A library of novel hydroxamic acids targeting the metallo-protease family: Design, parallel synthesis and screening

Article abstract of DOI:10.1016/j.bmc.2006.10.010

We report here the design and parallel synthesis of 217 compounds based on a malonic-hydroxamic acid template. These compounds are obtained via a two-step solution-phase procedure. The set of diverse building-blocks used makes this strategy suitable for t

Full text of DOI:10.1016/j.bmc.2006.10.010

Bioorganic & Medicinal Chemistry 15 (2007) 63–76
A library of novel hydroxamic acids targeting the metallo-protease
family: Design, parallel synthesis and screening
Marion Flipo, Terence Beghyn, Julie Charton, Virginie A. Leroux, Benoit P. Deprez*
and Rebecca F. Deprez-Poulain
Inserm, U761, University of Lille 2, Faculty of Pharmacy, Institut Pasteur Lille,
‘‘Biostructures and Drug Discovery’’ 3 rue du Pr Laguesse F-59006 Lille cedex, France
Received 3 July 2006; revised 4 October 2006; accepted 10 October 2006
Available online 12 October 2006
Abstract—We report here the design and parallel synthesis of 217 compounds based on a malonic–hydroxamic acid template. These
compounds are obtained via a two-step solution-phase procedure. The set of diverse building-blocks used makes this strategy suit-
able for the search of inhibitors of various metallo-proteases and for the investigation of the biological role of new metallo-prote-
ases. As a proof of concept, we screened this library on Neutral Aminopeptidase (APN; EC 3.4.11.2), the prototypal enzyme of the
M1 family. Several submicromolar inhibitors were identified.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In this context, we decided to design and screen a targeted
library of hydroxamates. Indeed, hydroxamate is a recog-
nized and well-characterized Zn2+ binding group.13,14
Diversely substituted hydroxamates should therefore be
useful to clarify functions of metallo-proteases by provid-
ing specific inhibitors. Furthermore, they could represent
potential starting points for the development of novel
therapeutic agents, as exemplified by current clinical tri-
als.15 To our knowledge, only one group reported the de-
sign of a chemical library explicitly designed to interact
with multiple members of the metallo-protease family.16
Here, we describe the preparation and first screening re-
sults of a library of malonylhydroxamic acids, based on
a simple convergent solution synthesis using diverse
amines and O-tert-butyl hydroxamic acids bearing a free
carboxylic acid function.
Proteases are important pharmacological targets for a
wide variety of therapeutic applications.1–3 Their drug
ability is illustrated by the development of potent inhib-
itors for the four classes of proteases (cysteine-, aspartyl-,
serine- and metallo-proteases). The most successful
examples are inhibitors of ACE (Angiotensin-converting
enzyme), as antihypertensive drugs, and inhibitors of the
aspartyl-protease of HIV.4,5 More recently, numerous
teams have focused on metallo-proteases and closely
related metallo-hydrolases for the treatment of cancer
or arthritis with inhibitors of TACE (TNF-a-converting
enzyme), HDAC (histone desacetylase) and MMPs (ma-
trix-metallo-proteases).6–9 More generally, metallo-pro-
teases represent about 35% of the proteases identified
in the sequenced human genome.10 They thus deserve
the development of inhibitors for the investigation of
their biological function.11 Nevertheless, the design of
such inhibitors using computational methods remains
challenging because of the key role of the metal ion in
the binding of inhibitors, as recently demonstrated by
Irwin et al.12
As a proof of concept, we have screened our library on
APN (an enkephalin-processing enzyme also called
APN/CD13), the prototypal metallo-protease of the
M1 family.
2. Library design
Keywords: Metallo-protease inhibitors; Hydroxamate; Malonic; Zn2+
binding group; Targeted library; Chemical biology.
2.1. Selection of hydroxamic acid as Zn-chelating moiety
*
Many metallo-enzyme inhibitors currently investigated
contain an hydroxamate to chelate the zinc ion present
Corresponding author. Tel.: +33 320 964 024; fax: +33 320 964
709; e-mail: benoit.deprez@univ-lille2.fr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.010

64
M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
in the catalytic site of hydrolases (Fig. 1).17,18 When tar-
geting metallo-proteases, an hydroxamate is convenient
to achieve high affinity at the screening stage. More spe-
cifically, N-acylamino-acid or N-succinylamino-acid de-
rived hydroxamates and naphthylthioacetic acid
hydroxamate were independently reported as APN
inhibitors.19–22 Hydroxamates have been reported to
have poor pharmacokinetic properties due to the hydro-
lysis of the hydroxamic acid to the corresponding short-
er and inactive carboxylic acids.23,24 Strategies have
been developed to eliminate the metabolic liability of
hydroxamate-containing inhibitors, which may limit
their usefulness as clinical agent, such as the incorpora-
tion of modifications at or near the hydroxamate moiety
in an attempt to decrease the rate of hydrolysis or biliary
excretion in vivo.9,25,26 Indeed, many successes, not yet
materialized with the launch of a new drug on the mar-
ket, are reported.27
Table 1. Chemical structures of hydroxamic acids in bioactive
compoundsa
Entry
Substructure search
% of total
O
O
N
H
H
O
1
2
3
4
5
6
7
32.6b
19.2
7.1
O
N
H
H
O
N
O
O
N
H
H
O
O
Ar
O
N
H
H
O
6.9b
5.5
O
2.2. Selection of the malonic template
S
N
H
H
O
In order to introduce diversity in our structures, we
wanted our template to possess both a carboxylic func-
tion that can be transformed into a hydroxamic acid,
and at least one other readily diversifiable function
(COOH or NH2). A survey of bioactive hydroxamates
in MDDR database (Table 1) showed that out of 1679
structures, hydroxamic acids derived from succinic acid
(substituted or not) have already been studied compre-
hensively (Entry 1). Indeed, due to a good flexibility,
succinyl hydroxamic acids display a conformational
space that overlaps significantly with the conformational
space of their amino-acid homologues, allowing a sub-
strate-like binding mode. In particular, this substructure
is found in many MMPs inhibitors.28 Amino-acid de-
rived hydroxamates are the second most represented
subfamily (Entry 2). Thus, in order to explore a more
virgin chemical space and to get out of the peptide field,
we chose as a core template of our library the malonic
template which has only been marginally studied (Entry
O
O
O
N
N
H
H
H
O
S
O
5.1
O
O
N
H
O
O
0.9
N
H
H
Atoms with no explicit hydrogens were allowed to bear any
substituents.
a Search updated in March 2006 within MDL Drug Data Report from
Prous Science Publisher.
b Queries 4 and 1 show some overlap.
7). Compared to succinyl hydroxamic acids, malonic
compounds are more constrained and less susceptible
to bind in a substrate-like binding mode to the targets.29
Moreover, various substituents can easily be introduced
at the malonic carbon. We have previously shown that
non-peptidic quinoline-based malonic hydroxamic acids
could inhibit proteases of the M1 family.30 A small ser-
ies of malonic-based hydroxamates were also described
as inhibitors of MMP-8 and ECE, two metallo-proteas-
es belonging to the M10 and M13 family,
respectively.29,31
O
N
O
O
HOHN
O
H
N
O
HOHN
N
N
H
OH
O
N
O
Trocade
N
Marimastat
O
O
H
O
N
O
N
HOHN
S
HOHN
2.3. Selection of inputs
O
O
Scheme 1 shows the general solution-phase method for
the parallel synthesis of library members, using the
malonic acids and amines presented in Figures 2 and
3. The malonic acids (1–7) were synthesized through dif-
ferent procedures to display a variety of substituents on
the malonic carbon: hydrogen, a polar group: amino
group, different hydrophobic groups: an isobutyl group
and four different aromatic groups, to target diverse
metallo-proteases.
CGS-27023
SAHA
O
S
N
O
O
O
HOHN
N
S
Prinomastat (AG-3340)
Figure 1. Hydroxamates in the clinic.

M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
65
R1
N
R2
R2
R1
NH
H
N
a
H
N
HO
+
R'1
O
O
R'1
O
O
O
O
b
R1
N
R2
H
N
R'1
OH
O
O
Scheme 1. Reagents and conditions: (a) i—acid 0.5 M/DMF (1 equiv), DIEA (1.1 equiv), CDI (1.1 equiv) 0.25 M/THF, 2 h, rt; ii—amine 0.1 M/
DMF (1 equiv), DIEA (1 equiv), rt, 2 h; (b) BTFA 0.5 M/TFA, 0.4% H2O (30 equiv), 0 ꢁC, 2 h to overnight.
According to Scheme 1, the first step of the library syn-
H
thesis consists in coupling an amine to a malonic acid
HO
HO
N
HO
HO
N
derivative using N,N0-carbonyldiimidazole (CDI). All
protections, tert-butyl based, were cleaved in acidic con-
ditions (boron tris(trifluoroacetate) (BTFA) in
TFA).34,35 Though BTFA is not easily synthesized and
handled, it was preferred over TFA/DCM solutions
since it yielded the desired deprotected products in
shorter times (2 h vs 72 h).
O
O
O
H
O
O
O
O
O
O
O
F
HO
N
H
N
H
N
O
O
H
O
O
O
O
N
N
NHBoc
The 217 (7 · 31) library members were synthesized in
deepwell plates at a 5 lmol scale. The library was ana-
lyzed by HPLC (detection at 215 nm) and MS. Out of
the 217 library members, 70% displayed purity above
80%, which fulfils our specifications for screening.36
HO
N
HO
N
H
O
O
H
O
O
O
O
Figure 2. Carboxylic acids inputs.
4. Screening
Amines were chosen in order to generate a diversity of
pharmacophore properties and geometries. Amines with
linear backbones of various lengths were selected (Series
A–E). Cyclic amines were also incorporated into the li-
brary (Series F), as well as amines displaying two phenyl
rings (Series G).
The whole library was screened against mammalian
Neutral Aminopeptidase (microsomal), in order to
prove that our library could deliver hits on a given zinc
metallo-enzyme and be a useful tool for the study of
other metallo-enzymes. Screening was performed at a
classical concentration of 10 lM.37 Enzyme activity
was assessed at 405 nm by the release of the chromogen-
ic p-nitro-aniline resulting from the cleavage of the sub-
strate Leu-p-nitro-anilide. A Z0 factor of 0.75 was
obtained using these conditions, which allowed us to
screen singlicate.38 Tables of inhibition percentages are
given in the Supporting Information Section. As expect-
ed from an array of hydroxamates, our library yielded a
significant number of hits on APN.
3. Synthesis32
Commercially available amines were purchased,
whereas amine 22 was synthesized from commercially
available 4-phenyl-butylamine via a reductive amina-
tion (Scheme 2).
Spiropiperidine 33, a privileged structure, was synthe-
sized by condensation of ortho hydroxy-acetophenone
and N-benzyl-piperidone.33 Reductive ring extension
was performed on the corresponding oxime with
diisobutylaluminium hydride (DIBAL-H). Finally,
the benzyl protection was removed to give 33
(Scheme 3).
5. Statistical results
Tables 2 and 3 display statistics on screening results
sorted by class of inputs. The contrast of activities ob-
served when results are sorted by malonic acid is consis-
tent with (1) the design of the library and (2) the binding
site of APN.
The synthesis of the malonic acids inputs proceeds in
three steps when the corresponding diethyl malonic ester
(45a–c) is commercially available (Scheme 4): mono-sa-
ponification of the diester, coupling of H2N–O–t-
BuÆHCl and saponification of the remaining ethyl
ester.29 Other substituted malonic acids were obtained
from N-tert-butoxy-malonamic acid ethyl ester (49)
and aldehydes. (Scheme 5). Malonic acid 1 was prepared
from 49 by saponification.
Binding of the hydroxamate to the Zn2+ ion is probably
the critical anchoring event and it is expected that the
enzyme discriminates more dramatically amongst sub-
stituents close to the hydroxamate than amongst those
remotely bound by a flexible chain. Carboxylic acid 2
bearing the isobutyl moiety gave the highest number

66
M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
A:
NH2
NH2
NH2
NH2
N
H
NH2
NH2
F
Cl
F
N
F
F
8
9
10
11
12
13
14
B:
NH2
NH2
NH2
N
15
16
17
C:
H
N
NH2
NH2
18
19
D:
E:
H
NH
NH2
N
20
21
22
N
N
NH2
NH2
N
NH2
23
24
25
F:
H
N
O
N
N
NH
NH
NH
NH
N
NH
NH
N
O
O
26
27
28
29
30
31
O
HN
NH
HN
N
NH
O
32
33
G:
H
NH2
N
NH2
NH
N
NH2
O
34
35
36
37
38
Figure 3. Amines inputs.
H
a,b
N
The amine substituents are supposed to bind to more re-
mote pockets relative to the Zn2+ ion. Only constrained
amines (series F) gave lower hit rates than the other
series. In order to confirm the overall behaviour of the
NH2
1.1
O
+
H
20
39
22
library, we resynthesized
compounds to establish primary structure–activity
relationships as described in the next section.
a
limited number of
Scheme 2. Reagents and conditions: (a) 0.4 equiv TEA, DCM,
˚
molecular sieves 4 A, 3 h, rt; (b) 5 equiv NaBH4, overnight, rt.
of hits above 80% of inhibition. This is consistent with
known substrate preferences for the pockets surround-
ing the Zn2+ ion in APN.19 Interestingly, an aromatic
substituent is also accepted.
6. Re-synthesis of selected hits and analogues
Library members displaying a percentage of inhibition
above 80% as well as some structurally similar, but less

M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
67
OH
N
H
N
O
O
HO
O
O
O
O
a
b
+
O
O
O
1
1.2
N
O
N
OH
N
d
40
41
42
43
H
N
a
O
Cl
O
c
O
O
O
H
49
H
48
N
O
N
b
d
R
O
N
H
NH
33
44
O
O
N
O
O
50a R = -p-fluorophenyl
50b R = -m-phenoxyphenyl
50c R = -(1-methyl)benzimidazol-2-yl
O
O
O
R
c
Scheme 3. Reagents and conditions: (a) 0.5 equiv pyrrolidine, MeOH,
reflux, overnight; (b) 2 equiv NH2OHÆHCl, 2 equiv pyridine, EtOH,
reflux, 2 h; (c) 5.8 equiv DIBAL-H, anhyd DCM, 0 ꢁC, 3 h then
MeOH, H2O, 20% H2SO4, 20 min, then 30% NaOH to pH 9; (d) H2,
Pd/C (10%), MeOH, 18 h, rt.
H
N
51a R = -p-fluorophenyl
O
51b R = -m-phenoxyphenyl
51c R = -(1-methyl)benzimidazol-2-yl
d
R
O
H
N
HO
R
R
O
a
4
5
7
R = -p-fluorophenyl
R = -m-phenoxyphenyl
R = -(1-methyl)benzimidazol-2-yl
O
OH
O
O
O
46a R = -iBu
46b R = -CH2C6H5
46c R = -NHBoc
O
O
O
O
Scheme 5. Reagents and conditions: (a) 1 equiv O-t-Bu-hydroxyl-
amineÆHCl, 2.1 equiv DIEA, DCM, 4 h, rt; (b) 1.1 equiv malonic
derivative, 1 equiv aldehyde, cat. piperidine, EtOH, 3–4 h, reflux; (c)
3.5–6 equiv NaBH3CN, acetic acid, acetonitrile, 3–24 h, rt; (d) 3 equiv
KOH, EtOH, cat. H2O, overnight, rt.
45a R = -iBu
45b R = -CH2C6H5
45c R = -NHBoc
b
R
H
O
N
O
Table 2. Percentage of library members derived from carboxylic acids
in the corresponding range of inhibition
O
O
47a R = -iBu
47b R = -CH2C6H5
c
47c R = -NHBoc
Range of % inhibition
at 10 lM
Carboxylic acid inputs
1
2
3
4
5
6
7
R
>90%
0
3
6
42
13
13
32
6
0
3
3
0
0
0
0
H
HO
N
80–90%
50–80%
<50%
13
29
52
42
42
13
O
39
58
16
84
45
55
O
O
2 R = -iBu
3 R = -CH2C6H5
91
6 R = -NHBoc
Scheme 4. Reagents and conditions: (a) 1 equiv KOH, EtOH, cat.
H2O, 2–3 h, rt; (b) 3 equiv oxalylchloride, DCM, cat. DMF, 1 h, 0 ꢁC
then rt, 1 equiv O-t-Bu-hydroxylamineÆHCl, 3 equiv DIEA, DCM, 2 h,
rt; or 1.1 equiv EDCI, 1.1 equiv HOBt, 1 equiv O-t-Bu-hydroxyl-
amineÆHCl, 2 equiv DIEA, THF, rt, overnight; (c) 3 equiv KOH,
EtOH, cat. H2O, overnight, rt.
Table 3. Percentage of library members derived from class of amine
inputs in the corresponding range of inhibition
Range of % inhibition
at 10 lM
Classes of amine inputs
A
B
C
D
E
F
G
>90%
6
10
14
33
43
15
7
10
10
29
51
5
4
10
10
32
48
80–90%
50–80%
<50%
12
37
45
19
19
57
6
14
76
21
57
active library members were selected for resynthesis and
further biological characterization (IC50). For some
compounds, O-tritylhydroxamate precursors were used
instead of O-tert-butylhydroxamates to avoid the use
of BTFA at a larger scale (Scheme 6).
We first analyzed data from compounds synthesized
from malonic acid 2 (R2 = isobutyl) among which most
of the hits were identified. In this class of compounds,
activity depends on the choice of the amine input. A
four carbon chain between the amide NH and the termi-
nal aromatic ring gives the best activity (compounds 64
and 65, IC50 = 82 and 452 nM, respectively). Com-
pounds 65 and 66 were useful to study the influence of
flexibility on the activity. Both compounds are less ac-
tive than 64. Interestingly, 66, the most constrained
compound, also a direct analogue of 61, is seven times
less active than 65. This suggests that the chain between
the nitrogen atom and the aryl moiety needs to be
Trityl is easily removed using TFA 2% in DCM in the
presence of triisopropylsilane at rt, in less than 5 min.
All re-synthesized compounds were controlled by
TLC, RP-HPLC, mass spectrometry and NMR.
7. Structure–activity relationships
IC50s obtained on resynthesized samples are given in
Table 4.

68
M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
R
R
R
activity could be to rigidify the carbon backbone be-
a
H
H
b
O
OH
O
N
Trt
HO
N
Trt
tween the hydroxamate and the aromatic ring. More
generally, we expect our library to be a useful tool in
our current attempt to elucidate the function of recently
identified metallo-proteases of the M1 family. Finally,
the diversity of the library, already revealed by our
screening results, suggests that it could be useful for
the study of other classes of Zn metallo-proteases.
O
O
O
O
O
O
O
O
46a R = -iBu
46b R = -CH2C6H5
52a R = -iBu
52b R = -CH2C6H5
53 R = -iBu
54 R = -CH2C6H5
R
O
R
O
c
d
O
O
O
O
O
O
O
O
O
O
57
55
e
56
R = -m-phenoxyphenyle
R
9. Experimental
9.1. Biology
O
OH
O
O
58
f
R
H
Microsomal neutral aminopeptidase (APN) from por-
cine kidney was purchased from Sigma. Inc. as an
ammonium sulfate suspension (3.5 M (NH4)2SO4 solu-
tion containing 10 mM MgCl2) 10–40 U/mg protein.
The enzyme suspension was diluted 600-fold in Tris–
HCl buffer (25 mM, pH 7.4) before use. The assays were
performed in 96-well plates. The compounds were tested
at the concentration of 10 lM. Thirty-three microliters
of purified APN was pre-incubated for 10 min at rt with
33 lL of the inhibitor (30 lM in Tris–HCl buffer, 0.3%
DMSO). Thirty-three microlitres of the substrate Leu-
cine–para-nitroanilide (Leu-pNA) (Km = 0.099 mM)
0.3 mM in Tris–HCl buffer was then added. The reac-
tion kinetics was followed on a UV-microplate reader
MultiskanRC (Labsystems, Finland) at 405 nm. The
control activity was determined by incubating the en-
zyme in the same conditions without inhibitor. Bestatin
was used as the reference inhibitor (IC50 = 2.7 lM). The
statistical Z0 factor for the test was 0.75, allowing activ-
ities to be determined with a single point with a 95%
confidence. Initial velocities are expressed in
lmol minꢀ1. Data were normalized to the controls that
represent Vmax. For the determination of IC50s, initial
velocities were plotted as a function of inhibitor concen-
tration, using XLfit software from IDBSꢂ.
R
O
g
H
N
HO
N
Trt
O
Trt
O
O
O
O
O
60
59
Scheme 6. Reagents and conditions: (a) i—1 equiv carboxylic acid, 2
equiv oxalyl chloride, cat. DMF, DCM, 1 h, rt; ii—1.2 equiv O-tert-
hydroxylamine, 1.2 equiv DIEA, DCM, 3 h, rt; (b) EtOH/DCM, 4
equiv KOH, cat. H2O, 24 h, rt; (c) 1 equiv diethyl malonate, 1 equiv 3-
phenoxybenzaldehyde, cat. piperidine, EtOH, 3 h 30, reflux; (d) 4 equiv
NaBH3CN, acetonitrile, 16 h, rt; (e) 1 equiv KOH, 1 equiv H2O,
EtOH, 4 h, rt; (f) i—1 equiv carboxylic acid, 1.2 equiv oxalyl chloride,
cat. DMF, DCM, 1 h, rt; ii—3 equiv DIEA, 0.85 equiv O-tert-
hydroxylamine, DCM, 4 h, rt; (g) 4 equiv KOH, 4 equiv H2O, EtOH,
24 h, rt.
extended to provide the required distance. The partial
loss of activity of compound 65 cannot be unambigu-
ously attributed to the conformational effect of the
piperidine ring since it has lost a NH functionality com-
pared to 64. Introduction of a chlorine or fluorine atom
in the para-position of the phenyl ring of 61, (com-
pounds 68 and 67) leads to a 3- or 5-fold loss of activity.
Interestingly, introduction of a phenoxy substituent in
the meta position of the phenyl ring of 61 (compound
69) improves activity that is now comparable to 65. This
confirms the need for a 4- to 5-atom chain between the
terminal aromatic ring and the amide NH.
9.2. Chemistry
As suggested by the screening results, compounds where
R2 is benzyl or 3-phenoxy-benzyle are less active, inde-
pendently of the chain length of the amine input (70
and 73 vs 61, and 71 and 74 vs 64). Interestingly, com-
pound 72, derived from (1-methyl-1H-benzo-imidazol-
2-ylmethyl)-malonamic acid and benzylamine, displays
a submicromolar IC50.
9.2.1. General information. NMR spectra were recorded
on a Bruker DRX-300 spectrometer. Chemical shifts are
in parts per million (ppm). The assignments were made
using one-dimensional (1D) 1H and 13C spectra and
two-dimensional (2D) HSQC and COSY spectra.
Mass spectra were recorded on a MALDI-TOF Voyag-
er-DE-STR spectrometer, or with a LC/MS/MS Varian
1200ws.
8. Conclusion
HPLC analysis was performed using a C18 TSK-GEL
Super ODS 2 lm particle size column, dimensions
50· 4.6 mm. A gradient starting from 100% H2O/0.05%
TFA and reaching 20% H2O/80% CH3CN/0.05% TFA
within 10 min at a flow rate of 1 mL/min was used. LCMS
gradient starting from 100% H2O/0.1% formic acid and
reaching 20% H2O/80% CH3CN/0.08% formic acid with-
in 10 min at a flow rate of 1 mL/min was used.
We designed and prepared via parallel synthesis a
library of 217 hydroxamic acids, targeting metallo-pro-
teases. As a proof of concept, these compounds were
tested for their ability to inhibit microsomal APN, a
prototypal aminopeptidase of the M1 family. We suc-
cessfully identified new inhibitors of this enzyme demon-
strating the usefulness of the library. Furthermore,
compound 64, displaying an IC50 of 82 nM, is a reliable
starting point for further optimization. As suggested by
our preliminary SAR analysis, a way to increase its
Melting points were determined on a Buchi B-540
¨
apparatus.

M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
Table 4. Inhibition of APN by compounds 61–74 (IC50 nM)
69
R1
N
R2
H
N
OH
R'
1
O
O
a
IC50 (nM)
Compound
R1R01N–
n
R2–
61
62
63
64
C6H5–(CH2)n–NH–
C6H5–(CH2)n–NH–
C6H5–(CH2)n–NH–
C6H5–(CH2)n–NH–
N
1
2
3
4
590
984
528
82
65
—
452
HN
66
67
—
1
3040
3000
4-F–C6H4–(CH2)n–NH–
4-Cl–C6H4–(CH2)n–NH–
68
69
70
71
72
73
1
1
1
4
1
1
4
2520
3-C6H5–O-C6H4–(CH2)n–NH–
C6H5–(CH2)n–NH–
C6H5–(CH2)n–NH–
C6H5–(CH2)n–NH–
C6H5–(CH2)n–NH–
C6H5–(CH2)n–NH–
443
>10,000
>10,000
718
N
N
>10,000
>10,000
O
74
a Bestatin IC50 (nM): 2700.
All commercial reagents and solvents were used without
further purification. Organic layers obtained after
extraction of aqueous solutions were dried over MgSO4
and filtered before evaporation in vacuo. Purification
yields were not optimized. Bond-Elut SAX-columns
were purchased from Varian.Inc. All the 5 lmol-scale
reactions were performed in 96-well (round-bottomed)
polypropylene plates (1200 lL).
Compound 22 was obtained as a yellow liquid. Yield
46%, purity 100%, NMR H (DMSO-d6) d ppm: 0.83
(d, J = 6.6 Hz, 6H), 1.35–1.43 (m, 2H), 1.52–1.64 (m,
3H), 2.26 (d, J = 6.7 Hz, 2H), 2.47 (t, J = 7.1 Hz, 2H),
2.55 (t, J = 7.5 Hz, 2H), 7.10–7.27 (m, 5H); MS (MH)+
m/z 206.
1
9.2.3. 4,5-Dihydro-3H-spiro[1,5-benzoxazepine-2,40-pi-
peridine] (33). N-Benzyl-4-piperidone (41) (6.8 g,
36 mmol, 1.2 equiv) and 2-hydroxyacetophenone (40)
(4.1 g, 30 mmol, 1 equiv) were dissolved in MeOH
(30 mL) under stirring, followed by addition of pyrroli-
dine (1.25 mL, 15 mmol, 0.5 equiv). The mixture was
heated overnight under refluxing conditions. The reac-
tion mixture was concentrated and dissolved in AcOEt
(200 mL). The solution was washed with Na2CO3 1 M
(3· 50 mL), water (50 mL), brine (50 mL), dried over
MgSO4 and concentrated to give a yellow solid. The res-
idue was recrystallized in methanol to give (42) as light
yellow crystals (10.5 g). Yield 98%, mp 99.6–100.9 ꢁC;
NMR 1H (CDCl3) ppm: 1.75 (m, 2H), 2.03 (d,
J = 17.5 Hz, 2H), 2.46 (td, J = 6.6 Hz, J = 2.7 Hz, 2H),
2.65 (dt, J = 11.9, 3.8 Hz, 2H), 2.7 (s, 2H), 3.5 (s, 2H),
9.2.2. N-Isobutyl-(4-phenylbutylamine) (22). In a round-
bottomed flask, 3.97 mL of 4-phenylbutylamine (20) (1
equiv), 2.50 mL of isopropyl-aldehyde (39) (1.1 equiv)
and 1.39 mL of triethylamine (0.4 equiv) were dissolved
˚
in 15 mL DCM. Molecular sieves (4 A) were added, and
the reaction mixture was stirred for 3 h at rt. The flask
was cooled in an ice-bath and 4.75 g NaBH4 (5 equiv)
was added slowly. The reaction mixture was stirred
overnight at rt and then quenched with 20 mL of water.
The reaction mixture was filtered and the aqueous phase
was washed three times with DCM. Organic phases were
joined, dried over MgSO4 and evaporated under re-
duced pressure. The desired product was purified via sil-
ica gel chromatography (95 DCm, 4 MeOH, 1 NH4OH).

70
M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
6.8 (ddd, J = 8.2, 6.2, 1.2 Hz, 2H), 7.3 (m, 5H), 7.47 (m,
1H), 7.84 (dd, J = 8.2, 1.8 Hz, 1H); MS (MH)+ m/z 308.
Compound 42 (8.33 g, 27.1 mmol, 1 equiv), hydroxyl-
amine hydrochloride (3.77 g, 54.2 mmol, 2 equiv) and
pyridine (4.38 mL, 54.2 mmol, 2 equiv) were mixed in
EtOH (25 mL). The mixture was then stirred under
refluxing conditions. After 2 h, the reaction mixture
was filtered and the filtrate was washed with water.
The residue was recrystallized in MeOH to give oxime
(43) as a white powder (5.6 g). Yield 64%, mp 229–
231 ꢁC; NMR 1H (DMSO-d6) d ppm: 1.6 (ddd,
J = 13.9, 13.0, 3.9 Hz, 2H), 1.73 (dm, J = 13.9 Hz, 2H),
2.3 (ddm, J = 13.0, 12.36 Hz, 2H), 2.5 (m, 2H), 2.75 (s,
2H), 3.45 (s, 2H), 6.85 (m, 2H), 7.2–7.3 (m, 6H), 7.75
(dd, J = 7.8, 1.6 Hz, 1H), NMR 13C (DMSO-d6) d
ppm: 32.1, 35.3, 40.5, 49.6, 63.2, 75.8, 119.0, 122.3,
124.6, 128.7, 129.2, 130.1, 132.2, 146.2, 155.7, 164.1;
MS (MH)+ m/z 323. Oxime (43) (3 g, 9.3 mmol, 1 equiv)
was dissolved in anhydrous DCM (20 mL). The mixture
was stirred at 0 ꢁC for 30 min, and 1 N diisobutylalu-
minium hydride in DCM (54 mL, 54 mmol, 5.8 equiv)
was added dropwise over 1 h. The mixture was stirred
for 3 h under nitrogen at 0 ꢁC. The reaction was
quenched by slowly adding MeOH (9 mL), followed
by distilled water (9 mL) and 20% sulfuric acid
(50 mL). The solution was stirred for a further 20 min.
The solution was basified to pH 9 using a 30% sodium
hydroxide solution. The resulting mixture was extracted
with AcOEt (2· 100 mL). The organic layer was dried
over MgSO4 and concentrated to give a yellow oil.
The residue was purified by chromatography on alumi-
na with cyclohexane/AcOEt (1:1, v/v) to give 1.5 g of
(44) a yellow oil, which crystallized. Yield 52%, mp
99–101 ꢁC; NMR 1H (CDCl3) d ppm: 1.54 (ddd,
J = 12.8, 12.6, and 4.6 Hz, 2H), 1.82 (t, J = 5.2 Hz,
2H), 1.86 (dm, J = 12.6 Hz, 2H), 2.40 (ddm, J = 12.6,
11.4 Hz, 2H), 2.51 (dm, J = 11.4 Hz, 2H), 3.18 (t,
J = 5.2 Hz, 2H), 3.47 (s, 2H), 6.53 (dd, J = 7.7,
1.6 Hz, 1H), 6.63 (td, J = 75, 1.6 Hz, 1H), 6.77 (td,
J = 7.5, 1.5 Hz, 1H), 6.87 (dd, J = 7.8, 1.5 Hz, 1H),
7.15–7.30 (m, 5H). NMR 13C (CDCl3) d ppm: 34.3,
40.2, 40.9, 48.7, 62.6, 117.7, 119.1, 123.0, 123.8, 126.2,
127.4, 127.4, 128.5, 137.7, 141.8; MS (MH)+ m/z 309.
Protected amine (44) (1 g, 3.1 mmol, 1 equiv) was dis-
solved in MeOH (5 mL). (10%) Pd/C (0.1 g) was added
and the mixture was stirred for 18 h at rt under hydro-
gen. The mixture was filtered on Celite, washed with
MeOH (20 mL) and the filtrate was dried over MgSO4
and concentrated to give 0.68 g of the free amine (33)
5%. The organic phase was extracted, dried and evapo-
rated under reduced pressure to give (49) as a brown oil.
Yield 95%, NMR 1H (CDCl3) d ppm: 1.26–1.31 (m,
12H), 3.36 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 9.05 (s,
NH), NMR 13C (CDCl3) d ppm: 14.4, 26.6, 40.5, 60.8,
82.8, 164.2, 169.0; MS (MH)+ m/z 204.2, (MNa)+ m/z
226.1.
In a 100 mL round-bottomed flask was dissolved
60 mmol KOH (3 equiv) in ethanol (50 mL) and water
(1 mL). Compound 49 was then added (20 mmol, 1
equiv) and the reaction mixture was stirred overnight
at rt. The solvent was removed under reduced pressure
and the crude product was dissolved in water. The aque-
ous phase was acidified with concd HCl and the product
was extracted (6·) with AcOEt. The organic phases were
joined, dried over MgSO4 and evaporated under re-
duced pressure to give (1) as a beige powder. Yield
1
77%, NMR H (DMSO-d6) d ppm: 1.14 (s, 9H), 3.03
(br s, 2H), 10.46 (s, 1H, NH), 12.46 (br s, 1H, COOH),
NMR 13C (DMSO-d6) d ppm: 26.5, 38.3, 82.9, 166.3,
169.4.
9.2.5. 2-tert-Butoxycarbamoyl-4-methyl-pentanoic acid
(2). In a 50 mL flask containing a solution of KOH
(898 mg, 1 equiv) in absolute ethanol (20 mL) was added
2-isobutyl-malonic diethyl ester (45a) (3.57 mL,
16 mmol, 1 equiv). The reaction mixture was stirred at
r t for 2 h. Ninety percent of the solvent was then evap-
orated under reduced pressure. A solution of NaHCO3
5% (20 mL) was added and the remaining diethyl ester
was extracted with AcOEt. The aqueous phase was acid-
ified with KHSO4 (powder), at 0 ꢁC. The expected prod-
uct was extracted with AcOEt. The organic phases were
joined, dried over MgSO4 and evaporated under re-
duced pressure (max temp 25 ꢁC) to give 2-isobutyl-
malonic acid monoethyl ester (46a) as a colourless oil.
Yield 75%, NMR 1H (CDCl3) d ppm: 0.93 (d,
J = 6.5 Hz, 6H), 1.28 (t, J = 7.1 Hz, 3H), 1.57–1.65 (m,
1H), 1.80–1.89 (m, 2H), 3.46 (t, J = 7.6 Hz, 1H), 4.22
(q, J = 7.1 Hz, 2H), 10.7 (br s, 1H, COOH). NMR 13C
(CDCl3) d ppm: 14.1, 22.2, 22.3, 26.2, 37.7, 50.2, 61.6,
169.7, 174.8. Compound 46a (8 mmol, 1 equiv) was then
added with N-ethyl-3-(3-dimethyaminopropyl)carbodii-
mide (EDCI) (1.684 g, 8.8 mmol, 1.1 equiv) and
N-hydroxybenzotriazole (HOBt) (1.187 g, 8.8 mmol,
1.1 equiv) to a solution of O-tert-butyl-hydroxyl-
amineÆHCl (1 g, 8 mmol, 1 equiv) in THF (20 mL) and
DIEA (2.77 mL, 16 mmol, 2 equiv). The reaction mix-
ture was stirred overnight at rt. The solvent was re-
moved under reduced pressure and the crude product
was solubilized in AcOEt. The organic phase was
washed with KHSO4 5% (5·), NaHCO3 1 M (5·). The
organic phases were dried and evaporated under re-
duced pressure to give 2-tert-butoxycarbamoyl-4-meth-
yl-pentanoic acid ethyl ester (47a) as a white powder.
1
as a white powder. Yield 100%, purity 97%, NMR H
(DMSO-d6) d ppm: 1.38–1.48 (m, 4H), 1.79–1.84 (m,
4H), 3.15–3.19 (m, 2H), 3.64–3.69 (m, 2H), 5.38–5.41
(m, 1H), 6.55 (td, J = 7.8, 1.8 Hz, 1H), 6.67 (dd,
J = 7.8, 1.5 Hz, 1H), 6.78 (td, J = 7.5, 1.5 Hz, 1H),
6.84 (dd, J = 7.8, 1.2 Hz, 1H); MS (MH)+ m/z 219.
1
9.2.4. N-tert-Butoxy-malonamic acid (1). In a round-bot-
tomed flask, 5 g of O-tert-butyl-hydroxyl-amineÆHCl
(40 mmol, 1 equiv) was dissolved in DCM (200 mL)
and DIEA (14.6 mL, 2.1 equiv). The reaction mixture
was cooled in an ice bath and the acid chloride (48)
(40 mmol, 5.06 mL) was added slowly. After stirring
for 4 h at rt, the reaction was quenched with NaHCO3
Yield 71%, purity 97%, NMR H (CDCl3) d ppm: 1.07
(d, J = 6.5 Hz, 6H), 1.38–1.42 (m, 12H), 1.70–1.80 (m,
1H), 1.92–1.98 (m, 2H), 3.52 (t, J = 7.5 Hz, 1H), 4.32
(q, J = 7.1 Hz, 2H). In a 50 mL flask containing a solu-
tion of KOH (840 mg, 15 mmol, 3 equiv) in absolute
ethanol (15 mL) was added (47a) (5 mmol, 1 equiv).
The reaction mixture was stirred at rt overnight. Ninety

M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
71
percent of the solvent was then evaporated under re-
duced pressure. A solution of NaHCO3 1 M (20 mL)
was added and the remaining ester was extracted with
AcOEt. The aqueous phase was acidified with KHSO4
(powder), at 0 ꢁC. The expected product was extracted
with AcOEt. The organic phases were joined, dried
and evaporated under reduced pressure (max temp
25 ꢁC) to give (2) as a white powder. Yield 70%, purity
added 4-fluorobenzaldehyde (32.0 mmol, 1 equiv). The
reaction mixture was refluxed for 3h30. Purification over
silica gel (DCM/MeOh, 98/2) allowed 7.05 g of (50a) as
1
a light yellow solid. Yield 71%, purity 85%, NMR H
(CDCl3)
d ppm: 1.21–1.29 (m, 12H), 4.24 (q,
J = 7.1 Hz, 2H), 7.01 (t, J = 8.6 Hz, 2H), 7.51–7.55 (m,
2H), 7.69 (s, 1H), 7.86 (s, NH); MS (MH)+ m/z 310.2
and (MNa)+ m/z 332.2. (50a) (6.94 mmol) was dissolved
in CH3CN (25 mL). NaBH3CN (41.6 mmol, 6 equiv)
and AcOH (3 mL) were added and the reaction mixture
was stirred at rt for 24 h. The solvent was removed in
vacuo, the residue dissolved in AcOEt and washed twice
with 5% NaHCO3 and twice with water. The organic
layer was dried over MgSO4, concentrated to give
1
97%, mp 129–132 ꢁC, NMR H (CDCl3) d ppm: 0.93–
0.96 (m, 6H), 1.26 (s, 9H), 1.63–1.69 (m, 1H), 1.79–
1.83 (m, 2H), 3.24 (t, J = 7.0 Hz, 0.73H), 3.80 (t,
J = 7.0 Hz, 0.27H), 8.65 (s, 1H, NH).
9.2.6. 2-Benzyl-N-tert-butoxy-malonamic acid (3). In a
round-bottomed flask, containing a solution of KOH
(1.68 g, 30 mmol, 1 equiv) dissolved in absolute ethanol
(45 mL), was added benzylmalonate diethyl ester (45b)
(7.5 g, 30 mmol, 1 equiv). The reaction mixture was stir-
red for 3 h at rt. The solvent was removed under reduced
pressure and the residue was partitioned between NaH-
CO3 5% and AcOEt. The aqueous phase was then acid-
ified to pH 1 and the expected product extracted with
AcOEt. The organic phase was dried and evaporated
under reduced pressure to give (46b) as a colourless
1
(51a) as a yellow oil. Yield 93%, purity 75%, NMR H
(CDCl3) d ppm: 1.09 (s, 9H), 1.15–1.23 (m, 3H), 3.15
(d, J = 7.8 Hz, 2H), 3.52 (t, J = 7.8 Hz, 1H), 4.02–4.11
(m, 2H), 6.91 (t, J = 8.6 Hz, 2H), 7.08–7.13 (m, 2H);
MS (MNa)+ m/z 334.2. (51a) (6.40 mmol) was dissolved
in absolute ethanol (65 mL) and KOH (19.2 mmol, 3
equiv) was added to the mixture. After stirring overnight
at r t, the solvent was removed and the mixture acidified
to pH 1 with HCl 1 N. The aqueous layer was extracted
four times with AcOEt. The organic layer was dried over
MgSO4 and concentrated to give (4) (1.50 g) as a white
solid. Yield 83%, purity 92%, mp 146–149 ꢁC, NMR
1H (DMSO-d6) d ppm: 0.99 (s, 9H), 2.92–3.01 (m,
2H), 3.38–3.45 (m, 1H), 7.04–7.11 (m, 2H), 7.18–7.23
(m, 2H), 10.47 (s, NH); MS (MH)+ m/z 284.1.
1
oil. Yield 62%, purity 100%, NMR H (DMSO-d6) d
ppm: 1.08 (t, J = 7.0 Hz, 3H), 3.05 (dd, J = 4.0, 7.2 Hz,
2H), 3.68 (dd, J = 7.4, 8.7 Hz, 1H), 4.05 (q, J = 7.1 Hz,
2H), 7.16–7.29 (m, 5H), 12.9 (s, 1H, COOH). Com-
pound 46b (4.1 g, 1 equiv, 18.4 mmol) was dissolved in
DCM (50 mL) with catalytic DMF (100 lL). The mix-
ture was cooled at 0 ꢁC (ice bath) and oxalyl chloride
(4.8 mL, 3 equiv) was added dropwise. The reaction
mixture was stirred for 1 h at rt and then evaporated un-
der reduced pressure. The residue was dissolved in DCM
9.2.8.
N-tert-Butoxy-2-(3-phenoxy-benzyl)-malonamic
acid (5). To a solution of N-tert-butoxy-malonamic acid
ethyl ester (49) (31.7 mmol, 1.1 equiv) and piperidine
(1 mL) in absolute ethanol (140 mL) was added 3-phen-
oxybenzaldehyde (28.9 mmol, 1 equiv). The reaction
mixture was refluxed for 3 h. The solvent was removed
in vacuo and the residue was dissolved in AcOEt,
washed with HCl 1 N, NaHCO3 5% and NaCl, dried
over MgSO4 and evaporated. Purification over silica
gel (cyclohexane/AcOEt, 80:20) allowed 5.49 g of (50b)
as a yellow oil. Yield 50%, purity 85%, NMR 1H
(DMSO-d6) d ppm: 1.17 (s, 9H), 1.25 (t, J = 7.1 Hz,
3H), 4.22 (q, J = 7.1 Hz, 2H), 6.99–7.06 (m, 3H), 7.13–
7.18 (m, 1H), 7.34–7.47 (m, 5H), 7.66 (s, 1H); MS
(MH)+ m/z 384.2 and (MNa)+ 406.2, (50b) (14.3 mmol)
was dissolved in CH3CN (60 mL). NaBH3CN
(85.8 mmol, 6 equiv) and AcOH (6 mL) were added
and the reaction mixture was stirred at rt for 24 h. The
solvent was removed in vacuo, the residue dissolved in
AcOEt and washed twice with 5% NaHCO3 and twice
with water. The organic layer was dried over MgSO4,
concentrated to give (51b) as a yellow oil. Yield 100%,
and O-tert-butyl-hydroxylamineÆHCl (18.4 mmol,
1
equiv) in suspension in DCM with DIEA (3 equiv)
was added. The reaction mixture was stirred for 2 h at
r t. The organic phase was washed with NaHCO3 5%
(3· 50 mL), with brine, dried and evaporated under re-
duced pressure. Trituration with Et2O/pentane allowed
to obtain (47b) as a beige powder. Yield 81%, purity
1
100%, NMR H (CDCl3) d ppm: 1.09–1.14 (m, 12H),
3.10–3.27 (m, 2H), 3.36 (t, J = 7.5 Hz, 1H), 4.06 (q,
J = 7.1 Hz, 2H), 7.11–7.23 (m, 5H). In a round-bot-
tomed flask was added a solution of KOH (2.150 g,
38.4 mmol, 3 equiv) in ethanol (100 mL), compound
47b (3.741 g, 12.8 mmol, 1 equiv). The reaction mixture
was stirred at rt overnight. The suspension obtained was
filtered and washed with AcOEt. The residue was dis-
solved in a small amount of water and the solution
was acidified with concd HCl at pH 1, and extracted
with AcOEt. The organic layers were joined, dried and
evaporated under reduced pressure to give (3) as a white
powder. Yield 59%, purity 95%, NMR 1H (DMSO-d6) d
ppm: 0.98 (s, 9H), 2.92–3.05 (m, 2H), 3.44 (dd, J = 5.6,
9.9 Hz, 1H), 7.12–7.27 (m, 5H), 10.45 (s, 1H, NH), 12.50
(br s, 1H, COOH), NMR 13C (DMSO-d6) d ppm: 26.9,
34.5, 51.5, 81.7, 127.0, 128.9, 129.7, 139.5, 166.6, 171.5.
1
purity 70%, NMR H (DMSO-d6) d ppm: 1.03 (s, 9H),
1.17 (t, J = 7.1 Hz, 3H), 2.92–3.00 (m, 1H), 3.07 (dd,
J = 5.8, 13.9 Hz, 1H), 3.54 (dd, J = 5.9, 9.7 Hz, 1H),
3.99–4.10 (m, 2H), 6.79–6.82 (m, 1H), 6.88–6.89 (m,
1H), 6.94–7.00 (m, 3H), 7.09–7.15 (m, 1H), 7.24–7.29
(m, 1H), 7.33–7.42 (m, 2H), 10.57 (s, NH); MS (MH)+
m/z 386.2 (MNa)+ m/z 408.2, (51b) (14 mmol) was dis-
solved in ethanol (150 mL) and KOH (42 mmol, 3
equiv) was added to the mixture. After stirring overnight
at rt, the solvent was removed and the mixture acidified
to pH 1 with 1 N HCl. The aqueous layer was extracted
9.2.7. N-tert-Butoxy-2-(4-fluoro-benzyl)-malonamic acid
(4). To a solution of N-tert-butoxy-malonamic acid
ethyl ester (49) (7.163 g, 35.2 mmol, 1.1 equiv) and
piperidine (1 mL) in absolute ethanol (150 mL) was

72
M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
three times with AcOEt. The organic layer was dried
over MgSO4 and concentrated to give pure (5) as a white
solid, yield 90%, purity 95%, NMR H (DMSO-d6) d
ppm: 1.01 (s, 9H), 2.90–3.07 (m, 2H), 3.37–3.47 (m,
1H), 6.78–6.81 (m, 1H), 6.88–6.89 (m, 1H), 6.95–6.98
(m, 3H), 7.09–7.15 (m, 1H), 7.24–7.29 (m, 1H), 7.34–
7.41 (m, 2H), 10.6 (s, NH); MS (MH)+ m/z 358.2,
(MNa)+ m/z 380.2, (MK)+ m/z 396.2.
1.72 mmol, 1.1 equiv) and piperidine (54 lL) in absolute
ethanol (12 mL) was added 1-methyl-2-formylbenzimi-
dazole (1.56 mmol, 1 equiv). The reaction mixture was
refluxed for 4 h. The solvent was removed in vacuo
and (50c) as a crude oil was directly used without any
purification. (50c) was dissolved in CH3CN (6.5 mL).
NaBH3CN (343 mg, 5.46 mmol, 3.5 equiv) and AcOH
(0.65 mL) were added and the reaction mixture was stir-
red at rt for 3 h. The solvent was removed in vacuo, the
residue dissolved in AcOEt and washed with NaHCO3
5% and water. The organic layer was dried over MgSO4,
concentrated and the residue was purified by thick layer
chromatography on silica gel (DCM/MeOH, 95:5) to
give (51c). Compound 51c was dissolved in ethanol
(12 mL) and H2O (0.11 mL, 6.2 mmol) and KOH
(348 mg, 6.2 mmol) was added to the mixture. After stir-
ring overnight at rt, the solvent was removed and the
mixture acidified to pH 1 with 1 N HCl. The aqueous
layer was extracted by DCM. The organic layer was
dried over MgSO4 and concentrated to give pure (7) as
1
9.2.9. N-tert-Butoxy-2-tert-butoxycarbonylamino-malo-
namic acid (6). In a 50 mL flask containing a solution
of KOH (440 mg, 1 equiv) in absolute ethanol (10 mL)
was added 2-tert-butoxycaronylamino-malonic acid
diethyl ester (45c) (2 mL, 7.84 mmol, 1 equiv). The reac-
tion mixture was stirred at rt for 2 h 30 min. Ninety per-
cent of the solvent was then evaporated under reduced
pressure. A solution of NaHCO3 1 M (20 mL) was add-
ed and the remaining diethyl ester was extracted with
AcOEt. The aqueous phase was acidified with KHSO4
(powder), at 0 ꢁC. The expected product was extracted
with AcOEt. The organic phases were joined, dried
and evaporated under reduced pressure (max temp
25 ꢁC) to give 2-tert-butoxycarbonylamino-malonic acid
monoethyl ester (46c) as a white powder. Yield 87%,
purity 100%, NMR 1H (CDCl3) d ppm: 1.32 (t,
J = 7.1 Hz, 3H), 1.44 (s, 9H), 4.24–4.33 (m, 2H), 4.78
(d, J = 4.8 Hz, 0.6H), 4.99 (d, J = 7.3 Hz, 0.4H), 5.67
(d, J = 6.9 Hz, 0.4H, NH), 6.65 (d, J = 4.4 Hz, 0.6H,
NH), 9.09 (br s, 1H, COOH). To a solution of O-tert-
butyl-hydroxylamineÆHCl (780 mg, 6.2 mmol, 1 equiv)
in THF (40 mL) and DIEA (2.1 mL, 12.4 mmol, 2
equiv) were added (46c) (6.2 mmol, 1 equiv), EDCI
(1.31 g, 6.8 mmol, 1.1 equiv) and HOBt (920 mg,
6.8 mmol, 1.1 equiv). The reaction mixture was stirred
overnight at r t. The solvent was removed under reduced
pressure and the crude product was dissolved in AcOEt
and the organic phase was washed with KHSO4 5%
(twice), NaHCO3 1 M (5·). The organic phases were
dried and evaporated under reduced pressure to give
(47c) as a yellow oil. Yield 75%, purity 100%, NMR
1H (CDCl3) d ppm: 1.28 (s, 9H), 1.29 (t, J = 7.2 Hz,
3H), 1.45 (s, 9H), 4.18–4.32 (m, 2H), 4.80 (d,
J = 7.5 Hz, 1H), 5.82 (d, J = 6.9 Hz, 1H, NH), 8.95 (br
s, 1H, CONHO). In a 50 mL flask containing a solution
of KOH (521 mg, 9.3 mmol, 3 equiv) in absolute ethanol
(10 mL) was added (47c) (990 mg, 3.1 mmol, 1 equiv).
The reaction mixture was stirred at rt overnight. Ninety
percent of the solvent was then evaporated under re-
duced pressure. A solution of NaHCO3 1 M (20 mL)
was added and the remaining ester was extracted with
AcOEt. The aqueous phase was acidified with KHSO4
(powder), at 0 ꢁC. The expected product was extracted
with AcOEt. The organic phases were joined, dried
and evaporated under reduced pressure (max temp
25 ꢁC) to give (6) as a white powder. Yield 40%, purity
1
a white solid (280 mg). Overall yield 56 %, NMR H
(DMSO-d6) d ppm: 1.00 (s, 9H), 3.66–3.38 (m, 2H),
4.01 (s, 3H), 4.07 (dd, J = 6.3 Hz and J = 9.0 Hz, 1H),
7.58–7.54 (m, 2H), 7.78 (d, J = 8.9 Hz, 1H), 7.93 (d,
J = 8.9 Hz, 1H), 11.04 (s, 1H, NH), NMR 13C
(DMSO-d6) d ppm: 26.6, 26.9, 31.9, 47.5, 81.9, 114.8,
126.0, 126.5, 133.2, 152.0, 165.3, 169.9; MS (MH)+ m/z
320.0.
9.2.11. Parallel synthesis at the scale of 5 lmol. On a
solution of carboxylic acid (0.5 M/DMF; 1 equiv) and
DIEA (1.1 equiv) was added N,N0-carbonyl-diimidazole
(0.25 M/THF; 1.1 equiv). The mixture was stirred for
2 h at rt. Then the appropriate amine (0.1 M/DMF; 1
equiv) and DIEA (1 equiv + 1 equiv if the amine was
a hydrochloride) were added and the mixture stirred
2 h at r t. Solvents were removed under reduced pressure
in a Genevacꢂ EZ-2. The residue was washed once with
HCl 10ꢀ2 M and solvents were reduced under pressure.
A suspension of BTFA (0.5 M/TFA; 30 equiv) with
0.4% H2O was added and the reaction mixture was stir-
red for 2 h to overnight at 0 ꢁC and then at rt. The reac-
tion mixture was evaporated under reduced pressure in a
Genevacꢂ EZ-2 and the residue was washed with cyclo-
hexane (3·) and dried in Genevacꢂ EZ-2.
9.2.12. 4-Methyl-2-trityloxycarbamoyl-pentanoic acid
(53). 2-Isobutyl-malonic acid monoethyl ester (46a)
(7.91 g, 42 mmol, 1 equiv) was dissolved in DCM
(140 mL) with DMF (280 lL). To this solution cooled
to 0 ꢁC was added dropwise oxalyl chloride (84 mmol,
7.2 mL, 2 equiv). The mixture was stirred at rt for 1 h
and evaporated under reduced pressure (max temp
30 ꢁC). The crude product was dissolved in DCM
(100 mL).
A
solution of O-tritylhydroxylamine
1
100%, mp 128–132 ꢁC; NMR H (CDCl3) d ppm: 1.21
(50 mmol, 13.88 g, 1.2 equiv) in DCM containing DIEA
(50 mmol, 8.3 mL, 1.2 equiv), was added dropwise. The
reaction mixture was stirred at rt for 3 h. The solution
was washed with NaHCO3 5% and the organic layer
was dried and evaporated under reduced pressure. The
crude product was dissolved in AcOEt and washed three
times with water. The organic layer was dried over
MgSO4 and evaporated to give a yellow solid. The solid
(d, J = 5.1 Hz, 9H), 1.39 (d, J = 4.3 Hz, 9H), 4.74 (d,
J = 5.9 Hz, 1H), 5.75 (d, J = 5.8 Hz, NH), 9.0 (1H,
CONHO).
9.2.10. N-tert-Butoxy-2-(1-methyl-1H-benzoimidazol-2-
yl methyl)-malonamic acid (7). To a solution of N-tert-
butoxy-malonamic acid ethyl ester (49) (350 mg,

M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
73
was washed with diethyl ether and filtered to provide
3.67 g of (52a) as a white powder. Yield 20%, purity
90%, mixture of 2 isomers (67:33) NMR H (CDCl3) d
6.93–6.96 (m, 2H), 7.11–7.12 (m, 3H), 7.29 (m, 15H),
10.48 (br, 1H, NH); MS (MH)ꢀ m/z 450.
1
ppm: 0.53–0.56 (m, 2H), 0.69 (d, J = 6.4 Hz, 4H), 1.08
(t, J = 7.1 Hz, 2H), 1.15–1.23 (m, 2H), 1.43 (t,
J = 7.6 Hz, 2H), 2.53–2.58 (m, 0.33H), 2.97 (t,
J = 7.6 Hz, 0.67H), 3.96 (q, J = 7.1 Hz, 1.33H), 4.06–
4.13 (m, 0.67H), 7.21–7.25 (m, 15H); MS (MH)+ m/z
444. Compound 52a (8.2 mmol, 3.67 g) was dissolved
in a mixture of absolute EtOH (70 mL) and DCM
(50 mL). KOH (32.8 mmol, 1.85 g, 4 equiv) and water
(250 lL) were added. The reaction mixture was stirred
at rt for 24 h and evaporated. The crude product was
dissolved in water and washed three times with AcOEt.
The aqueous phase was acidified with KHSO4 to pH 6
and extracted three times with AcOEt. The organic lay-
ers were dried over MgSO4, filtered and evaporated. The
product precipitated in Et2O to give 2.89 g of (53) as a
white powder. Yield 85%, purity 100%, NMR 1H
(DMSO-d6) d ppm: 0.62–0.66 (m, 6H), 0.84–0.91 (m,
1H), 1.12–1.27 (m, 2H), 3.08 (t, J = 7.3 Hz, 1H), 7.33
(s, 15H), 10.69 (br s, OH); MS (MH)+ m/z 416.
9.2.14. 2-(3-Phenoxy-benzyl)-N-trityloxy-malonamic acid
(60). To a solution of diethyl malonate (55) (50 mmol,
7.59 mL) in 100 mL of ethyl alcohol absolute were add-
ed 3-phenoxybenzaldehyde (50 mmol, 8.62 mL) and
piperidine (1 mL). The mixture was heated at reflux
for 3h30 and evaporated. The crude product was dis-
solved in ethyl acetate, washed twice with NaHCO3
5% and once with NaCl, dried over MgSO4, filtered
and evaporated to give (56) as a yellow oil. This product
was used in the next step without further purification.
(56) (50 mmol) was dissolved in acetonitrile (100 mL).
To this solution cooled to 0 ꢁC was slowly added
NaBH3CN (200 mmol, 12.6 g, 4 equiv). The mixture
was stirred at rt for 16 h and evaporated. The crude
product was dissolved in AcOEt, and the organic layer
was washed once with aq NaHCO3 5%, 10 times with
water and once with NaCl aq, dried over MgSO4, fil-
tered and evaporated. Purification over silica gel (cyclo-
hexane/ethyl acetate, 100:0–98:2) provided 5.14 g of (57)
as a yellow oil. Overall yield 30%, purity 100%, NMR
1H (DMSO-d6) d ppm: 1.09 (t, J = 7.0 Hz, 6H), 3.06
(d, J = 8.1 Hz, 2H), 3.81 (t, J = 8.1 Hz, 1H), 4.05 (q,
J = 7.0 Hz, 4H), 6.83–6.84 (m, 1H), 6.86–6.88 (m, 1H),
6.94–7.01 (m, 3H), 7.10–7.15 (m, 1H), 7.28 (t,
J = 7.8 Hz, 1H), 7.35–7.40 (m, 2H); MS (MH)+ m/z
343. Six hundred and seventy milligrams of KOH
(12 mmol, 1 equiv) was dissolved in ethyl alcohol abso-
lute (50 mL) with water (12 mmol, 220 lL). Compound
57 (12 mmol, 1 equiv) was added, the mixture was stir-
red at rt for 4 h and evaporated. The residue was dis-
solved in aq NaHCO3 5% and washed once with
AcOEt to get rid of the diethyl ester. The aqueous phase
was acidified with concn HCl to pH 1 and extracted four
times with AcOEt. The organic layers were dried over
MgSO4, filtered and evaporated to give 3.39 g of (58)
as a yellow oil. Yield 90%, purity 95%; MS (MH)+ m/z
315. Compound 58 (10 mmol) was dissolved in DCM
(30 mL) with DMF (70 lL). To this solution cooled to
0 ꢁC was added dropwise oxalyl chloride (1.0 mL,
12 mmol, 1.2 equiv). The mixture was stirred at rt for
1 h and evaporated under reduced pressure (max temp
30 ꢁC). The residue was dissolved in DCM (30 mL).
To this solution cooled to 0 ꢁC was added dropwise
DIEA (30 mmol, 4.96 mL, 3 equiv). Then, O-tritylhydr-
oxylamine (8.5 mmol, 2.34 g, 0.85 equiv) was added and
the mixture was stirred at rt for 4 h. The mixture was
washed once with aq NaHCO3 5% and the organic layer
was separated, dried over MgSO4 and evaporated. The
oil was precipitated in petroleum ether to give 3.88 g
of (59) as a beige powder. Yield: 80 %, purity 93%;
MS (MH)+ m/z 570. 1.8 g KOH (32 mmol, 4 equiv)
was dissolved in absolute EtOH (35 mL) with water
(32 mmol, 580 lL). Compound 59 (8 mmol) was added,
the mixture was stirred at rt for 24 h and evaporated.
The residue was dissolved in water and washed once
with AcOEt. The aqueous phase was acidified with
KHSO4 to pH 6 and extracted three times with AcOEt.
The organic layers were dried over MgSO4, filtered and
evaporated to give 3.90 g of (60) as a white powder.
9.2.13.
2-Benzyl-3-oxo-3-[(trityloxy)amino]-propanoic
acid (54). Three grams (13.5 mmol) of 2-benzyl-malonic
acid monoethyl ester (46b) was solubilized in 25 mL
DCM containing 200 lL of DMF. The flask was im-
merged in an ice bath. A solution of oxalyl chloride
(2.3 mL, 2 equiv) in 10 mL DCM was then added drop-
wise. The reaction mixture was stirred at rt for 1 h. Sol-
vents were removed under reduced pressure and the
crude product was dissolved in 25 mL DCM. A solution
of O-tritylhydroxylamine (1.2 equiv, 4.46 g) in 10 mL
DCM and 2.36 mL DIEA (1.2 equiv) was then added.
The reaction mixture was stirred at rt for 3 h. Solvents
were removed under reduced pressure and the crude
product was dissolved in 50 mL AcOEt. The organic
layer was washed with NH4Cl aq (pH 5), K2CO3 aq
(pH 11) and water, dried over MgSO4 and evaporated
under reduced pressure. The product was purified by
column chromatography (BPSUP 70 g Silice Flash-
smart) with DCM as the eluent. The product obtained
was then crystallized in a mixture of diethyl ether and
n-pentane to give (52b) as white powder. Yield 26%,
purity 96%, NMR 1H (DMSO-d6) d ppm: 0.99 (t,
J = 7.2 Hz, 3H), 2.5 (m, 1H), 2.81 (dd, J = 14.4,
8.7 Hz, 1H), 3.50 (dd, J = 8.1, 6.3 Hz, 1H), 3.80–3.97
(m, 2H), 7.00–7.03 (m, 2H), 7.16–7.18 (m, 3H), 7.31 (s,
15H), 10.50 (s, 1H, NH); MS (MH)+ m/z 480. 1.7 g
(3.5 mmol, 1 equiv) of (52b) was dissolved in 50 mL of
a mixture EtOH abs/DCM (4:1). Eight hundred milli-
grams of KOH (14 mmol, 4 equiv) and 71 lL of water
(1 equiv) were added and the mixture was stirred for
24 h at rt. The solvents were evaporated under reduced
pressure, the crude product was dissolved in water
(7.5 mL), and washed twice with 30 mL AcOEt. The
aqueous layer was acidified to pH 6 with KHSO4 and
the product was extracted with AcOEt. The organic
layer was dried over MgSO4 and evaporated under re-
duced pressure to give an oil that precipitated in pentane
to give (54) as a white powder. Yield 56%, purity 97%,
1
mp 149–151 ꢁC; NMR H (DMSO-d6) d ppm: 2.50 (m,
1H), 2.80 (dd, J = 13.8, 7.2 Hz, 1H), 3.21 (m, 1H),
1
Yield 90%, purity 95%, NMR H (DMSO-d6) d ppm:

74
M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
2.42 (dd, J = 13.6 Hz, J = 5.2 Hz, 1H), 2.81 (dd,
J = 13.5, 7.5 Hz, 1H), 3.15–3.19 (m, 1H), 6.72–6.76 (m,
2H), 6.95 (d, J = 8.4 Hz, 2H), 7.10–7.14 (m, 2H), 7.25–
7.40 (m, 18H); MS (MꢀH)ꢀ m/z 542.
9.2.18. Compound 62 method B. White powder; 18 mg,
yield 74%, purity 99%, NMR 1H DMSO-d6 d ppm:
0.82 (d, J = 6.6 Hz, 6H), 1.30–1.43 (m, 1H), 1.45–1.58
(m, 2H), 2.69 (t, J = 7.2 Hz, 2H), 2.94 (t, J = 7.5 Hz,
1H), 3.27 (q, J = 6.9 Hz, 2H), 7.18–7.31 (m, 5H), 7.68
(t, J = 5.7 Hz, NHCO), 8.94 (s, OH), 10.47 (s, CON-
HO), NMR 13C DMSO-d6 d ppm: 22.6, 22.9, 25.9,
35.5, 39.2, 41.0, 49.4, 126.5, 128.7, 129.1, 139.8, 167.3,
9.2.15. Method A: general procedure for the synthesis of
selected compounds using tertbutyle building-blocks. Car-
boxylic acid (0.7–0.8 mmol, 0.5 M/DMF, 1 equiv),
DIEA (1.2 equiv) and bromo-tris-pyrrolidinophospho-
nium-hexa-fluorophosphate (PyBrop) (0.2 M/DCm, 1.2
equiv) were stirred for 30 s at rt. Then the appropriate
amine (0.1 M/DMF, 1 equiv) and DIEA (2 equiv + 1
equiv if the amine is a hydrochloride) were added, the
mixture was stirred overnight at rt and then solvents
were removed under reduced pressure. The residue was
dissolved in DCM and the organic phase was washed
with aqueous HCl 1 N (3·), aq NaHCO3 5% (3·) and
water (once). The organic layer was dried over MgSO4
and evaporated under reduced pressure. The residue
was purified by thick layer chromatography on silica
gel (DCM/MeOH, 95:5). tert-Butyl intermediate was
dissolved in a suspension of BTFA (0.75 M/TFA, 25
equiv) with 0.4% H2O. The reaction mixture was stirred
for 4 h to 5 h 30 min at rt. Then, solvents were removed
under reduced pressure and the residue was dissolved in
water. The aqueous phase was basified with NaOH 1 M
to pH 7 and extracted three times with AcOEt. The
organic layers were dried over MgSO4, filtered and evap-
orated. The residue was precipitated in ether–pentane.
Yields given are those of the deprotection step.
169.3. tR
LCMS
(MNa)+ m/z 301, mp 154–155 ꢁC.
4.11 min; MS (MH)+ m/z 279 and
9.2.19. Compound 63 method B. White powder; 20 mg,
yield 75%, purity 99%, NMR 1H DMSO-d6 d ppm:
0.85 (d, J = 6.0 Hz, 6H), 1.38–1.51 (m, 1H), 1.55–1.73
(m, 4H), 2.48–2.52 (m, 2H), 2.98 (t, J = 7.5 Hz, 1H),
3.00–3.07 (m, 2H), 7.18–7.31 (m, 5H), 7.69 (br s,
NHCO), 8.93 (s, OH), 10.47 (s, CONHO), NMR 13C
DMSO-d6 d ppm: 22.6, 22.9, 26.1, 31.3, 32.9, 38.7,
38.9, 49.5, 126.2, 128.7, 128.8, 142.1, 167.3, 169.4. tR
4.63 min; MS (MH)+ m/z 293, mp 158–160 ꢁC.
LCMS
9.2.20. Compound 64 method A. The compound was
deprotected using TFA 80%/DCM, rt, 72 h. It was puri-
fied by Reverse-phase Preparative HPLC and lyophi-
lized, white powder; 10 mg, yield 7%, purity 99%,
NMR DMSO-d6 d ppm: 0.75 (dd, J = 1.2, 6.5 Hz, 6H),
1.28–1.35 (m, 3H), 1.40–1.50 (m, 4H), 2.48 (t,
J = 7.3 Hz, 2H), 2.87 (t, J = 7.5 Hz, 1H), 2.95–3.00 (m,
2H), 7.07–7.12 (m, 3H), 7.16–7.20 (m, 2H), 7.54 (t,
J = 5.7 Hz, NHCO), 8.82 (br s, OH), 9.30 (br s, CON-
HO). tR LCMS 5.10 min; MS (MH)+ m/z 307.
9.2.16. Method B: general procedure for the synthesis of
selected compounds using trityle building-blocks. Carbox-
ylic acid (0.3–0.4 mmol, 0.5 M/DMF, 1 equiv), DIEA (1.1
equiv) and N,N0-carbonyldiimidazole (0.25 M/THF, 1.1
equiv) were stirred 2 h at rt. Then the appropriate amine
(0.1 M/DMF, 1 equiv) and DIEA (2 equiv + 1 equiv if
the amine is a hydrochloride) were added, the mixture
was stirred for 2 h at rt and then solvents were removed
under reduced pressure. The residue was dissolved in
the minimum AcOEt and the organic phase was washed
with aqueous KHSO4 (pH 3) and water (3·). The organic
layer was dried over MgSO4 and evaporated under re-
duced pressure. The residue was purified on Bond-Elut
SAX column using DCM, to remove residual carboxylic
acid. The powder obtained was triturated in ether–pen-
tane. Trityle intermediate was dissolved in TFA 2%/
DCM (0.03 M) and triisopropylsilane was added drop
by drop until the yellow colour disappeared. The reaction
mixture was stirred for 5 min at rt, solvents were removed
under reduced pressure and the residue was washed with
ether–pentane (20:80). Yields given are those of the
deprotection step.
9.2.21. Compound 65 method B. White powder; 15 mg,
yield 30%, purity 99%, NMR H CD2Cl2 d ppm: 0.91–
0.98 (m, 6H), 1.11–1.22 (m, 2H), 1.55–1.59 (m, 1H),
1.61–1.82 (m, 5H), 2.52–2.59 (m, 3H), 3.03 (t,
J = 12.9 Hz, 1H), 3.73 (t, J = 7.0 Hz, 1H), 3.98 (d,
J = 13.2 Hz, 1H), 4.58 (d, J = 12.9 Hz, 1H), 7.16–7.24
1
(m, 3H), 7.29–7.34 (m, 2H). tR
(MH)+ m/z 333.
5.30 min; MS
LCMS
9.2.22. Compound 66 method B. White powder; 10 mg,
yield 24%, purity 99%, mixture of the cis and trans form
1
of the amide function; NMR H DMSO-d6 d ppm: 0.86
(d, J = 6.3 Hz, 6H), 1.42–1.86 (m, 7H), 2.64–2.82 (m,
2H), 3.01–3.08 (m, 1H), 4.91–4.94 (m, 1H), 7.09–7.17
(m, 4H), 7.95 (d, J = 8.4 Hz, 0.6H, NHCO), 7.99 (d,
J = 8.4 Hz, 0.4H, NHCO), 8.96 (s, OH), 10.42 (s, CON-
HO). tR LCMS 4.71 min; MS (MH)+ m/z 305.
9.2.23. Compound 67 method A. Beige powder; 7 mg,
yield 15%, purity 95%, NMR 1H DMSO-d6 d ppm:
0.85 (d, J = 5.4 Hz, 6H), 1.39–1.67 (m, 3H), 3.03 (t,
J = 7.5 Hz, 0.7H), 3.19 (t, J = 6.9 Hz, 0.3H), 4.24 (d,
J = 5.7 Hz, 2H), 7.12–7.17 (m, 2H), 7.25–7.29 (m, 2H),
8.17 (t, J = 5.7 Hz, 0.7H, NHCO), 8.40 (s, 0.3H,
9.2.17. Compound 61 method B. White powder; 23 mg,
yield 75%, purity 99%, NMR 1H DMSO-d6 d ppm:
0.85 (d, J = 6.0 Hz, 6H), 1.39–1.52 (m, 1H), 1.53–1.70
(m, 2H), 3.04 (t, J = 7.5 Hz, 1H), 4.26 (d, J = 5.7 Hz,
2H), 7.21–7.34 (m, 5H), 8.15 (t, J = 5.7 Hz, NHCO),
8.97 (s, OH), 10.53 (s, CONHO). NMR 13C DMSO-d6
d ppm: 22.6, 23.0, 26.0, 38.9, 42.7, 49.4, 127.2, 127.5,
NHCO), 8.96 (s, OH), 10.54 (s, CONHO). tR
LCMS
4.13 min; MS (MH)+ m/z 283, mp 140–152 ꢁC.
9.2.24. Compound 68 method A. White powder; 10 mg,
1
yield 30%, purity 95 %, NMR H DMSO-d6 d ppm:
0.85 (d, J = 6.3 Hz, 6H), 1.42-1.64 (m, 3H), 3.03 (t,
J = 7.5 Hz, 0.5H), 3.20 (t, J = 6.3 Hz, 0.5H), 4.24 (d,
128.7, 139.8, 167.2, 169.5. tR
3.90 min; MS
LCMS
(MH)+ m/z 265 and (MNa)+ m/z 287, mp 187–188 ꢁC.

M. Flipo et al. / Bioorg. Med. Chem. 15 (2007) 63–76
75
J = 6 Hz, 2H), 7.23–7.39 (m, 4H), 8.20 (t, J = 6.0 Hz,
Acknowledgments
0.5H, NHCO), 8.43 (m, 0.5H, NHCO), 8.96 (s, 1H,
OH), 10.55 (s, 1H, CONHO). tR LCMS 4.65 min; MS
(MH)+ m/z 299.
´
The authors would like to thank Professor Andre
Tartar for thoughtful discussions. We are grateful
to the institutions that support our laboratory (In-
´
serm, Universite de Lille2 and Institut Pasteur de
Lille). Marion Flipo received a grant from CNRS
9.2.25. Compound 69 method B. White powder; 11 mg,
yield 52%, purity 99%, NMR 1H DMSO-d6 d ppm:
0.82 (d, J = 6.3 Hz, 6H), 1.35–1.44 (m, 1H), 1.51–1.63
(m, 2H), 3.02 (t, J = 7.5 Hz, 1H), 4.24–4.27 (m, 2H),
6.85–6.90 (m, 2H), 6.98–7.01 (m, 3H), 7.14 (t,
J = 7.2 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.39 (t,
J = 8.1 Hz, 2H), 8.19 (t, J = 5.4 Hz, NHCO), 8.95 (s,
1H, OH), 10.52 (s, 1H, CONHO). tR LCMS 5.32 min;
MS (MH)+ m/z 357, mp 171–173 ꢁC.
´
and ‘Region Nord-Pas-de-Calais’. This work was spe-
cifically funded by ‘Region Nord-Pas-de-Calais’ and
´
EC. NMR spectra were recorded in the ‘Laboratoire
´
d’Application RMN (LARMN) at Universite de
Lille2, Faculte des Sciences Pharmaceutiques de Lille.
´
Data management was performed using Pipeline Pi-
lotꢂ from Scitegic.
9.2.26. Compound 70 method A. White powder; 30 mg,
yield 46%, purity 99%, NMR 1H DMSO-d6 d ppm:
3.00–3.08 (m, 2H), 3.30 (t, J = 6.75 Hz, 1H), 4.20 (dd,
J = 5.1, 15 Hz, 1H), 4.30 (dd, J = 5,4, 15 Hz, 1H),
7.10–7.23 (m, 10H), 8.20–8.23 (m, NHCO), 10.44 (s,
CONHO), NMR 13C DMSO-d6 d ppm: 35.1, 42.7,
52.7, 126.6, 127.1, 127.5, 128.6, 128.7, 129.3, 139.4,
139.6, 166.2, 168.6. tR LCMS 4.25 min; MS (MH)+ m/z
299, mp 167–169 ꢁC.
Supplementary data
Screening results are available via http://www.sciencedi-
rect.com. Supplementary data associated with this arti-
cle can be found, in the online version, at doi:10.1016/
j.bmc.2006.10.010.
9.2.27. Compound 71 method B. White powder; 13 mg,
yield 18%, purity 99%, NMR 1H DMSO-d6 d ppm:
1.29–1.39 (m, 2H), 1.43–1.53 (m, 2H), 2.53 (t,
J = 7.5 Hz, 2H), 2.98–3.06 (m, 4H), 3.19 (t,
J = 7.8 Hz, 1H), 7.16–7.25 (m, 10H), 7.69 (t,
J = 5.4 Hz, 1H, NHCO), 8.91 (s, 1H, OH), 10.4 (br
References and notes
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s, 1H, CONHO). tR
341.
5.20 min; MS (MH)+ m/z
LCMS
9.2.28. Compound 72 method A. Beige powder; 16 mg,
yield 44%, purity 98%, 1H NMR DMSO-d6 d ppm:
3.24 (dd, J = 15.9, 6.6 Hz, 1H), 3.40 (dd, J = 16.2,
8.1 Hz, 1H), 3.76 (s, 3H), 3.94 (t, J = 6.6 Hz, 1H), 4.18
(dd, J = 15.3, 5.4 Hz, 1H), 4.37 (dd, J = 15.6, 6.3 Hz,
1H), 7.27–7.13 (m, 7H), 7.56–7.50 (m, 2H), 8.33 (t,
J = 5.7 Hz, NHCO), 9.03 (s, 1H, OH), 10.54 (br s, 1H,
CONHO). tR LCMS 3.02 min; MS (MH)+ m/z 353, mp
142–145 ꢁC.
9.2.29. Compound 73 method B. Beige powder; 17 mg,
yield 89%, purity 99%, NMR 1H DMSO-d6 d ppm:
2.96–3.10 (m, 2H), 3.29–3.32 (m, 1H), 4.17 (dd,
J = 15.1, 5.2 Hz, 1H), 4.29 (dd, J = 15.1, 6.1 Hz, 1H),
6.81–7.39 (m, 14H), 8.26 (t, J = 5.7 Hz, NHCO), 8.95
(s, OH), 10.47 (s, CONHO). tR
5.47 min; MS
LCMS
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2359–2372.
(MH)+ m/z 391, mp 164–167 ꢁC.
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9.2.30. Compound 74 method B. White powder; 34 mg,
yield 88%, purity 99%, NMR 1H DMSO-d6 d ppm:
1.31–1.38 (m, 2H), 1.43–1.50 (m, 2H), 2.52–2.55 (m,
2H), 2.96–3.04 (m, 4H), 3.19 (t, J = 7.6 Hz, 1H),
6.78–6.97 (m, 5H), 7.10–7.39 (m, 9H), 7.69 (t,
J = 5.4 Hz, NHCO), 8.92 (s, OH), 10.39 (s, CONHO),
NMR 13C DMSO-d6 d ppm: 28.6, 29.0, 34.9, 35.2,
39.2, 52.6, 117.0, 118.8, 119.7, 123.7, 124.6, 126.1,
128.7, 128.8, 130.1, 130.4, 141.7, 142.5, 156.7, 157.2,
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166.2, 168.3. tR
mp 131–133 ꢁC.
6.26 min; MS (MH)+ m/z 433,
LCMS

76
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