ring
(9c) resulted in a slight loss of activity (Table 7, entry 6). Surpris-
ingly, the nitro substituent was less potent than the methoxy
group in meta position with this scaffold (9d, Table 7, entry 7).
Attempts to replace the nitro group by other electron withdrawing
substituents such as acetyl or methanesulfonyl groups generated
poorly active compounds (9a, 9e, Table 7, entries 4 and 8).
As could be expected, compound 9b bearing meta nitro and para
amino substituents was less active than 5e (Table 7, entry 5), in
part due to antagonistic electronic effects of the substituents. How-
ever compound 9f with meta methoxy and para amino groups was
more active than 9c (Table 7, entry 9), resulting from additional
effects of substituents.
of 272 lM).
Consideringthe other amide side chain bearing thepyridylgroup,
the results presented in Table 6 show that removal (5q) or shift (5o,
5p) of the pyridyl nitrogen provided inactive compounds (Table 6,
entries1–3). Placing thenitrogen out of the ring(7b) also proved del-
eterious for activity (Table 6, entry 4). Replacement of the 2-pyridi-
nyl group by a 2-furanyl (5r) led to a complete loss of activity (Table
6, entry5). In contrast the presenceof a 2-thiazolyl(5s) insteadof the
2-pyridinyl led only to a twofold loss of activity (table 6, entry 6),
thus reinforcing the importance of the nitrogen and its position in
R1. Whereas thepresence of an additionalphenylring (5t) fused with
the 2-pyridinyl only afforded a slight improvement compared to 5i
(Table 6, entry 7), it led to a tremendous gain in potency when it
was fused with the 2-thiazolyl group, compound 7c displaying an
Compounds 9g and 9h in which the phenyl ring was replaced by a
naphthyl or indolyl ring also showed interesting activities with IC50
of 0.48 lM and 0.33 lM, respectively (Table 7, entries 10 and 11).
These lastresults suggestedthatroom was availablearound thephe-
nyl ring, and in particular for a planar additional ring. Since the meta
substitution on the phenylring had alreadygiven potent compounds
with various substituents having different electronic properties, we
decided to prepare compound 5x with a pyrazole ring in meta posi-
tion (Scheme 10). Compound 5x was synthesized by Suzuki coupling
between the corresponding meta-bromo ester derivative and 1H-
pyrazole-3-boronic acid followed by saponification. This compound
displayed the best activity of our series of inhibitors with an IC50 of
IC50 of 0.27 lM (Table 6, entry 8).
Substitution on the phenyl ring was further investigated, with a
particular emphasis on the meta position. Morpholine derivative
5u and amides 5v and 5w were prepared from intermediates in
the synthesis of 5h and 5f (Scheme 8). Since the central oxazole
ring could be replaced by a thiazole, the straightforward route
depicted in Scheme 7 allowed the synthesis of analogues 9a–9h
(Schemes 8 and 9).
As seen in Table 7, replacement of the meta amino group of 5f
by a morpholine led to an increase of activity but remained less
active than 1 (5u, Table 7, entry 1). Interestingly introduction of
0.11 lM (Table 7, entry 12).
The IC50 of a subset of compounds towards HldE-kinase of
E. coli, RK of E. coli and HldA of N. meningitidis was studied (Table
8). ATP concentration in each of the 3 assays (0.2
and HldA, 3 M for RK) was below its apparent KM in the same con-
ditions (see Tables 2 and 3). The IC50 measured in these assays pro-
lM for HldE
l
N
N
R2
O
O
vided therefore
a
reasonable approximation of the relative
N
O
S
N
N
N
OH
OH
N
OH
O
O
O
NH
R2
=
O
O
O2N
H2N
O
N
O
N
O
O
N
HN
N
HN
S
5u
5v
5w
9a
9b
5x
Scheme 8. Meta and meta–para substitutions with R1 = pyridin-2-yl.
Scheme 10. Structure of 5x.

1282
N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
Table 8
atmosphere in oven-dried glassware with magnetic stirring or in
screwed-cap tubes with orbital shaking using an Advanced Chem-
tech PLS apparatus. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on a 400 MHz Brüker instrument, and
chemical shifts are reported in parts per million downfield from
the internal standard tetramethylsilane (TMS). NMR analysis dis-
played amide rotamers for most compounds. The following abbre-
viations are used to indicate signal multiplicities: s (singlet), d
(doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (dou-
blet of triplets), td (triplet of doublets), br (broad). J indicates the
NMR coupling constant measured in Hertz. CDCl3 is deuteriochlo-
roform, DMSO-d6 is hexadeuterio-dimethylsulfoxide, and CD3OD
is tetradeuteriomethanol. Mass spectra were obtained using elec-
trospray (ES) ionization techniques on an Agilent 1100 Series
LCMS. HPLC (analytical and preparative) were performed on an
Agilent 1100 HPLC with DAD (Diode Array Detection). Preparative
HPLC were performed at 0.7 mL/min on a ThermoElectron, Hyper-
Comparison of IC50 of compounds on HldE-kinase (E. coli), HldA (N. meningitidis) and
RK (E. coli)
a
a
Compound
HldE-K IC50
5.8 1.6
(
lM)
HldA IC50
(l
M)
RK IC50 (lM)
5e
5i
7c
9d
9g
9h
>300
>300
6.0 0.9
75 9.5
12 0.7
8.5 0.9
>300
>300
>300
>300
>33b
>100b
15
4
0.27 0.07
0.50 0.09
0.48 0.04
0.33 0.04
a
Values standard deviations are means of three experiments.
Signal quenching was observed above this concentration.
b
selectivity of the tested compounds towards the three enzymes. On
the basis of the compounds tested, we could conclude that the ser-
ies appeared selective with respect to RK. Actually, none of the
compounds tested did inhibit RK significantly at doses as high as
sil BDS C-18 column (250 ꢁ 4.6 mm, 5
lm) using a gradient of ace-
300 lM. HldA on the contrary was inhibited but only by the most
tonitrile in water with 0.1% TFA (50% in acetonitrile to 100% and
then back to 50%). Analtech Silica Gel GF and E. Merck Silica Gel
60 F-254 thin layer plates were used for thin layer chromatogra-
phy. Flash chromatography was carried out on Flashsmart Pack
cartridge, using silica gel columns prepacked with irregular silica
potent compounds and with a 10 to >100-fold higher IC50: the ser-
ies could therefore be potentially recognized by HldE/HldA
enzymes of Gram-negative bacteria but not equally.
Compounds of this series displayed no antibacterial activity
against strains of E. coli and other Gram-negative bacteria. Since
HldE is not essential for survival of Gram-negative bacteria under
normal growth conditions, further envisaged investigations
include characterization of the effect of our inhibitors on LPS bio-
synthesis and on the sensitivity towards antibiotics of Gram-nega-
tive bacteria.
40–60 lm or spherical silica 20–40 lm. The meaning of certain
abbreviations is given herein. ESI refers to electrospray ionization,
HPLC refers to high pressure liquid chromatography, LCMS refers
to liquid chromatography coupled with a mass spectrometer, M
in the context of mass spectrometry refers to the molecular peak,
MS refers to mass spectrometer, NMR refers to nuclear magnetic
resonance, pH refers to potential of hydrogen, TFA refers to triflu-
oroacetic acid, DIPEA refers to N,N-diisopropylethylamine, dppf
refers to 1,10-bis(diphenylphosphino)ferrocene, EDAC refers N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride,4-DMAP
refers to 4-(dimethylamino)pyridine, BINAP refers to 2,20-bis
(diphenylphosphino)-1,10-binaphthalene, TLC refers to thin layer
chromatography, Hepes refers to 4-(2-hydroxyethyl)piperazine-
1-ethanesulfonic acid, DTT refers to dithiothrietol, H-7-P refers to
5. Conclusion
HldE is a bifunctional enzyme involved in the synthesis of bac-
terial heptoses constituents of LPS. Gram-negative bacteria lacking
heptoses in their LPS display attenuated virulence and increased
sensitivity to human serum and to some antibiotics. Thus HldE rep-
resents a target of interest for the development of new Gram-neg-
ative antimicrobial agents.
High-throughput screening on HldE-kinase activity of E. coli led
to the discovery of inhibitors 1 and 2 with IC50 in the range of 50–
70 lM. These compounds were shown to be competitive reversible
D-glycero-D-manno-heptose-7-phosphate.
6.2. Chemistry protocols
inhibitors with respect to ATP in the reaction catalyzed by HldE-
kinase. The study of the structure–activity relationship of this series
resulted in a considerable improvement of potency with com-
6.2.1. Representative procedure for the synthesis of amines 3a–
i (Scheme 3, path a)
Under argon, a solution of 1,3-benzothiazol-2-ylmethylamine
pound 5x having an IC50 of 0.11 lM. Compounds of this series were
hydrochloride (600 mg, 3 mmol), ethyl chloroacetate (324
lL,
also able to inhibit HldA of N. meningitidis but were completely
inactive on ribokinase of E. coli. Considering the similarity between
HldE of E. coli and HldA of N. meningitidis, this series is likely to be
selective of HldE/HldA enzymes of Gram-negative bacteria.
As a result, this family of inhibitors represents a promising lead
series for the development of new Gram-negative antimicrobial
agents with antivirulence and membrane permeabilizing proper-
ties. Further chemical optimization and biological evaluation are
currently underway; these results will be presented in due course.
3 mmol) and triethylamine (916 L, 6.6 mmol) in anhydrous aceto-
l
nitrile (6 mL) was stirred at room temperature for 1.5 h, then at
50 °C overnight. The reaction mixture was evaporated under
reduced pressure. The crude product was purified by flash chroma-
tography on silica gel eluted with a gradient of methanol in
dichloromethane (0–5%) to afford ethyl [(1,3-benzothiazol-2-
ylmethyl)amino]acetate (3i, 436 mg, 58%) as a yellow oil. 1H
NMR (CDCl3): 7.97 (d, J = 8.4 Hz, 1H), 7.87 (dd, J = 8.0 Hz and
0.8 Hz, 1H), 7.49–7.43 (m, 1H), 7.40–7.35 (m, 1H), 4.37 (s, 2H),
4.20 (q, J = 7.2 Hz, 2H), 3.63 (s, 2H), 1.27 (t, J = 7.2 Hz, 3H). ESI-
MS m/z 251 (M+H)+.
6. Experimental
6.1. General
6.2.2. Representative procedure for the synthesis of amines 3a–
i (Scheme 3, path b)
Reagents and enzymes (except HldE, HldA and RK) for biochem-
ical assays were purchased from Sigma–Aldrich. Chemical reagents
were obtained from Sigma–Aldrich, Acros, Alfa Aesar, Maybridge
and solvents from Sigma–Aldrich or VWR, and were used without
further purification. Anhydrous solvents were purchased from
Sigma–Aldrich. Organometallic reagents were purchased from
Strem Chemicals, Inc. All reactions were conducted under argon
Under argon, acetic acid (275 lL, 4.8 mmol) was added to a
solution of thiazole-2-carbaldehyde (500 mg, 4.4 mmol) and gly-
cine ethyl ester hydrochloride (614 mg, 4.4 mmol) in anhydrous
dichloromethane (20 mL). The reaction mixture was stirred for
3 h at room temperature. The solution was then cooled to 0 °C
and sodium cyanoborohydride (418 mg, 6.6 mmol) was added por-
tionwise. After completion of the addition, the reaction mixture

N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
1283
was allowed to warm to room temperature and kept stirring over-
night. Then water was added at 0 °C and dichloromethane was
removed under reduced pressure. The aqueous solution was
extracted with ethyl acetate. The combined organic extracts were
washed with water, brine, dried over sodium sulfate, filtered and
evaporated. Purification by flash chromatography on silica gel
eluted with a gradient of methanol in dichloromethane (0–5%)
led to ethyl [(1,3-thiazol-2-ylmethyl)amino]acetate (3g, 120 mg,
14%) as a yellow oil. 1H NMR (CDCl3): 7.86 (d, J = 4.0 Hz, 1H),
7.30 (d, J = 4.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 4.2 (s, 2H), 3.50 (s,
2H), 1.31 (t, J = 7.2 Hz, 3H). ESI-MS m/z 201 (M+H)+.
(m, 2H of minor rotamer and 1H of both rotamers), 7.18–7.12
(m, 1H, minor rotamer), 7.02 (dd, J = 8.1 Hz and 1.8 Hz, 1H, major
rotamer), 6.95–6.92 (m, 1H, minor rotamer), 5.40 (s, 2H, minor rot-
amer), 5.24 (s, 2H, major rotamer), 4.61 (s, 2H, major rotamer), 4.36
(s, 2H, minor rotamer), 3.87 (s, 3H, major rotamer), 3.60 (s, 3H,
minor rotamer), 2.57 (3H). ESI-MS m/z 438 (M+H)+.
6.2.4. [{[2-(3-methoxyphenyl)-4-methyl-1,3-oxazol-5-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (7a)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 2-(3-methoxyphenyl)-4-methyl-
1,3-oxazole-5-carboxylic acid (following the procedure depicted
in Section 6.2.3). 1H NMR (DMSO-d6), 2 rotamers in a 2/1 ratio,
each chemical shift is for both rotamers except when stated: 8.74
(d, J = 5.2 Hz, 1H, major rotamer), 8.71 (d, J = 5.2 Hz, 1H, minor rot-
amer), 8.30–8.26 (m, 1H, major rotamer), 8.15–8.11 (m, 1H, minor
rotamer), 7.86 (d, J = 8.0 Hz, 1H, major rotamer), 7.76 (d, J = 8.0 Hz,
1H, minor rotamer), 7.72 (t, J = 6.4 Hz, 1H, major rotamer), 7.62–
7.60 (m, 1H), 7.51 (br s, 1H, major rotamer), 7.48 (t, J = 7.8 Hz,
1H, major rotamer), 7.29 (t, J = 7.8 Hz, 1H, minor rotamer), 7.17
(dd, J = 8.4 Hz and J = 2.0 Hz, 1H, major rotamer), 7.07 (dd,
J = 8.4 Hz and J = 2.0 Hz, 1H, minor rotamer), 7.00 (br s, 1H, minor
rotamer), 6.93 (d, J = 7.6 Hz, 1H, minor rotamer), 5.14 (s, 2H, minor
rotamer), 4.98 (s, 2H, major rotamer), 4.67 (s, 2H, major rotamer),
4.20 (s, 2H, minor rotamer), 3.85 (s, 3H, major rotamer), 3.72 (s, 3H,
minor rotamer), 2.42 (s, 3H). ESI-MS m/z 382 (M+H)+.
6.2.3. Representative procedure for the synthesis of compounds
7a–c (Scheme 4). ((1,3-benzothiazol-2-ylmethyl){[2-(3-
methoxyphenyl)-4-methyl-1,3-oxazol-5-
yl]carbonyl}amino)acetic acid (7c)
A solution of ethyl 2-chloroacetoacetate (1.45 mL, 10 mmol)
and 3-methoxybenzamide (1.55 g, 10 mmol) in anhydrous toluene
(3 mL) was stirred at 120 °C for 2 h, then at 140 °C for 2 h and at
120 °C overnight. An aqueous solution of ammonium chloride
was added at room temperature and the reaction mixture was
extracted with ethyl acetate. The combined organic extracts were
dried over sodium sulfate, filtered and evaporated. Purification
by flash chromatography on silica gel with a gradient of ethyl ace-
tate in cyclohexane (5–30%) led to ethyl 2-(3-methoxyphenyl)-4-
methyl-1,3-oxazole-5-carboxylate (1.28 g, 48%) as a white solid.
ESI-MS m/z 262 (M+H)+.
A solution of ethyl 2-(3-methoxyphenyl)-4-methyl-1,3-oxazole-
5-carboxylate (1.0 g, 3.83 mmol) and lithium hydroxide (275 mg,
11.5 mmol) in water (10 mL) and THF (10 mL) was stirred at room
temperature overnight. Then 1 N HCl was added and the reaction
mixture was extracted with diethyl ether and ethyl acetate. The
combined organic extracts were dried over sodium sulfate, filtered
and evaporated. The solid obtained was washed with 5% ethyl ace-
tate in cyclohexane and dried to give 2-(3-methoxyphenyl)-4-
methyl-1,3-oxazole-5-carboxylic acid (845 mg, 94%) as a white
solid. ESI-MS m/z 234 (M+H)+.
Under argon, ethyl 1,3-benzothiazol-2-ylmethyl)amino]acetate
(3i, 53.9 mg, 0.22 mmol) was added to a solution of 2-(3-methoxy-
phenyl)-4-methyl-1,3-oxazole-5-carboxylic acid (38.6 mg, 0.17 mmol),
EDAC (64 mg, 0.33 mmol) and 4-DMAP (62 mg, 0.51 mmol) in
anhydrous dichloromethane (3 mL). The resulting mixture was
stirred at room temperature for 5 h and then at 50 °C overnight.
Water was added and the reaction mixture was extracted with
dichloromethane. Combined organic extracts were washed with
1 N HCl, then dried over sodium sulfate, filtered and evaporated.
The crude product was purified by preparative TLC eluted with
5% methanol in dichloromethane to afford ethyl ((1,3-ben-
zothiazol-2-ylmethyl){[2-(3-methoxyphenyl)-4-methyl-1,3-oxa-
zol-5-yl]carbonyl}amino)acetate (57.7 mg, 75%) as a pale yellow
oil. ESI-MS m/z 466 (M+H)+.
6.2.5. {(2-aminobenzyl)[(4-methyl-2-phenyl-1,3-oxazol-5-
yl)carbonyl]amino}acetic acid (7b)
This compound was prepared from ethyl [(2-aminobenzyl)
amino]acetate (synthesized according to the procedure described
in Section 6.2.1) and 4-methyl-2-phenyl-1,3-oxazole-5-carboxylic
acid (synthesized according to the procedure described in Section
6.2.3). 1H NMR (DMSO-d6), 2 rotamers in a roughly 3/1 ratio, each
chemical shift is for both rotamers except when stated: 8.31–8.30
(m, 1H, minor rotamer), 8.07–8.03 (m, 1H of both rotamers and 1H
of major rotamer), 7.57–7.35 (m, 3H), 7.01–6.98 (m, 2H), 6.64–6.50
(m, 2H), 5.28 (br s, 2H, minor rotamer), 4.59 (br s, 2H, major rot-
amer), 3.81 (br s, 2H, major rotamer), 3.33 (2H of minor rotamer
under water peak), 2.40 (s, 3H, major rotamer), 2.34 (s, 3H, minor
rotamer). ESI-MS m/z 366 (M+H)+.
6.2.6. Representative procedure for the synthesis of compounds
5a–t (Scheme 4). [{[2-(3-Bromophenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5h)
Methyl 2-aminoacetoacetate hydrochloride (462 mg, 2.75
mmol) was added portionwise to a solution of 3-bromobenzoyl
chloride (400
stirred under argon at room temperature. Then triethylamine
(835 L, 6 mmol) was added dropwise. The reaction mixture was
lL, 3 mmol) in anhydrous dichloromethane (10 mL)
l
Lithium hydroxide (14.8 mg, 0.62 mmol) was added to a solu-
tion of ethyl ((1,3-benzothiazol-2-ylmethyl){[2-(3-methoxyphenyl)-
4-methyl-1,3-oxazol-5-yl]carbonyl} amino)acetate (57.7 mg, 0.12
mmol) in THF (1 mL) and water (1 mL). The reaction mixture was
stirred at room temperature overnight. Then 1 N HCl was added
and the reaction mixture was extracted with diethyl ether and
ethyl acetate. The combined organic extracts were dried over so-
dium sulfate, filtered and evaporated. Crystallization in a mixture
of dichloromethane and cyclohexane led to a solid which was fil-
tered and dried to give 7c (51 mg, 94%) as a pale yellow solid. 1H
NMR (CDCl3), 2 rotamers in a roughly 1/3 ratio, each chemical shift
is for both rotamers except when stated: 8.04 (d, J = 8.4 Hz, 1H,
minor rotamer), 7.99 (d, J = 8.1 Hz, 1H, major rotamer), 7.89 (d,
J = 7.5 Hz, 1H, major rotamer), 7.70 (d, J = 7.8 Hz, 1H, major rot-
amer), 7.62 (s, 1H, major rotamer), 7.55–7.41 (m, 2H), 7.37–7.26
stirred at room temperature overnight. Dichloromethane was
added and the organic layer was successively washed with 1 N
HCl, saturated NaHCO3 and water. The organic layer was dried over
sodium sulfate, filtered and evaporated. Purification by flash chro-
matography on silica gel eluted with a gradient of ethyl acetate in
dichloromethane (0–5%) led to methyl 2-[(3-bromobenzoyl)
amino]-3-oxobutanoate (454.9 mg, 53%) as a white solid. ESI-MS
m/z 314 and 316 (M+H)+.
To a solution of triphenylphosphine (760 mg, 2.90 mmol) and
iodine (736 mg, 2.90 mmol) in anhydrous dichloromethane (85 mL)
stirred under argon at room temperature were added triethyl-
amine (805 lL, 5.80 mmol) and a solution of methyl 2-[(3-bromo-
benzoyl)amino]-3-oxobutanoate (454.9 mg, 1.45 mmol) in dichlo-
romethane (15 mL). The resulting mixture was stirred at room
temperature overnight. Then the organic solution was successively

1284
N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
washed with saturated Na2S2O3, water, NH4Cl and water again. The
organic layer was dried over sodium sulfate, filtered and evapo-
rated. Purification by flash chromatography on silica gel eluted
with a gradient of ethyl acetate in dichloromethane (0–5%) gave
methyl 2-(3-bromophenyl)-5-methyl-1,3-oxazole-4-carboxylate
(344.6 mg, 80%) as a yellow solid. ESI-MS m/z 296 and 298 (M+H)+.
The remaining steps (saponification, coupling with amine and
saponification) were performed following the procedure described
in Section 6.2.3. Purification by preparative HPLC afforded 5h as its
trifluoroacetic salt. 1H NMR (CD3OD), 2 rotamers in a roughly 1/2
ratio, each chemical shift is for both rotamers except when stated:
8.73–8.68 (m, 1H), 8.38–8.32 (m, 1H), 8.17 (s, 1H, major rotamer),
8.01–7.95 (m, 2H, minor rotamer, 1H, both rotamers), 7.81–7.75
(m, 1H), 7.68–7.60 (m, 1H, major rotamer, 1H, both rotamers),
7.44 (t, J = 8.0 Hz, 1H, major rotamer), 7.35 (t, J = 8.0 Hz, 1H, minor
rotamer), 5.39 (s, 2H, minor rotamer), 5.03 (s, 2H, major rotamer),
4.83 (s, 2H, major rotamer), 4.35 (s, 2H, minor rotamer), 2.66 (s, 3H,
minor rotamer), 2.64 (s, 3H, major rotamer). ESI-MS m/z 430 and
432 (M+H)+.
described in Section 6.2.1) and 5-methyl-2-[2-(trifluoro-
methyl)phenyl]-1,3-oxazole-4-carboxylic acid (prepared according
to the procedure described in Section 6.2.6). 1H NMR (CDCl3), 2
rotamers in a roughly 2/3 ratio, each chemical shift is for both rota-
mers except when stated: 8.56 (br s, 1H), 8.16 (d, J = 8.0 Hz, 1H,
major rotamer), 8.03–7.95 (m, 1H), 7.80–7.45 (m, 1H of minor rot-
amer and 5H of both rotamers), 5.64 (s, 2H, minor rotamer), 5.01 (s,
2H, major rotamer), 4.89 (s, 2H, major rotamer), 4.28 (s, 2H, minor
rotamer), 2.66 (s, 3H, minor rotamer), 2.63 (s, 3H, major rotamer).
ESI-MS m/z 420 (M+H)+.
6.2.10. [{[2-(2-Bromophenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5d)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 2-(2-bromophenyl)-5-methyl-1,3-
oxazole-4-carboxylic acid (prepared according to the procedure
described in Section 6.2.6). 1H NMR (CDCl3), 2 rotamers in a
roughly 1/1 ratio, each chemical shift is for both rotamers except
when stated: 8.48–8.43 (m, 1H), 8.02 (d, J = 8.0 Hz, 1H, one rot-
amer), 7.82 (t, J = 8.0 Hz, 1H, one rotamer), 7.70–7.59 (m, 2H),
7.51–7.38 (m, 1H of one rotamer and 1H of both rotamers), 7.35–
7.16 (m, 1H of one rotamer and 2H of both rotamers), 5.39 (s,
2H, one rotamer), 4.85 (s, 2H, one rotamer), 4.78 (s, 2H, one rot-
amer), 4.08 (s, 2H, one rotamer), 2.61 (s, 3H, one rotamer), 2.56
(s, 3H, one rotamer). ESI-MS m/z 430 and 432 (M+H)+.
6.2.7. [{[5-Methyl-2-(2-nitrophenyl)-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5a)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 5-methyl-2-(2-nitrophenyl)-1,3-
oxazole-4-carboxylic acid (prepared according to the procedure
described in Section 6.2.6). 1H NMR (CDCl3), 2 rotamers in a
roughly 2/3 ratio, each chemical shift is for both rotamers except
when stated: 8.54–8.53 (m, 1H), 8.13 (d, J = 8.0 Hz, 1H, major rot-
amer), 7.95 (t, J = 8.0 Hz, 1H), 7.83–7.79 (m, 1H), 7.74–7.56 (m,
3H of both rotamers and 1H of minor rotamer), 7.45–7.42 (m,
1H), 5.46 (s, 2H, minor rotamer), 4.93 (s, 2H, major rotamer),
4,79 (s, 2H, major rotamer), 4.24 (s, 2H, minor rotamer), 2.64 (s,
3H, minor rotamer), 2.57 (s, 3H, major rotamer). ESI-MS m/z 397
(M+H)+.
6.2.11. [{[5-Methyl-2-(3-nitrophenyl)-1,3-oxazol-4-yl]
carbonyl}(pyridin-2-ylmethyl) amino]acetic acid (5e)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 5-methyl-2-(3-nitrophenyl)-1,3-
oxazole-4-carboxylic acid (prepared according to the procedure
described in Section 6.2.6).
1H NMR (CD3OD), 2 rotamers in a roughly 1/1 ratio, each chem-
ical shift is for both rotamers except when stated: 8.76 (br s, 1H,
one rotamer), 8.56–8.52 (m, 1H), 8.42 (br s, 1H, one rotamer),
8.39 (d, J = 8.0 Hz, 1H, one rotamer), 8.34–8.27 (m, 1H), 8.11 (d,
J = 7.2 Hz, 1H, one rotamer), 7.95–7.87 (m, 1H), 7.75 (t, J = 8.0 Hz,
1H, one rotamer), 7.69 (t, J = 8.0 Hz, 1H, one rotamer), 7.65 (d,
J = 8.0 Hz, 1H, one rotamer), 7.55 (d, J = 8.0 Hz, 1H, one rotamer),
7.41–7.35 (m, 1H), 5.26 (s, 2H, one rotamer), 4.93 (s, 2H, one rot-
amer), 4.61 (s, 2H, one rotamer), 4.23 (s, 2H, one rotamer), 2.67
(s, 3H). ESI-MS m/z 397 (M+H)+.
6.2.8. [{[2-(2-Aminophenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5b)
To a solution of [{[5-methyl-2-(2-nitrophenyl)-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5a, 40 mg,
0.1 mmol) in anhydrous methanol (4 mL), was added ferric chlo-
ride (2 mg, 5% by weight) and activated charcoal (2 mg, 5% by
weight). The reaction mixture was heated to 65 °C. Hydrazine hy-
drate (40 mg, 0.8 mmol) was added dropwise. The reaction mix-
ture was refluxed overnight and then cooled to room
temperature. Then the reaction mixture was filtered through a
pad of Celite and the filtrate was concentrated. Purification of the
crude product by preparative HPLC afforded 5b as its trifluoroace-
tic acid salt (15 mg, 31%). 1H NMR (CD3OD), 2 rotamers in a 1/2
ratio, each chemical shift is for both rotamers except when stated:
8.83–8.78 (m, 1H), 8.56–8.52 (m, 1H), 8.19 (d, J = 8.0 Hz, 1H, major
rotamer), 8.13 (d, J = 8.0 Hz, 1H, minor rotamer), 7.97–7.92 (m, 1H),
7.79 (d, J = 8.0 Hz, 1H, major rotamer), 7.68 (d, J = 8.0 Hz, 1H, minor
rotamer), 7.24 (t, J = 8.0 Hz, 1H, major rotamer), 7.15 (t, J = 8.0 Hz,
1H, minor rotamer), 6.91 (d, J = 8.0 Hz, 1H, major rotamer), 6.78
(t, J = 8.0 Hz, 1H, major rotamer), 6.72–6.68 (m, 2H, minor rot-
amer), 5.42 (s, 2H, minor rotamer), 5.08 (s, 2H, major rotamer),
4.87 (s, 2H, major rotamer), 4.31 (s, 2H, minor rotamer), 2.63 (s,
3H, minor rotamer), 2.59 (s, 3H, major rotamer). ESI-MS m/z 367
(M+H)+.
6.2.12. [{[2-(3-Aminophenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5f)
This compound was synthesized from 5e following the proce-
dure described in Section 6.2.8 for the synthesis of 5b. Purification
by preparative HPLC afforded 5f as its trifluoroacetic acid salt. 1H
NMR (CD3OD), 2 rotamers in a roughly 2/1 ratio, each chemical
shift is for both rotamers except when stated: 8.78–8.76 (m, 1H),
8.49–8.45 (m, 1H), 8.12–8.10 (m, 1H), 7.93–7.85 (m, 2H), 7.59–
7.42 (m, 3H), 5.49 (s, 2H, minor rotamer), 5.08 (s, 2H, major rot-
amer), 4.89 (s, 2H, major rotamer), 4.35 (s, 2H, minor rotamer),
2.66 (s, 3H, minor rotamer), 2.63 (s, 3H, major rotamer). ESI-MS
m/z 367 (M+H)+.
6.2.13. [({5-Methyl-2-[3-(trifluoromethyl)phenyl]-1,3-oxazol-4-
yl}carbonyl)(pyridin-2-ylmethyl)amino]acetic acid (5g)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 5-methyl-2-[3-(trifluoro-
methyl)phenyl]-1,3-oxazole-4-carboxylic acid (prepared according
to the procedure described in Section 6.2.6). Purification by pre-
parative HPLC afforded 5g as its trifluoroacetic acid salt. 1H NMR
6.2.9. [({5-Methyl-2-[2-(trifluoromethyl)phenyl]-1,3-oxazol-4-
yl}carbonyl)(pyridin-2-yl -methyl)amino]acetic acid (5c)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure

N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
1285
(CDCl3), 2 rotamers in a roughly 1/3 ratio, each chemical shift is for
6.2.18. [{[2-(4-Bromophenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5m)
both rotamers except when stated: 8.74 (br s, 1H), 8.30–8.25 (m,
1H), 8.20–8.15 (m, 2H, major rotamer), 8.02–7.95 (m, 1H), 7.85–
7.52 (m, 3H of both rotamers and 2H of minor rotamer), 5.56 (s,
2H, minor rotamer), 5.10 (s, 2H, major rotamer), 4.90 (s, 2H, major
rotamer), 4.34 (s, 2H, minor rotamer), 2.64 (s, 3H, minor rotamer),
2.61 (s, 3H, major rotamer). ESI-MS m/z 420 (M+H)+.
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 2-(4-bromophenyl)-5-methyl-1,3-
oxazole-4-carboxylic acid (prepared according to the procedure
described in Section 6.2.6). 1H NMR (CDCl3), 2 rotamers in a
roughly 1/3 ratio, each chemical shift is for both rotamers except
when stated: 8.51–8.50 (m, 1H), 7.93–7.89 (m, 3H), 7.60–7.53
(m, 3H), 7.42–7.37 (m, 1H), 5.50 (s, 2H, minor rotamer), 4.89 (s,
2H, major rotamer), 4.75 (s, 2H, major rotamer), 4.29 (s, 2H, minor
rotamer), 2.61 (s, 3H). ESI-MS m/z 430 and 432 (M+H)+.
6.2.14. [{[2-(3-Methoxyphenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5i)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 2-(3-methoxyphenyl)-5-methyl-
1,3-oxazole-4-carboxylic acid (prepared according to the procedure
described in Section 6.2.6). 1H NMR (CDCl3), 2 rotamers in a 1/3 ratio,
each chemical shift is for both rotamers except when stated: 8.52–
8.48 (m, 1H), 7.88–7.83 (m, 1H), 7.64 (s, 1H), 7.59 (d, J = 8.0 Hz,
1H), 7.51 (d, J = 8.0 Hz, 1H), 7.37–7.30 (m, 2H), 7.01–6.98 (m, 1H),
5.47 (s, 2H, minor rotamer), 4.86 (s, 2H, major rotamer), 4.72 (s,
2H, major rotamer), 4.27 (s, 2H, minor rotamer), 3.94 (s, 3H, major
rotamer), 3.83 (s, 3H, minor rotamer), 2.63 (s, 3H, minor rotamer),
2.61 (s, 3H, major rotamer). ESI-MS m/z 382 (M+H)+
6.2.19. [{[2-(4-Methoxyphenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5n)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 2-(4-methoxyphenyl)-5-methyl-
1,3-oxazole-4-carboxylic acid (prepared according to the proce-
dure described in Section 6.2.6). 1H NMR (CDCl3), 2 rotamers in a
roughly 1/3 ratio, each chemical shift is for both rotamers except
when stated: 8.47–8.46 (m, 1H), 7.94 (d, J = 8.0 Hz, 2H, major rot-
amer), 7.82 (t, J = 8.0 Hz, 1H, major rotamer), 7.74 (t, J = 8.0 Hz,
1H, minor rotamer), 7.65 (d, J = 8.0 Hz, 2H, minor rotamer), 7.49–
7.46 (m, 1H), 7.32–7.23 (m, 1H), 6.94 (d, J = 8.0 Hz, 2H, major rot-
amer), 6.86 (d, J = 8.0 Hz, 2H, minor rotamer), 5.38 (s, 2H, minor
rotamer), 4.84 (s, 2H, major rotamer), 4.70 (s, 2H, major rotamer),
4.17 (s, 2H, minor rotamer), 3.83 (s, 3H, major rotamer), 3.81 (s, 3H,
minor rotamer), 2.57 (s, 3H, major rotamer), 2.54 (s, 3H, minor rot-
amer). ESI-MS m/z 382 (M+H)+.
6.2.15. [{[5-Methyl-2-(4-nitrophenyl)-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5j)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 5-methyl-2-(4-nitrophenyl)-1,3-
oxazole-4-carboxylic acid (prepared according to the procedure
described in Section 6.2.6). 1H NMR (CD3OD), 2 rotamers in a
roughly 2/1 ratio, each chemical shift is for both rotamers except
when stated: 8.83–8.78 (m, 1H), 8.52–8.49 (m, 1H), 8.39–8.12
(m, 4H of both rotamers and 1H of minor rotamer), 7.95–7.90
(m, 1H), 7.84–7.81 (m, 1H, major rotamer), 5.49 (s, 2H, minor rot-
amer), 5.10 (s, 2H, major rotamer), 4.85 (s, 2H, major rotamer), 4.37
(s, 2H, minor rotamer), 2.71 (s, 3H, minor rotamer), 2.67 (s, 3H,
major rotamer). ESI-MS m/z 397 (M+H)+.
6.2.20. [[(5-Methyl-2-phenyl-1,3-oxazol-4-yl)carbonyl](pyridin-
3-ylmethyl)amino]acetic acid (5o)
This compound was prepared from ethyl [(pyridin-3-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 5-methyl-2-phenyl-1,3-oxazole-4-
carboxylic acid (prepared according to the procedure described in
Section 6.2.6). 1H NMR (CD3OD), 2 rotamers in a roughly 1/1 ratio,
each chemical shift is for both rotamers except when stated: 8.68
(s, 1H, one rotamer), 8.59 (s, 1H, one rotamer), 8.51–8.47 (m, 1H),
8.03–8.01 (m, 3H, one rotamer), 7.90 (d, J = 7.6 Hz, 1H, one rotamer),
7.85 (d, J = 7.2 Hz, 2H, one rotamer), 7.49–7.44 (m, 4H), 5.19 (s, 2H,
one rotamer), 4.85 (s, 2H, one rotamer), 4.48 (s, 2H, one rotamer),
3.98 (s, 2H, one rotamer), 2.65 (s, 3H). ESI-MS m/z 352 (M+H)+.
6.2.16. [{[2-(4-Aminophenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (5k)
This compound was synthesized from 5j following the proce-
dure described in Section 6.2.8 for the synthesis of 5b. 1H NMR
(CD3OD), 2 rotamers in a roughly 3/2 ratio, each chemical shift is
for both rotamers except when stated: 8.53 (br s, 1H), 7.95–7.91
(m, 1H), 7.73 (d, J = 8.0 Hz, 2H, major rotamer), 7.65 (d, J = 8.0 Hz,
1H, minor rotamer), 7.59 (d, J = 8.0 Hz, 1H, major rotamer), 7.49
(d, J = 8.0 Hz, 2H, minor rotamer), 7.43–7.39 (m, 1H), 6.72 (d,
J = 8.0 Hz, 2H, major rotamer), 6.65 (d, J = 8.0 Hz, 2H, minor rot-
amer), 5.32 (s, 2H, minor rotamer), 4.87 (s, 2H, major rotamer),
4.70 (s, 2H, major rotamer), 4.24 (s, 2H, minor rotamer), 2.58 (s,
3H, major rotamer), 2.56 (s, 3H, minor rotamer). ESI-MS m/z 367
(M+H)+.
6.2.21. [[(5-Methyl-2-phenyl-1,3-oxazol-4-yl)carbonyl](pyridin-
4-ylmethyl)amino]acetic acid (5p)
This compound was prepared from ethyl [(pyridin-4-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 5-methyl-2-phenyl-1,3-oxazole-4-
carboxylic acid (prepared according to the procedure described in
Section 6.2.6). 1H NMR (CD3OD), 2 rotamers in a roughly 1/1 ratio,
each chemical shift is for both rotamers except when stated: 8.55–
5.52 (m, 2H), 8.04–8.03 (m, 2H, one rotamer), 7.77 (d, J = 7.2 Hz,
2H, one rotamer), 7.51–7.40 (m, 5H), 5.23 (s, 2H, one rotamer),
4.87 (2H of one rotamer under water peak), 4.59 (s, 2H, one rotamer),
4.08 (s, 2H, one rotamer), 2.66 (s, 3H). ESI-MS m/z 352 (M+H)+.
6.2.17. [({5-Methyl-2-[4-(trifluoromethyl)phenyl]-1,3-oxazol-4-
yl}carbonyl)(pyridin-2-ylmethyl)amino]acetic acid (5l)
This compound was prepared from ethyl [(pyridin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.1) and 5-methyl-2-[4-(trifluoro-
methyl)phenyl]-1,3-oxazole-4-carboxylic acid (prepared according
to the procedure described in Section 6.2.6). 1H NMR (CDCl3) 2
rotamers in a roughly 1/3 ratio, each chemical shift is for both rota-
mers except when stated: 8.77 (br s, 1H), 8.38–8.32 (m, 1H), 8.16–
8.14 (m, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.85–7.75 (m, 1H), 7.67 (d,
J = 8.0 Hz, 2H), 5.82 (s, 2H, minor rotamer), 5.35 (s, 2H, major rot-
amer), 5.10 (s, 2H, major rotamer), 4.42 (s, 2H, minor rotamer),
2.70 (s, 3H). ESI-MS m/z 420 (M+H)+.
6.2.22. {Benzyl[(5-methyl-2-phenyl-1,3-oxazol-4-
yl)carbonyl]amino}acetic acid (5q)
This compound was prepared from ethyl (benzylamino)acetate
(synthesized according to the procedure described in Section 6.2.1)
and 5-methyl-2-phenyl-1,3-oxazole-4-carboxylic acid (prepared
according to the procedure described in Section 6.2.6). 1H NMR
(acetone-d6), 2 rotamers in a roughly 2/1 ratio, each chemical shift
is for both rotamers except when stated: 7.89–7.86 (m, 2H, major

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N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
rotamer), 7.82–7.79 (m, 2H, minor rotamer), 7.40–7.15 (m, 8H),
5.12 (s, 2H, minor rotamer), 4.68 (s, 2H, major rotamer), 4.46 (s,
2H, major rotamer), 3.93 (s, 2H, minor rotamer), 2.55 (s, 3H, major
rotamer), 2.51 (s, 3H, minor rotamer). ESI-MS m/z 351 (M+H)+.
8.47 (s, 1H), 7.82 (br s, 1H), 7.59–7.46 (m, 2H), 7.31–7.16 (m, 3H),
7.06–7.02 (m, 1H), 5.17 (s, 2H, one rotamer), 4.87 (2H of one rot-
amer under water peak), 4.47 (s, 2H, one rotamer), 4.07 (s, 2H,
one rotamer), 3.81 (s, 4H), 3.18 (s, 4H, one rotamer), 3.01 (s, 4H,
one rotamer), 2.58 (s, 3H). ESI-MS m/z 437 (M+H)+.
6.2.23. {(2-Furylmethyl)[(5-methyl-2-phenyl-1,3-oxazol-4-
yl)carbonyl]amino}acetic acid (5r)
6.2.27. {[(2-{3-[(Cyclopropylcarbonyl)amino]phenyl}-5-methyl-
1,3-oxazol-4-yl)carbonyl](pyridin-2-ylmethyl)amino}acetic
acid (5w)
This compound was prepared from ethyl [(2-furylmeth-
yl)amino]acetate (synthesized according to the procedure
described in Section 6.2.2) and 5-methyl-2-phenyl-1,3-oxazole-4-
carboxylic acid (prepared according to the procedure described in
Section 6.2.6). 1H NMR (DMSO-d6), 2 rotamers in a roughly 2/1
ratio, each chemical shift is for both rotamers except when stated:
7.94 (br s, 2H), 7.56–7.52 (m, 4H), 6.40 (s, 2H), 5.15 (s, 2H, minor
rotamer), 4.71 (s, 2H, major rotamer), 4.51 (s, 2H, major rotamer),
4.07 (s, 2H, minor rotamer), 2.61 (s, 3H, major rotamer), 2.58 (s, 3H,
minor rotamer). ESI-MS m/z 341 (M+H)+.
Under argon, cyclopropanecarbonyl chloride (17 lL, 0.2 mmol)
was added to a solution of ethyl [{[2-(3-aminophenyl)-5-methyl-
1,3-oxazol-4-yl]carbonyl}(pyridin-2-ylmethyl)amino]acetate (52 mg,
0.13 mmol, prepared as in Section 6.2.12) and triethylamine
(54 lL, 0.4 mmol) in anhydrous dichloromethane (2 mL) cooled
to 0 °C. The reaction mixture was allowed to warm to room tem-
perature and stirred at room temperature for 30 min. The
reaction was quenched with water. The layers were separated
and the aqueous layer was extracted with dichloromethane. The
combined organic layers were washed with brine, dried over
anhydrous sodium sulfate, filtered and concentrated. The crude
product was purified by flash chromatography on silica gel eluted
with a gradient of methanol in dichloromethane (0–10%) to obtain
ethyl {[(2-{3-[(cyclopropylcarbonyl)amino]phenyl}-5-methyl-1,3-
oxazol-4-yl)carbonyl](pyridin-2-ylmethyl)amino}acetate (31 mg,
51%) as a colorless oil. ESI-MS m/z 463 (M+H)+.
Saponification of ethyl {[(2-{3-[(cyclopropylcarbonyl) amino]-
phenyl}-5-methyl-1,3-oxazol-4-yl)carbonyl] (pyridin-2-ylmethyl)
amino}acetate performed according to the procedure described in
Section 6.2.3 followed by purification by preparative HPLC led to
5w as its trifluoroacetic acid salt. 1H NMR (CD3OD), 2 rotamers in a
roughly 1/1 ratio, each chemical shift is for both rotamers except
when stated: 8.83–8.79 (m, 1H, one rotamer), 8.78–8.72 (m, 1H,
one rotamer), 8.49–8.41 (m, 1H), 8.31 (s, 1H, one rotamer), 8.23 (s,
1H, one rotamer), 8.11–8.07 (m, 1H), 7.89–7.84 (m, 1H), 7.77–7.75
(m, 1H, one rotamer), 7.65–7.63 (m, 1H, one rotamer), 7.46–7.35
(m, 2H), 5.47 (s, 2H, one rotamer), 5.05 (s, 2H, one rotamer), 4.87
(2H under water peak), 4.34 (s, 2H, one rotamer), 2.66 (s, 3H, one rot-
amer), 2.62 (s, 3H, one rotamer), 1.81–1.78 (m, 1H), 1.00 (br s, 4H,
one rotamer), 0.91 (br s, 4H, one rotamer). ESI-MS m/z 435 (M+H)+.
6.2.24. [[(5-Methyl-2-phenyl-1,3-oxazol-4-yl)carbonyl] (1,3-
thiazol-2-ylmethyl)amino]acetic acid (5s)
This compound was prepared from ethyl [(1,3-thiazol-2-
ylmethyl)amino]acetate (3i, synthesized as in Section 6.2.2) and
5-methyl-2-phenyl-1,3-oxazole-4-carboxylic
acid
(prepared
according to the procedure described in Section 6.2.6). 1H NMR
(CDCl3), 2 rotamers in roughly 2/3 ratio, each chemical shift is for
both rotamers except when stated: 8.00–7.97 (m, 2H), 7.81–7.76
(m, 1H), 7.48–7.41 (br s, 4H), 5.66 (s, 2H, minor rotamer), 5.12 (s,
2H, major rotamer), 4.73 (s, 2H, major rotamer), 4.28 (s, 2H, minor
rotamer), 2.72 (s, 3H, minor rotamer), 2.69 (s, 3H, major rotamer).
ESI-MS m/z 358 (M+H)+.
6.2.25. [{[2-(3-Methoxyphenyl)-5-methyl-1,3-oxazol-4-
yl]carbonyl}(quinolin-2-ylmethyl)amino]acetic acid (5t)
This compound was prepared from ethyl [(quinolin-2-
ylmethyl)amino]acetate (synthesized according to the procedure
described in Section 6.2.2) and 5-methyl-2-phenyl-1,3-oxazole-4-
carboxylic acid (prepared according to the procedure described in
Section 6.2.6). 1H NMR (CD3OD), 2 rotamers in a roughly 3/2 ratio,
each chemical shift is for both rotamers except when stated: 9.01–
8.97 (m, 1H), 8.30–8.21 (m, 2H), 8.15–8.07 (m, 2H), 7.92–7.88 (m,
1H), 7.62–7.60 (m, 2H, major rotamer), 7.42 (t, J = 8.0 Hz, 1H, major
rotamer), 7.19 (t, J = 8.0 Hz, 1H, minor rotamer), 7.10–7.06 (m, 1H),
6.95–6.92 (m, 1H, minor rotamer), 6.85 (br s, 1H minor rotamer),
5.64 (s, 2H, minor rotamer), 5.25 (s, 2H, major rotamer), 4.97 (s,
2H, major rotamer), 4.48 (s, 2H, minor rotamer), 3.89 (s, 3H, major
rotamer), 3.58 (s, 3H, minor rotamer), 2.66 (s, 3H, minor rotamer),
2.61 (s, 3H, major rotamer). ESI-MS m/z 432 (M+H)+.
6.2.28. [({2-[3-(Acetylamino)phenyl]-5-methyl-1,3-oxazol-4-
yl}carbonyl)(pyridin-2-ylmethyl)amino]acetic acid (5v)
This compound was prepared following the same procedure as
in Section 6.2.27 using acetyl chloride instead of cyclopropanecar-
bonyl chloride. Final purification by preparative HPLC led to 5v as
its trifluoroacetic acid salt. 1H NMR (CD3OD), 2 rotamers in a
roughly 1/1 ratio, each chemical shift is for both rotamers except
when stated: 8.81–8.80 (m, 1H, one rotamer), 8.72 (br s, 1H, one
rotamer), 8.53–8.43 (m, 1H), 8.25 (br s, 1H, one rotamer), 8.12–
8.09 (m, 1H of both rotamers and 1H of one rotamer), 7.92–7.84
(m, 1H), 7.71–7.69 (m, 1H, one rotamer), 7.56–7.54 (m, 1H, one rot-
amer), 7.40–7.25 (m, 2H), 5.43 (s, 2H, one rotamer), 5.01 (s, 2H, one
rotamer), 4.81 (s, 2H, one rotamer), 4.30 (s, 2H, one rotamer), 2.61
(s, 3H, one rotamer), 2.56 (s, 3H, one rotamer), 2.17 (s, 3H). ESI-MS
m/z 409 (M+H)+.
6.2.26. [({5-Methyl-2-(3-morpholin-4-ylphenyl)-1,3-oxazol-4-
yl}carbonyl)(pyridin-2-ylmethyl)amino]acetic acid (5u)
Under argon, a solution of ethyl 2-(3-bromophenyl)-5-methyl-
1,3-oxazole-4-carboxylate (500 mg, 1.61 mmol, prepared as in Sec-
tion 6.2.6), morpholine (175 lL, 2 mmol), cesium carbonate
(2.25 mmol, 735 mg), Pd2dba3 (29.5 mg, 0.03 mmol), BINAP
(30.1 mg, 0.05 mmol) in degassed toluene (6.5 mL) was stirred at
110 °C for 2 days. The reaction mixture was filtered through a
pad of Celite and evaporated. The crude product was purified by
flash chromatography on silica gel with a gradient of ethyl acetate
in cyclohexane (5–40%) to give ethyl 5-methyl-2-(3-morpholin-4-
ylphenyl)-1,3-oxazole-4-carboxylate (330 mg, 65%). ESI-MS m/z
317 (M+H)+.
The remaining steps (saponification, coupling with amine and
saponification) were performed following the procedure described
in Section 6.2.3. Purification by preparative HPLC afforded 5u as its
trifluoroacetic salt. 1H NMR (CD3OD), 2 rotamers in a roughly 1/1
ratio, each chemical shift is for both rotamers except when stated:
6.2.29. ((1,3-Benzothiazol-2-ylmethyl){[5-methyl-2-(3-(2H-
pyrazol-3-yl)-phenyl)-1,3-oxazol-4-yl]carbonyl}amino)acetic
acid (5x)
Under argon,
a solution of ethyl ((1,3-benzothiazol-2-yl-
methyl){[2-(3-bromo)-phenyl)-5-methyl-1,3-oxazol-4-yl]carbonyl}
amino)acetate (50.5 mg, 0.10 mmol, prepared from ethyl [(1,3-
thiazol-2-ylmethyl)amino]acetate 3i, synthesized as in Section
6.2.2 and 2-(3-bromophenyl)-5-methyl-1,3-oxazole-4-carboxylic
acid synthesized as in Section 6.2.6), 1H-pyrazole-3-boronic acid
(22 mg, 0.20 mmol), sodium carbonate (31.1 mg, 0.29 mmol) and

N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
1287
Pd(PPh3)4 (34 mg, 0.03 mmol) in degassed anhydrous DMF
(0.7 mL) was stirred overnight at 100 °C. Cold HCl 1 N was added
and the solution was extracted with ethyl acetate. The combined
organic extracts were dried over sodium sulfate, filtered and
evaporated. Purification by preparative TLC eluted with 10%
methanol in dichloromethane led to ethyl ((1,3-benzothiazol-2-
ylmethyl){[5-methyl-2-(3-(2H-pyrazol-3-yl)-phenyl)-1,3-oxazol-4
-yl]carbonyl}amino)acetate. The latter compound was dissolved in
THF (1 mL) and water (1 mL). LiOH (9.4 mg, 0.39 mmol) was added
and the reaction mixture was stirred overnight at room tempera-
ture. Then HCl 1N was added and the solution was extracted with
diethyl ether and ethyl acetate. The combined organic extracts
were dried over sodium sulfate, filtered and evaporated. The crude
product was purified by preparative TLC eluted with 10% methanol
in dichloromethane to afford 5x (26.7 mg, 57%) as a white solid. 1H
NMR (DMSO-d6), 2 rotamers in a roughly 2/1 ratio, each chemical
shift is for both rotamers except when stated: 8.38 (br s, 1H, major
rotamer), 8.32 (br s, 1H, minor rotamer), 7.81–7.43 (m, 8H), 6.81
(br s, 1H, major rotamer), 6.60 (br s, 1H, minor rotamer), 5.51 (s,
2H, minor rotamer), 5.07 (s, 2H, major rotamer), 4.68 (br s, 2H,
major rotamer), 4.15 (br s, 2H, minor rotamer), 2.70 (s, 3H, minor
rotamer), 2.64 (s, 3H, major rotamer). ESI-MS m/z 474 (M+H)+.
carboxylic acid, ethyl [(pyridin-2-ylmethyl)amino]acetate (synthe-
sized according to the procedure described in Section 6.2.1) and
3-acetylphenylboronic acid. 1H NMR (CD3OD) 2 rotamers in a 1/1
ratio, each chemical shift is for both rotamers except when stated:
8.46–8.34 (m, 2H), 8.08–7.98 (m, 2H), 7.83–7.71 (m, 1H), 7.55–7.45
and 7.33–7.26 (m, 3H), 4.84–4.80 (m, 2H), 4.20–4.16 (m, 2H), 2.57
(s, 3H, one rotamer), 2.55 (s, 3H, one rotamer), 2.40 (br s, 3H). ESI-
MS m/z 410 (M+H)+.
6.2.32. [{[2-(4-Amino-3-nitrophenyl)-4-methyl-1,3-thiazol-5-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (9b)
According to the procedure described in Section 6.2.30, this
compound was prepared from 2-bromo-4-methyl-1,3-thiazole-5-
carboxylic acid, ethyl [(pyridin-2-ylmethyl)amino]acetate (synthe-
sized according to the procedure described in Section 6.2.1) and
2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline. 1H
NMR (CD3OD): 8.58–8.53 (m, 2H), 7.86–7.82 (m, 2H), 7.40–7.35
(m, 2H), 7.02 (d, J = 8.2 Hz, 1H), 4.87 (2H under water peak),
4.29–4.24 (m, 2H), 2.44 (s, 3H). ESI-MS m/z 428 (M+H)+.
6.2.33. ((1,3-Benzothiazol-2-ylmethyl){[2-(3-methoxy phenyl)-
4-methyl-1,3-thiazol-5-yl]carbonyl}amino)acetic acid (9c)
According to the procedure described in Section 6.2.30, this
compound was prepared from {(1,3-benzothiazol-2-ylmethyl)[(2-
bromo-4-methyl-1,3-thiazol-5-yl)carbonyl]amino}acetate and 3-
methoxyphenylboronic acid. 1H NMR (DMSO-d6): 8.12 (d,
J = 8.0 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.53–7.40 (m, 5H), 7.10–
7.08 (m, 1H), 5.10 (br s, 2H), 4.27–4.17 (m, 2H), 3.83 (br s, 3H),
2.48 (s, 3H). ESI-MS m/z 454 (M+H)+.
6.2.30. Representative procedure for the synthesis of
compounds 9a–h (Scheme 6). ((1,3-Benzothiazol-2-
ylmethyl){[2-(1H-indol-5-yl)-4-methyl-1,3-thiazol-5-
yl]carbonyl}amino)acetic acid (9h)
Under argon, to a solution of 2-bromo-4-methyl-1,3-thiazole-5-
carboxylic acid (80.5 mg, 0.36 mmol) in anhydrous dichlorometh-
ane (2 mL) at 0 °C, were successively added a solution of oxalyl
bromide (2 M solution in dichloromethane, 190
l
L, 0.38 mmol)
6.2.34. ((1,3-Benzothiazol-2-ylmethyl){[4-methyl-2-(3-
and N,N-dimethylformamide (1 drop). The reaction mixture was
stirred for 2 h allowing the temperature to rise to room
temperature. Then at 0 °C, a solution of ethyl [(1,3-benzothiazol-
2-ylmethyl)amino]acetate (3i, 86.9 mg, 0.35 mmol) in dichloro-
methane (1 mL) was added followed by N,N-diisopropylethylamine
(0.2 mL, 1.1 mmol). The resulting mixture was stirred overnight
allowing the temperature to rise to room temperature. Water
was added and the reaction mixture was extracted with
dichloromethane. The combined organic extracts were dried over
sodium sulfate, filtered and evaporated. The crude product was
purified by flash chromatography on silica gel eluted with a
gradient of ethyl acetate in cyclohexane (0–30%) to afford ethyl
{(1,3-benzothiazol-2-ylmethyl)[(2-bromo-4-methyl-1,3-thiazol-5-
yl)carbonyl]amino}acetate (124.5 mg, 79%) as a yellow oil. ESI-MS
m/z 454 and 456 (M+H)+.
Under argon, a solution of {(1,3-benzothiazol-2-ylmethyl)[(2-
bromo-4-methyl-1,3-thiazol-5-yl)carbonyl]amino}acetate (35.2mg,
0.077 mmol), tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-y)-1H-indole-1-carboxylate (35.6 mg, 0.10 mmol), cesium car-
bonate (50.5 mg, 0.15 mmol), and PdCl2dppf (3.2 mg, 0.004 mmol)
in degassed 1,4-dioxane (0.5 mL) and water (0.15 mL) was stirred
at 110 °C for 2 days. The reaction mixture was filtered through a
bed of Celite and rinsed with dichloromethane, methanol and ethyl
acetate. The solvents were evaporated and the crude product was
purified by preparative TLC eluted with 10% methanol in dichloro-
methane to give 9h (9.6 mg, 27%) as a beige solid. 1H NMR
(CD3OD): 8.23–8.13 (m, 1H), 8.04 (t, J = 7.2 Hz, 2H), 7.75–7.65 (m,
1H), 7.85 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.37 (d,
J = 2.4 Hz, 1H), 6.60 (br s, 1H), 5.26–5.18 (m, 2H), 4.87 (2H under
water peak), 2.58 (br s, 3H). ESI-MS m/z 463 (M+H)+.
nitrophenyl)-1,3-thiazol-5-yl]carbonyl}amino)acetic acid (9d)
According to the procedure described in Section 6.2.30, this
compound was prepared from {(1,3-benzothiazol-2-ylmethyl)[(2-
bromo-4-methyl-1,3-thiazol-5-yl)carbonyl]amino}acetate and 3-
nitrophenylboronic acid. 1H NMR (CD3OD): 8.85–8.75 (m, 1H),
8.41–8.26 (m, 2H), 8.05 (t, J = 8.6 Hz, 2H), 7.82–7.75 (m, 1H), 7.59
(t, J = 7.4 Hz, 1 H), 7.51 (t, J = 7.6 Hz, 1H), 5.27–5.20 (m, 2H), 4.87
(2H under water peak), 2.63 (br s, 3H). ESI-MS m/z 469 (M+H)+.
6.2.35. ((1,3-Benzothiazol-2-ylmethyl){[2-(3-methanesulfonyl-
phenyl)-4-methyl-1,3-thiazol-5-yl]carbonyl}amino)acetic acid
(9e)
According to the procedure described in Section 6.2.30, this
compound was prepared from {(1,3-benzothiazol-2-ylmethyl)[(2-bro-
mo-4-methyl-1,3-thiazol-5-yl)carbonyl]amino}acetate and 3-(meth-
anesulfonyl)phenylboronic acid. 1H NMR (CDCl3): 8.44–8.36 (m, 1H),
8.06–7.75 (m, 4H), 7.60–7-30 (m, 3H), 5.25–5.05 (m, 2H), 4.35–4.15
(m, 2H), 3.09 (br s, 3H), 2.57 (br s, 3H). ESI-MS m/z 502 (M+H)+.
6.2.36. ((1,3-Benzothiazol-2-ylmethyl){[2-(4-amino-3-methoxy
phenyl)-4-methyl-1,3-thiazol-5-yl]carbonyl}amino)acetic acid
acid (9f)
According to the procedure described in Section 6.2.30, this
compound was prepared from {(1,3-benzothiazol-2-ylmethyl)[(2-
bromo-4-methyl-1,3-thiazol-5-yl)carbonyl]amino}acetate and 2-
methoxy-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-aniline.
1H NMR (CD3OD): 8.01–7.97 (m, 2H), 7.53 (td, J = 8.0 Hz and 1.2 Hz,
1H), 7.45 (td, J = 8.0 Hz and 1.2 Hz, 1H), 7.39–7.26 (m, 2H), 6.72–
6.70 (m, 1H), 5.16 (br s, 2H), 4.39 (br s, 2H), 3.90 (s, 3H), 2.48 (s,
3H). ESI-MS m/z 469 (M+H)+.
6.2.31. [{[2-(3-Acetylphenyl)-4-methyl-1,3-thiazol-5-
yl]carbonyl}(pyridin-2-ylmethyl)amino]acetic acid (9a)
According to the procedure described in Section 6.2.30, this
compound was prepared from 2-bromo-4-methyl-1,3-thiazole-5-
6.2.37. ((1,3-Benzothiazol-2-ylmethyl){[4-methyl-2-(2-
naphthyl)-1,3-thiazol-5-yl]carbonyl}amino)acetic acid (9g)
According to the procedure described in Section 6.2.30, this
compound was prepared from {(1,3-benzothiazol-2-ylmethyl)

1288
N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
[(2-bromo-4-methyl-1,3-thiazol-5-yl)carbonyl]amino}acetate and
2-naphthylboronic acid. 1H NMR (DMSO-d6): 8.53–8.47 (m, 1H),
8.11–7.96 (m, 6H), 7.59 (br s, 2H), 7.52 (t, J = 7.6 Hz, 1H), 7.45 (t,
J = 7.6 Hz, 1H), 5.13 (s, 2H), 4.30 (s, 2H), 2.50 (s, 3H). ESI-MS m/z
474 (M+H)+.
1H, minor rotamer), 8.50 (br s, 1H, major rotamer), 7.82–7.69 (m,
2H), 7.46–7.26 (m, 6H), 7.13–7.04 (m, 2H), 5.01 (br s, 2H, minor rot-
amer), 4.74 (br s, 2H, major rotamer), 4.10 (br s, 2H, major rotamer),
3.97 (br s, 2H, minor rotamer). ESI-MS m/z 337 (M+H)+.
6.2.43. [[(5-Methyl-3-phenylisoxazol-4-yl)carbonyl] (pyridin-2-
ylmethyl)amino]acetic acid (15)
6.2.38. [[(4-Methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]
(pyridin-2-ylmethyl)amino]acetic acid (10)
The title compound was obtained from commercially available
5-methyl-3-phenylisoxazole-4-carboxylic acid (Sigma–Aldrich)
and amine 3a, following the same procedures as in Section 6.2.3
for coupling with amine and saponification. 1H NMR (DMSO-d6),
2 rotamers in a roughly 1/1 ratio, each chemical shift is for both
rotamers except when stated: 8.56 (d, J = 4.0 Hz, 1H, one rotamer),
8.43 (d, J = 4.0 Hz, 1H, one rotamer), 7.83–7.64 (m, 3H), 7.48–7.40
(m, 3H), 7.35–7.22 (m, 1H of both rotamers and 1H of one rot-
amer), 7.13 (d, J = 7.6 Hz, 1H, one rotamer), 4.58 (br s, 2H, one rot-
amer), 4.11 (br s, 2H, one rotamer), 3.71 (br s, 2H, one rotamer),
3.33 (2H of one rotamer under water peak), 2.45 (s, 3H, one rot-
amer), 2.48 (s, 3H, one rotamer). ESI-MS m/z 338 (M+H)+.
The title compound was obtained from commercially available
4-methyl-2-phenyl-1,3-thiazole-5-carboxylic acid (Maybridge)
and amine 3a, following the same procedures as in Section 6.2.3
for coupling with amine and saponification. 1H NMR (DMSO-d6),
2 rotamers in a 2/1 ratio, each chemical shift is for both rotamers
except when stated: 8.56–8.50 (m, 1H), 7.94–7.84 (m, 2H), 7.82–
7.72 (m, 1H), 7.49 (br s, 3H), 7.38–7.34 (m, 1H, major rotamer),
7.32–7.22 (m, 1H of minor rotamer and 1H of both rotamers),
4.75 (br s, 2H, major rotamer), 4.70 (br s, 2H, minor rotamer),
3.92 (br s, 2H, minor rotamer), 3.68 (br s, 2H, major rotamer),
2.48 (s, 3H, minor rotamer), 2.42 (s, 3H, major rotamer). ESI-MS
m/z 368 (M+H)+.
6.3. Plasmid construction, expression and purification of HldE,
HldA and RK
6.2.39. [(2-Methyl-5-phenyl-3-furoyl)(pyridin-2-yl
methyl)amino]acetic acid (11)
The title compound was obtained from commercially available
2-methyl-5-phenyl-3-furoic acid (Maybridge) and amine 3a, fol-
lowing the same procedures as in Section 6.2.3 for coupling with
amine and saponification. 1H NMR (DMSO-d6), 2 rotamers in a 2/
1 ratio, each chemical shift is for both rotamers except when sta-
ted: 8.54 (br s, 1H, minor rotamer), 8.48 (br s, 1H, major rotamer),
7.80–7.73 (m, 1H), 7.64–7.55 (m, 2H), 7.42–7.24 (m, 5H), 6.97 (s,
1H, major rotamer), 6.84 (s, 1H, minor rotamer), 4.73 (br s, 2H),
3.89 (br s, 2H, minor rotamer), 3.79 (br s, 2H, major rotamer),
2.48 (br s, 3H). ESI-MS m/z 351 (M+H)+.
Genes encoding HldE, RK and HldA were amplified by PCR from
the genomic DNA of E. coli O18:K1 C7 strain (hldE gene), E. coli K12
MG1655 strain (ribokinase gene) or N. meningitidis 2C43 strain
(hldA gene) by using pfu polymerase (Promega) and the following
primers: rk forward primer (CACCATGCAAAACGCAGGCAGC) and
reverse primer (TCACCTCTGCCTGTCTAAAAATG), hldA forward pri-
mer (CACCATGTCCGCCAAGTTCCAACAAGAAAC) and reverse primer
(CTACATTGTTGATTGCCCTGACAATGCCTT), hldE forward primer
(CACCATGAAAGTAACGCTGC) and reverse primer (AACCGCTTTCCG
GTTAGCCT). Blunt-ended PCR fragments were cloned into pET100
expression vector (Invitrogen) and resulting plasmids were used
to transform E. coli Top10 (Invitrogen). Finally, purified plasmids
were introduced into E. coli BL21 (Invitrogen) by transformation.
Nucleotide sequencing of the various cloned fragment revealed
no mutation.
6.2.40. [[(1-Methyl-3-phenyl-1H-pyrazol-5-yl)carbonyl]
(pyridin-2-ylmethyl)amino]acetic acid (12)
The title compound was obtained from commercially available
1-methyl-3-phenyl-1H-pyrazole-5-carboxylic acid (Maybridge)
and amine 3a, following the same procedures as in Section 6.2.3
for coupling with amine and saponification. 1H NMR (DMSO-d6),
2 rotamers in a 1/1 ratio, each chemical shift is for both rotamers
except when stated: 8.58 (d, 1H, J = 3.6 Hz, one rotamer), 8.55 (d,
1H, J = 4.4 Hz, one rotamer), 7.82–7.64 (m, 3H), 7.47–7.28 (m,
5H), 6.95 (br s, 1H, one rotamer), 6.82 (br s, 1H, one rotamer),
4.79 (br s, 2H), 4.24 (br s, 2H, one rotamer), 4.12 (br s, 2H, one rot-
amer), 3.88 (br s, 3H). ESI-MS m/z 351 (M+H)+.
Recombinant proteins were expressed and purified as
previously described.30 Briefly, exponential phase culture
(OD600 = 0.5–0.7) were induced for expression by the addition of
isopropyl-b-D-glucopyranoside (IPTG) (0.5 mM final concentration)
and incubated for further 3 h. Bacteria containing over-expressed
recombinant proteins were collected by centrifugation and bacte-
rial lysis was achieved by sonication. After centrifugation and
removal of bacterial debris or unbroken cell, Ni-NTA agarose (Sig-
ma) was added to the supernatant (soluble fraction) and incuba-
tion at 4 °C was performed to maximize contact of recombinant
proteins with gel matrix. His-tagged proteins were eluted stepwise
by competition with imidazole (Sigma). The various fractions were
analyzed by SDS–PAGE and fractions containing recombinant pro-
tein were pooled and concentrated by ultrafiltration (Amicon
Ultra-15-Millipore). Protein concentration was determined by
Bradford Method (Invitrogen). Protein solutions were adjusted to
50% glycerol and stored at ꢀ20 °C until used.
6.2.41. [(1,10-Biphenyl-4-ylcarbonyl)(pyridin-2-
ylmethyl)amino]acetic acid (13)
The title compound was obtained from commercially available
1,10-biphenyl-4-carboxylic acid (Sigma–Aldrich) and amine 3a, fol-
lowing the same procedures as in Section 6.2.3 for coupling with
amine and saponification. 1H NMR (DMSO-d6) 2 rotamers in a
roughly 1/1 ratio, each chemical shift is for both rotamers except
when stated: 8.58 (br s, 1H, one rotamer), 8.53 (br s, 1H, one rot-
amer), 7.82–7.31 (m, 12 H), 4.76 (s, 2H, one rotamer), 4.66 (s, 2H,
one rotamer), 4.02 (s, 2H, one rotamer), 3.85 (s, 2H, one rotamer).
ESI-MS m/z 347 (M+H)+.
6.4. Activity assays
6.4.1. HldE-kinase luminescent assay
6.2.42. [(5-Phenyl-2-furoyl)(pyridin-2-ylmethyl)amino]acetic
acid (14)
This assay was especially used for HTS and IC50 measurements.
The luminescent assay was based on the detection of ATP deple-
tion by the luciferase/luciferin system. The assay buffer (AB)
adjusted to pH 7.5 contained Hepes (50 mM), MnCl2 (1 mM), KCl
(25 mM), Triton-X100 (0.012%) and DTT (1 mM) in deionized
water. The following components were added in a white polysty-
The title compound was obtained from commercially available 5-
phenyl-2-furoic acid (Sigma–Aldrich) and amine 3a, following the
same procedures as in Section 6.2.3 for coupling with amine and
saponification. 1H NMR (DMSO-d6) 2 rotamers in a 2/1 ratio, each
chemical shift is for both rotamers except when stated: 8.59 (br s,
rene Costar plate up to a final volume of 31 lL:3 lL of inhibitor

N. Desroy et al. / Bioorg. Med. Chem. 17 (2009) 1276–1289
1289
dissolved in DMSO (or only DMSO), and 28
30 min of pre-incubation at room temperature, 29
mixture (ATP and H-7-P) in AB were added in each well to a final
volume of 60 L. This reaction mixture was then composed of HldE
(3 nM), inhibitor to be tested (various concentrations), H-7-P
(0.2 M) in AB. After
M, in house synthesis)31 and ATP (0.2
40 min of incubation at room temperature, 200 L of the revelation
mixture (luciferase, -luciferin and N-acetylcysteamine) were
added to give a final volume of 260 L, including the following con-
stituents at the respective final concentrations: luciferase (4 nM),
-luciferin (30 M) and N-acetylcysteamine (100 M). Lumines-
cence intensity was immediately measured on an Analyst-HT
(Molecular Devices) or Microbeta 1450 (Wallac) and converted
into inhibition percentages.
For IC50 determinations, the inhibitor is tested at 6–10 different
concentrations, and the related inhibitions are fitted to a classical
Langmuir equilibrium model using XLFITÒ (IDBS).
l
L of HldE in AB. After
6.4.4. RK luminescent assay
lL of substrates
This assay was basically identical to the HldE-kinase lumines-
cent assay, with the following differences: H-7-P (0.2
replaced by -ribose (3 M), HldE (3 nM) by RK (0.6 nM), and a
concentration of 3 M ATP was used instead of 0.2 M.
lM) was
l
D
l
l
l
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References and notes
D
1. Croft, A. C.; D’Antoni, A. V.; Terzulli, S. L. Med. Sci. Monit. 2007, 13, RA 103.
2. Payne, D.; Tomasz, A. Curr. Opin. Microbiol. 2004, 7, 435.
3. (a) Ralph, E.; Christoffersen, R. E. Nat. Biotechnol. 2006, 24, 1512; (b) Fernandes,
P. Nat. Biotechnol. 2006, 24, 1497.
4. Datamonitor. Commercial Insights: Antibacterials—Pharma strikes antibac
(Datamonitor, New York, 2005).
5. Alanis, A. J. Arch. Med. Res. 2005, 36, 697.
6. Kumar, A.; Schweizer, H. P. Adv. Drug Deliv. Rev. 2005, 57, 1486.
7. Talbot, G. H. Expert. Rev. Anti-Infect. Ther. 2008, 6, 39.
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8. Stakeholder Opinion: Nosocomial Infections—The need for new Gram-negative
drugs (Datamonitor, New York, 2007).
9. Escaich, S. Curr. Opin. Chem. Biol. 2008, 12, 400.
10. (a) Marra, A. Drugs R. D. 2006, 7, 1; (b) Marra, A. Expert. Rev. Anti-Infect. Ther. 2004,
2, 61; (c) Lee, Y.; Almqvist, F.; Hultgren, J. Curr. Opin. Pharmacol. 2003, 3, 513.
11. (a) Raetz, C. R. H.; Whitfield, C. Annu. Rev. Biochem. 2002, 71, 635; (b) Kadrmas,
J. L.; Raetz, C. R. H. J. Biol. Chem. 1998, 273, 2799.
12. (a) Coleman, W. G.; Leive, L. J. Bacteriol. 1979, 139, 899; (b) Nikaido, H. Drug
Resist. Updates 1998, I, 93.
13. Vongsouthi, V.; Metais, A.; Floquet S.; Escaich, S. Abstracts N° B-798, 44th
Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC),
Oct 30–Nov 2, 2004; Washington, DC.
14. In addition, a viable E. coli strain lacking Kdo residues was recently disclosed:
Meredith, T. C.; Aggarwal, P.; Mamat, U.; Lindner, B.; Woodard, R. W. ACS Chem.
Biol. 2006, 1, 33.
15. Kim, C. H. Mol. Cells 2003, 15, 226.
16. Stojiljkovic, I.; Hwa, V.; Larson, J.; Lin, L.; So, M.; Nassif, X. FEMS Microbiol. Lett.
1997, 151, 41.
17. Grizot, S.; Salem, M.; Vongsouthi, V.; Durand, L.; Moreau, F.; Dohi, H.; Vincent,
S.; Escaich, S.; Ducruix, A. J. Mol. Biol. 2006, 363, 383.
6.4.2. HldE-kinase fluorescent assay
This assay was more specifically used for steady-state kinetics
and ATP competition experiments.
The fluorescent assay was based on the detection of the product
ADP by the coupled enzymatic assay using pyruvate kinase (PK)
and lactate dehydrogenase (LDH). The assay buffer (AB) adjusted
to pH 7.5 contained Hepes (50 mM), MnCl2 (1 mM), KCl
(25 mM), Triton-X100 (0.012%) and DTT (1 mM) in deionised
water. The following components were added in a black polysty-
rene Costar plate up to a final volume of 50
dissolved in DMSO (or only DMSO) and 45
30 min of pre-incubation at room temperature, 50
l
l
L:5
L HldE in AB. After
L of sub-
lL of inhibitor
l
18. Moreau, F.; Desroy, N.; Genevard, J. M.; Vongsouthi, V.; Gerusz, V.; Le Fralliec,
G.; Oliveira, C.; Floquet, S.; Denis, A.; Escaich, S.; Wolf, K.; Busemann, M.;
Aschenbrenner, A. Bioorg. Med. Chem. Lett. 2008, 18, 4022.
19. (a) Valvano, M. A.; Messner, P.; Kosma, P. Microbiology 2002, 148, 19793; (b)
Kneidinger, B.; Marolda, C.; Graninger, M.; Zamyatina, A.; McArthur, F.; Kosma,
P.; Valvano, M. A.; Messner, P. J. Bacteriol. 2002, 184, 363.
20. (a) McArthur, F.; Andersson, C. E.; Loutet, S.; Mowbray, S. L.; Valvano, M. A. J.
Bacteriol. 2005, 187, 5292; (b) De Leon, G. P.; Elowe, N. H.; Koteva, K. P.;
Valvano, M. A.; Wright, G. D. Chem. Biol. 2006, 13, 437.
strates-revelation mixture (ATP, H-7-P, pyruvate kinase, phospho-
enolpyruvate, lactate dehydrogenase and NADH) in AB were added
in each well to a final volume of 100
then composed of HldE (66 pM), inhibitor to be tested (various
concentrations), H-7-P (1 M), ATP (50 M), pyruvate kinase
(5 u/mL), phosphoenolpyruvate (50 M), lactate dehydrogenase
(5 u/mL) and NADH (2.5 M) in AB. Fluorescence intensity of
lL. This reaction mixture was
l
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21. Caroff, M.; Karibian, D. Carbohydr. Res. 2003, 338, 2431.
22. Andersson, C. E.; Mowbray, S. L. J. Mol. Biol. 2002, 315, 409.
23. Chuvikovsky, D. V.; Esipov, R. S.; Skoblov, Y. S.; Chupova, L. A.; Muravyova, T. I.;
Miroshnikov,A.I.;Lapinjoki,S.;Mikhailopulo,I.A. Bioorg. Med. Chem. 2006,14, 6327.
24. Feng, B. Y.; Shoichet, B. K. Nat. Protocol. 2006, 1, 550.
25. Sigrell, J. A.; Cameron, A. D.; Jones, T. A.; Mowbray, S. L. Structure 1998, 6, 183.
26. Combet, C.; Blanchet, C.; Geourjon, C.; Deléage, G. TIBS 2000, 25, 147.
27. Tarzia, G.; Schiatti, P.; Selva, D.; Favara, D.; Ceriani, S. Eur. J. Med. Chem.—Chim.
Ther. 1976, 11, 263.
NADH (kex = 360 nm, kem = 520 nm) was immediately measured
kinetically by a Fluostar Optima (BMG). All calculations/fits were
done in ExcelÒusing XLFITÒ (IDBS).
Alternatively, H-7-P, inhibitor to be tested, ATP and NADH were
varied on relevant scales in order to accommodate competition and
saturation experiments.
28. Wipf, P.; Miller, C. P. J. Org. Chem. 1993, 58, 3604.
6.4.3. HldA luminescent assay
29. Sznaidman, M. L. et al Bioorg. Med. Chem. Lett. 2003, 13, 1517.
30. Durant, L.; Metais, A.; Soulama, C.; Genevard, J. M.; Nassif, X.; Escaich, S. Infect.
Immun 2007, 75, 1916.
This assay was basically identical to the HldE-kinase lumines-
cent assay. HldE (3 nM) was replaced by HldA (20 nM), other com-
ponents were used at the same concentrations as in the
luminescent assay for HldE-kinase.
31. A synthesis of D-glycero-b-D-manno-heptose-7-phosphate is described in the
following article: Zamyatina, A.; Gronow, S.; Puchberger, M.; Graziani, A.;
Hofinger, A.; Kosma, P. Carbohydr. Res. 2003, 338, 2571.