Tetra-substituted imidazoles as a new class of inhibitors of the p53-MDM2 interaction

Article abstract of DOI:10.1016/j.bmcl.2014.03.039

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53-MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein-protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line.

Full text of DOI:10.1016/j.bmcl.2014.03.039

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2110–2114
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetra-substituted imidazoles as a new class of inhibitors
of the p53–MDM2 interaction
⇑
Andrea Vaupel , Guido Bold, Alain De Pover, Thérèse Stachyra-Valat, Joanna Hergovich-Lisztwan,
⇑
Joerg Kallen, Keiichi Masuya, Pascal Furet
Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide
inhibitors of the p53–MDM2 interaction, we have discovered another new class of compounds able to
disrupt this protein–protein interaction, an important target in oncology drug research. The new inhib-
itors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency
in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us
to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent
MDM2 amplified cell line.
Received 4 February 2014
Revised 13 March 2014
Accepted 15 March 2014
Available online 22 March 2014
Keywords:
Protein–protein interaction inhibitors
Structure-based design
Ó 2014 Elsevier Ltd. All rights reserved.
p53
MDM2
The p53 tumor suppressor protein plays a key role in the control
of cellular integrity. Loss of function of the p53 gene by mutations
or deletions is observed in almost 50% of all human cancer tissues.
In other cancer tissues, still expressing the wild-type form, the nor-
mal function of p53 is altered by overexpression or amplification of
MDM2 (or HDM2), the main negative regulator of the tumor sup-
pressor. In this situation, MDM2 mainly functions as a p53 specific
ubiquitin ligase which by binding to the N-terminal transactivation
domain of p53 triggers its proteasomal degradation. Cancerous
cells having elevated MDM2 levels are thus protected against
p53 dependent apoptosis and cell cycle arrest mechanisms.
To restore normal p53 function in such tumor cells, one can
envisage disrupting the p53–MDM2 interaction by small mole-
cules having high affinity for the p53 binding pocket of MDM2.
This attractive therapeutic concept has raised a lot of interest in
anticancer drug research and some molecules exerting an anti-pro-
liferative activity by this mechanism have entered clinical
evaluation.1,2
This octapeptide was designed on the basis of the available crystal
structure of MDM2 in complex with a 15-mer peptide derived from
the natural sequence of p53.4
We have pursued this effort by the search for non-peptide
inhibitors of this critical protein–protein interaction. Along this
line, we recently reported a structure-based design concept that
led to the identification of a promising new class of small molecule
inhibitors of the p53–MDM2 interaction.5 Key to the success of this
concept was the observation that residue Val 93 occupies a central
position in the p53 binding pocket of MDM2, a peculiar topological
feature of this protein cavity. We realized that placement of a pla-
nar aromatic or hetero-aromatic core moiety within van der Waals
distance of Val 93 afforded appropriate exit vectors to attach sub-
stituents that efficiently occupy the three essential sub-pockets of
the MDM2 cleft involved in the recognition of residues Phe 19, Trp
23 and Leu 26 of the transactivation domain of p53.
Following the same concept, we have discovered a second class
of p53–MDM2 interaction inhibitors that use a tetra-substituted
imidazole ring as core structure. We report here the design of these
compounds and the optimization of their biochemical and cellular
potency.
Several years ago, we initiated an effort in this direction that led
to the identification of a very potent octapeptide inhibitor of the
p53–MDM2 interaction incorporating non-natural amino-acids.3
In our previous work on the identification of 3-imidazolyl in-
dole p53–MDM2 inhibitors based on the central valine concept, a
crystal structure of MDM2 in complex with compound 1 (Fig. 1)
was obtained.6 In this structure (Fig. 2) one observes an aromatic
p–p stacking interaction between MDM2 residue His 96 and the
benzylic chlorophenyl ring of the compound. We noticed that
⇑
Corresponding authors. Address: Novartis, WKL-136.3.94, CH-4002 Basel,
Switzerland. Tel.: +41 61 696 78 67; fax: +41 61 696 62 46 (A.V.). Address:
Novartis, WKL-136.P.12, CH-4002 Basel, Switzerland. Tel.: +41 61 696 79 90; fax:
+41 61 696 48 70 (P.F.).
E-mail addresses: andrea.vaupel@novartis.com (A. Vaupel), pascal.furet@
novartis.com (P. Furet).
besides this stacking interaction the side chain of His 96 offered
http://dx.doi.org/10.1016/j.bmcl.2014.03.039
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

A. Vaupel et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2110–2114
2111
Cl
NH
NH
O
O
N
N
Cl
Cl
N
N
N
N
O
N
O
Cl
N
NH
Cl
H
Cl
3
0.31 µM
)
(
2
1.1 µM
)
1 (0.03 µM)
(
Figure 1. Chemical structures of compounds 1–3. Inhibitory activity in the TR-FRET biochemical assay is indicated in parentheses.
Figure 3. The design of the prototype compounds is illustrated here by a model of
compound 3 (yellow) binding in the MDM2 pocket. The model is based on the
Figure 2. Crystal structure of MDM2 in complex with compound 1 (PDB code:
4DIJ). The stacking interaction with His 96 and the proximity of the 2-position
of the compound imidazole ring with the hydrogen bond donor nitrogen of the side
chain of this residue are highlighted. The three MDM2 sub-pockets are labeled PHE
(Phe 19), TRP (Trp 23) and LEU (Leu 26).
p–p
crystal structure of MDM2 in complex with compound
1 (PDB code: 4DIJ).
Compound 1 (beige) is also represented as reference. For compound 3, the putative
hydrogen bond formed with His 96 appears as a dashed pink line while the putative
p–p stacking interaction is represented by the dashed pink arrow.
the possibility to form a hydrogen bond with the inhibitor. Closer
examination of the crystal structure suggested creating this new
interaction by placing a hydrogen bond accepting group in position
2 of the imidazole ring of 1.7 In particular, in one molecular mod-
eling experiment, we probed an amide group directly attached at
this position of the imidazole ring to interact with His 96. However,
in the resulting model the amide oxygen atom was too far from the
side chain imidazole ring of His 96 to actually form a hydrogen
bond. Further modeling experiments indicated that this hydrogen
bond could be created if two other modifications of the 3-imidaz-
olyl indole inhibitor were made: firstly replace the indole moiety
by a para- or meta-chlorophenyl ring to fill the Trp 23 sub-pocket
and secondly replace the benzylic group by a meta-chlorophenyl
ring directly attached to the imidazole core for stacking with His
96. These modifications allowed the formation of the targeted
hydrogen bond by a slight motion of the molecule towards His
96 upon energy minimization (Fig. 3).8 After consideration of syn-
thetic access, we settled on prototype molecules 2 and 3 (Fig. 1) to
strengthening of this interaction being expected as the conse-
quence of replacing a neutral hydrogen bond acceptor group by
an ionized negatively charged one. These promising results
prompted us to initiate a chemistry program aimed at optimizing
the potency of the new chemotype with compound 4 as the start-
ing point.
Guided by our model of the compound docked in the MDM2
cavity (Fig. 4), we designed several modifications of 4 with the
objective of either enhancing its conformational preorganization
for MDM2 binding or improving its interactions with the protein.
This effort resulted in the synthesis of compounds 5–8 (Table 1).
Concerning the former aspect, we noticed that in the binding mod-
el (Fig. 4) the meta-chlorophenyl ring of 4 occupying the Trp 23
sub-pocket had a nearly perfect perpendicular orientation with re-
spect to the plane of the core imidazole ring. However, when the
compound was energy minimized outside the cavity, the angle be-
tween the two ring planes was smaller assuming a value of 50°.10
To stabilize the higher energy perpendicular conformation induced
by the cavity, we envisaged the introduction of substituents at the
positions of the chlorophenyl ring ortho to its attachment point.
Modeling suggested that either a methyl group at the 6-position
or a fluorine atom at the 2-position of the ring would facilitate,
by intramolecular steric hindrance, the full deconjugation of the
chlorophenyl and imidazole rings without creating adverse inter-
actions with the binding site. It was expected that the 6-methyl
substituent would face solvent while the 2-fluorine atom would in-
crease the favorable contact surface area with MDM2 through
additional hydrophobic interactions with residues Val 93, Phe 91
and Ile 99. Remarkably, with compounds 5 and 6, designed on
the basis of this concept, the biochemical potency of 4 could be im-
proved 11- and 17-fold, respectively. We sought to further increase
probe the idea of engaging His 96 for both a
p–p stacking and
hydrogen bond interaction while conforming to our central valine
ligand design concept.
Compounds 2 and 3 were synthesized and tested in our bio-
chemical TR-FRET assay measuring the ability of a compound to in-
hibit the binding of a p53-derived peptide to MDM2.9 Gratifyingly,
with IC50 values of 1.1 and 0.31 lM respectively, 2 and 3 showed
significant activity in this assay. The encouraging sub-micromolar
activity of 3 incited us to also test its carboxylic acid synthetic
intermediate 4 (Table 1). The latter compound turned out to be
3-fold more potent than 3. The observed improvement was consis-
tent with our hypothesis that the prototype compounds establish a
hydrogen bond with the side chain imidazole ring of His 96, a

2112
A. Vaupel et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2110–2114
Table 1
Biochemical and cellular activity of compounds 4–8
OH
R3
Cl
O
N
R4
N
R1
R2
Cl
Compound
R1
R2
R3
R4
TR-FRET
SJSA-1
IC50
SAOS-2
IC50 (lM)
IC50
(lM)
(
l
M)
4
5
6
7
8
H
H
H
F
H
F
H
H
H
H
H
H
H
F
0.12
n.d.
n.d.
>30
>30
>30
>30
Methyl
H
Methyl
H
0.011
0.007
0.008
0.003
16.1
12.2
10.2
14.0
Methyl
H
The IC50 values are averages of at least 2 separate determinations.
Figure 4. Binding model of compound
4 (yellow) in the MDM2 pocket. The
positions of the compound where substituents were introduced to enhance its
potency are indicated by pink arrows.
Figure 5. Crystal structure of MDM2 in complex with compound 7 (PDB code:
4OQ3). The side chain of the central valine 93 is shown in CPK representation. The
hydrogen bond and
p–p stacking interactions with His 96 are indicated in green
the inhibitory activity of these first nanomolar compounds of the
series by optimizing interactions in the Phe 19 and Leu 26 sub-
pockets of MDM2. Two modifications were designed to this end
based on the docking model. On the one hand, we introduced a
methyl group in meta position of the phenyl ring interacting with
the Phe 19 sub-pocket. This substituent was meant to fill a small
empty hydrophobic space left in this part of the cavity between
the side chains of residues Ile 61, Tyr 67 and Val 75. On the other
hand, a fluorine atom was added at the 4-position of the chloro-
phenyl ring occupying the Leu 26 sub-pocket to make favorable
van der Waals contacts with the side chains of residues Leu 54
and Tyr 100. Compounds 7 and 8 are representative of these de-
signs. Comparing the biochemical IC50 values of the pairs 5, 7
and 6, 8 indicates that, as expected, some gain in binding affinity,
although modest, was achieved by seeking additional interactions
in the Phe 19 and Leu 26 sub-pockets. With compound 8, low sin-
gle digit nanomolar potency was thus reached. In addition, com-
pound 7 provided the first success in our attempts to obtain an
X-ray crystal structure of MDM2 in complex with a member of
the new chemotype series.11 As shown in Figure 5, this structure
fully validated the design principles used for the identification of
and pink colors, respectively. As expected from the docking models, the plane of the
inhibitor core imidazole ring and that of its chlorophenyl ring occupying the Trp 23
sub-pocket are nearly perpendicular, an angle of 80° being observed. MDM2
residues Lys 51, Leu54 and Phe 55 facing position 6 of the latter chlorophenyl ring
appear in stick representation.
conforms to our central valine concept with the imidazole ring of
the compound in van der Waals interaction with residue Val 93
and the three attached phenyl groups projecting in the three cru-
cial p53 binding sub-pockets of MDM2.
Having obtained very potent inhibitors of the p53–MDM2 inter-
action at the biochemical level, we next turned our attention to the
optimization of cellular activity. We assessed it by measuring the
ability of compounds to inhibit the proliferation of SJSA-1 cells.
These are p53 positive cancer cells in which the MDM2 gene is
amplified. For control, inhibition of the proliferation of the p53-
null SAOS-2 cells was also measured.13
We were pleased to note that compounds 5–8 (Table 1) showed
inhibition of the proliferation of the p53-dependent SJSA-1 cells
while having no effect on the p53-null SAOS-2 cells in the same
range of concentrations. However, limited to the double digit
micromolar level, this inhibition was weak in sharp contrast with
the high biochemical potency achieved. Although p53–MDM2
inhibitors are expected to lose potency going from a biochemical
to a cellular setting because of the existence of the p53–MDM2
the new class of p53–MDM2 inhibitors. The p–p stacking interac-
tion between the meta-chlorophenyl ring and His 96 is indeed ob-
served in the Leu 26 sub-pocket. So is the hydrogen bond between
this residue and the carboxylic acid group in position 4 of the com-
pound imidazole ring.12 Moreover, the binding mode of 7 overall

A. Vaupel et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2110–2114
2113
auto-regulatory feedback loop in cells,14 we believed that the pres-
ence of a carboxylic acid group in the compounds, by hampering
their cell permeability, could be an important contributor to the
observed discrepancy. To explore this possibility, we envisaged
bioisosteric replacements of the carboxylic acid group. Since, in
the perspective of ADME properties optimization, we also wanted
to reduce aromaticity in our lead series, replacement of the carbox-
ylic group was carried out on 9 (Table 2), the analogue of 8 in
which the phenyl group fitting the Phe 19 sub-pocket was replaced
by a cyclohexyl ring. A docking experiment suggested that such a
modification would be tolerated and indeed 9 turned out to be as
potent as 8, both in the biochemical and cellular assay.
The carboxylic acid in 9 was first converted to a primary amide to
give compound 10 (Table 2). As expected, this alteration caused a
drop in biochemical potency because of the weakening of the hydro-
gen bond formed with the side chain of His 96. Nevertheless, the de-
sired improvement in cellular activity was obtained, compound 10
Consequently, compound 13 was selected as the starting point
for a new round of cellular potency optimization. Looking for ways
to further improve the intrinsic binding affinity of the compound
for MDM2 that might translate in better cellular activity, we no-
ticed that extending a side chain from position 6 of the phenyl moi-
ety occupying the Trp 23 sub-pocket allowed the creation of
interactions with residues Leu 54, Lys 51 and Phe 55 (see Fig. 5).
In particular, with an acetamide group attached to this position,
it was possible to form a hydrogen bond with the backbone car-
bonyl group of Leu 54, recapitulating an interaction made by the
side chain of the p53 residue Trp 23. 14 was the first compound
synthesized following this concept. Besides this methyl derivative,
analogues bearing a larger substituent on the amide group (com-
pounds 15–17) were also prepared to target the side chains of
the three aforementioned residues for hydrophobic interactions.
From the data in Table 3 it can be seen that this strategy met with
some success, three compounds of the series, those having an ali-
phatic substituent (14, 16 and 17), being more active both bio-
chemically and cellularly than the parent compound 13.
Importantly, the tert-butyl derivative 17 reached sub-micromolar
having an IC50 value of 4.1
pared to a value of 13.2
l
M in the SJSA-1 proliferation assay com-
l
M for 9. We next tried the classical tetra-
zole carboxylic acid isostere. The resulting compound 11, being
able to interact with His 96 in an ionized form, regained potency
in the TR-FRET assay compared to 10 but was not more active at
the cellular level. Going back to a neutral group as carboxylic acid
substitute, a 2-methyl-1,3,4-oxadiazole ring was designed (com-
pound 12).15 In the binding model, one of the ring nitrogen atoms
could establish the hydrogen bond with His 96. Again, replacement
of the carboxylic acid by a neutral isostere led to a reduction of bio-
chemical potency while improving activity in the cellular assay.
Reasoning that a strong electron donating substituent on the oxadi-
azole ring would make the nitrogen atom interacting with His 96 a
better hydrogen bond acceptor, we synthesized compound 13. Con-
sistent with this notion, 13 showed enhanced biochemical activity
compared to 12, reaching the low nanomolar levels of 9 or 11. In
potency (IC50 = 0.5
ing favorably with the micromolar activity (IC50 = 1.9
l
M) in the SJSA-1 proliferation assay, compar-
M) of the
l
well established p53–MDM2 inhibitor Nutlin-3a in this assay.
A representative synthesis for compound 17 is shown in
Scheme 1.16 Commercially available 2-fluoro-3-chloro aniline
was subjected to iodination with NIS, providing a regioisomeric
mixture of 4-and 6-iodo products which were separable by flash
chromatography. The desired 6-iodo regioisomer was reacted with
cyclohexane carbonitrile in the presence of trimethyl aluminum to
form the corresponding benzamidine 18 which underwent a
smooth cyclization with ethylbromo pyruvate under mild basic
conditions (NaHCO3; rt). Water elimination was effected by addi-
tion of p-toluene sulfonic acid and heating to 120 °C to furnish
the imidazole core. Selective Sonogashira coupling of the iodine
with trimethyl silyl acetylene provided intermediate 19. Conver-
sion of the acetylene side chain to the desired acid was achieved
by hydroboration (cyclohexene/borane-dimethylsulfide complex)
and oxidative workup. Efficient and selective bromination of the
imidazole core was effected by treatment with NBS in acetonitrile
at room temperature, providing the suitable substrate for a Suzuki
coupling with commercially available 3-chloro-4-fluoro boronic
acid. Orthogonal ester protection/deprotection steps provided acid
20 which was converted to the 2-amino-oxadiazole in a two steps
sequence (HATU promoted hydrazone formation and ring closure
with BrCN). Finally deprotection of the tert-butyl ester 21 liberates
carboxylic acid which was converted to the corresponding carbox-
amide 17 using Propsal™ as a coupling reagent.
addition, with an IC50 value of 3.5 lM, 13 represented the most po-
tent compound of the series in the SJSA-1 proliferation assay.
Table 2
Biochemical and cellular activity of compounds 9–13
R
N
Cl
F
N
F
Cl
Compound
R
TR-FRET
IC50 M)
SJSA-1
SAOS-2
(
l
IC50
(l
M)
IC50
(lM)
Table 3
OH
Biochemical and cellular activity of compounds 14–17
9
0.004
0.018
13.2
>30
N
O
NH2
H2N
N
O
Cl
N
10
4.1
29
F
O
N
N
N
O
R
F
11
12
13
0.003
0.025
0.006
4.4
5.9
3.5
>30
>30
>30
N
N
H
N
NH
Cl
N
Compound
R
TR-FRET
IC50 M)
SJSA-1
SAOS-2
IC50 (lM)
(
l
IC50
(
l
M)
O
N
14
15
16
17
Methyl
Phenyl
0.004
0.006
0.002
0.002
1.4
2.5
1.4
0.5
24
30
28
23
N
Cyclohexyl
tert-Butyl
O
NH2
The IC50 values are averages of at least 2 separate determinations.
The IC50 values are averages of at least 2 separate determinations.

2114
A. Vaupel et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2110–2114
Scheme 1. Reactions and conditions: (a) NIS, CH2Cl2, rt, 5d; (b) cyclohexane carbonitrile, Me3Al, toluene, 110 °C; (c) (1) ethyl bromopyvurate, NaHCO3, rt, 2 h, (2) p-TSA,
toluene, 120 °C, 1 h; (d) trimethylsilylacetylene, Pd(OAc)2, PPh3, NEt3, rt, 20 h; (e) (1) cyclohexene, Me2S-BH3, THF, rt, 3 h, (2) NaHCO3, H2O2, THF, rt; (f) NBS, CH3CN, rt, 8 h; (g)
3-chloro,4-fluoro phenylboronic acid, Pd(PPh3)4, K3PO4, dioxane/H2O, 100 °C, 18 h; (h) tert-butyl 2,2,2-trichloroacetimidate, BF3ÁEt2O, CH2Cl2/cyclohexene, rt, 3 h; (i) LiOH,
H2O/dioxane, 70 °C, 3 h; (j) hydrazine, HATU, N-methylmorpholine, DMF, rt, 2 h; (k) BrCN, NaHCO3, H2O/dioxane, rt, 6 h; (l) HCl/dioxane, rt, 3 days; (m) tert-butyl amine,
Propsal™, NEt3, DMAP, DMF, rt, 2 h.
5. Furet, P.; Chène, P.; De Pover, A.; Stachyra-Valat, T.; Hergovich-Lisztwan, J.;
Kallen, J.; Masuya, K. Bioorg. Med. Chem. Lett. 2012, 22, 3498.
6. The PDB code of this crystal structure is 4DIJ.
In conclusion, building on our previous work on the discovery of
3-imidazolyl indole inhibitors of the p53–MDM2 interaction, we
have identified by structure-based design another new class of
inhibitors of this interaction. By appropriate derivatization strate-
gies guided by structural information, we were able to optimize
the MDM2 affinity of these tetra-substituted imidazole inhibitors
to the low nanomolar range. The most potent compounds of the
series show significant and specific anti-proliferative activity in
cultures of p53-dependent cancer cells. These results warrant fur-
ther evaluation of the new inhibitors towards the goal of develop-
ing anti-cancer agents to fight tumors harboring an overexpressed
or amplified MDM2 gene.
7. We assumed that in the crystal structure the Ne2 nitrogen atom of the side
chain of His 96 is protonated because it is within hydrogen bond distance of the
backbone carbonyl oxygen atom of Val 93.
8. Modeling and docking were performed with
a version of MacroModel
enhanced for graphics by A. Dietrich. MacroModel: Mohamadi, F.; Richards,
N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.;
Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440. The compounds
were manually constructed and docked in the MDM2 pocket (PDB structure
4DIJ) and the resulting ligand-protein complexes energy-minimized using the
AMBER⁄/H2O/GBSA force field.
9. For
a detailed description of the TR-FRET (Time Resolved Fluorescence
Resonance Energy Tranfer) biochemical assay used see: Berghausen, J.;
Buschmann, N.; Furet, P.; Gessier, F.; Hergovich Lisztwan, J.; Holzer, P.;
Jacoby, E.; Kallen, J.; Masuya, K.; Pissot Soldermann, C.; Ren, H.; Stutz, S. PCT
Int. Appl. WO 2011076786, 2011; Chem. Abstr. 2011, 155, 152537. In this assay
the donor fluorophore is MDM2 (amino acid residues 2–188) tagged with a C-
terminal biotin moiety in combination with a Europium labeled streptavidin.
The acceptor fluorophore is a p53 derived peptide (amino acid sequence 18–26
of p53: TFSDLWKLL) labeled with the fluorescent dye Cy5. The IC50 values given
are means of at least two measurements. For reference, the p53–MDM2
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inhibitor Nutlin-3A has an IC50 of 0.01 lM in this assay.
10. The minimization of the isolated molecule was performed in MacroModel
using the AMBER⁄/H2O/GBSA force field.
11. Kallen, J. Crystallographic coordinates and structural factors of compound 7 in
complex with the N-terminal domain of MDM2 have been deposited with the
Protein Data Bank (entry code 4OQ3). The structure was solved at 2.3 Å
resolution.
12. A search in the Protein Data Bank indicates that other classes of p53–MDM2
inhibitors originating from independent efforts are able to form these
beneficial interactions with His 96. Both the stacking interaction and the
hydrogen bond are observed in PDB structures 4HBM, 4ERE, 4ERF, 4OAS
(Amgen piperidinone inhibitors) and 4JVR, 4JSC (Roche spiroindoline
inhibitors) while the stacking interaction only is observed in structures 3JZK
(Amgen chromenotriazolopyrimidine inhibitors) and 1T4E (Johnson & Johnson
benzodiazepinedione inhibitors).
13. The cellular SJSA-1 and SAOS-2 proliferation assays are based on YO-PROÒ-1
iodide staining (J. Immunol. Methods 1995, 185, 249). To test the effect of
compounds on cell growth, SJSA-1 cells (p53 wild-type cells) and SAOS-2 cells
(p53 null cells) are plated out into 96-well micro-titer plates and treated with
decreasing concentrations of the compounds. After a 72 h incubation period,
2.5
performed using
l
M YO-PROÒ-1 iodide is directly added to the cells and a first read-out is
a
standard fluorescence plate reader (filter setting 485/
530 nm) revealing the relative number of apoptotic cells. Subsequently, cells
are permeabilized by directly adding lysis buffer containing the detergent
NP40, EDTA and EGTA to obtain final concentrations of 0.01% and 5 mM,
respectively. After complete permeabilization, the total cell number is
quantified during a second read using the fluorescence plate reader with the
same settings.
14. Lahav, G. In Cellular Oscillatory Mechanisms; Maroto, M., Monk, N. A. M., Eds.;
Landes Bioscience and Springer Science, 2008; pp 28–38.
15. Bergström, J.; Hogner, A.; Llinàs, A.; Wellner, E.; Plowright, A. T. J. Med. Chem.
1817, 2012, 55.
16. The preparation of the compounds presented in this work is detailed. In: Bold,
G.; Furet, P.; Gessier, F.; Kallen, J.; Hergovich Listwan, J.; Masuya, K.; Vaupel, A.
PCT Int. Appl. WO 2011023677.
3. García-Echeverría, C.; Chène, P.; Blommers, M. J. J.; Furet, P. J. Med. Chem. 2000,
43, 3205.
4. Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.;
Pavletich, N. P. Science 1996, 274, 948 (PDB code: 1YCR).