Structure-based design, synthesis, and biological evaluation of novel 1,4-diazepines as HDM2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.02.018

Crystallographic analysis of ligands bound to HDM2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic the α-helix of p53 peptide and may represent a promising scaffold to develop HDM2-p53 antagonists. To verify this hypothesis, we synthesized and biologically evaluated 5-[(3S)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-7-phenyl-1, 4-diazepin-1-yl]valeric acid (10) and 5-[(3S)-7-(2-bromophenyl)-3-(4- chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-yl] valeric acid (11). Preliminary in vitro testing shows that 10 and 11 substantially antagonize the binding between HDM2 and p53 with an IC ₅₀ of 13 and 3.6 μM, respectively, validating the modeling predictions. Taken together with the high cell permeability of diazepine 11 determined in CACO-2 cells, these results suggest that 1,4-diazepine-2,5-diones may be useful in the treatment of certain cancers.

Full text of DOI:10.1016/j.bmcl.2005.02.018

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1857–1861
Structure-based design, synthesis, and biological evaluation of
novel 1,4-diazepines as HDM2 antagonists
*
Pierre Raboisson, Juan Jose Marugan, Carsten Schubert, Holly K. Koblish, Tianbao Lu,
´
Shuyuan Zhao, Mark R. Player, Anna C. Maroney, Rolanda L. Reed,
´
Norman D. Huebert, Jennifer Lattanze, Daniel J. Parks and Maxwell D. Cummings*
Departments of Medicinal Chemistry, Structural Biology and Molecular Design and Informatics, Drug Discovery,
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, PA 19341, USA
Received 28 July 2004; revised 6 February 2005; accepted 7 February 2005
Abstract—Crystallographic analysis of ligands bound to HDM2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic
the a-helix of p53 peptide and may represent a promising scaffold to develop HDM2–p53 antagonists. To verify this hypothesis, we
synthesized and biologically evaluated 5-[(3S)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-7-phenyl-1,4-diazepin-
1-yl]valeric acid (10) and 5-[(3S)-7-(2-bromophenyl)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-
yl]valeric acid (11). Preliminary in vitro testing shows that 10 and 11 substantially antagonize the binding between HDM2 and
p53 with an IC50 of 13 and 3.6 lM, respectively, validating the modeling predictions. Taken together with the high cell permeability
of diazepine 11 determined in CACO-2 cells, these results suggest that 1,4-diazepine-2,5-diones may be useful in the treatment of
certain cancers.
ꢀ 2005 Published by Elsevier Ltd.
The hdm2 oncogene product (HDM2) is an ubiquitin
protein ligase1,2 that suppresses the transcriptional
activity of the tumor suppressor p53 and promotes its
rapid degradation through the ubiquitin proteolysis
pathway.3–5 Since p53 is the most frequently inactivated
protein in human cancers,6 small molecule antagonists
of the HDM2–p53 protein–protein interaction appear
to offer an attractive strategy for cancer therapy, be-
cause it offers the possibility to up-regulate the p53 re-
sponse.7–9 After the recent publication by Roche
scientists of the ÔnutlinsÕ,10 a series of imidazoline anta-
gonists of HDM2–p53 interaction (compounds 1–3,
Chart 1) found to activate the p53 pathway in cancer
cells, the p53/HDM2 target has received increasing
attention in the scientific community.
ited since compounds 1–3 were found to activate the
p53 pathway in cancer cells, leading to cell arrest, apop-
tosis and growth inhibition.10 Crystallographic analysis
established that the reported imidazolines 1–3 bind to
HDM2 in the p53-helix binding site.10
Independently, from a library of a-(acylamino)amides
screened with our proprietary high-throughput minia-
turized thermal denaturation assay ThermoFluorꢁ,11
compound 4 (Chart 1) emerged as a new small molecule
antagonist
of
the
HDM2–p53
interaction
(IC50 = 15 lM).12 This hit 4 was shown to bind to
HDM2 in the p53-binding pocket using a fluorescence
polarization (FP) peptide displacement assay.13 The
rigidification of this highly flexible open structure by
incorporation of an amide linkage between two of the
three phenyl rings of compound 4 (Fig. 1) led to the dis-
covery of the 1,4-benzodiazepine-2,5-dione 5, which is
Moreover, disruption of the protein–protein interaction
with low-molecular weight compounds has been revis-
approximately 10-fold more active than 4 (IC50
=
1.7 lM).12,14 Further structure–activity relationship
studies led to the identification of benzodiazepine 6 as
one of the most potent HDM2 antagonists reported so
far (IC50 = 220 nM).14,15 Cinnamoyl amide 7 was also
identified in our library to significantly antagonize the
binding between HDM2 and p53 (IC50 = 30 lM).12
Keywords: HDM2–p53; Diazepine; Selenium; Cancer.
*
Corresponding authors. Tel.: +1 610 458 5264x6539; fax: +1 610 458
8249 (J.J.M.); tel.: +1 610 458 5264x6581; fax: +1 610 458 8249
(M.D.C.); e-mail addresses: jmaruga1@prdus.jnj.com; mcumming@
prdus.jnj.com
0960-894X/$ - see front matter ꢀ 2005 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2005.02.018

1858
P.Raboisson et al./ Bioorg.Med.Chem.Lett.15 (2005) 1857–1861
HO
O
O
Cl
Cl
Br
Cl
Cl
O
O
O
N
N
NH
N
N
N
N
N
N
O
O
O
N
N
N
O
O
O
Br
2
1
3
Chart 1. HDM2 antagonists.10
Cl
O
O
O
COOH
COOH
I
I
I
N
N
N
COOH
CF3
Cl
N
N
H
O
H
O
Amide linkage
CF3
Rigidification
5, IC50 = 1.7 µM
6, IC50 = 0.22 µM
4, IC50 = 15 µM
Structural
optimisation
Cl
Cyclisation
O
N
NH2
O
N
O
R'
Cl
Cl
N
R
O
7, IC50 = 30 µM
Cl
8, R = H, R' = H
9, R = H, R' = Br
10, R = (CH2)4COOH, R' = H
11, R = (CH2)4COOH, R' = Br
Figure 1. Design of HDM2 antagonists.
Following the same ÔrigidificationÕ strategy, which
successfully led to the identification of the benzodiazep-
inediones 5 and 6, we hypothesized that the 1,4-diazep-
inedione 8 (Chart 1) might act as a constrained version
of 7 and thereby lock the structure in the desired bioac-
tive conformation. Alternatively, 8 could be viewed as a
seco analogue of compound 6, where the phenyl ring of
the benzodiazepine system has been detached from the
diazepine ring. In this communication we report the
rational design, synthesis and in vitro evaluation of an
original series of potent diazepine antagonists of the
HDM2–p53 binding. The CACO-2 cell permeability
was also assessed for the target compounds 10 and 11.
phenyl rings of 6 into the regions occupied by the
Phe19, Trp23, and Leu26 side chains in the peptide com-
plex.15 As such, the benzodiazepinedione scaffold serves
as an a-helix mimetic, and other diazepine-based scaf-
folds are also of interest in this context. Although 4
can access the analogous conformation to that observed
for 6 bound to HDM2, it is reasonable to presume that
some or all of the difference observed in binding potency
is due to the increased flexibility of 4 versus the con-
strained analog 6 (Chart 1). Alternative modes of cycli-
zation or rigidification are of interest, since the 1,4-
benzodiazepine-2,5-dione does present inherent limita-
tions given the observed binding mode. One such limita-
tion relates to access to the pocket occupied by Phe19 in
the peptide complex. The orientation and rigidity of the
iodophenyl ring precludes access to the bottom of the re-
gion occupied by the Phe19 side chain (Fig. 2). The ob-
served SAR for phenyl substituents and alternative
fused rings supports this contention.12,14,15 Figure 2b
shows that the orientation of Phe19 differs from that
of the iodophenyl ring of 6, and highlights the inaccessi-
bility of the space immediately below the plane of the
iodophenyl ring.
Recent crystallographic studies of HDM2 complexed
with a p53-derived helical peptide have defined a deep
hydrophobic binding pocket.16 Side chains from the
hydrophobic face of two turns of the p53 helix, Phe19,
Trp23, and Leu26 (i, i + 4, i + 7), occupy this hydropho-
bic pocket on the HDM2 surface. The crystal structure
of our lead compound 6 bound to the same site on
HDM2 (PDB17 ID: 1T4E) establishes that the
(benzo)diazepinedione scaffold projects the three halo-

P.Raboisson et al./ Bioorg.Med.Chem.Lett.15 (2005) 1857–1861
1859
O
N
1) PhSeBr, AgOTf,
DMF, CH3CN
O
NH2
N
O
NH
Ph
O
2) H2O2, THF
Ph
12
13
Scheme 1. Synthesis of diazepine 13 by the phenylselenium-induced
lactamization of 12.18
tity and stereochemistry of compound 13 have been
unambiguously determined by X-ray diffraction analy-
sis.18 Thus, the organoselenium-induced lactamization
reported by Chung et al.18 was found to be a suitable
reaction for the synthesis of the desired HDM2 antago-
nists 10 and 11. These two diazepines (10 and 11) were
synthesized according to the procedure outlined in
Scheme 2.
Alkylation of the (R)-benzylamine 14 with ethyl a-bro-
mo-4-chlorophenylacetate 15 afforded the ester 16,
which was successively acylated with cinnamoyl chlo-
rides and saponified to give the desired acids 19 and
20. Reaction of 19 and 20 with ammonium chloride,
HOBT
and
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate afforded,
after separation from the undesired diastereomers by sil-
ica gel chromatography, the two key amide substrates 7
and 21 required for the organoselenium-induced lacta-
mization reaction. The identity of the diastereomers 7
and 21 was investigated using a series of NMR experi-
ments in conjunction with molecular modeling, which
have established that the active diastereomer as having
the (R,S) configuration. Treatment of electron-deficient
olefins 7 and 21 with phenylselenium bromide, silver tri-
flate and dimethylformamide in dry acetonitrile gave the
desired seleno seven-membered bislactams 22 and 23.
The oxidative intramolecular deselenylation of com-
pounds 22 and 23 with hydrogen peroxide in tetra-
hydrofuran afforded the target diazepines 8 and 9.
Introduction of the acid solubilizing group was achieved
by the alkylation of 8 and 9 with 5-bromovaleric acid
tert-butyl ester and subsequent deprotection of the acid
moiety to afford the desired final diazepine products 10
and 11. The new diazepines 10 and 11 (Fig. 1) prepared
in the present study were first tested for their ability to
antagonize the binding between HDM2 and p53 in the
fluorescence peptide (FP) displacement assay as de-
scribed earlier.13,14 The polarization of a fluorescein-
labeled p53 peptide analog was measured by exciting
at 485 nm and monitoring emission at 530 nm. The
change in polarization upon displacement of the peptide
from HDM2 (residues 17–125) by an antagonist was ex-
pressed as a percent with respect to the fluoresceinated
peptide control.13 Results are summarized in Table 1.
Although compounds 8 and 9 are not soluble enough
to be tested in this standard assay, the diazepine 10
was found to be twofold more potent than the corre-
sponding open structure 7, validating the rigidification
approach suggested by the modeling experiments. This
effect was even stronger with the more constrained bro-
mo derivative 11, which was found to be approximately
Figure 2. Superposition of a model of the low energy conformer of 1,4-
diazepine-2,5-dione 10 (color-by-atom) onto the benzodiazepinedione
6 (purple) bound to HDM2. In the lower panel the Phe sidechain of a
bound peptide from a different complex is also shown (light blue).
The diazepinedione scaffold embodied in 8–11 (Fig. 1)
was designed as an alternative to the benzodiazepinedi-
one parent. The former essentially represents the parent
with the iodophenyl ring removed and modified at the
newly unfused position (Fig. 1). Conformational analy-
sis indicated that the preference for the bound confor-
mation observed for 6 would be maintained in the
modified scaffold. This new scaffold would therefore
provide an alternate mode of access to the Phe19-bind-
ing region, while maintaining the orientation of the
other two side chain mimics of the parent 6 (Fig. 1).
We conjectured that the limited flexibility of the phen-
yl–diazepinedione linkage might allow for improved
(or alternative) access to the deepest part of the Phe19
binding region that is not achievable with the benzodia-
zepinedione framework. In the binding models that were
explored, the acid solubilizing moieties of 10 and 11 are
expected to be largely solvent exposed and to not have a
major impact on binding. This hypothesis was previ-
ously verified in the benzodiazepine series: compounds
bearing a valeryl solubilizing group in position 1 instead
of position 4 were synthesized and found to be equipo-
tent with 6.12 With the aim of verifying this model-based
hypothesis, 10 and 11 were synthesized and tested for
their ability to antagonize the HDM2–p53 protein–pro-
tein interaction. To the best of our knowledge, there is
only one example of the ÔnonfusedÕ 1,4-diazepine-2,5-
dione reported in the literature.18 This cyclic proline
derivative 13 was generated by the cyclofunctionaliza-
tion of an olefinic (S)-N-(a,b-unsaturated)acylprolin-
amide 12 using an interesting organoselenium-induced
cyclofunctionalization (Scheme 1). The structural iden-

1860
P.Raboisson et al./ Bioorg.Med.Chem.Lett.15 (2005) 1857–1861
COOMe
NH2 Br
COOH
COOMe
COOMe
N
N
i
N
H
ii
iv
iii
+
O
Cl
Cl
O
Cl
Cl
Cl
Cl
14
15
16
Cl
O
Cl
Cl
19, R = H
20, R = Br
17, R = H
18, R = Br
R
R
Cl
O
N
Cl
Cl
O
CONH2
N
v
vii
O
vi
Cl
N
O
Se
N
N
Cl
Cl
Cl
R
N
N
H
O
H
O
O
R
R
R
7, R = H
21, R = Br
8, R = H
9, R = Br
22, R = H
23, R = Br
OR'
24, R = H, R' = t-Bu
25, R = Br, R' = t-Bu
10, R = H, R' = H
11, R = Br, R' = H
viii
Scheme 2. Reagents and conditions: (i) K2CO3, TBAI, CH3CN; (ii) PhCH@CHCOCl, pyridine, CHCl3; (iii) NaOH, MeOH, H2O, THF; (iv) NH4Cl,
HOBT, O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate, DIPEA, DMF; (v) PhSeBr, AgOTf, DMF, CH3CN; (vi)
H2O2, THF; (vii) Br(CH2)4COOt-Bu, K2CO3, DMF; (viii) TFA, CH2Cl2.
(Papp apical to basolateral = 27.1 · 10ꢀ6 cm/s). These
promising preliminary results have encouraged us to
Table 1. Binding competition between HDM2 and p53 determined
with the fluorescence peptide (F1 9mer) assay13
Compound
HDM2-FP1 binding IC50 (lM)
undertake systematic structural optimization of the
three phenyl groups in positions 3, 4, and 7 of the diaze-
pine heterocycle, the results of which will be reported in
due course together with cell-based activity.
512
612
712
10
1.7
0.2
30
13
11
3.6
More generally, these results strongly support the
hypothesis that, like the benzodiazepine-2,5-dione scaf-
fold, the 1,4-diazepine-2,5-dione seco bioisoster is mim-
icking the a-helix of the natural p53 protein with respect
to the HDM2 binding interaction. These and related
scaffolds could serve in the future as templates for the
design of novel, nonpeptidic small molecule a-helix pro-
teomimetics in the quest for antagonists of protein–pro-
tein interactions.
10-fold more potent than 7 (IC50 of 3.6 and 30 lM,
respectively). Furthermore, compound 11 was tested in
a CACO-2 cell monolayer assay,19 a widely accepted
in vitro model to predict intestinal drug permeability
in humans.20 The apparent permeability coefficient
(Papp) from the apical to the basolateral side was deter-
mined to be 27.1 · 10ꢀ6 cm/s. Based on this excellent
Papp coefficient, compound 11 is expected to have good
gastrointestinal permeability.19,20
References and notes
In summary, we have described the structure-based de-
sign of an original series of 1,4-diazepine-2,5-diones that
act as antagonists of the HDM2–p53 protein–protein
interaction. These compounds were synthesized enantio-
merically pure, using the previously reported organose-
lenium-induced cyclofunctionalization followed by a
reductive elimination of the seleno moiety and the sub-
sequent introduction of the acid solubilizing group in
position 1. Conformational constraint yielded improved
potency, with the IC50 decreasing from 30 lM for
unconstrained analogue (S)-a-(4-chlorophenyl)-a-[(R)-
N-[1-(4-chlorophenyl)ethyl]-N-(cinnamoyl)amino]acet-
amide (7) to 3.6 lM for the constrained version 5-[(3S)-
7-(2-bromophenyl)-3-(4-chlorophenyl)-4-[(R)-1-(4-chloro-
phenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-yl]valeric acid (11),
providing one of the most potent series of small mole-
cule HDM2 antagonists reported to date. Moreover,
diazepine 11 exhibits good CACO-2 cell permeability
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