Benzimidazole-2-one: A novel anchoring principle for antagonizing p53-Mdm2

Article abstract of DOI:10.1016/j.bmc.2012.06.020

Herein we propose the benzimidazole-2-one substructure as a suitable tryptophan mimic and thus a reasonable starting point for the design of p53 Mdm2 antagonists. We devise a short multicomponent reaction route to hitherto unknown 2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamides by reacting mono N-carbamate protected phenylenediamine in a Ugi-3CR followed by base induced cyclisation. Our preliminary synthesis and screening results are presented here. The finding of the benzimidazolone moiety as a tryptophan replacement in mdm2 is significant as it offers access to novel scaffolds with potentially higher selectivity and potency and improved biological activities. Observing low μM affinities to mdm2 by NMR and fluorescence polarization we conclude that the 2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamide scaffold might be a good starting point to further optimize the affinities to Mdm2.

Full text of DOI:10.1016/j.bmc.2012.06.020

Bioorganic & Medicinal Chemistry xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzimidazole-2-one: A novel anchoring principle for antagonizing p53-Mdm2
Wei Wang a,b, Haiping Cao a, Siglinde Wolf c, Miguel S. Camacho-Horvitz d, Tad A. Holak c,e
,
Alexander Dömling a,f,
⇑
a University of Pittsburgh, 3501 Fifth Avenue, BST3 11019, Pittsburgh, PA 1526, USA
b Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and Wuhan University, School of Pharmaceutical Sciences, Wuhan 430071, PR China
c Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
d Pittsburgh Allderdice High School, 2409 Shady Avenue Pittsburgh, PA 15217, USA
e Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Cracow, Poland
f University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
Herein we propose the benzimidazole-2-one substructure as a suitable tryptophan mimic and thus a rea-
sonable starting point for the design of p53 Mdm2 antagonists. We devise a short multicomponent reac-
tion route to hitherto unknown 2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamides by reacting
mono N-carbamate protected phenylenediamine in a Ugi-3CR followed by base induced cyclisation. Our
preliminary synthesis and screening results are presented here. The finding of the benzimidazolone moi-
ety as a tryptophan replacement in mdm2 is significant as it offers access to novel scaffolds with poten-
Keywords:
Protein–protein interaction
ANCHOR
Tryptophan
Multicomponent reaction
Ugi
tially higher selectivity and potency and improved biological activities. Observing low
lM affinities to
mdm2 by NMR and fluorescence polarization we conclude that the 2-(2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)acetamide scaffold might be a good starting point to further optimize the affinities
to Mdm2.
p53 mdm2
Selectivity
Ó 2012 Published by Elsevier Ltd.
1. Introduction
deeply buried in the Mdm2 receptor, thus enabling the accommo-
dation of a small molecule. (2) The dimension of the binding site is
The protein protein interaction (PPI) between the transcription
factor p53 and the oncogene Mdm2 is of major recent interest as a
novel non genotoxic anti cancer target.1 Although PPIs are gener-
ally believed to be difficult to treat by small molecules, p53-
Mdm2 is an exception and several potent compounds have been
recently disclosed.2 Additionally one compound RG-7112 currently
undergoes clinical trials and interim results are promising.3
As part of our ongoing interest in the rational design of small
molecular weight PPI antagonists we describe here the discovery
and screening of novel p53-Mdm2 antagonists based on a new var-
iation of the three component Ugi reaction leading to substituted
1-oxo-benzimidazoles.2,4
of similar size as typically seen in small molecules; the distance be-
tween the C- ’s of F19 and L26 is 12 Å. (3) Additionally, the inter-
a
action is highly hydrophobic in nature, thus potentially facilitating
the transport of the small molecule to the intracellular and -nucle-
ar target site. The characteristics of benign PPIs for discovering
drug-like small molecule inhibitors have been recently discussed
by Fry.6
Several potent small molecules have been recently structurally
characterized in Mdm2.4m,n,7 Currently, all well characterized
small molecules have been shown to competitively inhibit the
PPI and in fact bind into the same p53 Mdm2 site. Thus most
known p53-Mdm2 antagonizing small molecule scaffolds clearly
mimic the hot-spot amino acids F19Y23L26. Amongst the hot-spot
amino acids, Y23 is the most buried and hydrophobic. Presumably
much binding energy can be gained for the small molecules by
properly mimicking this moiety. Additionally, the indole-NH of
p53-W23 is forming a hydrogen bond to the backbone carbonyl
of Mdm2-L54. Currently there are several p53-W23 mimics used
in small molecules (Fig. 2). The para-halo phenyl residue is the
most popular used in the majority of active compounds (1, 2, 5)
to mimic W23. Remarkably, in these compounds the p53-W23
Mdm2-L54 hydrogen bond is not reconstituted suggesting that it
is not needed to achieve high affinity to the receptor. Compound
In his seminal work on the ‘structure of the mdm2 oncoprotein
bound to the p53 tumor suppressor transactivation domain’ Pavle-
tich described the ‘hot spot’ of this protein–protein interaction
(PPI).5 The interface of this PPI is formed by a small
a-helical part
of the N-terminal p53 interacting with the deep and spatially con-
fined binding site on the N-terminal Mdm2 (Fig. 1).
Thus p53-Mdm2 differs from many other PPI in several re-
spects: (1) the binding site is deep and concave as opposed to flat
and convex or planar; the hot spot amino acids F19W23L26 are
⇑
Corresponding author.
E-mail addresses: asd30@pitt.edu, a.s.s.domling@rug.nl (A. Dömling).
3
is mimicking W23 with an oxindole moiety. A 6-chloro
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.06.020
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.06.020

2
W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
Figure 1. The hot spot interaction of the transcription factor p53 and the oncogene Mdm2 (PDB ID 1YCR). Left: Frontal view of the p53 amino acids F19W23L26 (orange
sticks) mounted on the amphipathic -helix are deeply buried in the Mdm2 receptor (grey surface); Right: Cut away view to show the hydrophobic floor below the anchor
amino acid p53W23, which is typically filled with bulky halogen atoms in the inhibitor molecules.
a
substituent, similarly to all other compounds considerably en-
hances the affinity to the receptor by filling a hydrophobic pocket
at the bottom of the W23. Compounds 4 and 6 most closely mimic
the W23 by providing a 6-chloroindole substructure. Both com-
pounds were discovered by an approach that makes use of the
most buried amino acid side chain of a PPI, serving as a focal point
With this short 2-step reaction sequence in hand we designed a
small compound library of 2-(2-oxo-2,3-dihydro-1H-benzo[d]imi-
dazol-1-yl)acetamides based on modeling virtual compounds into
the p53/Mdm2 PDB structure 1YCR using the software Moloc.13
In order to obtain compounds with good affinity to Mdm2 it is well
established that indoles with a chlorine in 6-position can be used
to fortunately fill a void and very hydrophobic subpocket at the
bottom of p53’s Trp23.14 Thus we included a chlorine at the 6-po-
sition of our phenylenediamine starting material (Fig. 3, S1). Next
in order to fill the crucial Leu26 and Phe19 pockets suitable sub-
stituents in the isocyanide and aldehyde part had to be chosen.
Based on the pseudo symmetry of the p53 binding pocket in
Mdm2 we included into our design bulky aliphatic side chains such
as tert-butyl, cyclohexyl, iso-butyl and aromatic side chains such
as benzyl and phenylethyl (Fig. 3, S12–S16).2b
4k
for synthesis. A very large virtual library containing indole sub-
structures based on multicomponent reaction chemistry was
screened using the freeware ANCHOR.QUERY.8 In the following
we want to introduce the benzimidazole-2-one substructure as a
new anchor moiety useful for the design of p53 mdm2 antagonists.
2. Results
We envisioned that a 1-oxo-benzimidazole moiety might func-
tion in a similar way to tryptophan exhibiting similar size, shape,
hydrophobicity and H-bond donor capacity and bind into the
W23 pocket of Mdm2. To test this idea and efficiently access sam-
ple compounds we screened the synthetic literature for short and
convergent access to the benzimidazole-2-one moiety. However
all syntheses of complex molecules incorporating this moiety were
of considerable length involving several sequential steps or were
leading to compounds of insufficient diversity.9 Therefore we
decided to investigate convergent and short multicomponent reac-
tion (MCR) approaches towards the benzimidazole-2-one struc-
ture. In fact Hulme et al. reported an elegant and versatile MCR
towards hydantoins and cyclic ureas using mono protected bifunc-
tional building blocks and CO2 in methanol in an Ugi 5-CR followed
by cyclisation (Scheme 1).10 Unfortunately when using o-phenyl-
enediamine the expected product could not be isolated.
We therefore designed a straightforward and general MCR ap-
proach involving the two step sequence Ugi 3-CR of mono carba-
mate protected o-phenylenediamine followed by base induced
cyclisation (Scheme 2). The required mono carbamate protected
phenylenediamines can be readily accessed by reduction of the
corresponding nitro aniline precursor.11
There are many experimental conditions described to run the
U-3CR including a very recent improvement by List et al. using tol-
uene as a solvent and phenylphosphinic acid as an acid catalyst un-
der refluxing conditions.12 These variations of the U-3CR, however,
in our hands did not work and we had to adapt it to our starting
materials by changing the solvent and reaction temperature.
Screening several reaction conditions with the three components
methyl 2-amino-5-chlorophenylcarbamate (S1), phenylacetalde-
hyde (S3) and phenylethylisocyanide (S12), we found that the
combination of the solvent THF and the catalyst phenylphosphinic
acid works well for the U-3CR of our starting materials. The cycli-
sation can be performed in good to acceptable yields using triaza-
bicyclodecene (TBD) as a strongly basic catalyst (Table 1).
In order to physically measure the interaction of our small mol-
ecules to the Mdm2 receptor we performed a fluorescence polari-
zation (FP) assay as previously described by us. 4n The compounds’
affinities screened as racemic mixtures are between no binding
(>60 lM) and 16 lM (Table 1).
For the most active compound 10 we separated the enantio-
mers using preparative supercritical CO2 HPLC. The separation
was fast and very efficient (Fig. 4 1 and 2). The slower dextrorota-
tory (½a 2D0:5
ꢀ
ꢁ69.6 c = 0.98 g/100 ml, MeOH) enantiomer 10 B was
M) then the faster levorotatory enantiomer
+66.54, c = 0.98 g/100 ml, MeOH) (>60 M) and the
more active in FP (4
l
10 A (½a 2D0:5
ꢀ
l
separated enantiomers served for further binding studies using
orthogonal physicochemical screening systems. A NMR-based
competition assay for the discovery of PPI antagonists was recently
introduced by us (AIDA_NMR for antagonist induced dissociation
4b-d,i
assay-NMR).
In this one dimensional experiment the antago-
nist is titrated to the preformed p53-peptide Mdm2 complex. The
release of p53 can be observed in a concentration dependent man-
ner by observing the indole-NH of W23 of p53 (ꢂ10 ppm, Fig. 4.4).
According to this AIDA experiments enantiomer 10 B has a Ki of
ꢂ2
lM whereas enantiomer 10 A is inactive (>60 lM). As a third
screening system we performed the well-known heteronuclear
single quantum coherence (HSQC) experiment where the enantio-
mer 10 B is titrated to the 15N labeled Mdm2 and the ligand-in-
duced perturbations in NMR chemical shifts are observed
resulting in a Ki of 5 l
M.15 Dependent on the binding site of the
small molecule on the receptor the NMR cross peaks are shifted
in a concentration dependent manner. Moreover, the strong bind-
ing of compounds to Mdm2, with KD of less than 1 lM (and a slow
16,4k,n
chemical exchange), is indicated by signals doubling.
Enantiomer 10 B is showing binding to Mdm2 in all three
screening systems and gratifyingly the binding constants are com-
parable (Ki ꢂ2–5
lM). We also screened the compound for Mdm4
activity by FP but could not find any binding. Based on the different
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W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
3
Figure 2. The W23-mimicking anchors of different cocrystallized p53 Mdm2 antagonists based on six chemotypes shown as structural alignments of the indicated PDBs. In
2D the anchor residues are marked in blue. Center: Anchor residue p53-W23 shown as grey sticks, p53-L26 and –F19 shown as red lines, Mdm2-L54 shown as green lines,
hydrogen bond indicated with yellow dotted line (1YCR). First row left: nultin derivative 1 as brown sticks (1RV1); middle: benzodiazepindione 2 as green sticks (1T4E);
right: spirooxindole 3 as yellow sticks (3LBL, the morpholinoethyl side chain has no electron density due to flexibility); lowest row right: imidazole 4 as pink sticks (3LBK);
chromenotriazolopyrimidine 5 as purple sticks (3JZK); a-aminoacylamide 6 as cyan sticks (3TU1).
biophysical screening results and the structurally well character-
ized p53 Mdm2 structure we docked the most potent compound
10 into the Mdm2 binding site using the Moloc software (Fig. 5).13
According to this model the benzimidazolone moiety binds into
the W27 binding site displaying a very similar size, shape and
hydrophobicity as the indole ring. The benzimidazolidinone NH
forms a similar hydrogen bond to the rim L54 carbonyl. In addition
the 6-chloro substituent fills the hydrophobic subpocket at the
bottom of W23 as seen in all co crystal structures (Figs. 1 and 2).
The cyclohexyl substituent resulting from the isocyanide input in
the Ugi reaction mimics the F19 and fills the hydrophobic pockets
exhaustively. The benzyl group introduced via the phenylacetalde-
hyde is pointing deeply into the L26 pocket.
3. Discussion
Amongst all, the largest and most hydrophobic amino acid
tryptophan plays an extraordinary role in PPIs and is the most
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4
W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
R
O
(i) TFA
(ii) Na2CO3 (sat.).
H
N
H
N
R
O
H
N
NC
NC
CHO
CHO
R
N
CO2
CO2
+
+
+
+
+
+
O
H2N
Pg
R
R
R
R
MeOH
MeOH
R
N
O
NH
O
HN
Pg
H
O
R
N
H
Pg
N
R
N
O
NH2
O
HN
Pg
Scheme 1.
R1NC
R2CHO
NHCOOMe
X
O
NH2
H
N
NHCOOMe
NO2
NHCOOMe
X
ClCOOMe
Ugi-3CR
R1
NO2
Zn
N
H
H2N
R2
reduction
r.t THF
X
X
O
R2
H
TBD or
K2CO3 130
NHCOOMe
X
O
H
N
N
°
C microwave
R1
R1
N
NH
X
N
H
O
cyclization
R2
Scheme 2.
abundant interface amino acid.17 Typically tryptophan it is highly
buried and the indole substructure is anchored in deep hydropho-
bic pockets and can thus form a formidable starting point for the
design of small molecular weight antagonists. Our ongoing interest
in the rational design of small molecule antagonists of the p53
Mdm2 and Mdm4 interactions has prompted us to design scaffolds
with novel tryptophan mimicking features.4k While Wang et al.
introduced the oxindole anchor, we introduced indole and others
are using p-substituted phenyl groups, we wanted to explore here
the benzimidazolidinone moiety as a tryptophan mimicking moi-
ety.18 For reasons of resources we restricted our search of suitable
benzimidazolidinone scaffolds to multicomponent reactions as op-
posed to sequential multi step syntheses. We developed a novel
Ugi 3-CR variant using N-methylcarbamate monoprotected
o-phenylenendiamines followed by a base-induced cyclisation to
efficiently access the target scaffold in great diversity and only
two steps from abundantly accessible starting materials. These
reactions suited us well to investigate initial binding activities to
the oncogene Mdm2 by using two NMR assays and the fluores-
cence polarization assay. The use of several orthogonal screening
systems is of high importance to avoid false positives especially
when screening protein protein interactions with strongly hydro-
similar shape and hydrophobicity characteristics exist.
FxxxW(F)xxL sequence motif query in the PDB, for example, results
almost 5000 hits and many of them are parts of -helix motifs
A
a
(query Dec. 2011). The question of selectivity of the small mole-
cules addressing such interaction thus arises.21 We hypothesize
that small molecules interacting with their target exclusively
based on shape and hydrophobicity features might be ambiguous
and show binding to a range of similar hydrophobic binding sites.
On the other hand it is well established that hydrogen bonds are
spatially directed, while van der Waals interactions are not.22
Whereas several currently pursued p53 Mdm2 antagonizing scaf-
folds rely on exploration of shape and hydrophobic characteristics
(e.g. 1, 2, 5), we believe that a mix of hydrogen bonds and hydro-
phobic interactions with the receptor increases the likelihood for
more selective compounds (e.g. 3, 4, 6). On the other hand the pur-
posely design of polypharmacological features can be beneficial as
often seen in the kinase field.23 This however requires a detailed
structural and biochemical target class understanding which is cer-
tainly currently not given in the PPI field. Therefore we believe that
novel tryptophan mimicking anchors in addition to the established
indole and oxindole moieties will advance the field of design of
specific and potent small molecular weight PPI inhibitors for p53
Mdm2 and Mdm4 specifically, but also generally for other trypto-
phan mediated PPIs. Although the herein disclosed benzimidazo-
lidinones show weak initial activity they are suitable starting
points for future optimization.
phobic interfaces.19 Initially found activities ranged from >60
(inactive) to 16 M for racemic compound 10 in a small set of syn-
lM
l
thesized compounds. We note on the side that the compounds of
this series—although synthesized as racemic mixtures—can be con-
veniently and fast separated on a preparative scale using supercrit-
ical fluid CO2 HPLC. The more active enantiomer of compound 10
revealed similar Ki’s of 2–5 lM in the three convergent binding
assay.
4. Experimental
4.1. Synthesis
Strikingly, a very large subset of PPIs is mediated by hydropho-
bic interactions involving an amphipathic
a
-helix.20 Typically, a
All reagents were purchased from commercial sources and
used without further purification. The reactions were conducted
under air atmosphere unless otherwise indicated. Analytical
thin-layer chromatography (TLC) was performed on SiO2
string of hydrophobic amino acids side chains (i, i + 3, i + 7 and
higher) of the donor protein interacts with a suitably formed
hydrophobic binding groove of the receptor protein. Based on the
shire number of
a-helix mediated PPIs it is likely that PPIs with
plates 250 lm on Alumina from Whatman. Visualization was
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W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
5
accomplished by UV irradiation at 254 nm. The purification was
conducted using Preparative Silica gel TLC plates (1000 m,
2.1 ꢃ 50 mm, 3.0
l
m, 0.2 ml/min). Acetonitrile/water mixtures
l
were used as mobile phase for reverse-phase HPLC coupled to
electrospray ionization-mass spectrometry (ESI-MS). The enantio-
mers of 10 were separated by chiral separation via Supercritical
Fluid Chromatography (SFC). Experiments were performed using
the analytical SFC-MS Resolution System (Waters 2998 Photodi-
ode Array Detector, Waters 3100 Mass Detector), and the pre-
parative TharSFC System (Waters 2998 Photodiode Array
Detector). Both analytical and preparative SFC columns were
operating at 40 °C in Analytical-2-Prep Column Oven. Carbon
dioxide supplied by BDS 500 Gas Delivery System was used as
the primary mobile phase for SFC. The optical rotation of 10
20 ꢃ 20 cm). Proton and carbon NMR spectra were determined
on Bruker 400 or 600 MHz NMR spectrometer. Chemical shifts
are reported as d values in parts per million (ppm) as referenced
to residual solvent. 1H NMR spectra are tabulated as follows:
chemical shift, number of protons, multiplicity (s = singlet, br
s = broad singlet, d = doublet, t = triplet, q = quartet, m = multi-
plet), and coupling constant. High Resolution Mass spectra were
obtained at the University of Pittsburgh, Mass Spectrometry facil-
ity. LC–MS analysis was performed on an SHIMADZU instrument,
using an analytical C18 column (Dionex Acclaim 120 Å,
Table 1
Yields and bioactivities of the synthesized structures
No.
Starting materials
Structure Ugi products
Yieldsa
Structure cyclized Products
Yieldsa
Kib
cLogPc
O
O
Cl
NH HN
O
N
HN
1
S1, S3, S12
66%
65%
60
4.93
Cl
O
N
HN
H
O
8
7
O
HN
N
Cl
NH
O
O
N
H
2
S1, S3, S13
67%
49%
16
4.71
O
HN
N
H
Cl
Cl
Cl
O
10
9
O
O
N
HN
O
Cl
NH HN
O
3
4
5
S1, S3, S14
S1, S4, S13
S1, S3, S15
65%
80%
60%
61%
44%
36%
43
3.97
4.31
4.64
N
HN
H
O
12
11
O
O
Cl
NH HN
O
N
HN
O
n.i.d
N
H
HN
O
14
13
O
N
O
N
H
O
Cl
NH HN
O
33
N
Cl
H
HN
O
16
15
(continued on next page)
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6
W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
Table 1 (continued)
No.
Starting materials
Structure Ugi products
Yieldsa
Structure cyclized Products
Cl
Yieldsa
Kib
cLogPc
Cl
HN
HN
O
O
N
NH
O
6
S1, S5, S13
45%
31%
n.i.
5.03
O
N
H
Cl
Cl
Cl
N
H
O
18
17
Cl
O
O
N
H
N
H
NH
N
O
7
S1, S5, S12
52%
45%
n.i.
4.47
O
Cl
N
H
O
NH
Cl
19
20
NH
Cl
NH
Cl
O
O
Cl
Cl
8
S1, S6, S13
S1, S7, S13
S1, S8, S13
S1, S9, S13
42%
41%
n.i.
5.63
N
Cl
NH
O
Cl
O
N
H
HN
O
22
21
NH
NH
O
O
9
63%
41%
48%
43%
41%
32%
n.i.
35
54
5.41
5.30
5.19
Cl
Cl
Cl
NH
O
N
Cl
Cl
Cl
O
N
H
HN
O
24
23
NH
NH
O
F
O
F
F
F
10
NH
O
F
N
F
O
N
H
HN
O
26
25
NH
NH
O
O
Br
Br
11
NH
O
N
O
N
H
HN
O
28
27
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W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
7
Table 1 (continued)
No. Starting materials
Structure Ugi products
Yieldsa
Structure cyclized Products
Yieldsa
Kib
cLogPc
NH
O
NH
O
F
F
Cl
Cl
NH
O
12
S1, S10, S13
52%
31%
n.i.
4.57
N
HN
Cl
O
O
N
H
29
30
Cl
O
O
N
H
F
NH
O
N
F
N
13
S2, S5, S13
43%
30%
fe
4.57
H
O
N
H
O
N
H
31
32
NH
NH
F
O
O
F
Cl
Cl
N
NH
O
14
S1, S5, S16
45%
33%
n.i.
4.01
O
N
H
HN
O
34
33
Cl
Cl
O
O
Cl
NH HN
15
S1, S11, S13
48%
40%
n.i.
5.60
N
HN
NH
O
Cl
O
N
O
H
35
36
a
b
c
Yields after chromatographic isolation of the products are given for both reactions (Ugi, cyclisation) seperately.
Ki based on fluorescence polarisation.
cLogP are predicted by Instant J. Chem 2.4.3.1, 2011, ChemAxon (http://www.chemaxon.com).
n.i.-no interaction.
f-fluorescent.
d
e
was measured on the Schmidt & Haensch Polartronic MH8 using
a 1 dm qs cuvette at T = 20.5 °C in the solvent methanol.
5-Chloro-2-nitroaniline (1.0 g, 5.8 mmol) added into 5 ml methyl
chloroformate, the reaction mixture was refluxed for 9 h. Evapo-
rated and the residue purified by recrystallization with EtOAc and
hexane. Methyl 5-chloro-2-nitrophenylcarbamate was obtained as
solid (1.2 g, 90%).24
5. General procedures
5.1. N-Protection
5.2. Methyl 5-chloro-2-nitrophenylcarbamate
C8H7ClN2O4, Mw: 230.60 g/mol; HRMS (ESI-TOF) m/z (calcd):
230.0094, (found) [M+H]+: 230.0102; 1H NMR (600 MHz,
CDCl3): 9.87 (s, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.10 (d,
J = 9.0 Hz, 1H), 7.03 (dd, J = 2.4, 9.0 Hz, 1H), 3.80 (s, 3H). 13C
NMR (150 MHz, CDCl3): 151.13, 142.61, 136.26, 133.93,
127.06, 122.48, 119.93, 53.01
NO2
Cl
NO2
Cl
H
N
O
NH2
O
+
Cl
O
O
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8
W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
Amine Component
O
O
O
O
Cl
NH
NH2
NH
NH2
F
S1
S2
Aldehyde Component
CHO
Cl
Cl
Cl
CHO
CHO
CHO
CHO
S3
S4
S5
S6
S7
F
F
Cl
F
Br
F
CHO
CHO
CHO
CHO
S8
S9
S10
S11
Isocyanide Component
NC
NC
NC
NC
NC
S12
S13
S14
S15
S16
Figure 3. Starting materials used in Ugi 3-CR reaction.
5.3. Reduction
5.4. Methyl 2-amino-5-chlorophenylcarbamate
C8H9ClN2O2, Mw: 200.62 g/mol; HRMS (ESI-TOF) m/z (calcd):
200.0353, (found) [M+Na]+: 200.0361; 1H NMR (600 MHz, CDCl3):
7.39 (br, 1H), 6.98 (dd, J = 1.8, 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H),
6.48 (br, 1H), 3.79 (s, 3H), 3.69 (br, 2H). 13C NMR (150 MHz, CDCl3):
154.81, 137.98, 126.10, 125.33, 124.35, 118.63, 52.78
NO2
H
NH2
Cl
H
N
N
O
O
+
+ FeCl3
Zn
O
O
5.5. Ugi 3-CC reaction
Cl
O
O
P
NH2
Cl
Cl
H
H
H
N
N
H
N
O
O
NC
+
+
O
O
HN
O
O
To a solution of methyl 2-amino-5-chlorophenylcarbamate (20 mg,
0.1 mmol), 2-phenylacetaldehyde (12 mg, 0.1 mmol), phenylphos-
phinic acid (7 mg, 0.05 mmol) in 0.5 ml THF was added (2-isocyano-
ethyl)benzene (13 mg, 0.1 mmol). After stirring at room
temperature for 24 h, 4 ml water was added the reaction mixture.
The mixture was neutralized with 20% NaOH. The solution was ex-
tracted with ether (3 ꢃ 5 ml). The combined organic layer was
washed with brine, dried and concentrated. The residue was puri-
fied by column chromatography to afford methyl 5-chloro-2-(1-
oxo-1-(phenethylamino)-3-phenylpropan-2-ylamino)phenylcarba-
mate (20 mg, 66%).
A mixture of ferric chloride (2.8 g, 172 mmol), Zinc dust (3.4 g,
52.4 mmol) and methyl 5-chloro-2-nitrophenylcarbamate (1.2 g,
52 mmol) was added to the mixed solvent dimethyl formamide
and water (1: I, 80 ml) and heated in water-bath (100 °C). After
completion of the reaction (0.5 h, monitored by TLC), the reaction
mixture filtered and filtrate was diluted with water (80 ml) and
basified by adding saturated solution of sodium carbonate. It was
extracted with dichloromethane (3 ꢃ 140 ml). The combined organ-
ic layer was dried and concentrated. The residue was purified by
column chromatography to afford methyl 2-amino-5-chlorophe-
nylcarbamate (0.88 g, 84%).
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W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
9
1
O
N
H
O
N
NH
Cl
Figure 4. (1): Structure of benzimidazolone 10; (2): preparative SFC trace (UV 220 nM) of the separation of 10 on the RegisCell (#784104, OD) column with baseline
separation after 3 min; (3): Superposition of NMR HSQC spectra of 15N-labeled Mdm2 titrated against enantiomer 10 B. The spectrum of free Mdm2 is shown in red. The
spectrum of Mdm2–10 B (intermediate ratio, 1: 7.56, respectively) is shown in blue, and the spectrum of Mdm2–10 B (final ratio, 1:2) is shown in green; (4): AIDA NMR of the
enantiomers of compound 10. (A and D) p53-Mdm2 reference, (B) enantiomer 10 A ratio 1:2, (C) enantiomer 10 A ratio 1:8, (E) enantiomer 10 B ratio 1:2, (F) enantiomer 10 B
ratio 1:8.
5.6. Cyclization
Two methods:
A: For cyclization of Ugi products 7, 9, 11, 13, 15, 17, 19
Cl
O
H
N
TBD
THF
N
H
N
H
N
O
HN
O
O
N
H
Cl
O
20 mg Methyl 5-chloro-2-(1-oxo-1-(phenethylamino)-3-phenyl-
propan-2-ylamino)phenylcarbamate and 1 mg of triazabicyclode-
cene in 1 ml THF were refluxed for 4 h. 12 mg (65%) of 8 was
obtained after preparative TLC separation.
B: For cyclization of Ugi products 21, 23, 25, 27, 29, 31, 33, 35
Figure 5. Docking pose of (S)-10 into the superimposed p53 Mdm2 structure
suggesting the binding of the 1-oxo-benzimidazole, the cyclohexyl and the benzyl
moieties into the W23, F19 and L26 pockets, respectively (PDB ID 1YCR).
NH
NH
K2CO3
O
O
microwave
and evaporated. The residue was purified by preparative TLC to give
product 22 19 mg (43%).
N
Cl
NH
DMSO
Analytical Data of Ugi products and cyclization products:
O
Cl
O
N
HN
H
O
5.7. Methyl 5-chloro-2-(1-oxo-1-(phenethylamino)-3-
phenylpropan-2-ylamino)phenylcarbamate (7)
46 mg of methyl 5-chloro-2-(2-(cyclohexylamino)-1-(naphthalen-
1-yl)-2-oxoethylamino)phenyl carbamate and 13 mg of K2CO3 were
heated under 130 °C for 30 min. Next it was diluted with 5 ml EtOAc
and washed with 5 ml water, the organic layer was collected, dried
C25H26ClN3O3, Mw:451.95 g/mol; HRMS (ESI-TOF), m/z (calcd):
449.1273, (found) [M+Na]+:474.1581; 1H NMR (600 MHz, CDCl3):
7.25–7.32 (m, 3H), 7.14–7.21 (m, 6H), 7.02 (dd, J = 1.8, 9.0 Hz,
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10
W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
1H), 6.99 (d, J = 6.6 Hz, 2H), 6.86 (br, 1H), 6.49 (d, J = 9.0 Hz, 1H),
6.45 (br, 1H), 4.11 (s, 1H), 4.02 (s, 1H), 3.66 (s, 3H), 3.48–3.50 (m,
1H), 3.37–3.42 (m, 1H), 3.17–3.20 (m, 1H), 3.11–3.12 (m, 1H),
2.65–2.71 (m, 2H), 13C NMR (150 MHz, CDCl3): 172.21, 154.90,
139.67, 138.66, 136.29, 129.22, 128.81, 128.63, 128.44, 127.57,
127.23, 126.30, 125.89, 124.38, 123.36, 114.17, 58.97, 52.87,
40.45, 38.83, 35.32
5.12. N-tert-Butyl-2-(5-chloro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)-3-phenylpropanamide (12)
C20H22ClN3O2, Mw: 371.86 g/mol; HRMS (ESI-TOF), m/z (calcd):
371.1401, (found) [M+Na]+: 394.1288; 1H NMR (600 MHz, CDCl3):
8.50 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.12–7.18 (m, 3H), 7.08 (dd,
J = 1.8, 8.4 Hz, 1H), 7.02–7.03 (m, 3H), 5.95 (s, 1H), 5.05 (dd,
J = 6.0, 9.6 Hz, 1H), 3.56 (dd, J = 6.0, 13.8 Hz, 1H), 3.31 (dd,
J = 10.2, 13.8 Hz, 1H), 1.29 (s, 9H). 13C NMR (150 MHz, CDCl3):
167.53, 154.72, 136.59, 128.94, 128.50, 128.18, 127.62, 127.45,
126.92, 121.88, 111.44, 109.94, 51.78, 34.67, 30.96, 28.53.
5.8. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-
N-phenethyl-3-phenylpropanamide (8)
C
24H22ClN3O2, Mw: 419.90 g/mol; HRMS (ESI-TOF), m/z
5.13. Methyl 5-chloro-2-(1-(cyclohexylamino)-4-methyl-1-
oxopentan-2-ylamino)phenylcarbamate (13)
(calcd):419.1401, (found) [M+Na]+: 442.1263; 1H NMR
(600 MHz, CDCl3): 9.80 (s, 1H), 7.10–7.19 (m, 7H), 7.06 (dd,
J = 1.8, 8.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 2H), 6.99 (d, J = 1.8 Hz,
1H), 6.97 (d, J = 7.2 Hz, 2H), 6.42 (t, J = 6.0 Hz, 1H), 5.10 (dd,
J = 6, 10.2 Hz, 1H), 3.54–3.60 (m, 2H), 3.43–3.48 (m, 1H), 3.35
(dd, J = 4.2, 14.4 Hz, 1H), 2.73 (t, J = 6.6 Hz, 2H). 13C NMR
(150 MHz, CDCl3): 168.39, 155.28, 138.34, 136.50, 128.87,
128.59, 128.52, 127.72, 127.20, 126.95, 126.50, 121.83, 111.33,
110.24, 57.98, 40.84, 35.31, 34.49.
C20H30ClN3O3, Mw: 395.92 g/mol; HRMS (ESI-TOF) m/z
(calcd): 395.1976, (found) [M+Na]+: 418.1908; 1H NMR
(600 MHz, CDCl3): 7.38 (br, 1H), 7.17 (s, 1H), 7.02 (dd, J = 2.4,
9.0 Hz, 1H), 6.95 (br, 1H), 6.51 (d, J = 9.0 Hz, 1H), 4.23 (d,
J = 4.8 Hz, 1H), 3.77 (s, 3H), 3.62–3.65 (m, 1H), 1.72–1.78 (m,
3H), 1.51–1.65 (m, 5H), 1.17–1.27 (m, 3H), 1.02–1.11 (m, 2H),
0.95 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.0 Hz, 3H). 13C NMR
(150 MHz, CDCl3): 172.98, 155.43, 140.01, 127.20, 125.79,
124.33, 122.75, 113.62, 57.29, 52.87, 48.02, 42.68, 32.67, 32.61,
25.34, 25.05, 24.81, 24.67, 23.19, 21.60.
5.9. Methyl 5-chloro-2-(1-(cyclohexylamino)-1-oxo-3-
phenylpropan-2-ylamino)phenylcarbamate (9)
5.14. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-N-cyclohexyl-4-methylpentanamide (14)
C
23H28ClN3O3, Mw:429.94 g/mol; HRMS (ESI-TOF), m/z (calcd):
429.1819, (found) [M+Na]+: 452.1707; 1H NMR (600 MHz, CDCl3):
7.38–7.25 (m, 3H), 7.19 (d, J = 7.2 Hz, 2H), 7.11 (s, 1H), 7.03 (d,
J = 9.0 Hz, 1H), 6.85 (br, 1H), 6.73 (br, 1H), 6.51 (d, J = 9.0 Hz, 1H),
4.20 (s, 1H), 4.12 (s, 1H), 3.68–3.69 (m, 1H), 3.64 (s, 3H), 3.13–
3.20 (m, 2H), 1.74 (d, J = 10.8 Hz, 1H), 1.54–1.64 (m, 4H), 1.21–
1.29 (m, 2H), 0.99–1.06 (m, 2H), 0.84–0.88 (m, 1H). 13C NMR
(150 MHz, CDCl3): 171.06, 154.98, 139.87, 136.16, 129.40, 128.70,
127.43, 127.16, 126.04, 124.24, 122.88, 113.86, 58.63, 52.81,
48.16, 38.65, 32.58, 32.50, 25.31, 24.83, 24.69.
C19H26ClN3O2, Mw: 363.88 g/mol; HRMS (ESI-TOF), m/z
(calcd):363.1714, (found) [M+Na]+: 386.1647; 1H NMR (600 MHz,
CDCl3): 10.23 (s, 1H), 7.22 (d, J = 9.0 Hz, 1H), 7.12 (s, 1H), 7.06 (d,
J = 8.4 Hz, 1H), 6.25 (s, 1H), 4.99 (dd, J = 5.4, 10.2 Hz, 1H), 3.73–
3.79 (m, 1H), 2.15–2.17 (m, 1H), 2.03–2.07 (m, 1H), 1.88–1.93
(m, 1H), 1.78–1.80 (m, 1H), 1.57–1.69 (m, 3H), 1.30–1.39 (m,
3H), 1.02–1.14 (m, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.6 Hz,
3H). 13C NMR (150 MHz, CDCl3): 168.38, 155.58, 128.70, 127.69,
127.37, 121.82, 111.44, 110.20, 55.27, 48.61, 37.34, 32.80, 25.39,
24.98, 24.73, 24.65, 22.99.
5.10. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-N-cyclohexyl-3-phenylpropanamide (10)
5.15. Methyl 2-(1-(benzylamino)-1-oxo-3-phenylpropan-2-
ylamino)-5-chlorophenylcarbamate (15)
C
22H24ClN3O2, Mw: 397.90 g/mol; HRMS (ESI-TOF), m/z (calcd):
397.1557, (found) [M+Na]+: 420.1466; 1H NMR (600 MHz, CDCl3):
9.95 (s, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.13 (t, J = 7.2 Hz, 2H), 7.06–
7.09 (m, 2H), 7.00–7.02 (m, 3H), 6.23 (d, J = 7.8 Hz, 1H), 5.13 (dd,
J = 6.0, 10.2 Hz, 1H), 3.74–3.81 (m, 1H), 3.60 (dd, J = 6.0, 14.4 Hz,
1H), 3.35 (dd, J = 10.2, 14.4 Hz, 1H), 1.79–1.84 (m, 2H), 1.57–1.67
(m, 3H), 1.29–1.36 (m, 2H), 0.99–1.13 (m, 3H). 13C NMR
(150 MHz, CDCl3): 167.45, 155.48, 136.55, 128.90, 128.59, 128.48,
127.70, 127.32, 126.93, 121.75, 111.49, 110.27, 58.33, 48.75,
34.83, 32.80, 32.71, 25.36, 24.74, 24.67.
C24H24ClN3O2, Mw: 437.92 g/mol; HRMS (ESI-TOF), m/z (calcd):
437.1506, (found) [M+Na]+: 460.1408; 1H NMR (600 MHz, CDCl3):
7.05–7.25 (m, 12H), 6.56 (d, J = 7.8 Hz, 1H), 4.30–4.42 (m, 2H),
4.11–4.16 (m, 1H), 3.60 (s, 3H), 3.21 (s, 2H). 13C NMR (150 MHz,
CDCl3): 172.17, 154.91, 139.81, 137.92, 136.11, 129.30, 128.82,
128.55, 128.40, 127.67, 127.38, 127.17, 126.00, 124.31, 123.41,
114.40, 59.00, 52.84, 43.17, 38.74.\
5.11. Methyl 2-(1-(tert-butylamino)-1-oxo-3-phenylpropan-2-
ylamino)-5-chlorophenylcarbamate (11)
5.16. N-Benzyl-2-(5-chloro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)-3-phenylpropanamide (16)
C
21H26ClN3O3, Mw: 403.90 g/mol; HRMS (ESI-TOF), m/z
C
23H20ClN3O2, Mw: 405.88 g/mol; HRMS (ESI-TOF), m/z (calcd):
(calcd): 403.1663, (found) [M+Na]+: 426.1546; 1H NMR
(600 MHz, CDCl3): 7.31 (t, J = 7.2 Hz, 2H), 7.25–7.28 (m, 1H),
7.20 (d, J = 6.6 Hz, 2H), 7.16 (s, 1H), 7.04 (dd, J = 1.2, 9.0 Hz,
1H), 6.69 (br, 1H), 6.53 (d, J = 9.0 Hz, 1H), 6.37 (br, 1H), 4.20 (s,
1H), 3.82–3.85 (m, 1H), 3.66 (s, 3H), 3.14 (m, 2H), 1.22 (s, 9H).
13C NMR (150 MHz, CDCl3): 171.34, 154.82, 139.55, 136.47,
129.39, 128.72, 128.53, 127.14, 125.66, 124.75, 123.28, 114.37,
59.92, 52.73, 51.13, 38.82, 28.38.
405.1244, (found) [M+Na]+: 428.1143; 1H NMR (600 MHz, CDCl3):
8.68 (s, 1H), 7.25–7.26 (m, 3H), 7.21 (d, J = 8.4 Hz, 1H), 7.12–7.17
(m, 5H), 7.03–7.07 (m, 3H), 6.96 (s, 1H), 6.70 br, 1H), 5.17 (dd,
J = 6.0, 10.2 Hz, 1H), 4.38–4.52 (m, 2H), 3.63 (dd, J = 6.0, 14.4 Hz,
1H), 3.39 (d, J = 4.2, 13.8 Hz, 1H). 13C NMR (150 MHz, CDCl3):
168.35, 154.83, 137.49, 136.35, 128.91, 128.70, 128.55, 128.18,
127.73, 127.67, 127.62, 127.40, 126.98, 121.90, 111.31, 110.04,
58.25, 43.76, 34.63.
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W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
11
5.17. Methyl 5-chloro-2-(2-(cyclohexylamino)-1-(1-(3-
fluorophenyl)cyclopropyl)-2-oxoethylamino)phenyl carbamate
(17)
32.4, 32.6, 48.2, 52.5, 59.2, 113.1, 120.4, 125.7, 127.8, 129.2,
130.7, 131.1, 131.6, 141.0, 155.5, 168.7.
5.22. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-N-cyclohexyl-2-(3,4-dichlorophenyl)acetamide (22)
C
22H25Cl2N3O3, Mw: 450.36 g/mol; HRMS (ESI-TOF), m/z
(calcd): 449.1273, (found) [M+H]+: 472.1166; HPLC-MS rt: 11.41,
m/z [M]+ 450.2. 1H NMR (CDCl3, 400 MHz): d = 0.95 (1H, m),
1.14–1.20 (2H, m), 1.22–1.36 (2H, m), 1.60 (2H, m), 1.71 (2H, m),
1.89 (2H, m), 3.76 (1H, m), 3.80 (3H, m), 4.76 (1H, s), 6.47 (1H,
s), 6.57 (2H, d, J = 7.2 Hz), 7.04 (1H, dd, J1 = 2.4 Hz, J2 = 8.8 Hz),
7.32 (1H, s), 7.38 (4H, s). 13C NMR (CDCl3, 100 MHz): d = 24.6,
24.7, 25.3, 32.6, 32.8, 48.5, 52.9, 62.7, 114.8, 124.1, 125.4, 126.8,
128.7, 129.4, 134.6, 136.7, 138.8, 155.0, 169.3.
C21H20Cl3N3O2, Mw: 452.76 g/mol; HRMS (ESI-TOF), v (calcd):
451.0621, (found) [M+Na]+: 474.0525; 1H NMR (CDCl3,
400 MHz): d = 1.11–1.17 (3H, m), 1.20–1.40 (3H, m), 1.61–1.74
(3H, m), 1.89 (d, 1H, J = 12 Hz), 2.0 (d, 1H, J = 12 Hz), 3.88 (s, 1H),
6.11 (s, 1H), 6.39 (1H, d, J = 7.6 Hz), 6.99 (1H, d, J = 8.4 Hz), 7.08
(1H, s), 7.19 (1H, d, J = 8.4 Hz), 7.45 (1H, t, J = 6.4 Hz), 9.55 (1H,
s). 13C-NMR (CDCl3, 100 MHz): d = 24.7, 32.7, 32.8, 49.2, 58.6,
110.3, 111.8, 122.0, 127.1, 127.3, 128.0, 128.5, 130.0, 130.8,
133.0, 133.2, 134.3, 135.1, 165.7.
5.18. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-2-(4-chlorophenyl)-N-cyclohexylacetamide (18)
5.23. Methyl 5-chloro-2-(2-(cyclohexylamino)-1-(naphthalen-1-
yl)-2-oxoethylamino)phenylcarbamate (23)
C21H21Cl2N3O2, Mw: 418.32 g/mol; HRMS (ESI-TOF), m/z
(calcd): 417.1011, (found) [M+H]+: 440.0908; HPLC-MS rt: 11.18,
m/z [M+H]+ 418.1. 1H NMR (MeOD, 400 MHz): d = 1.2–1.42 (6H,
m), 1.65 (1H, d, J = 12.4 Hz), 1.78 (2H, m), 1.87 (1H, d,
J = 11.2 Hz), 1.99 (1H, d, J = 12 Hz), 3.78 (1H, m), 6.26 (1H, s),
6.72 (1H, d, J = 8.4 Hz), 6.87 (1H, d, J = 8.8 Hz), 7.06 (1H, s), 7.32
(2H, d, J = 8.4 Hz), 7.41 (2H, d, J = 8 Hz). 13C-NMR (MeOD,
100 MHz): d = 24.7, 25.2, 47.8, 58.1, 109.1, 112.2, 120.4, 126.8,
128.0, 128.6, 129.4, 129.5, 133.6, 134.0, 155.2, 167.4.
C
26H28ClN3O3, Mw: 465.97 g/mol; HRMS (ESI-TOF), m/z (calcd):
465.1819, (found) [M+H]+: 466.1903; HPLC-MS rt: 11.53, m/z
[M+H]+466.0. 1H NMR (MeOD, 400 MHz): d = 1.15–1.19 (2H, m),
1.34–1.39 (3H, m), 1.64-1.68 (3H, m), 1.78 (1H, m), 1.95 (1H, m),
3.67 (3H, s), 3.79 (1H, m), 5.72 (1H, s), 6.74 (1H, d, J = 8.8 Hz),
7.09 (1H, dd, J1 = 2.8 Hz, J2 = 8.4 Hz), 7.26 (1H, d, J = 1.6 Hz), 7.50
(1H, dd, J1 = 7.2 Hz, J2 = 8 Hz), 7.53 (2H, m), 7.60 (1H, dd,
J1 = 0.8 Hz, J2 = 7.2 Hz), 7.92 (1H, d, J1 = 8.4 Hz), 7.93 (1H, dd,
J1 = 1.2 Hz, J2 = 7.2 Hz), 8.11 (1H, d, J = 7.2 Hz). 13C NMR (MeOD,
100 MHz): d = 24.5, 24.6, 25.1, 32.0, 32.2, 48.7, 51.6, 58.9, 122.8,
122.2, 122.9, 124.96, 125.0, 125.6, 126.4, 128.6, 128.8, 131.6,
133.7, 124.2, 156.2, 171.6.
5.19. Methyl 5-chloro-2-(1-(4-chlorophenyl)-2-
(isobutylamino)-2-oxoethylamino)phenylcarbamate (19)
C
20H23Cl2N3O3, Mw: 424.32 g/mol; HRMS (ESI-TOF), m/z
5.24. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-N-cyclohexyl-2-(naphthalen-1-yl)acetamide (24)
(calcd): 423.1116, (found) [M+Na]+: 446.1006; HPLC-MS rt:
11.06, m/z [M]+424.1. 1H NMR (MeOD, 400 MHz): d = 0.87 (6H, d,
J = 6 Hz), 1.01 (rotamer, d, J = 6.8 Hz), 1.80 (1H, m), 3.00 (1H, m),
3.14 (1H, m), 3.85 (3H, s), 5.04 (1H, s), 6.58 (1H, d, J = 8.8 Hz),
7.06 (1H, d, J = 4.8 Hz), 7.31 (1H, s), 7.45 (2H, d, J = 8 Hz), 7.58
(2H, d, J = 8 Hz), 8.42 (1H, s). 13C-NMR (MeOD, 100 MHz): d
= 18.9, 28.2, 47.1, 51.7, 61.2, 113.5, 122.2, 125.4, 1256.0, 128.5,
128.6, 133.7, 137.3, 139.5, 156.3, 171.8.
C25H24ClN3O2, Mw: 433.93 g/mol; HRMS (ESI-TOF), m/z (calcd):
433.1557, (found) [M+Na]+: 456.1487; 1H NMR (CDCl3, 400 MHz):
d = 1.19–1.38 (2H, m), 1.40–1.0 (3H, m), 1.64 (1H, m), 1.76 (1H, m),
1.91 (1H, d, J = 2.0 Hz), 2.05 (1H, d, J = 2.0 Hz), 3.84 (1H, m), 6.52
(1H, d, J = 8.4 Hz), 6.66 (1H, dd, J1 = 2 Hz, J2 = 8.4 Hz), 6.97 (1H, d,
J = 2 Hz), 7.49 (1H, m), 7.59 (1H, dd, J1 = 7.6 Hz, J2 = 8.4 Hz), 7.77
(1H, d, J = 7.2 Hz), 7.90–7.99 (3H, m). 13C NMR (CDCl3, 100 MHz):
d = 24.6, 25.2, 32.1, 32.2, 48.9, 56.8, 109.0, 111.8, 120.3, 122.4,
124.4, 126.0, 126.3, 126.8, 127.0, 126.7, 128.7, 129.2, 129.7,
130.3, 131.8, 134.1, 155.0, 168.7.
5.20. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-2-(4-chlorophenyl)-N-isobutylacetamide (20)
C19H19Cl2N3O2, Mw: 392.28 g/mol; HRMS (ESI-TOF), m/z
(calcd): 391.0854, (found) [M+Na]+: 414.0746; HPLC-MS rt:
10.84, m/z [M+H]+392.. 1H NMR (MeOD, 400 MHz): d = 0.93 (6H,
d, J = 6.8 Hz), 1.85 (1H, m), 3.12 (2H, m), 6.29 (1H, s), 6.74 (1H, d,
J = 8.4 Hz), 6.89 (1H, dd, J1 = 2 Hz, J2 = 10.4 Hz), 7.07 (1H, d,
J = 2 Hz), 7.34 (2H, d, J = 8.4 Hz), 7.41 (2H, d, J = 8.4 Hz). 13C NMR
(MeOD, 100 MHz): d = 19.1,19.1, 28.1, 58.3, 109.2, 111.9, 120.5,
126.9, 128.0, 129.4, 129.7, 133.4, 134.1, 155.2, 168.4.
5.25. Methyl 5-chloro-2-(2-(cyclohexylamino)-2-oxo-1-(4-
(trifluoromethyl)phenyl)ethylamino)phenyl carbamate (25)
C23H25ClF3N3O3, Mw: 483.91 g/mol; HRMS (ESI-TOF), m/z
(calcd): 483.1537, (found) [M+H]+: 484.1602; HPLC-MS rt:
11.55, m/z [M+H]+484.2. 1H NMR (DMSO, 400 MHz): d = 1.08–
1.25 (5H, m), 1.51–1.60 (3H, m), 1.63–1.67 (1H, m), 1.78 (1H,
m), 3.5 (1H, m), 3.69 (3H, s), 5.13 (1H, d, J = 7.6 Hz), 5.85 (1H,
d, J = 7.6 Hz), 6.27 (1H, d, J = 8.8 Hz), 6.96 (1H, dd, J = 2.4 Hz,
J = 8.4 Hz), 7.24 (1H, s), 7.72 (2H, d, J = 8.8 Hz), 7.75 (2H, d,
J = 8.8 Hz), 8.32 (1H, d, J = 8 Hz), 9.25 (1H, s). 13C-NMR (DMSO,
100 MHz): d = 24.6, 24.8, 25.5, 32.4, 32.7, 48.2, 52.5, 60.0, 113.1,
120.4, 123.3, 125.7, 125.8, 126.0, 128.1, 128.5, 128.8, 139.3,
144.4, 155.6, 168.8.
5.21. Methyl 5-chloro-2-(2-(cyclohexylamino)-1-(3,4-
dichlorophenyl)-2-oxoethylamino)phenylcarbamate (21)
C22H24Cl3N3O3, Mw: 484.80 g/mol; HRMS (ESI-TOF), m/z
(calcd): 483.0883, (found) [M+Na]+: 506.0785; HPLC-MS rt:
11.78, m/z [M+H]+484.1. 1H NMR (DMSO, 400 MHz): d = 1.05–
1.25 (5H, m), 1.52–1.58 (3H, m), 1.67 (1H, m), 1.78 (1H, m), 3.50
(1H, m), 3.70 (3H, s), 5.04 (1H, d, J = 7.6 Hz), 5.81 (1H, d,
J = 7.6 Hz), 6.28 (1H, d, J = 8.8 Hz), 6.97 (1H, dd, J1 = 2.4 Hz,
J2 = 8.8 Hz), 7.24 (1H, s), 7.52 (1H, dd, J1 = 2 Hz, J2 = 8.4 Hz), 7.63
(1H, d, J = 8.4 Hz), 7.80 (1H, d, J = 1.6 Hz), 8.28 (1H, d, J = 8 Hz),
9.22 (1H, s). 13C NMR (DMSO, 100 MHz): d = 24.6, 24.8, 25.5,
5.26. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-N-cyclohexyl-2-(4-(trifluoromethyl)phenyl) acetamide (26)
C
22H21ClF3N3O2, Mw: 451.87 g/mol; HRMS (ESI-TOF), m/z
(calcd): 451.1274, (found) [M+Na]+: 474.1148; HPLC-MS rt:
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.06.020

12
W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
11.36, m/z [M+H]+ 452.2. 1H NMR (MeOD, 400 MHz): d = 1.2–1.5
(6H, m), 1.65–1.90 (4H, m), 2.02 (1H, d, J = 12.4 Hz), 3.81 (1H,
m), 6.37 (1H, s), 6.77 (1H, d, J = 8.4 Hz), 6.89 (1H, dd,
J1 = 2 Hz, J2 = 8.4 Hz), 7.09 (1H, d, J = 2 Hz), 7.52 (2H, d,
J = 8.4 Hz), 7.72 (2H, d, J = 8.4 Hz). 13C NMR (MeOD, 100 MHz):
d = 23.2, 23.7, 23.8, 30.6, 30.7, 3108, 47.5, 56.8, 107.7, 110.5,
119.0, 121.2, 123.8, 125.4, 126.4, 127.0, 127.9, 128.5, 128.8,
137.8, 153.7, 165.5.
5.31. Methyl 2-(1-(4-chlorophenyl)-2-(cyclohexylamino)-2-
oxoethylamino)-4-fluorophenylcarbamate (31)
C22H25ClFN3O3, Mw: 433.90 g/mol; HRMS (ESI-TOF), m/z
(calcd): 433.1568, (found) [M+H]+: 434.1653; HPLC-MS rt:
11.12, m/z [M+H]+434.1. 1H NMR (MeOD, 400 MHz): d = 1.24–
1.17 (2H, m), 1.35–1.45 (3H, m), 1.68–1.76 (3H, m), 1.84 (1H, d,
J = 12.4 Hz), 1.95 (1H, d, J = 11.2 Hz), 3.70 (1H, m), 3.83 (1H, s),
5.03 (1H, s), 6.29 (1H, d, J = 11.2 Hz), 6.47 (1H, t, J = 8.4 Hz),
7.16 (1H, t, J = 7.2 Hz), 7.45 (2H, d, J = 8 Hz), 7.57 (2H, d,
J = 8 Hz). 13C-NMR (MeOD, 100 MHz): d = 24.5, 24.6, 25.1, 31.9,
32.2, 51.6, 60.4, 99.4 (d = 27 Hz), 103.1 (d = 23 Hz), 119.5
(d = 2 Hz), 128.4, 128.5, 133.7, 137.1, 143.3 (d = 11 Hz), 157,
161.5 (d = 340 Hz), 170.4.
5.27. Methyl 2-(1-(4-bromophenyl)-2-(cyclohexylamino)-2-
oxoethylamino)-5-chlorophenylcarbamate (27)
C22H25BrClN3O3, Mw: 494.81 g/mol; HRMS (ESI-TOF), m/z
(calcd): 493.0768, (found) [M+Na]+: 516.0669; HPLC-MS rt:
11.49, m/z [M+H]+ 494.0. 1H NMR (DMSO, 400 MHz): d = 1.10–
1.28 (5H, m), 1.54–1.80 (3H, m), 1.80–1.82 (2H, m), 3.36 (1H, s),
3.52 (1H, m), 3.72 (3H, s), 5.02 (1H, d, J = 7.6 Hz), 5.79 (1H, d,
J = 7.6 Hz), 6.30 (1H, d, J = 8.8 Hz), 7.00 (1H, dd, J = 2.4 Hz,
J2 = 8.4 Hz), 7.26 (1H, s), 7.50 (2H, d, J = 8.4 Hz), 7.70 (2H, d,
J = 8.4 Hz), 8.26 (1H, d, J = 7.6 Hz), 9.25 (1H, s). 13C NMR (DMSO,
100 MHz): d = 24.7, 24.8, 25.6, 32.4, 32.7, 48.1, 52.4, 59.7, 113.1,
120.2, 121.2, 125.6, 129.5, 131.7, 139.0, 155.5, 169.1.
5.32. 2-(4-Chlorophenyl)-N-cyclohexyl-2-(6-fluoro-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol-1-yl)acetamide (32)
C21H21ClFN3O2, Mw: 401.86 g/mol; HRMS (ESI-TOF), m/z
(calcd): 401.1306, (found) [M+H]+: 402.1383; HPLC-MS rt: 10.86,
m/z [M+H]+ 402.1. 1H NMR (MeOD, 400 MHz): d = 1.2–1.43 (6H,
m), 1.66 (1H, d, J = 12.8 Hz), 1.78 (2H, m), 1.88 (1H, d,
J = 11.2 Hz), 2.00 (1H, d, J = 11.6 Hz), 3.79 (1H, m), 6.28 (1H, s),
6.55 (1H, d, J = 9.2 Hz), 6.78 (1H, dd, J1 = J2 = 9.2 Hz), 7.00 (1H,
dd, J1 = 4.8 Hz, J2 = 8.4 Hz), 7.33 (2H, d, J = 8 Hz), 7.42 (2H, d,
J = 8 Hz). 13C-NMR (MeOD, 100 MHz): d = 24.6, 25.2, 32.1, 32.2,
48.9, 58.2, 99.3 (d, J = 30 Hz), 107.7 (d, J = 24 Hz), 109.3 (d,
J = 10 Hz), 124.5, 128.0 (m), 128.7, 129.5, 133.5, 134.1, 155.7,
158.0 (d, J = 240 Hz), 167.4.
5.28. 2-(4-Bromophenyl)-2-(5-chloro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)-N-cyclohexylacetamide (28)
C21H21BrClN3O2, Mw: 462.77 g/mol; HRMS (ESI-TOF), m/z
(calcd): 461.0506, (found) [M+Na]+: 484.0356; HPLC-MS rt: 11.3,
m/z [M+H]+462.1. 1H NMR (MeOD, 400 MHz): d = 1.2–1.5 (6H, m),
1.65–1.90 (4H, m), 2.01 (1H, d, J = 12.4 Hz), 3.78 (1H, m), 6.24
(1H, s), 6.72 (1H, d, J = 8.8 Hz), 6.87 (1H, dd, J1 = 2 Hz,
J2 = 8.4 Hz), 7.06 (1H, d, J = 2 Hz), 7.25 (2H, d, J = 8 Hz), 7.56 (2H,
d, J = 8.4 Hz). 13C-NMR (MeOD, 100 MHz): d = 24.7, 24.7, 25.2,
32.1, 32.2, 49.0, 58.2, 109.1, 112.2, 120.4, 122.0, 126.8, 129.0,
129.4, 129.8, 131.6, 134.1, 155.2, 167.3
5.33. Methyl 2-(1-(4-chlorophenyl)-2-(isobutylamino)-2-
oxoethylamino)-4-fluorophenylcarbamate (33)
C20H23ClFN3O3, Mw: 407.87 g/mol; HRMS (ESI-TOF), m/z
(calcd): 407.1412, (found) [M+H]+: 408.1467; HPLC-MS rt: 10.78,
m/z [M+H]+408.3. 1H NMR (DMSO, 400 MHz): d = 0.74 (6H, d,
J = 6.4 Hz), 0.85 (2H, d, J = 6.8 Hz), 1.65 (1H, m), 2.93 (2H, m),
3.66 (3H, s), 5.05 (1H, d, J = 6.4 Hz), 5.88 (1H, d, J = 7.2 Hz), 6.05
(1H, dd, J1 = 2.4 Hz, J2 = 11.6 Hz), 6.38 (1H, m), 7.07 (1H, m), 7.72
(2H, d, J = 6.8 Hz), 7.57 (2H, d, J = 6.8 Hz), 8.02 (1H, s), 8.31 (1H,
s), 8.97 (1H, s). 13C-NMR (DMSO, 100 MHz): d = 20.3, 20.4, 28.5,
45.0, 46.6, 52.3, 59.4, 98.7, 98.9, 102.6, 102.8120.4, 128.3, 128.9,
129.3, 132.8, 138.5, 156.0 m, 161.4, 170.0.
5.29. Methyl 5-chloro-2-(2-(cyclohexylamino)-1-(4-
fluorophenyl)-2-oxoethylamino)phenylcarbamate (29)
C22H25ClFN3O3, Mw: 433.90 g/mol; HRMS (ESI-TOF), m/z
(calcd): 433.1568, (found) [M+H]+: 434.1653; 1H NMR (DMSO,
400 MHz): d = 1.07–1.25 (5H, m), 1.51–1.54 (3H, m), 1.60–1.82
(2H, m), 3.49 (1H, s), 4.04 (3H, s), 5.00 (1H, d, J = 7.6 Hz), 5.75
(1H, d, J = 7.6 Hz), 6.28 (1H, d, J = 8.8 Hz), 6.95 (1H, dd,
J = 2.4 Hz, J2 = 8.4 Hz), 7.15–7.23 (3H, m), 7.55 (2H, dd,
J = 5.2 Hz, J = 8.8 Hz), 8.21 (1H, d, J = 7.6 Hz), 9.22 (1H, s). 13C-
NMR (DMSO, 100 MHz): d = 24.7, 24.8, 25.6, 32.4, 32.7, 48.0,
52.4, 59.6, 113.1, 115.6 (d, J = 21 Hz), 120.1, 125.6, 129.3 (d,
J = 9 Hz), 135.7, 155.5, 169.4 .
5.34. 2-(4-Chlorophenyl)-2-(6-fluoro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)-N-isobutylacetamide (34)
C19H19ClFN3O2, Mw: 375.82 g/mol; HRMS (ESI-TOF), m/z
(calcd): 375.115, (found) [M+Na]+: 398.1056; 1H NMR (MeOD,
400 MHz): d = 0.91 (6H, d, J = 2.8 Hz), 1.84 (1H, m), 3.11 (2H, dd,
J1 = 2.4 Hz, J2 = 2.4 Hz), 6.27 (1H, s), 6.55 (1H, d, J = 9.6 Hz), 6.74
(1H, dd, J1 = 9.2 Hz, J2 = 9.2 Hz), 6.96 (1H, dd, J1 = 8.4 Hz,
J2 = 5.6 Hz), 7.33 (2H, d, J = 8 Hz), 7.41 (2H, t, J = 8 Hz), 13C NMR
(MeOD, 100 MHz): d = 19.1, 28.1, 47.8, 58.3, 99.0 (d, J = 0 Hz),
107.7 (d, J = 22 Hz), 109.3 (d, J = 11 Hz), 124.6, 128.6, 129.6,
129.8, 133.4, 134.2, 155.8 (d, J = 127 Hz), 159.3, 168.4.
5.30. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-N-cyclohexyl-2-(4-fluorophenyl)acetamide (30)
C21H21ClFN3O2, Mw: 401.86 g/mol; HRMS (ESI-TOF), m/z
(calcd): 401.1306, (found) [M+H]+: 402.1419; HPLC-MS rt: 10.83,
m/z [M+H]+ 402.2. 1H NMR (DMSO, 400 MHz): d = 1.1–1.4 (5H,
m), 1.56 (1H, d, J = 12.8 Hz), 1.69 (3H, m), 1.86 (1H, d,
J = 11.2 Hz), 3.66 (1H, m), 6.18 (1H, s), 6.66 (1H, d, J = 8.8 Hz),
6.84 (1H, dd, J1 = 2 Hz, J2 = 8.4 Hz), 6.98 (1H, d, J = 2 Hz), 7.20
(1H, m), 7.28 (1H, m), 8.49 (1H, d, J = 4.8 Hz), 11.21 (1H, s). 13C-
NMR (DMSO, 100 MHz): d = 25.0, 25.6, 32.6, 32.7, 48.5, 57.7,
109.1, 112.6, 115.9, 116.1, 120.3, 125.4, 128.7, 130.0, 130.4,
130.5, 132.3, 154.6, 161.5, 166.7.
5.35. Methyl 5-chloro-2-(1-(1-(4-chlorophenyl)cyclopropyl)-2-
(cyclohexylamino)-2-oxoethylamino)phenyl carbamate (35)
C
25H29Cl2N3O3, Mw: 490.42 g/mol; HRMS (ESI-TOF), m/z
(calcd): 489.1586, (found) [M+H]+: 490.1665; HPLC-MS rt: 11.88,
m/z [M+H]+490.2. 1H NMR (CDCl3, 400 MHz): d = 0.79–1.05 (4H,
m), 1.18–1.41 (4H, m), 1.42–1.60 (2H, m), 1.65–1.76 (3H, m),
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.06.020

W. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
13
1.93–197 (1H, m), 3.88 (3H, s), 6.45 (2H, d, J = 8.8 Hz), 7.04–7.08
(1H, m), 7.27–7.34 (4H, m). 13C NMR (CDCl3, 100 MHz): d = 11.8,
12.4, 24.7, 24.8, 25.0, 25.3, 28.3, 32.4, 33.1, 47.1, 48.1, 53.0, 65.6,
113.8, 122.8, 123.7, 126.0, 127.7, 128.2, 128.4, 129.6, 132.1,
132.3, 132.5, 133.4, 139.0, 140.0, 154.8, 160.2, 163.5, 169.5.
was introduced to eliminate differences in the transmission of ver-
tically and horizontally polarized light. All fluorescence polariza-
tion values were expressed in millipolarization units (mP). The
binding affinities of the fluorescent p53-derived peptide of Hu
et al. (the P4 peptide)25 towards MDM2 was determined in the buf-
fer which contained 50 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA,
10% DMSO. Competition binding assays were performed using the
10 nM fluorescent P4 peptide and 100 nM MDM2. Binding constant
and inhibition curves were fitted using the SigmaPlot (SPSS Science
Software). The obtained curves and data points for all compounds
are shown below.
5.36. 2-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-2-(1-(4-chlorophenyl)cyclopropyl)-N-cyclohexylacetamide
(36)
C24H25Cl2N3O2, Mw: 458.38 g/mol; HRMS (ESI-TOF), m/z
(calcd):457.1324, (found) [M+H]+: 458.1409; 1H NMR 1H NMR
(CDCl3, 400 MHz): d = 0.84–1.21 (4H, m), 1.22–1.52 (4H, m),
1.52–1.80 (4H, m), 1.85–1.90 (1H, m), 3.77 (1H, m), 5.07 (1H, s),
6.35 (1H, s), 6.69 (1H, d, J = 8.4 Hz), 6.87 (1H, dd, J1 = 0.8 Hz,
J2 = 8.4 Hz), 6.70 (1H, d, J1 = 8.4 Hz), 7.07 (1H, d, J1 = 8.4 Hz), 7.21
(1H, d, J1 = 8.4 Hz), 9.08 (1H, S). 13C-NMR (CDCl3, 100 MHz): d
= 11.2, 12.4, 24.7, 24.8, 25.4, 26.3, 29.7, 32.8,48.8, 63.2, 109.6,
112.6, 121.4, 127.4, 127.8, 127.9, 128.2, 132.2, 133.1, 140.1,
155.2, 165.7.
8. NMR methods
All NMR spectra were acquired at 298 K on a Bruker DRX
600 MHz spectrometer equipped with a cryoprobe. Typically,
NMR samples contained 0.1–0.2 mM protein in 50 mM KH2PO4
and 50 mM Na2HPO4, pH 7.4., containing 150 mM NaCl and
5 mM b-mercaptoethanol. Water suppression was carried out
using the WATERGATE sequence. NMR data were processed using
the Bruker program Xwin-NMR version 3.5. NMR ligand binding
experiments were carried out in an analogous way to those previ-
ously described.4a,i The maximum concentration of DMSO at the
end of titration experiments was less than 1%. The pH was main-
tained constant during the entire titration. The 1H–15N-HSQC
spectra were recorded using fast HSQC pulse sequence.26 The max-
imum concentration of DMSO at the end of titration experiments
was less than 1%.
6. Protein expression and purification
The vector pET-20b containing the MDM2 crystallization con-
struct (residues 18–111) as well as the pET-11a vector containing
the MDM2 construct (residues 1–125) used for NMR-spectroscopy
and FP-assay were transformed into the Bl21 Codon Plus (DE3) RIL
or BL21 Arctic Express (DE3) expression cells (Stratagene). The
cells were grown until the OD600 reached 0.8 and were then
induced with 1 mM IPTG. After 4 h post induction the cells were
harvested by centrifugation at 5500g for 15 min. The pellets were
re-suspended in the PBS buffer. Lysis was performed by sonication
followed by centrifugation at 10,000g for 30 min. The pellets
were re-suspended in 10 ml of the solubilization buffer (6 M
guanidinium chloride, 100 mM Tris–HCl pH 8.0, 1 mM EDTA,
10 mM b-mercaptoethanol) and incubated for 2 h on a rotator at
4 °C. Dialysis in 4 M guanidinium chloride, 10 mM b-mercap-
toethanol and pH 3.5 was performed overnight, followed by
refolding at 4 °C for 8 h in 10 mM Tris–HCl pH 7.0, 1 mM EDTA,
10 mM b-mercaptoethanol. Ammonium sulfate was added to a fi-
nal concentration of 2 M and incubated overnight at 4 °C. The solu-
tion was centrifuged at 11,000g for 30 min. Buthylsepharose 6 Fast
Flow (GE Healthcare) was added to the supernatant and incubated
for 2 h. The protein was eluted with 100 mM Tris, 10 mM
b-mercaptoethanol and pH 7.0, concentrated and loaded onto the
HiLoad 16/60 Superdex75 or Superdex 200 pg gel filtration col-
umns (Amersham Biosciences). The gel filtration was run with fol-
lowing buffer: 50 mM KH2PO4, 50 mM Na2HPO4, 150 mM NaCl,
5 mM DTT, pH 7.4. The purity and folding of the protein was con-
firmed by SDS–gel electrophoresis and NMR spectroscopy.
Acknowledgements
This work was supported by the NIH grant P41 GM094055–02
and 1R01GM097082–01 (to AD), and by a Marie Curie FP7-Reinte-
gration-Grants within the 7th European Community Framework
Programme and by a Project operated within the Foundation for
Polish Science TEAM Programme, co-financed by the EU European
Regional Development Fund (to TAH) and by a grant of the Qatar
National Science Foundation NPRP 4–319–3-097 (to AD and TAH) .
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.020.
References and notes
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2. (a) Khoury, K.; Popowicz, G. M.; Holak, T. A.; Dömling, A. MedChemComm 2011,
2, 246; (b) Popowicz, G. M.; Dömling, A.; Holak, T. A. Angew. Chem., Int. Ed. 2011,
50, 2680.
3. Andreeff, M.; Kojima, K.; Padmanabhan, S.; Strair, R.; Kirschbaum, M.; Maslak,
P.; Hillmen, P.; O’Brien, S.; Samaniego, F.; Borthakur, G.; Konopleva, M.;
Vassilev, L.; Nichols, G. American Society of Hematology Annual Meeting; San
Diego: CA, 2010.
7. Fluorescence polarization (FP) assay
4. (a) Stoll, R.; Renner, C.; Hansen, S.; Palme, S.; Klein, C.; Belling, A.; Zeslawski,
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All FP experiments were performed as described.25 Briefly, the
fluorescence polarization experiments were read on an Ultra Evo-
lution 384-well plate reader (Tecan) with the 485 nm excitation
and 535 nm emission filters. The fluorescence intensities parallel
(Intparallel) and perpendicular (Intperpedicular) to the plane of
excitation were measured in parallel perpendicular black 384-well
NBS assay plates (Corning) at room temperature (ꢂ20 °C). The
background fluorescence intensities of blank samples containing
the references buffer were subtracted and steady-state
fluorescence polarization was calculated using the equation:
P = (Intparallel ꢁ Gint erpendicular)/(Intparallel + Gint erpendicu-
lar), and the correction factor G (G = 0.998 determined empirically)
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.06.020

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